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Natural Killer Cell Education and the Response to Infection and Cancer Therapy: Stay Tuned

Natural Killer Cell Education and the Response to Infection and Cancer Therapy: Stay Tuned | Immunology | Scoop.it
The functional capacities of natural killer (NK) cells differ within and between individuals,
reflecting considerable genetic variation. ‘Licensing/arming’, ‘disarming’, and ‘tuning’
are models that have been proposed to explain how interactions between MHC class I
molecules and their cognate inhibitory receptors – Ly49 in mice and KIR in humans
– ‘educate’ NK cells for variable reactivity and sensitivity to inhibition. In this
review we discuss recent progress toward understanding the genetic, epigenetic, and
molecular features that titrate NK effector function and inhibition, and the impact
of variable NK cell education on human health and disease.

Via Krishan Maggon
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JCI - The innate immune receptor TREM-1 promotes liver injury and fibrosis

JCI - The innate immune receptor TREM-1 promotes liver injury and fibrosis | Immunology | Scoop.it
TREM-1 enhances the development of liver fibrosis. A single dose of CCl4 induces centrilobular necrosis and reversible injury that triggers a wound-healing response (23). Repetitive administration of CCl4 promotes progressive fibrogenesis, cirrhosis, and, finally, HCC. To determine the role of TREM-1 in liver injury and fibrogenesis, Trem1–/– and WT mice were injected with 12 doses of CCl4 over a 6-week period that in WT mice induced marked hepatic fibrosis. Trem1–/– mice showed significantly less fibrosis than did WT mice, as analyzed by (a) macroscopic appearance, (b) collagen deposition (Picrosirius red staining, 12.2% ± 1.5% positive area for WT mice, 6.3% ± 0.5% for Trem1–/– mice), and (c) expression of α–smooth muscle actin (α-SMA) (IHC, 33.7% ± 1.6% for WT mice and 17.2% ± 1.5% for Trem1–/– mice, Figure 1, A–E). A significant decrease of hepatic α-SMA expression in Trem1–/– mice compared to WT mice was also observed by immunoblot analyses of total liver proteins (Figure 1, F and G). We also examined fibrosis development using bismuth oxide nanoparticles (Mvivo BIS), a contrast agent designed for small animal liver micro-CT imaging. Following administration, low doses of Mvivo BIS are rapidly taken up by the reticuloendothelial system in the liver, enabling high-definition imaging. After 6 weeks of CCl4 treatment, WT mice showed significantly less uptake of Mvivo BIS nanoparticles in the liver than did Trem1–/– mice (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI98156DS1). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured as indicators of CCl4-induced liver injury. Both groups of mice showed elevated ALT and AST levels 6 weeks after CCl4 treatment. However, ALT and AST levels increased by more than 2-fold and 3-fold, respectively, in WT mice by week 6, while no significant increase was seen in Trem1–/– mice, indicating a greater sensitivity to the development of liver injury in WT mice (Supplemental Figure 2). CCl4-treated WT mice showed significantly higher expression levels of fibrogenic genes that are upregulated in hepatic fibrosis than did Trem1–/– mice, including α1 type 1 collagen (Col1a1), α2 type 1 collagen (Col1a2), α1 type 3 collagen (Col3a1), α1 type 5 collagen (Col5a1), TGF-β1 (Tgfb1), and α-SMA (Acta2) (Supplemental Figure 3). Figure 1 Deletion of Trem1 attenuates hepatic fibrogenesis. (A) Representative macroscopic images of livers from WT and Trem1–/– control mice (oil-injected, n = 3/group, top) and WT and Trem1–/– mice treated with 12 injections of CCl4 over a 6-week period (n = 6–7/group, bottom). Arrowheads indicate fibrotic nodules visible on CCl4-treated WT mice. (B) Collagen deposition was evaluated with Picrosirius red staining. Representative images of liver sections from WT and Trem1–/– control mice (n = 3/group, top) and from WT and Trem1–/– mice treated with CCl4 (n = 6–7/group, bottom). Original magnification, ×20; scale bars: 50 μm. (C) Quantification (percentage) of Picrosirius red–positive areas. (D) Representative images of liver sections from WT and Trem1–/– control mice (n = 3/group, top) and from WT and Trem1–/– mice treated with CCl4 (n = 5–6/group, bottom) stained with anti–α-SMA antibody. Original magnification, ×10; scale bars: 100 μm. (E) Quantification of α-SMA–positive areas (percentage). (F) Immunoblot analysis of α-SMA in liver lysates from the indicated mice (n = 3/group). β-Actin was used as a loading control. The full, uncut gels are shown in the supplemental material. (G) Quantification of α-SMA expression (n = 3 mice/group). Results are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-tailed Student’s t test (C, E, and G). Experiments shown in A, B, and D are representative of 2 independent experiments. TREM-1 is essential for HSC activation. HSCs are the major collagen-producing cells in the fibrotic liver (1). Upon chronic liver injury, HSCs are activated to promote fibrogenesis by a wide range of signals from injured hepatocytes, activated Kupffer cells, inflammatory cells, and liver sinusoidal endothelial cells (LSECs). Upon activation, HSCs release vitamin A and lipid droplets and differentiate into myofibroblasts, which are elongated cells with fibrogenic and contractile activities (24, 25). In control (oil-treated) WT and Trem1–/– mice, HSCs exhibit a quiescent phenotype and store vitamin A and lipid droplets, which display fading blue-green autofluorescence when excited with a light of approximately 405 to 407 nm and detected with a 450- to 50-nm bandpass filter (26) (Figure 2A, top). After 12 doses of CCl4, HSCs from WT mice showed characteristics of activated HSCs (Figure 2A, bottom left). In contrast, most HSCs from CCl4-treated Trem1–/– mice maintained a nonactivated vitamin A–rich round morphology (Figure 2A, bottom right). The role of TREM-1 in HSC activation was confirmed by the observation that mRNA expression of genes upregulated during cell activation and fibrogenesis (Col5a1, Acta2, Mmp10, and Timp1) was significantly higher in CCl4-treated WT mice than in Trem1–/– mice (Figure 2B). Conversely, the transcript levels of genes that are downregulated in activated HSCs (Hhip encoding hedgehog-interacting protein and Plxnc1-encoding plexin C1) were lower in CCl4-treated WT mice than in Trem1-–/– mice (Figure 2C). Figure 2 Deletion of Trem1 attenuates HSC activation and differentiation. HSCs were isolated from oil-injected (Oil) control WT and Trem1–/– mice (n = 3/group) and from WT and Trem1–/– mice treated with CCl4 for 6 weeks (n = 3–4/group). (A) Representative images of freshly isolated HSCs from the indicated mice (oil-injected, top; CCl4-treated, bottom), visualized using a merging of phase-contrast microscopy and retinoid fluorescence (blue channel), show that HSCs from WT CCl4-injured mice differentiated into myofibroblasts and lost their retinoic acid droplets. Original magnification, ×40; scale bars: 25 μm. Images shown are representative of 2 independent experiments. (B and C) Total RNA was isolated from HSC fractions from WT or Trem1–/– mice treated with CCl4 for 6 weeks (n = 3–4/group). Col1a1, Col5a1, Acta2, Mmp10, Edn1, Birc5, Timp1, Hhip, and Plxnc1 mRNA levels were determined by RT-qPCR and are represented as the fold induction. Results are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test. TREM-1 enhances hepatic inflammation during fibrogenesis. Treatment of WT mice with 12 doses of CCl4 elicited extensive changes in liver morphology, as observed in H&E-stained tissue sections, including significantly increased cell infiltration surrounding islands of hepatocytes. In contrast, CCl4-treated Trem1–/– mice showed significantly reduced hepatic cell infiltration compared with WT mice (Figure 3, A and B). Fluorescence immunohistology showed, after CCl4 treatment, a substantially lower number of F4/80-positive cells in the livers of Trem1–/– mice than in WT mice (Figure 3C). Nonparenchymal cells isolated from WT and Trem1–/– livers were analyzed by flow cytometry to evaluate the CCl4-induced accumulation of myeloid cells that are required for HSC activation and hepatic fibrosis (27). While the number of cells expressing the pan-myeloid marker CD11b was similarly upregulated in both mouse strains by CCl4 treatment, the number of F4/80+ cells was markedly reduced in CCl4-treated Trem1–/– mice compared with the number in WT mice (Figure 3, D and E). Liver-associated F4/80+ cells were composed of 2 populations: F4/80+CD11b– cells, corresponding to resident Kupffer cells, and F4/80+CD11b+ cells. Analysis of the cell-surface markers Ly6C and Ly6G revealed that most F4/80+CD11b+ cells were inflammatory monocyte–derived macrophages (F4/80+CD11b+Ly6ChiLy6Glo) at different stages of differentiation (Figure 3D). The number of Kupffer cells and F4/80+CD11b+ cells was not significantly different in the livers of untreated WT and Trem1–/– mice, but after CCl4 treatment, the numbers of both cell types increased and reached significantly higher levels in WT mice than in Trem1–/– mice (Figure 3E). Most F4/80–CD11b+ cells expressed a high level of surface Ly6G (Ly6GhiLy6Clo) (Figure 3D) that identified them as neutrophils. These neutrophils were increased to a similar degree by CCl4 treatment in WT and Trem1–/– mice (Figure 3E). The significant increase in monocyte-derived macrophages (F4/80+CD11b+) in CCl4-treated WT mice compared with Trem1–/– mice was confirmed in situ using a fluorescence multiplexed IHC assay (Supplemental Figure 4, A and B). In both groups of CCl4-treated mice, the abundance of adaptive immune cells such as T cells (CD4+, CD8+) and B cells (B220+) was minimal (Supplemental Figure 4C). These data demonstrate that during fibrosis development, TREM-1 signaling modulates myeloid hepatic inflammation, inducing an increased accumulation of monocyte-derived macrophages and resident Kupffer cells, but not affecting the number of neutrophils. Figure 3 TREM-1 is essential for the recruitment and differentiation of monocyte-derived macrophages during hepatic fibrogenesis. WT and Trem1–/– mice were treated with 12 injections of CCl4 for 6 weeks. (A) Representative images of H&E-stained liver sections stained from WT and Trem1–/– control mice (oil-injected, n = 3/group, top) and WT and Trem1–/– CCl4-injured mice (n = 6/group, bottom). Histology images revealed important mononuclear cell infiltration of fibrotic livers. Original magnification, ×20; scale bars: 50 μm. (B) Quantification (percentage) of cell infiltration areas. (C) Representative images of FITC-conjugated anti-F4/80 antibody–stained liver sections from control (n = 3/group, top) and CCl4-injured (n = 6–7/group, bottom) mice. Original magnification, ×20; scale bars: 50 μm. (D) Flow cytometric dot plots of cells for identification of Kupffer cells (F4/80+CD11b–), monocyte-derived macrophages (F4/80+CD11b+-Ly6ChiLy6Glo), and neutrophils (F4/80–CD11b+Ly6GhiLy6Clo) in liver sections from control WT and Trem1–/– mice (n = 3/group) and CCl4-injured WT and Trem1–/– mice (n = 4/group). Control staining was performed with IgG isotype (gray histograms). APC, allophycocyanin. (E) Percentage of liver-infiltrated cell populations in WT and Trem1–/– mice, analyzed by flow cytometry, after 6 weeks of oil or CCl4 treatment. Results are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test (B and E). Images in A and C are representative of 2 independent experiments. TREM-1 controls the mobilization of inflammatory cells in response to injury and consequently enhances liver damage. To analyze the role of TREM-1 in mediating the recruitment of inflammatory cells during fibrogenesis, we examined the early stage of CCl4-induced liver injury. CCl4 is quickly metabolized by liver cytochrome P450 enzymes into trichloromethyl free radicals that initiate a lipid peroxidation chain reaction leading to hepatocyte death and liver damage (28). Necrotic hepatocytes release damage-associated molecular pattern (DAMP) signaling molecules, including high-mobility group box 1 (HMGB1) protein and HSP70, that induce the activation, proliferation, and recruitment of inflammatory cells (21, 29–32), thus amplifying liver injury and establishing chronic inflammation. Both liver-resident cells and cells that are recruited in response to injury produce proinflammatory signals that contribute to the apoptotic and necrotic damage of hepatocytes. Injection of a single dose of CCl4 increases ALT and AST activity almost equivalently in WT and Trem1–/– mice at 6 hours and 12 hours (Figure 4, A, B, D, and E). However, while these increased levels were maintained in WT mice, they decreased and were reduced at 72 hours in Trem1–/– mice to the levels seen in untreated mice (Figure 4, C and F). These data indicate that TREM-1 signaling contributes to the persistence of the inflammatory response following CCl4 treatment and enhances liver injury. Figure 4 Deletion of Trem1 decreases liver injury at early stages of liver fibrogenesis. WT and Trem1–/– mice were analyzed 6 hours, 12 hours, and 72 hours after injection of a single dose of CCl4. Serum ALT (A–C) and AST (D–F) levels for the indicated mice were measured with colorimetric assay (n = 3–10 mice/time point/group). Results are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test. The bone marrow is one of the sensors of liver injury. A single dose of CCl4 treatment induced only a modest perturbation of the number of total CD11b+ cells, neutrophils, and monocytes in the bone marrow of Trem1–/– and WT mice (Supplemental Figure 5, A–D, and Supplemental Figure 6A). We analyzed peripheral blood cells 12 hours after CCl4 treatment and found a similar increase of CD11b-positive myeloid cells, including neutrophils and monocytes, in both groups of mice. However, 72 hours after CCl4 treatment, the frequency of myeloid cells in the blood was significantly higher in WT mice than in Trem1–/– mice (Supplemental Figure 5, E–H, and Supplemental Figure 6B). A much higher blood accumulation of patrolling monocytes (CD11b+F4/80+CCR2–CX3CR1+) (33) was induced by CCl4 injection into WT mice compared with Trem1–/– mice (Supplemental Figure 5H). However, we observed the most dramatic changes in the livers of CCl4-treated mice. The number of myeloid cells, and especially neutrophils (Supplemental Figure 6C), progressively increased at both 12 hours and 72 hours and was significantly higher in livers from WT mice than in those from Trem1–/– mice (Figure 5, A–C). The number of monocyte-derived macrophages (CD11b+F4/80+Ly6Chi) (Supplemental Figure 6C) was greatly increased in the injured livers of WT mice, but these monocyte-derived macrophages were almost absent in the livers of Trem1–/– mice (Figure 5D). These cells were CCR2+CX3CR1lo and produced IL-1β, TNF, and TGF-β1 (Figure 5A), which characterized them as infiltrating inflammatory monocyte–derived macrophages (34). The expression of genes involved in the recruitment and maintenance of inflammatory cells, including Ccl2, Ccl7, Cxcl10, Tnf, Il1b, and Il6, was induced by CCl4 (12 hours and 72 hours after CCl4 injection) in the livers of WT mice, but not in those of Trem1–/– mice (Supplemental Figure 7). Thus, our data reveal a key role of TREM-1 in the mobilization, recruitment, and differentiation of inflammatory cells to the site of inflammation and injury. Figure 5 Deletion of Trem1 reduces inflammatory cell infiltration at early stages of liver fibrogenesis. (A) Flow cytometric dot plots of liver cells from control WT and Trem1–/– mice (oil-injected, n = 3/group) and WT and Trem1–/– mice 12 hours and 72 hours after CCl4 injury (n = 7 mice/time point/group). Cells were stained with anti-F4/80, anti-CD11b, anti-CCR2, anti-CX3CR1, anti–IL-1β, anti-TNF, and anti–TGF-β1 antibodies. Liver cells from WT mice were stained and analyzed 72 hours after CCl4 injection for the expression of CCR2 and CX3CR1 (n = 5 mice) and for the intracellular expression of IL-1β, TNF, and TGF-β1 (n = 7 mice). Flow cytometric histograms represent cells gated on a F4/80+CD11b+ population. Control staining was performed with IgG isotype (gray histograms). (B–D) Percentage of CD11b+ cells, neutrophils, and inflammatory monocyte–derived macrophages in liver from WT and Trem1–/– mice quantified after 12 hours and 72 hours of oil injection (n = 3 mice/time point/group) or CCl4 treatment (n = 7 mice/time point/group). Results are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test (B, C, and D). TREM-1 expression in the liver and the profibrogenic signature of activated Kupffer cells increase during fibrogenesis. Total RNA was isolated from the livers of oil- or CCl4-injected WT mice and analyzed by real-time quantitative PCR (RT-qPCR). Trem1 mRNA accumulation was increased at 12 hours, 72 hours, and 6 weeks after CCl4 treatment, with a maximum 20-fold induction observed at 12 hours (Figure 6A). TREM-1 protein levels in the liver were also increased by CCl4 injection, with a peak detected at 72 hours (Figure 6, B and C). Fluorescence immunostaining of liver from WT mice at different time points confirmed the increase in TREM-1 expression in response to CCl4 (Figure 6D). To determine the cell types that express TREM-1 in normal liver, RT-PCR was performed on total RNA isolated from whole liver, purified hepatocytes, Kupffer cells, and HSCs from WT mice; we found that only Kupffer cells expressed Trem1 (Figure 6E). Flow cytometric analysis confirmed that TREM-1 protein was present only on Kupffer cells, and its density on these cells was increased at 12 hours, and to an even greater degree at 72 hours, in CCl4-induced liver injury (Figure 6, F and G). The increased levels of TREM-1 expression on Kupffer cells following CCl4 treatment may be responsible for the production by Kupffer cells of inflammatory chemokines, cytokines, and growth factors. Figure 6 Expression of Trem1 in liver during fibrogenesis. (A) WT mice were treated with oil (control, n = 3) or a single dose of CCl4 and analyzed after 12 hours and 72 hours (n = 5 mice/time point). Another group of WT mice was treated with 12 injections of CCl4 and analyzed after 6 weeks (n = 7 mice). mRNA levels of Trem1 in liver were assessed by RT-qPCR. Expression was normalized to the average of 3 different control genes (Actb, Gapdh, and Hprt1) and is represented as the fold induction. (B) Representative immunoblot analysis for TREM-1 in WT mouse liver lysates at different time points. β-Actin was used as a loading control. The full, uncut gels are shown in the supplemental material. (C) Quantification of TREM-1 expression in liver from WT control mice (n = 3) and CCl4-treated mice at 12 hours (n = 5), 72 hours (n = 3), and 6 weeks (n = 3). (D) Representative phycoerythrin-conjugated (PE-conjugated) anti–TREM-1 antibody–stained immunofluorescence images of liver sections from WT control oil-injected mice (n = 3, top) and CCl4-treated mice at 12 hours (top), 72 hours, and 6 weeks (n = 5–7 mice/time point/group, bottom). Original magnification, ×20; scale bars: 50 μm. Images are representative of 2 independent experiments. (E) RT-qPCR was performed to assess Trem1 mRNA expression in whole liver as well as in purified hepatocyte, Kupffer cell, and HSC fractions from WT mice (n = 3 mice/cell fraction). (F) Flow cytometric dot plots of WT control mouse liver cells (n = 9 mice) stained with anti-F4/80 and anti-CD11b antibodies. Flow cytometric histograms represent TREM-1 expression on a gated F4/80–CD11b– cell population and on gated F4/80+CD11b– Kupffer cells (n = 9 mice). Control staining was performed with IgG isotype (gray histogram). (G) Flow cytometric dot plots and histograms of TREM-1 expression on gated F4/80+CD11b– Kupffer cells isolated from oil-injected WT control mice (n = 9) and from mice treated with CCl4 (n = 7 mice/time point/group) for 12 hours and 72 hours. Control staining was performed with IgG isotype (gray histogram). (H) Flow cytometric histograms of intracellular expression of IL-1β, TNF, and TGF-β1 on gated F4/80+CD11b– Kupffer cells from CCl4-injured (72 h) WT (n = 4) and Trem1–/– (n = 5) mice. Control staining was performed with IgG isotype (gray histograms). (I) Quantification of mean fluorescence intensity of the indicated cytokines. Results are displayed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by ANOVA followed by Bonferroni’s post hoc test (A and C) and 2-tailed Student’s t test (I). RNA-sequencing (RNA-seq) data showed that CCl4 treatment of WT mice resulted in a major shift of the transcriptomic profile of Kupffer cells, with approximately 1,000 genes differentially expressed, of which approximately 360 were increased and 640 decreased. Interestingly, the large majority of the genes that were induced or decreased by CCl4 treatment in WT mice were not modified or were modified to a significantly lesser degree than was seen in Trem1–/– mice (Supplemental Figure 8A). The similarity between Kupffer cells from untreated WT mice and those from CCl4-treated Trem1–/– mice was further demonstrated by correlation analysis of log2-transformed ratios of Kupffer cell transcripts from CCl4-treated WT mice versus those from CCl4-treated Trem1–/– mice or untreated WT mice. The R value between the 2 ratios was 0.76, with a P value of less than 10–4. Among the genes encoding growth factors that were upregulated by Kupffer cells in WT but not in Trem1–/– mice after CCl4 treatment were factors already shown to play a role in liver fibrosis, such as Tgfb1, Jag1 (jagged 1), and Osm (oncostatin M), as well as other factors, such as β-cellulin (Btc, a member of the EGF family), angiotensinogen (Agt), inhibin β A (Inhba), and macrophage-stimulating 1, hepatocyte growth factor–like (Mst1). The expression of genes encoding cytokines and chemokines was upregulated in WT mice, but not in Trem1–/– mice. These included the cytokine gene Il23a and the chemokine genes Ccl9, Cxcl2, and Cxcl3, whose expressed proteins attract inflammatory and immune cell migration via CCR1 and CXCR2 and affect their processes of differentiation and function (Supplemental Figure 8B). In addition, total liver expression of TGF-β1 was significantly higher in WT mice than in Trem1–/– mice (Supplemental Figure 9). These data support the notion that TGF-β1 is the major regulator of liver fibrogenesis. We detected significantly decreased TNF and TGF-β1 production in Kupffer cells from Trem1–/– mice 72 hours after CCl4 injury, as shown in Figure 6, H and I. Lack of upregulation of the Ccl9, Cxcl2, Cxcl3, Il1f9, and Il23a genes, as well as the decreased levels of TNF and TGF-β1 in Trem1–/– mice suggest that deletion of the Trem1 gene alters Kupffer cell activation during the CCl4-induced inflammatory response. Adoptive transfer of Trem1-sufficient Kupffer cells into Trem1-deficient mice reconstitutes the impaired myeloid inflammatory response following CCl4-induced injury. To test whether the impaired activation of Kupffer cells in Trem1–/– mice plays a major role in the reduced response of liver injury and recruitment of inflammatory cells to CCl4-induced liver damage, we depleted Kupffer cells in WT and Trem1–/– mice by treatment with clodronate-containing liposomes (Figure 7A, top), followed by intravenous reconstitution with Kupffer cells isolated from WT mice and CCl4 injection (Figure 7A, bottom). We observed that a similar number of transferred Kupffer cells migrated at 18 hours into the livers of depleted WT and Trem1–/– recipient mice. Trem1–/– mice in which Trem1-sufficient Kupffer cells were transferred recovered the ability to increase serum ALT and AST enzymes 72 hours after CCl4 injection at comparable levels, as observed in WT mice (Figure 4, C and F, and Figure 7, B and C). Adoptively transferred Trem1-sufficient Kupffer cells produced comparable amounts of TNF and TGF-β1 in WT and Trem1–/– recipient mice following CCl4 treatment (Figure 7, D–F). The recruitment of peripheral blood neutrophils after CCl4 treatment was comparable between Trem1–/– and WT mice reconstituted with Trem1-sufficient Kupffer cells (Supplemental Figure 10, A and B). Also, CCl4 treatment of Trem1–/– mice reconstituted with Trem1-sufficient Kupffer cells induced a similar increase in the number of CD11b+F4/80+ patrolling monocytes/macrophages at 72 hours, as observed in CCl4-injured WT mice (Supplemental Figure 10, A and C). The numbers of inflammatory monocytes and neutrophils recruited 72 hours after CCl4 treatment to the livers of Trem1–/– mice reconstituted with Trem1-sufficient Kupffer cells were comparable to those observed in WT mice, although the numbers were lower than in WT mice reconstituted with Trem1-sufficient Kupffer cells (Figure 7, D, G, and H). Conversely, adoptive transfer of Trem1-deficient Kupffer cells into WT mice resulted in reduced CCl4-induced liver injury and altered recruitment of neutrophils as well as of patrolling and inflammatory monocytes/macrophages (Supplemental Figure 11, A–H). These data indicate that transferred Trem1-sufficient Kupffer cells were activated in Trem1–/– mice in response to CCl4 exposure and could induce liver injury, while the transfer of Trem1-deficient Kupffer cells into WT mice protected them from CCl4-induced liver injury. Figure 7 Adoptive transfer of Kupffer cells from WT mice increases liver injury and recruitment of inflammatory cells in Trem1–/– mice. (A) Representative images of FITC-conjugated anti-F4/80 antibody–stained liver sections from the indicated mice 72 hours after injection of clodronate-containing liposomes (n = 3 mice/group, top), followed by reconstitution of predepleted livers with WT Kupffer cells (n = 3 mice/group). Adoptively transferred cells were observed at 72 hours by immunofluorescence staining. Original magnification, ×40; scale bars: 20 μm. Images shown are representative of 2 independent experiments. Levels of (B) ALT and (C) AST in serum from WT and Trem1–/– mice with adoptive transfer of WT Kupffer cells followed by a single dose of CCl4 treatment were measured 72 hours after CCl4 injury by colorimetric assay (n = 3–5 mice/group). (D) Macrophage-depleted WT and Trem1–/– mice were adoptively transferred with WT Kupffer cells following treatment with a single dose of CCl4. Flow cytometric dot plots of liver cells stained with anti-F4/80, anti-CD11b, anti-TNF, anti–TGF-β1, anti-Ly6C, anti-Ly6G, anti-CCR2, and anti-CX3CR1 antibodies (n = 3 mice/group). Flow cytometric histograms of Ly6C, Ly6G, CCR2, and CX3CR1 expression shown on gated F4/80+CD11b+ and F4/80–CD11b+ cells (n = 3 mice/group). Control staining was performed with IgG isotype (gray histograms). Mean fluorescence intensity of TNF (E) and TGF-β1 (F) in the indicated mice is shown for gated F4/80+CD11b– Kupffer cells (n = 3 mice/group). (G and H) Percentage of liver-infiltrated neutrophils and inflammatory monocytes under the indicated experimental conditions in WT and Trem1–/– mice (n = 3–7/group). Results are displayed as the mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test (B, C, E, and F) and ANOVA followed by Bonferroni’s post hoc test (G and H). AT, adoptive transfer. Increased liver infiltration with TREM-1–positive cells, including Kupffer cells and monocytes/macrophages, in patients with hepatic fibrosis. Human liver tissues from a control group (n = 6) and from patients with liver fibrosis (n = 5) were analyzed for fibrosis biomarkers, TREM-1 expression, and phenotype. We found that collagen deposition, evaluated by Masson’s trichrome staining, and α-SMA expression levels were significantly increased in patients with advanced liver fibrosis (Figure 8, A–C, left panels, and D, and Supplemental Figures 12 and 13). In normal liver, most TREM-1–positive cells were found in and around the hepatic sinusoid, which is the primary location of Kupffer cells and of the morphologically and functionally unique LSECs (Figure 8C, middle and right panels). Since LSECs play a major role in liver regeneration after liver injury, it will be interesting to determine the role of TREM-1 in LSEC functions, especially in their crosstalk with HSCs during fibrogenesis. Healthy (noncapillarized) LSECs prevent and reverse the activation of HSCs (35, 36). However, during fibrosis, capillarized LSECs lose the ability to antagonize HSC activation (36). We detected a similar number of TREM-1–positive cells in normal liver tissues from the control group and in nonfibrotic areas from patients with advanced liver fibrosis (31.47% ± 13.47% and 27.25% ± 6.8%, respectively) (Figure 8C, middle panels). In contrast, we observed an increase in the number of TREM-1–positive cells in fibrotic areas compared with nonfibrotic areas (82.04% ± 9.68%, Figure 8, C, middle panels, and E, and Supplemental Figure 14). TREM-1–positive cells that express CD68, a marker of Kupffer cells, were significantly increased in fibrotic areas (Figure 8, F and H, and Supplemental Figure 15). Other TREM-1–positive cells that were also substantially increased in fibrotic areas expressed the CD11b marker and were most markedly increased in these fibrotic areas; the majority of these cells were myeloid cells, including monocytes and monocyte-derived macrophages (Figure 8, F and G, and Supplemental Figure 16). Together, these data suggest that human liver fibrosis is associated with the recruitment and differentiation of TREM-1–positive Kupffer cells and monocytes and monocyte-derived macrophages. Figure 8 Increased TREM-1–, CD11b-, and CD68-positive cell infiltration in human liver samples from patients diagnosed with advanced fibrosis. (A) Representative images of human liver samples from a control subject (Metavir = F0, n = 6) and a patient diagnosed with advanced fibrosis (Metavir = F3/F4, n = 4). Samples were stained with Masson’s trichrome to assess collagen deposition. Original magnification, ×10; scale bars: 100 μm. (B) Quantification (percentage) of Masson’s trichrome–positive areas in liver samples from a control subject (n = 10 areas) and a patient with advanced fibrosis (n = 13 areas). (C) Fluorescence multiplexed IHC of human liver samples. Samples from a control subject and a patient with advanced fibrosis were stained for α-SMA (red), TREM-1 (green), and DAPI (blue). Original magnification, ×20; scale bars: 50 μm. (D) Quantification of α-SMA–positive cells (percentage) from a control subject and a patient with advanced fibrosis (n = 10 areas vs. n = 15 areas, respectively. (E) Quantification (percentage) of TREM-1–positive cells from a control subject and a patient with advanced fibrosis (n = 9 and n= 14 areas, respectively). (F) Samples from a control subject and a patient with advanced fibrosis were stained for CD11b (orange), CD68 (magenta), TREM-1 (green), and DAPI (blue). Original magnification, ×20; scale bars: 50 μm. (G) Quantification (percentage) of CD11b-positive cells from a control subject and a patient with advanced fibrosis (n = 10 vs. n = 15 areas, respectively). (H) Quantification (percentage) of CD68-positive cells from a control subject and a patient with advanced fibrosis (n = 8 and n = 12 areas, respectively). Data are displayed as the mean ± SEM. ****P < 0.0001, by 2-tailed Student’s t test (B, D, E, G, and H).
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The 10,000 Immunomes Project: Building a Resource for Human Immunology

Zalocusky et al. report the development of a data resource comprising curated, integrated,
and normalized immunology measurements from all healthy normal human subjects in the
ImmPort database.
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Transient T-bet expression functionally specifies a distinct T follicular helper subset

Transient T-bet expression functionally specifies a distinct T follicular helper subset | Immunology | Scoop.it
T follicular helper (Tfh) cells express transcription factor BCL-6 and cytokine IL-21. Mature Tfh cells are also capable of producing IFN-γ without expressing the Th1 transcription factor T-bet. Whether this IFN-γ–producing Tfh population represents a unique Tfh subset with a distinct differentiation pathway is poorly understood. By using T-bet fate–mapping mouse strains, we discovered that almost all the IFN-γ–producing Tfh cells have previously expressed T-bet and express high levels of NKG2D. DNase I hypersensitivity analysis indicated that the Ifng gene locus is partially accessible in this “ex–T-bet” population with a history of T-bet expression. Furthermore, multicolor tissue imaging revealed that the ex–T-bet Tfh cells found in germinal centers express IFN-γ in situ. Finally, we found that IFN-γ–expressing Tfh cells are absent in T-bet–deficient mice, but fully present in mice with T-bet deletion at late stages of T cell differentiation. Together, our findings demonstrate that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in this Th1-like Tfh cell subset.
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Viruses | Free Full-Text | Interplay between Cellular Metabolism and Cytokine Responses during Viral Infection

Viruses | Free Full-Text | Interplay between Cellular Metabolism and Cytokine Responses during Viral Infection | Immunology | Scoop.it
Metabolism and immune responses are two fundamental biological processes that serve to protect hosts from viral infection. As obligate intracellular pathogens, viruses have evolved diverse strategies to activate metabolism, while inactivating immune responses to achieve maximal reproduction or...
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JCI - Inhibition of neogenin fosters resolution of inflammation and tissue regeneration

Research ArticleImmunologyInflammation Free access | 10.1172/JCI96259 Inhibition of neogenin fosters resolution of inflammation and tissue regeneration Martin Schlegel,1 Andreas Körner,1 Torsten Kaussen,2 Urs Knausberg,1 Carmen Gerber,1 Georg Hansmann,2 Hulda Soffia Jónasdóttir,3 Martin Giera,3 and Valbona Mirakaj1 First published September 17, 2018 - More info Abstract The resolution of inflammation is an active process that is coordinated by endogenous mediators. Previous studies have demonstrated the immunomodulatory properties of the axonal guidance proteins in the initial phase of acute inflammation. We hypothesized that the neuronal guidance protein neogenin (Neo1) modulates mechanisms of inflammation resolution. In murine peritonitis, Neo1 deficiency (Neo1–/–) resulted in higher efficacies in reducing neutrophil migration into injury sites, increasing neutrophil apoptosis, actuating PMN phagocytosis, and increasing the endogenous biosynthesis of specialized proresolving mediators, such as lipoxin A4, maresin-1, and protectin DX. Neo1 expression was limited to Neo1-expressing Ly6Chi monocytes, and Neo1 deficiency induced monocyte polarization toward an antiinflammatory and proresolving phenotype. Signaling network analysis revealed that Neo1–/– monocytes mediate their immunomodulatory effects specifically by activating the PI3K/AKT pathway and suppressing the TGF-β pathway. In a cohort of 59 critically ill, intensive care unit (ICU) pediatric patients, we found a strong correlation between Neo1 blood plasma levels and abdominal compartment syndrome, Pediatric Risk of Mortality III (PRISM-III) score, and ICU length of stay and mortality. Together, these findings identify a crucial role for Neo1 in regulating tissue regeneration and resolution of inflammation, and determined Neo1 to be a predictor of morbidity and mortality in critically ill children affected by clinical inflammation. Introduction There are only a few targeting therapies for critically ill patients in the intensive care unit (ICU) who are suffering from complex and potentially life-threatening illnesses, such as acute respiratory distress syndrome and multiple organ failure. Hence, severe inflammation is recognized as a considerable problem in the care of these critically ill patients when resolution of inflammation fails to induce homeostasis (1). It is evident that nonresolving inflammation may lead to the activation of chronic inflammatory processes and ultimately to the development of organ dysfunction and the incurrence of comorbidities (2, 3). The initiation of this pivotal process is guided by diverse classes of mediators including cytokines, chemokines, and lipid mediators (3, 4). These mediators initiate the influx of proinflammatory cells that cause tissue injury. Following the initiation of an inflammatory response, when self-limited, a superfamily of endogenous mediators (SPMs) is generated to activate processes for resolution, indicating that the resolution of inflammation is a process that is distinct from antiinflammatory mechanisms (5). The key steps in this phase include (a) cessation of further PMN influx, (b) normalization of chemokine/cytokine gradients, (c) apoptosis of PMNs, (d) activation of macrophage (MΦ) phagocytosis and efferocytosis, and (e) generation of endogenous proresolving mediators (i.e., SPMs). A paradigm for neuronal guidance proteins (NGPs) and their target receptors exists in the developing nervous system, where neuronal movement is mediated by the interplay of both attractive and repulsive signals. Analogies with axonal migration have postulated that these NGPs play an important role outside the central nervous system in guiding leukocyte migration (6–11). Neo1, a type I transmembrane protein and receptor for Netrin-1 and the repulsive guidance molecules (RGMs), is recognized to be essential in neurogenic and embryonic processes, in which it contributes to chondrogenesis, myogenesis, organ-specific development of the mammary gland, and neural tube formation (12–14). Recent studies have shown Neo1 to have pivotal nonneuronal functions during the onset of acute inflammation (9, 15, 16). However, the primary issue with inflammation is not the frequency of its initiation, but rather the formation of excessive or unresolved processes (2, 5). This notion, coupled with its immunomodulatory attributes, led us to question whether Neo1 might contribute to local inflammation resolution mechanisms and tissue regeneration processes. Our studies revealed that functional inhibition of Neo1 induced apoptosis of neutrophils, which is a key feature of the initiation of the inflammation resolution mechanism (17) and ultimately shortened the neutrophil lifespan. Functional inhibition of Neo1 activated eat-me and find-me signals and G protein–coupled receptors (GPCRs) in human apoptotic PMNs or macrophages (MΦ) to mediate proresolving actions. In a model of murine peritonitis, we found that deficiency of Neo1 led to antiinflammatory, proresolving, and proregenerative effects, as shown by reduced PMN infiltration to the site of inflammation, increased neutrophil apoptosis, enhanced local clearance via phagocytosis of apoptotic cells, and the biosynthesis of local endogenous proresolving lipid mediators (i.e., lipoxin A4 [LXA4], maresin-1 [Mar1], and protectin DX [PDX]). Neo1 expression was particularly restricted to the inflammatory peritoneal Ly6Chi cells, and Neo1 deficiency induced monocyte polarization toward the proresolving and prohealing Ly6Clo phenotype. Bone marrow transplant chimeric mouse experiments showed hematopoietic Neo1 repression to be crucial for the reduction of Ly6Chi monocytes, the increase of Ly6Clo monocytes, and finally, the increase in clearance. In our analysis, we found Neo1–/– monocytes to activate the PI3K/AKT pathway and suppress the TGF-β pathway, both of which are critical in restricting proinflammatory and promoting antiinflammatory responses and activating the monocyte and monocyte-derived MΦ polarization toward the proresolving phenotype. In line with these results, in an observational clinical study that included 59 critically ill ICU pediatric patients suffering from, in part, intraabdominal hypertension (IAH), abdominal compartment syndrome (ACS), internal cardiac and oncological diseases, or being cared for after surgical interventions, we found a strong correlation among plasma Neo1 levels and IAH, ACS, severity of illness, ICU length of stay, and survival. Our studies indicate a critical role for Neo1 in controlling processes involved in the inflammation resolution and tissue regeneration phases, and they may represent an advance in our understanding of the pathways that can restrain or promote the resolution of inflammation. In turn, our data may help identify new potential targets in diseases of major global health significance. Results Impact of Neo1 on human PMN apoptosis and macrophage efferocytosis. It is now evident that the failed clearance of dying cells alters immune tolerance and promotes nonresolving inflammation (2, 17). In the early phase of inflammation, apoptosis of neutrophils induces neutrophil functional shutdown, which is a key feature of the initiation of inflammation resolution mechanisms (18, 19). We therefore sought to investigate whether Neo1 plays a role in the apoptosis of neutrophils. Human PMNs were stimulated with vehicle or LPS (100 ng/ml) and/or anti–Neo1 antibody (Ab) and then allowed to undergo apoptosis for 20 hours. Functional inhibition of Neo1 induced the apoptosis of neutrophils, suggesting that anti-Neo1 treatment shortened the neutrophil lifespan (Figure 1A). In apoptotic PMNs, blockade of Neo1 induced the expression of the decoy receptor IL-1R2, which is known for its strong impact on limiting the proinflammatory effects of IL-1β (Figure 1A) (20). It is evident that apoptotic neutrophils induce their own clearance by expressing find-me and eat-me signals (17, 21). Therefore, we sought to determine the expression of CX3CL1, a critical protein contributing as a find-me signal in MΦ, and its receptor CX3CR1, which is crucial for sensing chemokines and recruiting monocytes (22). We found that blockade of Neo1 markedly increased both the CX3CL1 mRNA in apoptotic PMNs and the CX3CR1 mRNA in MΦ. These data were substantiated by increased levels of one of the most crucial eat-me receptors, TIM4, which mediates the direct recognition of phosphatidylserine by MΦ. (Figure 1B). In addition to neutrophil apoptosis, MΦ efferocytosis is a key feature of resolution programs. We therefore set out to investigate the expression of Neo1 in human MΦ and PMNs (Figure 1C and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI96259DS1). Our data revealed a strong induction of Neo1 expression in MΦ following IL-1β stimulation. We found that the functional inhibition of Neo1 significantly increased the phagocytosis rate in a dose-dependent manner. These results were confirmed by immunofluorescence analysis (Figure 1D). In subsequent E. coli efferocytosis studies as a surrogate for infection-resolving actions, we were able to corroborate our results (Figure 1E). Given that GPCRs such as ALX/FPR2 and DRV1/GPR32 have been shown to mediate proresolving actions (23), we demonstrated that stimulation with anti-Neo1 plus IL-1β significantly augmented the mRNA levels of these 2 receptors in MΦ (Figure 1F). These findings showed that functional inhibition of Neo1 plays an important regulatory role in resolving inflammation, by sensing and detecting dying and apoptotic neutrophils in the early stages of inflammation and by phagocytizing and subsequently removing them in later stages of inflammation. Figure 1 Role of Neo1 on human PMN apoptosis and MΦ efferocytosis. (A) Apoptosis of human PMNs following LPS and/or anti-Neo1 stimulation was determined by flow cytometry and the expression of CX3CL1 mRNA and IL-1R2 mRNA was evaluated by RT-PCR. (B) Human MΦ were stimulated with IL-1β and/or anti-Neo1 antibody and the CX3CR1 mRNA and TIM4 mRNA levels were determined by RT-PCR. (C) Neo1 protein expression was assessed by immunofluorescence staining (n = 3/condition, magnification ×630, scale bar 20 μm). (D) The dose-dependent impact of anti-Neo1 treatment on MΦ clearance of the apoptotic PMNs and the corresponding immunofluorescence images (n = 3/condition, magnification ×400, scale bar 20 μm). (E) MΦ efferocytosis of E. coli. (F) mRNA expression of the ALX/FPR2 and GPR32 receptors in human MΦ. Results represent 2 independent experiments and are expressed as median ± 95% CI (n = 6–8 per group). Statistical analysis was done by ANOVA followed by Bonferroni’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001. Mice deficient in Neo1 display a reduction in PMN recruitment, enhancement of neutrophil apoptosis, and augmentation of efferocytosis. Based on the results described above, we hypothesized that Neo1 is a major player in the active resolution of acute inflammatory responses. As mentioned before, key characteristics of resolution are the cessation of neutrophil migration, the enhancement of uptake and clearance of apoptotic cells and microorganisms in inflamed tissues, and the biosynthesis of proresolving mediators (5). Using mice deficient in Neo1 (Neo1–/–), we modeled a self-limited resolving murine peritonitis and examined the cellular events in both the early phase and the resolution phase. In a time series (4 hours, 12 hours, 24 hours, and 48 hours), wild-type (WT) littermates displayed maximal PMN infiltration at 4 hours in the peritoneal exudates (Figure 2A), followed by a reduction, providing a resolution interval (Ri) of 27 hours. In Neo1–/– mice, we found a strong reduction in leukocyte recruitment, with a shift of maximal PMNs to 12 hours and a resolution interval of 9 hours (Figure 2B). Additionally, TNF-α and IL-1β, 2 well-known proinflammatory cytokines that mediate the inflammatory response and contribute to apoptotic cell death, were significantly decreased (Figure 2C). Neutrophil apoptosis, as an important control point in resolution processes, was enhanced in Neo1–/– mice compared with the littermate control animals (Figure 2D). This was accompanied by a significant reduction in the classical Ly6Chi monocytes and an increase in the alternatively activated Ly6Clo monocytes and peritoneal MΦ Neo1–/– mice versus controls (Figure 2E). In this context, the phagocytosis of apoptotic neutrophils was strongly enhanced in Neo1–/– mice, suggesting that Neo1 affects a delayed-resolution phenotype in acute peritonitis (Figure 2F). To underline the influence of Neo1 on the resolution phase, we then measured the levels of IL-6, KC, MIP2, and MCP-1 within the peritonitis lavages collected 12 hours after zymosan A (ZyA) injection and found significantly decreased levels in Neo1-deficient exudates compared with WT controls (Supplemental Figure 2A). To further substantiate the temporal pattern of Neo1-mediated influence on efferocytosis and tissue homeostasis, we collected peritoneal MΦ either from WT or Neo1–/– mice, and the phagocytosis of fluorescent ZyA particles was determined at 2, 4, and 6 hours after injection. Our data demonstrate that depletion of Neo1 markedly increases the phagocytosis rate at the indicated time points (Supplemental Figure 2B). Together, these data point to a role for Neo1 in the initiation and resolution of inflammatory processes, particularly in the removal of apoptotic cells. Figure 2 Targeted deletion of Neo1 promotes resolution of acute inflammation. Neo1–/– mice and their littermate controls were exposed to ZyA-induced peritonitis, and peritoneal lavages were collected at 4, 12, 24, and 48 hours. (A) The leukocyte total was enumerated by light microscopy and the PMNs were determined by flow cytometry. (B) Resolution indices, as previously defined (41). (C) The IL-1β and TNF-α levels were measured in the peritoneal fluids (4 hours after ZyA injection). (D) Apoptotic PMNs (12 hours after ZyA injection), (E) classical Ly6Chi monocytes, nonclassical Ly6Clow monocytes, F4/80+ peritoneal MΦ, and (F) monocyte-derived MΦ efferocytosis were determined by flow cytometry. The results represent 2 independent experiments and are expressed as mean ± SEM (A, E–F) and as median ± 95% CI (C, D) (n = 8–12 per group). Statistical analysis was done by unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. Neo1 expression is confined to peritoneal Ly6Chi monocytes. After demonstrating that Neo1 controls apoptosis and phagocytosis programs in vitro and in vivo, we next aimed to investigate more precisely the role of Neo1 in the regulatory mechanisms underlying these processes. It is evident that monocytes derived from precursors in the bone marrow circulate first in the blood, and from there into tissues to mature to macrophages (24). Knowing that Ly6C is mainly expressed on the migrating inflammatory monocyte population with less expression on the alternatively activated monocytes (25), we determined the Ly6C expression in bone marrow monocytes (BMMs) and the peritoneal monocytes in WT and Neo1–/– mice 12 hours after ZyA injection. Interestingly, we found that proinflammatory Ly6Chi monocytes had increased Ly6C expression after leaving the bone marrow and migrating into the peritoneal cavity (Figure 3, A and B). This increase in Ly6C expression was significantly reduced in peritoneal Neo1–/– monocytes compared with littermate controls. We then examined the Neo1 expression in BMMs and peritoneal monocytes and found Neo1 expression to be specifically restricted to the peritoneal inflammatory Ly6Chi monocytes (Figure 3C). Figure 3 Neo1-dependent monocyte intracellular signaling. WT and Neo1–/– bone marrow monocytes and WT and Neo1–/– peritoneal monocytes were collected 12 hours after ZyA injection. (A) Schematic illustration of maturation patterns in WT and Neo1–/– monocytes. (B) Ly6C expression and (C) Neo1 expression in bone marrow monocytes and peritoneal monocytes were determined by flow cytometry. The results represent 2 independent experiments and are expressed as median ± 95% CI (n = 8–12 per group). Statistical analysis was done by 1-way ANOVA followed by Bonferroni’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001. To more precisely determine the migration patterns of Ly6Chi monocytes, we generated chimeric animals through bone marrow transplantation between Neo1+/+ and Neo1–/– mice and vice versa, with WT to Neo1+/+ and Neo1–/– to Neo1–/– transplanted animals as controls for nonspecific radiation effects. We then exposed the chimeric animals to ZyA peritonitis and analyzed the cellular events in defined time intervals (4 hours and 12 hours). Bone marrow chimeric animals with hematopoietic Neo1 repression demonstrated a strong reduction in the classical Ly6Chi, an increase in the nonclassical Ly6Clo monocytes, and finally an increase of the MΦ phagocytosis of apoptotic PMNs in both time points (Supplemental Figure 3, A and B). Ly6C, known to be mainly expressed on migrating proinflammatory monocytes, was significantly reduced in peritoneal Neo1–/– monocytes compared with littermate controls (Figure 3B). This effect was also reflected in the bone marrow chimeric animals with hematopoietic Neo repression (Supplemental Figure 3C). We were able to show that Ly6C MFI is strongly decreased in Ly6Chi cells in bone marrow chimeric animals with hematopoietic Neo repression, suggesting that Neo1 impacts the proinflammatory Ly6Chi monocytes. These findings suggest that upon activation and migration to the site of inflammation, Ly6Chi monocytes induce Neo1 expression, enabling them to contribute to the immune response. Neo1-dependent monocyte intracellular signaling. To investigate the mechanisms by which Neo1 impedes the resolving/regenerative effects, peritoneal monocytes from WT and Neo1–/– mice were collected for microarray analysis 12 hours after ZyA induction of peritonitis (Figure 4 and Figure 5). Analysis of protein microarray data demonstrated that Neo1–/– monocytes activate the PI3K/AKT pathway, which is crucial in restricting proinflammatory responses, promoting antiinflammatory responses, and activating the monocyte differentiation and polarization toward a proresolving phenotype (Figure 4, Figure 5B, Supplemental Figure 1, and Supplemental Table 1) (26, 27). The expression and/or phosphorylation of enzymes such as AKT1, AKT2, MAPK1, MAPK3, mTOR, PIK3R1, and PIK3R3, which are required for AKT activity, were increased in Neo1–/– monocytes. The TGF-β pathway plays divergent roles in the innate immune system (28). Our microarray data on Neo1–/– monocytes revealed TGF-β signaling to be activated specifically in the context of cell apoptosis, whereas in WT monocytes TGF-β signaling is induced and associated with proinflammatory monocyte migration, fibrosis, chronic inflammation, and cell survival (Figure 5A, Supplemental Figure 4, and Supplemental Table 1). Collectively, these findings provide evidence that deficiency of Neo1 contributes to proresolving and proregenerative actions in monocytes and ultimately in MΦ, and this action is associated with the PI3K/AKT/mTOR and TGF-β signaling pathways. Figure 4 Neo1-dependent monocyte intracellular signaling in the PI3K/AKT pathway. The PI3K/AKT signaling pathway was assessed in peritoneal monocytes by using a protein microarray. Samples were pooled from 4 mice in each group for each experiment. Figure 5 Neo1-dependent monocyte intracellular signaling in the TGF-β pathway. (A) The TGF-β signaling pathway was assessed in peritoneal monocytes by using a protein microarray. (B) Representative flow cytometry plot and bar chart showing the peritoneal monocytes used for protein profiling. Samples were pooled from 4 mice in each group for each experiment. Impact of Neo1 on lipid mediator biosynthesis. The SPMs — namely, lipoxins, resolvins, protectins, and maresins — have been identified as important determinants of inflammation resolution (5). To examine whether Neo1 impacts the generation of SPMs during inflammation resolution, we carried out liquid chromatography–tandem mass spectrometry–based (LC-MS/MS-based) profiling. In inflammatory peritoneal exudates obtained from Neo1–/– mice and their littermate controls, we identified SPMs as well as their precursors and pathway markers. Specifically, we identified arachidonic acid–derived LXA4 (Figure 6, A and D), docosahexanoic acid–derived (DHA-derived) PDX (also referred to as 10S,17S-diHDHA), and Mar1 to be increased in Neo1–/– (Figure 6, B and D). It is well appreciated that prostanoids such as PGD2, PGE2, and PGI2 elicit immunomodulatory and antiinflammatory effects (23, 29–31). In particular, PGD2 and PGE2, which are known to induce the inflammatory response, subsequently stimulate antiinflammatory effects by activating the 15-LOX in neutrophils to ultimately promote lipid mediator class-switching during the resolution of acute inflammation. Our data demonstrate the enhanced production of PGD2 and PGE2 in the initial phase, suggesting that the mediator class switch is implemented in the resolution phase (Figure 6A and Supplemental Table 2) (31). We also found enhanced levels of the arachidonic acid–derived products 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-HETE, and the eicosapentaenoic acid–derived (EPA-derived) 15-hydroxyeicosapentaenoic acid (15-HEPE) and 18-HEPE in Neo1–/– (Figure 6, A and C). Furthermore, metabolites 14,15-diHETE and 19,20-DiHDPA produced by cytochrome P450 epoxygenases, and the actions of soluble epoxidehydrolase (sEH), thus belonging to a different class of antiinflammatory and proresolving lipids, were also significantly increased in Neo1-deficient mice (Figure 6, A and B). Knowing that the enzymes 5-LOX and 12/15-LOX contribute to the generation of proresolving mediators and finally to increased resolution effects, we incubated peritoneal MΦ from WT or 12/15-LOX–deficient mice with Neo1 Ab and found a reduced efferocytosis rate of fluorescence-labeled ZyA particles after stimulation with Neo1 Ab (Supplemental Figure 6D). In a second set of experiments, we incubated peritoneal MΦ from WT or Neo1–/– mice with 5-LOX and 12/15-LOX inhibitors baicalein or cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and found a reduced ZyA efferocytosis rate in Neo1–/– cells (Supplemental Figure 6E). To substantiate these data we incubated human MΦ with Neo1 Ab and baicalein or CDC. The impact of Neo1 inhibition on MΦ phagocytosis was significantly reduced when costimulated with 5-LOX and 12/15-LOX inhibitors, suggesting that the Neo1 effects on resolution are 5-LOX and 12/15-LOX dependent (Supplemental Figure 6F). This result is consistent with the increased biosynthesis of the 5-LOX– and 12/15-LOX–dependent proresolving mediators, such as LXA4, Mar1, and PDX in the Neo1–/– mice. Figure 6 Endogenous deficiency of Neo1 activates proresolving lipid mediator biosynthesis. Neo1–/– and WT mice were challenged with ZyA peritonitis. Peritoneal lavages were collected at 4 hours and analyzed using LC-MS/MS. (A) Lipid mediators and precursors derived from arachidonic acid (AA), (B) docosahexaenoic acid (DHA), and (C) eicosapentaenoic acid (EPA). (D) Corresponding MS/MS spectra and the multiple reaction monitoring chromatograms (MRM) for the identified lipid mediators. Results represent 3 independent experiments and are expressed as median ± 95% CI (n = 8–12 mice/group). Statistical analysis was done by unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. All results are reported as ng/107 peritoneal cells. Taken together, these results strongly highlighted that targeted deletion of Neo1 modulates the lipid mediator profile in murine exudates toward an antiinflammatory and proresolving state. Genetic deletion of Neo1 contributed to tissue regeneration mechanisms in vivo. After demonstrating that genetic deletion of Neo1 promoted key resolution features, we turned our attention to the influence of Neo1 on tissue repair/regeneration and found increased levels of IL-10 and TGF-β peritonitis exudate, 2 parameters contributing to peritoneal tissue repair and regeneration in Neo1–/– mice (Figure 7A) (2, 18, 32). This is in line with our cellular data that shows Neo1–/– monocytes activate the PI3K/AKT pathway for the induction of cell polarization toward the M2 phenotype. To substantiate this proregenerative impact, we performed a staining for proliferating cell–nuclear antigen (PCNA) within the peritoneum, which displayed an index increase of approximately 20% in Neo1–/– peritonitis compared with the WT group (Figure 7B). Since our data revealed Neo1 to be a negative regulator in the resolving and regenerative processes, we next elucidated the temporal regulation of Neo1 during the initiation and resolution phase (Figure 7C). Exudate Neo1 was markedly increased between 4 hours and 24 hours and subsequently decreased at the end of the resolution phase, suggesting that Neo1 impacts processes during initiation and resolution/regeneration of acute inflammation. To clarify whether Neo1 expression is cell type–specific and not only related to the cell trafficking events, we determined the Neo1 expression on cellular exudates and found Neo1 to be strongly increased between 4 hours and 12 hours, followed by a decrease at the end of the resolution phase (Supplemental Figure 5). Figure 7 Functional inhibition of Neo1 promotes the resolution and regeneration processes. Neo1–/– and WT littermate controls were exposed to ZyA, and peritoneal lavages were collected at 4, 12, 24, and 48 hours. (A) Exudate levels of IL-10 (after 4 hours) and TGF-β (after 12 hours). (B) Regeneration of the peritoneum was evaluated by PCNA immunohistochemistry staining of the peritoneum (24 hours after ZyA) (n = 4/condition, magnification ×630, scale bar 200 μm) and the calculated index. (C) Temporal regulation of Neo1 in ZyA-induced peritonitis exudates measured by ELISA. The results represent 2 independent experiments and are expressed as median ± 95% CI (A) and mean ± SEM (C) (n = 8–12 per group). Statistical analysis was done by unpaired Student’s t test, *P < 0.05, **P < 0.01. Functional inhibition of Neo1 promotes the resolution and regeneration processes. Having shown that endogenous deletion of Neo1 initiated the resolution of acute inflammation by inducing the apoptosis of PMNs, the cessation of PMN influx, efficient clearance of PMNs, and the biosynthesis of SPMs, we next sought to investigate whether anti-Neo1 has any therapeutic efficacy in acute inflammation (e.g., potentially resolving processes such as peritonitis). When a functional anti-Neo1 Ab was given as a prophylactic treatment (in parallel with ZyA injection) for murine peritonitis, WT mice displayed reduced PMN infiltration and shortened resolution interval, from 26 hours to 7 hours (Figure 8, A and B). Furthermore, administration of an anti-Neo1 Ab decreased classical Ly6Chi monocytes and increased nonclassical Ly6Clo monocytes and MΦ, which led to strong enhancement of MΦ clearance of apoptotic PMNs (Figure 8C). Also, the inflammation-initiated cytokines, such as TNF-α, IL-1β, IL-6, and keratinocyte chemoattractant (KC, IL-8 in humans), were reduced (Figure 8D). In a second set of experiments, we investigated the therapeutic administration of an anti-Neo1 Ab. The agent was given at the peak of inflammation as monitored by maximal neutrophil recruitment, and peritoneal lavages were collected at 12, 24, and 48 hours. As expected, we found activation of cardinal signs of resolution with a shortening of the resolution interval from 23 hours to only 16 hours (Figure 9, A and B), suggesting a stronger treatment effect when anti-Neo1 Ab was given at the onset of inflammation. To further validate the proresolving attributes of the functional inhibition of Neo1, we examined the exudate IL-10 and TGF-β levels, which contribute to resolution and regenerative programs (18) (Figure 9C). Here, we found increased levels of both cytokines following anti-Neo1 Ab administration. To corroborate these results, we performed immunohistochemical characterization of PCNA, and found improved responses in tissue repair (Figure 9D). Finally, to clarify whether the loss of Neo1 with genetic deletion or with anti-Neo1 Ab treatment may blunt the initial inflammatory response, giving the false appearance of improved resolution, we first examined the biosynthesis of the lipid mediators specific to the resolution processes at a later time point (12 hours after ZyA injection), and found increased levels of specifically arachidonic acid–derived LXA4 and DHA-derived PDX in Neo1–/– (Supplemental Figure 6, A–C). Then we exposed WT mice to ZyA peritonitis and this time the anti-Neo1 Ab was given in the resolution phase 6 hours after ZyA injection (e.g., regression of the neutrophil infiltration). The samples were collected 12 hours after ZyA injection. As expected, the collected data demonstrate that supplementation of anti-Neo1 6 hours after ZyA injection promotes the resolution/regeneration mechanism by decreasing the classical Ly6Chi monocytes and increasing the nonclassical (M2) Ly6Clo monocytes and macrophages that indicate a strong enhancement of macrophage clearance of apoptotic PMNs (Supplemental Figure 7). Studies have revealed Neo1 to be a specific receptor for 2 ligands, namely Netrin-1 and RGM-A (12, 13). We incubated peritoneal MΦ from WT and Neo1–/– mice with RGM-A or Netrin-1 to determine a possible influence on MΦ efferocytosis of fluorescent ZyA particles. Collected data revealed that RGM-A did not increase MΦ clearance in the Neo1–/– cells. When MΦ were stimulated with Netrin-1, efferocytosis was not significantly affected, suggesting that the actions of Neo1 are RGM-A dependent (Supplemental Figure 8). Figure 8 Exogenous inhibition of Neo1 attenuates inflammation and fosters resolution programs. WT mice were challenged with ZyA and subsequently injected with either IgG or a Neo1 inhibitory Ab, and lavages were collected at 4, 12, 24, and 48 hours. (A) The total leukocytes were enumerated by light microscopy, and the PMNs were determined by flow cytometry. (B) The resolution index was calculated as previously described (41). (C) Classical, nonclassical, peritoneal MΦ, and monocyte-derived MΦ efferocytosis were assessed by flow cytometry. (D) Proinflammatory cytokines TNF-α, IL-1β, IL-6, and KC 4 hours after ZyA injection. The therapeutic potential of Neo1 blockade was evaluated by application of anti-Neo1 Ab 4 hours after ZyA peritonitis induction at the peak of inflammation and peritoneal lavages were collected at 12, 24, and 48 hours. Results represent at least 2 independent experiments and are expressed as mean ± SEM (A, C) and median ± 95% CI (D) (n = 8–12 per group). Statistical analysis was done by unpaired Student’s t test *P < 0.05; **P < 0.01; ***P < 0.001. Figure 9 Exogenous therapeutic inhibition of Neo1 attenuates inflammation and fosters resolution programs. The therapeutic potential of Neo1 blockade was evaluated by application of anti-Neo1 Ab 4 hours after ZyA peritonitis induction at the peak of inflammation, and peritoneal lavages were collected at 12, 24, and 48 hours. (A) Total leukocyte cell numbers were enumerated and PMNs, classical and nonclassical monocytes, peritoneal MΦ, and monocyte-derived MΦ efferocytosis were measured by FACS. (B) Resolution index. (C) IL-10 and TGF-β levels 12 hours after ZyA injection. (D) PCNA immunohistochemistry images (n = 4/condition, magnification ×630, scale bar 200 μm) and the calculated PCNA index. Results represent at least 2 independent experiments and are expressed as mean ± SEM (A) and median ± 95% CI (D) (n = 8–12 per group). Statistical analysis was done by unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. These results indicate a critical role for the functional inhibition of Neo1 in controlling inflammation processes in the resolution and regeneration phases, and might demonstrate a possible therapeutic approach. Plasma Neo1 is increased in critically ill pediatric patients with IAH or ACS and it is associated with clinical outcome. To translate our preclinical findings to humans, we investigated the association between Neo1 blood plasma levels and IAH grade, ACS, severity of illness, pediatric ICU (PICU) length of stay, and survival in a cohort of 59 critically ill pediatric patients partly suffering from abdominal compartment syndrome (ACS). In brief, we prospectively enrolled medical and surgical patients ranging in age from newborn to 17 years old with cardiac or oncological diseases, or after surgical interventions. In all enrolled subjects, intensive care monitoring was urgently indicated (i.e., admission to PICU). Due to the different severities of the patients’ illnesses, a selective division into 3 test groups was possible. The criteria were Pediatric Risk of Mortality III score (PRISM-III score), organ dysfunction, and intraabdominal pressure (IAP) level. Patient demographic and clinical data are shown in Figure 10, A and B, Table 1, Table 2, Supplemental Table 3, and Supplemental Table 4. The severity of illness was assessed by the PRISM-III score. The vital signs, other cardiorespiratory parameters, drug administration, IAP, and fluid balances were recorded continuously. With regard to ACS associated with substantial morbidity, such as renal failure and multiorgan dysfunction syndrome (MODS), and mortality in critically ill patients (33), our data revealed 1.8-fold higher Neo1 plasma concentrations in children with ACS versus those without ACS, and 1.7-fold higher plasma levels in children with ACS versus control ICU patients (Figure 10B). When comparing the Neo1 plasma levels with the severity of illness, we found that significantly increased levels of Neo1 in affected children correlated with PRISM-III score, IAH grade, and clinically established laboratory parameters such as serum C-reactive protein (CRP), lactate, creatinine, and bilirubin (Figure 10C). We also compared conventional laboratory inflammatory parameters with the above-mentioned organ and outcome parameters. Only CRP showed correlations comparable to those of Neo1 (related to Rho and P values). In contrast to Neo1, to our knowledge CRP has never shown any prognostic value with regard to the development of IAH or ACS. Only D-lactate was identified in the context of 2 animal studies as a biomarker for the development of ACS in the past (34, 35). In the pediatric literature, there is no study that would have identified a biomarker for the development of an AKS. Procalcitonin (PCT; n = 6) did show higher correlation coefficients (especially for creatinine, PICU length of stay, and PRISM-III scores); however, these results did not reach statistical significance, probably because PCT was only determined in 6 children on admission to our PICU (Supplemental Table 5). To clarify whether increased cell lysis might have caused an increase in membrane-bound Neo1 entering the plasma, we subsequently analyzed the serum concentrations of lactate dehydrogenase (LDH). All children enrolled into our study had a mild (non–critically ill [NCI] PICU controls) to marked increase (critically ill [CI] groups) in LDH serum concentration (normal reference values age-dependent, approximately <344 U/l). The difference in circulating LDH between controls and test groups was significant (P = 0.03). On the other hand, there was no significant difference between CI-ACS and CI+ACS (P = 0.69). Since plasma levels of Neo1 were significantly higher in the CI+ACS group than in the CI-ACS group, the results of LDH analysis thus indicate that the main source of Neo1 most likely does not arise from cell lysis (Supplemental Table 6). Moreover, the PICU length of stay was also correlated with increased levels of Neo1 (Figure 10C). Since mortality is one of the most reliable endpoints of clinical management in the ICU, we investigated whether plasma Neo1 could be a mortality predictor in critically ill children. We found Neo1 to be 5.0-fold higher in nonsurvivors compared with survivors and 5.7-fold higher in nonsurvivors compared with ICU controls. Receiver operating curve (ROC) analysis comparing survivors with nonsurvivors demonstrated a strong specificity (92.86%, sensitivity 66.67%) for Neo1 plasma levels greater than 36.777 pg/ml, indicating that Neo1 can be a valid predictor of mortality (Figure 10D). Figure 10 Neo1 in PICU patients with ACS. Plasma samples from 59 children with and without ACS were collected within 24 hours after admission to the PICU of Hannover Medical School (MHH). (A) Photos displaying the intraoperative situs during open abdomen therapy and the postoperative condition after the establishment of a belly-widening vacuum-assisted closure (VAC) wound dressing in children with fulminant ACS. (B) Value of Neo1 at the admission to the PICU was investigated by comparing the control group (Prism-III score <8), critically ill children without ACS (Prism-III score ≥8), and critically ill children meeting the 2013 WSACS definitions (38) for ACS. (C) Correlation between Neo1 and clinical parameters of all PICU patients enrolled. The Spearman’s rank correlation coefficient Rho and the corresponding 95% CI interval are shown. (D) The predictive value of Neo1 at the admission to the PICU was investigated comparing PICU control patients (PRISM-III score <8), critically ill survivors (PRISM-III score ≥8), and critically ill nonsurvivors. An ROC curve was calculated comparing Neo1 in critically ill survivors with nonsurvivors. Patient characteristics for survivors and nonsurvivors. Results are displayed as median ± 95% CI. Statistical analysis was done by nonparametric Kruskal-Wallis test followed by Dunns post hoc test; correlation was tested using Spearman’s rank correlation test, *P < 0.05, **P < 0.01. Table 1 Correlation between Neo1 and ACS in PICU patients with ACS Table 2 Correlation between Neo1 and survival in PICU patients with ACS Discussion Results from the present report reveal that the neuronal guidance protein Neo1 contributed not only to the onset of an inflammatory response but also to local inflammation resolution mechanisms and tissue regeneration processes. In this report, we demonstrated that genetic deletion or functional inhibition of Neo1 led to a reduction in neutrophil recruitment at the injury sites and abbreviation of neutrophil lifespan by increasing apoptosis and ultimately inducing MΦ clearance. The biosynthesis of endogenous SPMs and their pathway markers was enhanced in Neo1–/– mice, and the regeneration of tissue injury was improved. Treatment with an anti-Neo1 Ab demonstrated reduced inflammatory status, indicating acceleration in resolution and promotion of tissue repair. In a cohort that included critically ill PICU patients, we found associations between plasma Neo1 and IAH grade, ACS, PRISM-III score, PICU length of stay, and survival. Together these data point toward a crucial role of Neo1 in the initiation and resolution of inflammatory and resolving/regenerative processes. Inflammation driven by tissue injury or infection is characterized as modular in its temporal sequence of events, and this is critical for survival. An acute inflammatory response is divided into distinct phases: the initiation phase and the resolution/regeneration phase. The initiation phase (proinflammatory early state) comprises the migration of key inflammatory cells to the site of inflammation, guided by chemical messengers such as cytokines, chemokines, and lipid mediators (23, 36). The resolution of the inflammatory response is a precisely controlled active process governed by local SPMs that mediate the clearance and killing, efferocytosis, and phagocytosis of apoptotic PMNs to restore tissue homeostasis after injury and phlogistic processes (5). A group of guidance proteins was originally recognized for its role in the developing nervous system. Considering the similarities between neuronal cell guidance and leukocyte trafficking, recent studies have pointed to additional roles for NGPs in modulating the inflammatory response outside the CNS (6–8, 10, 37, 38). A crucial target receptor in mediating NGP function in the CNS is the Neo1 receptor. Its function is best described in the nervous system where it guides cell and axon migration during embryonic development (14). Neo1 provides chemoattractive and chemorepulsive attributes, depending on its ligand binding (12). More recently, Lee et al. demonstrated a crucial role for Neo1 in the control of junctional stability during epithelial morphogenesis (39). Neo1 has been studied at the onset of inflammation (9, 15, 38) in peripheral organs; however, its role during the resolution and regeneration phases, particularly the understanding of pathways that can promote or mitigate the resolution of inflammation, remains unclear. Considering that failed clearance of apoptotic cells modifies immune tolerance and promotes nonresolving inflammation (17) (i.e., sepsis), we investigated the role of Neo1 in the apoptosis of neutrophils. The inhibition of Neo1 led to an increase of the find-me signals CX3CL1 and CX3CR1, the decoy receptor IL-1R2, and the eat-me receptor TIM4 in human apoptotic PMNs or MΦ, and the efferocytosis of apoptotic PMNs was substantially enhanced. In murine peritonitis, Neo1–/– deficiency displayed enhanced neutrophil apoptosis. This was accompanied by decreased recruitment of classical Ly6Chi cells and an increased population of alternatively activated Ly6Clo and conclusively enhanced efferocytosis of apoptotic PMNs. When investigating the role of Neo1 more precisely, we found that in bone marrow and peritoneal monocytes there was a strong reduction of the migratory classical Ly6Chi cells in the peritoneal cavity of Neo1–/– mice. Neo1 expression was restricted to the peritoneal inflammatory Ly6Chi cells, suggesting that the lack of Neo1 induced a phenotypic switch toward the antiinflammatory and proresolving M2 type. To show in more detail that Neo1 has a direct influence on classical and nonclassical monocytes and efferocytosis, we carried out additional experiments. We exposed bone marrow chimeric mice to ZyA peritonitis and analyzed the leukocytes and clearance at 4 hours and 12 hours after ZyA injection. As expected, bone marrow chimeric animals with hematopoietic Neo1 repression demonstrated a reduction in classical Ly6Chi cells, an increase in nonclassical Ly6Clo monocytes, and finally a strong enhancement of the efferocytic capacity at both time points. As demonstrated before, Ly6C, which is known to be mainly expressed on the migrating proinflammatory monocytes, was markedly decreased in peritoneal Neo1–/– monocytes compared with littermate controls. This influence was also noted in the bone marrow chimeric animals with hematopoietic Neo repression. Ly6C MFI was markedly decreased in Ly6Chi cells in bone marrow chimeric animals with hematopoietic Neo repression, implying that Neo1 may impact the proinflammatory Ly6Chi monocytes. At the signaling level, we were able to demonstrate the activation of the PI3K/AKT pathway and the suppression of the TGF-β pathway in Neo1–/– monocytes. Specifically, the activation of the PI3K/AKT pathway has been reported to be a crucial step toward the proresolving M2 phenotype (26). Since the resolution of inflammation is induced to a large extent by SPMs, the generation of several of these endogenous classes of SPMs and their pathway markers (i.e., LXA4, Mar1, and PDX) were markedly enhanced in Neo1–/– exudates. Likewise, the metabolites 13,14-diHETE and 19,20-diHDPA, which belong to a different class of antiinflammatory and proresolving lipids and are produced by cytochrome P450, were strongly increased in Neo1-deficient mice compared with their littermate controls. Also, the resolution interval was shortened from 27 hours to 9 hours. The decreased levels of IL-6, KC, MIP2, and MCP-1 within the Neo1–/– peritonitis lavages collected in the resolution phase 12 hours after ZyA injection underlined the effect of Neo1 on the resolution phase. When determining the therapeutic efficacy of Neo1 in resolving processes, we found that both the prophylactic and the therapeutic functional inhibition of Neo1 activated inflammation resolution programs and promoted tissue regeneration as demonstrated by reduced PMN recruitment, decreased classical Ly6Chi monocytes, and increased nonclassical Ly6Clo monocytes and MΦ, which led to the strong enhancement of MΦ clearance of apoptotic PMNs. The exudate IL-10 and TGF-β levels that contributed to the resolution and regenerative programs (18) were strongly enhanced. The improved tissue repair responses analyzed by PCNA corroborate the critical role for Neo1 in the resolution and regenerative processes. Finally, to clarify the question of whether the loss of Neo1 with genetic deletion or with anti-Neo1 Ab treatment may blunt the initial inflammatory response, giving the false appearance of improved resolution, we injected the anti-Neo1 Ab in the resolution phase 6 hours after ZyA injection, and found anti-Neo1 treatment to have a strong impact on key characteristics of resolution. It is evident that critically ill patients might display severe comorbidities that are a major threat to global health. In fact, clinical trials may be the most effective way to investigate the usefulness of novel predictive indications. Therefore, in a cohort of critically ill PICU patients suffering in part from ACS, we found a strong correlation between Neo1 plasma levels and ACS, IAH grade, PRISM-III disease severity score, ICU length of stay, and survival. In conclusion, our study reveals a key role for Neo1 in controlling inflammation resolution and regeneration programs. Our findings demonstrate that deficiency of Neo1 directly promotes antiinflammatory, proresolving effects (i.e., shortening of resolution phase, activating SPM generation, reducing PMN influx, activating PMN apoptosis, and increasing MΦ phagocytosis of apoptotic PMNs). Moreover, our data revealed that Neo1 correlates with ACS, PRISM-III score, ICU length of stay, and survival in critically ill children, and might therefore evolve as a new clinical marker and therapeutic target in inflammatory conditions. Methods Animals. This project was approved by the institutional review board and the Regierungspräsidium Tübingen. WT (C57BL/6N), Neo1–/– (C57BL/6N-Neo1Gt(KST265)Byg), and littermate control mice (C57BL/6N) were bred and genotyped as previously described (8). At 8 to 10 weeks old, mice of either sex were assigned to the respective study time points and/or experimental interventions at random. Murine peritonitis. All animal protocols were performed in accordance with the regulations of the Regierungspräsidium Tübingen and the local ethics committee. All trials took into account Directive 2010/63/EU adopted by the European Parliament and Council. The mice were intraperitoneally injected with 1 ml zymosan A (ZyA; 1 mg/ml; Sigma-Aldrich, catalog Z4250) and subsequently intravenously with either IgG control (Santa Cruz Biotechnology, catalog sc-2028) or 2 μg Neo1 blocking antibody (R&D Systems, catalog AF-1079) in a total volume of 150 μl. Peritoneal fluids and tissues were obtained at 4, 12, 24, and 48 hours and prepared as previously described (37). The collected exudates were washed, suspended in PBS (MilliporeSigma), and counted. Differential leukocyte counts, FACS analysis, and cytokines. Exudate cells from the murine peritonitis models were prepared to determine their cellular composition. The cells were blocked with mouse anti-CD16/CD32 (Biolegend, catalog 101320) antibodies for 10 minutes at room temperature and then stained with anti-mouse APC-Ly6G (BioLegend, catalog 127614), e450-F4/80 (eBioscience, catalog 48-4801-82), and FITC-Ly6C (BioLegend, catalog 128006) antibodies for 30 minutes at 4°C. To analyze the MΦ phagocytosis of apoptotic PMNs in vivo, the cells were permeabilized using a fixation and permeabilization kit (BD Biosciences, catalog 554714) prior to staining with PerCP-Cy5.5–conjugated anti-Ly6G (BioLegend, catalog 127616) for 30 minutes at 4°C. The cells were acquired on a FACSCanto II (BD Biosciences) and analyzed with FlowJo (TreeStar). Cytokines were measured in the murine peritoneal exudates using standard ELISA (R&D Systems). Lipid mediator lipidomics. LC-MS/MS analysis was carried out as previously described with some modifications (40, 41). Peritoneal lavage samples were thawed, and internal standards were added and subsequently extracted twice using methanol. The combined organic extracts were cleaned up using solid-phase extraction according to published protocols (41). LC-MS/MS analysis was carried out using a 6500 QTrap LC-MS/MS system as previously described (40). For a detailed description of the analytical procedure please refer to the Supplemental Material. Antibody array for protein expression. Peritoneal monocytes/macrophages from WT and Neo–/– mice were used following 12 hours of ZyA-induced peritonitis. Protein and phosphorylation (TGF-β Phospho Antibody Array, Full Moon BioSystems, catalog PTG176) profiling of peritoneal monocytes (pooled lavages from 4 mice/condition) was carried out according to the manufacturer’s instructions. The images were acquired by the manufacturer. For each antibody, the average signal intensity of 6 replicates was normalized to the median signal of all antibodies on the array. The presented fold change represents the ratio of the normalized signal from Neo1–/– mice compared with WT littermate controls. GAPDH and beta-actin were used as housekeeping proteins. Data analysis was performed with IPA software (Qiagen). Pathways were substantiated and updated with recent literature, the KEGG database (HSA 04350, HAS 04151; https://www.genome.jp/kegg/), and the Reactome database (R-HSA-198203, R-HSA-2173789; https://reactome.org/). Data were deposited in the NCBI’s Gene Expression Omnibus database (GEO GSE117137) (42). Human PMN apoptosis and MΦ efferocytosis. PMNs and human peripheral blood monocytes were isolated from healthy volunteers or human leukapheresis collars from the Blood Bank of Eberhard Karls University of Tübingen by gradient centrifugation using Histopaque-1077 (MilliporeSigma). Monocytes were cultured in RPMI 1640 medium (MilliporeSigma) with 10 ng/ml human recombinant GM-CSF (Milteny Biotec, catalog 130-093-866 ) at 37°C in 5% CO2 for 7 days. Human PMNs were labeled with carboxyfluorescein diacetate (10 μM, 30 minutes at 37°C; Molecular Probes) and allowed to undergo apoptosis in serum-free RPMI 1640 medium (Gibco) for 16 to 18 hours. GM-CSF–differentiated MΦ (0.1 × 106 cells/well) were then incubated with human Neo1 Ab (R&D Systems, catalog AF1079) or IgG control (Santa Cruz Biotechnology, catalog sc-2028) for 15 minutes. Apoptotic PMNs were added in a 1:3 ratio (MΦ/PMNs) and incubated at 37°C for 60 minutes to allow phagocytosis. In a separate experiment, GM-CSF–differentiated MΦ were incubated with Neo1 Ab (R&D Systems, catalog AF1079) or IgG control (Santa Cruz Biotechnology, catalog sc-2028) for 15 minutes at 37°C and then incubated with fluorescently labeled (BacLight; Thermo Fisher Scientific, catalog B35000) E. coli at a 1:50 ratio for 60 minutes. Efferocytosis was determined using a fluorescent plate reader (Tecan). To evaluate PMN apoptosis, PMNs were incubated in RPMI 1640 (Gibco) plus 10% FCS in the presence or absence of LPS (MilliporeSigma, catalog LA4391) and/or Neo1-antibody (R&D Systems, catalog AF1079) for 20 hours at 37°C in 5% CO2. Apoptosis was measured by FACS analysis using an Annexin V PE apoptosis detection kit with 7-AAD (BioLegend, catalog 640934) according to the manufacturer’s instructions, and transcriptional analysis was performed. Transcriptional analysis of human MΦ and PMNs. Human GM-CSF–differentiated MΦ were incubated in RPMI 1640 (Gibco) in the presence or absence of IL-1β (Promokine, catalog C-61120) and/or anti-Neo1 antibody (R&D Systems, catalog AF1079) for 4 hours prior to transcriptional analysis. Human 18S expression as a housekeeping gene was evaluated with the sense primer 5′-GTAACCCGTTGAACCCCATT-3′ and antisense primer 5′-CCATCCAATCGGTAGTAGCG-3′. The following primers were used: Cx3cl1: 5′-CGGTGTGACGAAATGCAACA-3′, 5′-CTCCAAGATGATTGCGCGTTT-3′; Il1r2: 5′-GTGAGCAACAAGGCC A-3′, 5′-TACCAACACGTACAAGCGCA-3′; Cx3cr1: 5′-GAGGCGTTTAAGTTGGCAGA-3′, 5′-ATGGTGAAGGCCCCACT-3′; Tim4: 5′-ACAGGACAGATGGATGGAATACCC-3′, 5′-AGCCTTGTGTGTTTCTGCG-3′; Gpr32: 5′-GGGCCTGCAAACTCTACA-3′, 5′-GGAGGCAGTTACTGGCAA-3′; Alx/Fpr: 5′-TGTTCTGCGGATCCTCCCATT-3′, 5′-CTCCCATGGCCATGGAGACA-3′. Cytology, immunofluorescence, and immunohistochemistry staining. GM-CSF–differentiated human MΦ were stimulated for 4 hours with IL-1β (Promokine, catalog C-61120) prior to labeling with rabbit anti-Neo1 (Santa Cruz Biotechnology, catalog sc-15337) and rhodamine phalloidin (Invitrogen, catalog R415). An IgG isotype control antibody (Santa Cruz Biotechnology, catalog sc-2027) was used as a negative control. Alexa Fluor 488–conjugated goat anti-rabbit (Life Technologies, catalog A27034) was used as the secondary antibody. DAPI (4′,6-diamidino-2-phenylindole; Invitrogen, catalog P36931) was employed for nuclear counterstaining. For immunofluorescence analysis of human MΦ efferocytosis of fluorescently labeled PMNs, MΦ were stained with mouse-anti-CD14 (Santa Cruz Biotechnology, catalog sc-58951) and A594 goat-anti-mouse secondary antibody (Thermo Fisher Scientific, catalog A-11005). DAPI (Invitrogen, catalog P36931) was employed for nuclear counterstaining. Immunofluorescence images were acquired using a confocal microscope (LSM 510 Meta fluorescence microscope, Carl Zeiss) and ZEN software (Carl Zeiss). To perform immunohistochemical staining for PCNA, paraffin-embedded peritoneal tissues were stained with an anti-PCNA antibody (Santa Cruz Biotechnology, catalog sc-56) using a Vectastain ABC Kit (Vector Labs, catalog PK-4004) and DAB peroxidase substrate (Sigma-Aldrich, catalog E109) according to the manufacturers’ instructions. As the secondary antibody, a biotin-conjugated horse-anti-mouse antibody (Vector Labs, catalog BA-2000) was used. The sections were then counterstained with hematoxylin. Light microscopy images were acquired with a DM IRM microscope (Leica) equipped with an AxioCam MRc (Carl Zeiss) using AxioVision software (Carl Zeiss). Pediatric ICU patient samples with and without ACS. A total of 59 plasma samples were taken from patients from the PICU of Hannover Medical School (MHH) within 24 hours after admission. The 2013 WSACS definitions (43) (with respect to IAP and ACS; www.wsacs.org) were used to define the ACS. Severity of illness in the ICU children was measured using PRISM-III scoring (44). Vital and cardiorespiratory parameters (including ventilation parameters), drug administration, intraabdominal pressure (measured via gastric Spiegelberg monitoring system) (45), and fluid balances were recorded continuously via the digital patient data management system (mlife, mediside). A Neo1 ELISA (Cusabio, catalog CSB-EL015712HU) was performed according to manufacturer’s instructions. For a detailed description of the criteria for patient selection and monitoring please refer to the Supplemental Material. Statistics. Statistical analysis of murine and in vitro data was performed using ANOVA with Bonferroni’s multiple comparisons test. An unpaired 2-tailed Student’s t test was used to compare 2 independent groups. Experimental data are reported as mean ± SEM. Statistical analyses of data from PICU patients were performed using the nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data are reported as median ± 95% CI. Correlation of clinical data was tested using Spearman’s rank correlation test. For all tests, a P value less than 0.05 was considered statistically significant. Analyses were performed using GraphPad Prism5 (GraphPad Software) and JMP 13 (SAS). Study approval. Critically ill children newborn to age 17 years were enrolled between January and August 2015 after informed written consent was obtained from the parents or guardians of each child. The study was approved by the local ethics committee (Ethikkommission der MHH, Hannover, Germany) of Hannover Medical School (MHH 6677) and internationally registered (WHO-ICTRP DRKS00006556). Animal experiments were approved by the institutional review board and the Regierungspräsidium Tübingen (Tübingen, Germany). Author contributions MS, AK, CG, and UK performed the experiments, and collected and analyzed the data. TK and GH performed the clinical experiments in patients. HSJ and MG performed the targeted lipidomic and lipid mediator analysis studies. All authors contributed to manuscript preparation and figure preparation. VM carried out overall experimental design, conceived of the overall research, and wrote the manuscript. Supplemental material View Supplemental data Acknowledgments We thank Alice Mager, Hannes Frohnmeyer, and Marieke Heijink for their technical support. This work was supported by grants from the Interdisziplinäres Zentrum für Klinische Forschung (IZKF; 2110-0-0) and the Deutsche Forschungsgemeinschaft (DFG-MI 1506/4-1 and DFG-MI 1506/5-1) to VM; by IZKF fortüne grants to MS (2299-0-0) and AK (2377-0-0); and by an intramural grant from the Hannover Medical School (Junge Akademie/MHH Young Academy grant 9790012) to TK. Footnotes Conflict of interest: The authors have declared that no conflict of interest exists. Reference information: J Clin Invest. 2018;128(10):4711–4726.https://doi.org/10.1172/JCI96259. References Ferreira FL, Bota DP, Bross A, Mélot C, Vincent JL. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA. 2001;286(14):1754–1758. View this article via: PubMed CrossRef Google Scholar Nathan C, Ding A. Nonresolving inflammation. 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View this article via: PubMed CrossRef Google Scholar Buckley CD, Gilroy DW, Serhan CN. Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity. 2014;40(3):315–327. View this article via: PubMed CrossRef Google Scholar Jakubzick CV, Randolph GJ, Henson PM. Monocyte differentiation and antigen-presenting functions. Nat Rev Immunol. 2017;17(6):349–362. View this article via: PubMed CrossRef Google Scholar Quintar AA, Hedrick CC, Ley K. Monocyte phenotypes: when local education counts. J Exp Med. 2015;212(4):432. View this article via: PubMed CrossRef Google Scholar Manning BD, Toker A. AKT/PKB signaling: navigating the network. Cell. 2017;169(3):381–405. View this article via: PubMed CrossRef Google Scholar Covarrubias AJ, et al. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation. Elife. 2016;5:e11612. View this article via: PubMed Google Scholar Chen W, Ten Dijke P. Immunoregulation by members of the TGFβ superfamily. Nat Rev Immunol. 2016;16(12):723–740. View this article via: PubMed CrossRef Google Scholar Zhang Y, et al. Tissue regeneration. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration. Science. 2015;348(6240):aaa2340. View this article via: PubMed CrossRef Google Scholar Dalli J, Serhan C. Macrophage proresolving mediators-the when and where. Microbiol Spectr. https:/doi.org/10.1128/microbiolspec.MCHD-0001-2014 View this article via: PubMed Google Scholar Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. Lipid mediator class switching during acute inflammation:
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One-step generation of monoclonal B cell receptor mice capable of isotype switching and somatic hypermutation

One-step generation of monoclonal B cell receptor mice capable of isotype switching and somatic hypermutation | Immunology | Scoop.it
We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation.
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Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis

Extensive, high-dimensional characterization of T cells in breast cancer reveals activated TRM population and a gene signature associated with improved prognosis.
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PreB cells are moving on

PreB cells are moving on | Immunology | Scoop.it
In this issue of JEM , Fistonich et al. (<https://doi.org/10.1084/jem.20180778>) address how the bone marrow microenvironment supports diverse lineages through multiple developmental stages. Differential motility between pro- and preB cells results in differential IL-7 exposure, and, intriguingly, stromal cells respond to abnormal B cells by reducing Il7 .
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JCI - The innate immune receptor TREM-1 promotes liver injury and fibrosis

JCI - The innate immune receptor TREM-1 promotes liver injury and fibrosis | Immunology | Scoop.it
Inflammation occurs in all tissues in response to injury or stress and is the key process underlying hepatic fibrogenesis. Targeting chronic and uncontrolled inflammation is one strategy to prevent liver injury and fibrosis progression. Here, we demonstrate that triggering receptor expressed on myeloid cells-1 (TREM-1), an amplifier of inflammation, promotes liver disease by intensifying hepatic inflammation and fibrosis. In the liver, TREM-1 expression is limited to liver macrophages and monocytes and is highly upregulated on Kupffer cells, circulating monocytes, and monocyte-derived macrophages in a mouse model of chronic liver injury and fibrosis induced by carbon tetrachloride (CCl4) administration. TREM-1 signaling promotes pro-inflammatory cytokine production and mobilization of inflammatory cells to the site of injury. Deletion of Trem1 reduced liver injury, inflammatory cell infiltration, and fibrogenesis. Reconstitution of Trem1-deficient mice with Trem1-sufficient Kupffer cells restored recruitment of inflammatory monocytes and severity of liver injury. Markedly increased infiltration of liver fibrotic areas with TREM-1-positive Kupffer cells and monocytes/macrophages was found in patients with hepatic fibrosis. Our data support a role of TREM-1 in liver injury and hepatic fibrogenesis and suggests that TREM-1 is a master regulator of Kupffer cell activation, which escalates chronic liver inflammatory responses, activates hepatic stellate cells, and reveals a novel mechanism of promotion of liver fibrosis.
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Induction of innate immune memory: the role of cellular metabolism - ScienceDirect

Induction of innate immune memory: the role of cellular metabolism - ScienceDirect | Immunology | Scoop.it
The paradigm that only adaptive immunity can develop immunological memory has recently been challenged by studies showing that cells from the innate i…
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Roles of the RANKL–RANK axis in antitumour immunity — implications for therapy

Roles of the RANKL–RANK axis in antitumour immunity — implications for therapy | Immunology | Scoop.it
Intriguing evidence suggests that expression of RANK or RANKL by various cells of the tumour microenvironment modulates the anticancer immune response. Herein, the authors review this evidence, discuss the current preclinical and clinical data supporting a potential of RANKL inhibition to improve...
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Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation | Scientific Reports

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BSI at Parliament to discuss ‘The Future of Immunology’ | British Society for Immunology

BSI at Parliament to discuss ‘The Future of Immunology’ | British Society for Immunology | Immunology | Scoop.it
The BSI is co-hosting our first ever Parliamentary event this week on 'The Future of Immunology'.
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Upregulation of HLA‐E by dengue and not Zika viruses - Drews - 2018 - Clinical & Translational Immunology - Wiley Online Library

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Seminars in Cancer Biology | Immuno-oncological biomarkers | ScienceDirect.com

Read the latest articles of Seminars in Cancer Biology at ScienceDirect.com, Elsevier’s leading platform of peer-reviewed scholarly literature
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Checking into the germinal centers: PD-1 regulates entry

Checking into the germinal centers: PD-1 regulates entry | Immunology | Scoop.it
PD-1 functions on T follicular helper cells to dictate localization within lymph node germinal centers.

 

Program cell death protein 1 (PD-1) is an inhibitory B7 family member; it has been extensively studied for its role in inhibiting CD8+ T cell function within tumors or during chronic infections. This work has led to successful clinical use of checkpoint inhibitors to rejuvenate the antitumor T cell response. PD-1 is also a defining marker of CD4+ T follicular helper (TFH) cells, which are responsible for guiding B cell antibody production. What role it plays on TFH cell function is not well understood. Consistent with its inhibitory function, PD-1 does in fact constrain TFH cell development. However, recent work from Shi et al. demonstrates that PD-1 is also required to fine-tune the TFH-B cell humoral response by regulating TFH cell positioning within lymph nodes (LNs). As CD4+ T cells approach the follicle, they are rebuffed by bystander B cells expressing a ligand for PD-1, which dampens phosphoinositide-3 kinase (PI3K) signaling downstream of the chemokine receptor CXCR5 and thus slows T cell motility. As inducible T-cell costimulator (ICOS) enhances PI3K signals, theoretically only T cells with the highest level of ICOS can overcome this and make it through the inhibitory ring of B cells to the germinal center. PD-1 expression also blocks developing TFH cell distraction from chemokines expressed outside of the follicle by negatively regulating CXCR3 expression. All together then, PD-1 expression by recently activated CD4+ T cells helps to enforce a TFH cell concentration within the antigen-reactive B cell germinal center. An unresolved immunologic mystery is how naïve T cells—after early antigenic stimulation—“choose” a particular differentiation fate. In particular, T helper type 2 (TH2) and TFH cells require similar costimulatory signals from antigen presenting cells, express higher levels of CXCR5 than other T effector fates, and are concentrated in the same region of the LN during activation—the T-B border—where they are activated by type 2 conventional dendritic cells (cDC2s). Based on this recent work, differential expression of ICOS might be the tipping point toward TFH rather than TH2 differentiation. By overcoming PD-1 inhibition, strong ICOS signaling allows passage into the germinal center where the final stage of TFH differentiation occurs.

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    1. J. Shi
    2. S. Hou
    3. Q. Fang
    4. X. Liu
    5. X. Liu
    6. H. Qi
    PD-1 controls follicular T helper cell positioning and function.Immunity 49264274 (2018).
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Infographic: The Omentum's Role in Health and Disease

Infographic: The Omentum's Role in Health and Disease | Immunology | Scoop.it
Belly fat helps fight infection, but is also a common site of metastasis.
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Advanced model systems and tools for basic and translational human immunology | Genome Medicine | Full Text

Advanced model systems and tools for basic and translational human immunology | Genome Medicine | Full Text | Immunology | Scoop.it
There are fundamental differences between humans and the animals we typically use to study the immune system. We have learned much from genetically manipulated and inbred animal models, but instances in which these findings have been successfully translated to human immunity have been rare.
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Transient T-bet expression functionally specifies a distinct T follicular helper subset

Transient T-bet expression functionally specifies a distinct T follicular helper subset | Immunology | Scoop.it
T follicular helper (Tfh) cells express transcription factor BCL-6 and cytokine IL-21. Mature Tfh cells are also capable of producing IFN-γ without expressing the Th1 transcription factor T-bet. Whether this IFN-γ–producing Tfh population represents a unique Tfh subset with a distinct differentiation pathway is poorly understood. By using T-bet fate–mapping mouse strains, we discovered that almost all the IFN-γ–producing Tfh cells have previously expressed T-bet and express high levels of NKG2D. DNase I hypersensitivity analysis indicated that the Ifng gene locus is partially accessible in this “ex–T-bet” population with a history of T-bet expression. Furthermore, multicolor tissue imaging revealed that the ex–T-bet Tfh cells found in germinal centers express IFN-γ in situ. Finally, we found that IFN-γ–expressing Tfh cells are absent in T-bet–deficient mice, but fully present in mice with T-bet deletion at late stages of T cell differentiation. Together, our findings demonstrate that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in this Th1-like Tfh cell subset.
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From stability to dynamics: understanding molecular mechanisms of regulatory T cells through Foxp3 transcriptional dynamics - Bending - - Clinical & Experimental Immunology - Wiley Online Library

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Pain and the immune system: emerging concepts of IgG-mediated autoimmune pain and immunotherapies

The immune system has long been recognised important in pain regulation through inflammatory cytokine modulation of peripheral nociceptive fibres. Recently, cytokine interactions in brain and spinal cord glia as well as dorsal root ganglia satellite glia have been identified important— in pain...
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Cardioimmunology: the immune system in cardiac homeostasis and disease

Cardioimmunology: the immune system in cardiac homeostasis and disease | Immunology | Scoop.it
Recent studies have characterized complex interactions between resident and infiltrating immune cells in the heart and cardiac cells, including cardiomyocytes, fibroblasts and endothelial cells. This Review explores the role of immune cells in cardiac development and physiological function, as...
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Researchers find elusive source of most abundant immune cell

Researchers find elusive source of most abundant immune cell | Immunology | Scoop.it
Neutrophils—short-lived, highly mobile and versatile—outnumber all other immune cells circulating through the blood stream. Yet, despite the cells' abundance, the progenitor cell that only gives rise to neutrophils had eluded all efforts to track it down. Now, researchers at La Jolla Institute for Allergy and Immunology identified a progenitor of neutrophils in the bone marrow of mice and humans and tied it to cancer-promoting activities.
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JCI - The role of the complement system in cancer

Review Free access | 10.1172/JCI90962 The role of the complement system in cancer Vahid Afshar-Kharghan First published March 1, 2017 - More info Abstract In addition to being a component of innate immunity and an ancient defense mechanism against invading pathogens, complement activation also participates in the adaptive immune response, inflammation, hemostasis, embryogenesis, and organ repair and development. Activation of the complement system via classical, lectin, or alternative pathways generates anaphylatoxins (C3a and C5a) and membrane attack complex (C5b-9) and opsonizes targeted cells. Complement activation end products and their receptors mediate cell-cell interactions that regulate several biological functions in the extravascular tissue. Signaling of anaphylatoxin receptors or assembly of membrane attack complex promotes cell dedifferentiation, proliferation, and migration in addition to reducing apoptosis. As a result, complement activation in the tumor microenvironment enhances tumor growth and increases metastasis. In this Review, I discuss immune and nonimmune functions of complement proteins and the tumor-promoting effect of complement activation. Introduction The complement system is a cascade of serine proteases encoded by genes originating from the same ancestral genes as coagulation proteins (1). Like the coagulation system, complement activation involves several steps, is tightly regulated, and requires both plasma and membrane proteins (2, 3). Many complement proteins possess dual functions that provide crosstalk between the complement system and other effector and regulatory systems. As a result, the complement system participates in adaptive immunity, hemostasis, neuroprotection and synaptic pruning, and organ development in addition to its role in innate immunity. It is also involved in a diverse array of pathologic conditions, such as thrombotic disorders, autoimmune disorders, schizophrenia, alloimmune responses including allograft rejection and graft-versus-host disease, and cancer. The complement system’s role in fighting invasive pathogens has been extensively studied (4, 5), but recent discoveries provide new perspectives on the complement system’s function in the extravascular and interstitial tissue compartment. These discoveries illustrate an important role for complement proteins in cell-cell and stroma-cell communications. In this Review, I briefly discuss activation, regulation, immune, and nonimmune functions of the complement system to provide a framework for examining the role of complement in cancer. Activation of the complement system The complement system is activated by three major pathways: the classical pathway, via antigen-antibody complexes; the alternative pathway, via any permissive surfaces; and the lectin pathway, via binding of pattern-recognizing mannose-binding lectins (MBLs) to carbohydrate ligands on the surface of pathogens (Figure 1 and refs. 6–9). The convergence point for all complement activation pathways is the formation of the C3 convertase complex on the surface of targeted cells, summarized in Figure 1, A–C. After forming C3 convertase, complement is able to carry out its effector functions. Figure 1 Complement activation. (A) The classical pathway is initiated by a complement-fixing antibody binding to an antigen on targeted cells. C1q binds to the antibody’s Fc domain in the antibody-antigen complex. C1r and C1s assemble on C1q, C1r cleaves and activates C1s, and activated C1s cleaves C4 and C2 into C4b and C2a, respectively. C4b and C2a form the C3 convertase C4bC2a. (B) In the lectin pathway, MBL binds to repetitive sugar moieties such as mannose. MBL and MASP2 then form a C1-like complex. Activated MASP2 in MBL-MASP2 complex cleaves C4 and C2 and generates C3 convertase (C4bC2a). (C) In the alternative pathway, small amounts of hydrolyzed plasma C3 [C3(H2O)] bind to factor B, which forms the C3(H2O)Bb complex with help from factor D. C3(H2O)Bb cleaves additional plasma C3 to generate highly active C3b, which binds to cell the surface. On a complement-activating surface, C3b binds Bb (produced by factor D–mediated cleavage of factor B) and generates C3bBb (the alternative pathway’s C3 convertase). (D) Regardless of the initiation steps, C3 convertase deposits additional C3b molecules and generates C3a. If it remains intact, C3 convertase binds to additional C3b to generate C5 convertase. C5 convertase cleaves C5 to generate C5b. (E) C5b binds to C6, C7, and C8, forming a C5b-8 complex, which polymerizes several C9 molecules, forming the cytolytic MAC. In all three complement activation pathways, C3 convertase complex cleaves C3 molecules to C3a, one of the two major anaphylatoxins, and to C3b, a potent opsonin. Binding of C3b molecules to the surface of cells or cell debris in a process called opsonization marks them for phagocytosis by macrophages. Surface-bound C3b and its degradation products are ligands for complement receptors CR1, CR3, and CR2 that are expressed on myelomonocytic cells, lymphocytes, and follicular dendritic cells. Binding of C3b and its degradation products to correspondent receptors are crucial to cell-cell interactions in the innate and adaptive immune responses and in the removal of complement-coated apoptotic and necrotic cells. Propagation of complement activation by C3 convertase results in the generation of the C5 convertase complex on the cell surface. C5 convertase then cleaves C5 to C5a and C5b. C5a is a potent anaphylatoxin and recruits neutrophils to areas of inflammation and tissue damage. C5b forms a complex with C6 and C7 that may insert into cell membrane, and subsequently be joined by C8 and multiple C9 to form the membrane attack complex (MAC or C5b-9 complex; Figure 1D). Deposition of an adequate number of MACs disrupts the phospholipid bilayer of the cell membrane, leading to massive calcium influx, loss of mitochondrial membrane potential, and cell lysis. However, MAC deposition at sublytic concentrations on cell membrane has a different result, activating intracellular signal transduction and cell proliferation (10). Eukaryotic cells have developed several defense mechanisms to counteract the dire consequences of MAC accumulation at the cell surface, including expression of complement regulatory proteins (CRPs) that disassemble MAC (i.e., CD59, vitronectin, and clusterin), and endocytosis or shedding of MAC from the cell surface. Thus, the three main consequences of complement activation are tagging of cells by C3b degradation products for phagocytosis; chemotaxis of inflammatory cells in response to C3a and C5a; and MAC-mediated cell lysis. As described below, complement activation end products affect tumor growth by altering cancer cell behavior and modulating the immune response to the tumor. Regulation of the complement system The complement system’s ability to cause cellular damage is strictly controlled at several steps, both in the fluid phase and on the cell surface (6). In the classical and lectin pathways, C1 inhibitor (C1INH) binds to and inactivates C1r, C1s, and MBL-associated serine proteases (MASPs). The activities of other CRPs can be categorized into two major groups: (a) decay-accelerating activity, which breaks up the C3 convertase complex, as can be seen in C4-binding protein (C4bp), CR1, decay-accelerating factor (DAF, also known as CD55), and factor H; and (b) membrane cofactor activity, which acts as a cofactor for the factor I–mediated cleavage of C3b or C4b to their inactive degradation products, iC3b and iC4b, respectively. CRPs with membrane cofactor activity include C4bp, CR1, membrane cofactor protein (MCP, or CD46), and factor H. Another important CRP is CD59, which is expressed on many different cell types and prevents assembly of MAC on the cell membrane. The anaphylatoxins C3a and C5a are complement activation products that are rapidly inactivated in plasma by carboxypeptidases, particularly carboxypeptidase N (11). CRPs are overexpressed by many cancer cells and may be used as potential therapeutic targets. Immune function of the complement system The complement system is an ancient defense mechanism preceding adaptive immunity (12). It can be activated by pattern-recognition molecules and natural antibodies (13). Complement system activation and generation of anaphylatoxins orchestrate an inflammatory response to pathogens (12, 14). Anaphylatoxins activate macrophages, neutrophils, mast cells, basophils, and eosinophils, resulting in their degranulation and the production of cytokines, which in turn causes vasodilation, increases vascular permeability, and enhances neutrophil extravasation and chemotaxis (13). The complement system links innate immunity to adaptive immunity. Complement deficiency impairs both B and T cell responses (15). The effect of complement on the B cell response is mediated by CR2 on B cells and follicular dendritic cells. Activation of the classical pathway on the surface of an antigen tags that antigen with C3d, enabling its binding to CR2 on B cells. CR2, CD19, and CD81 form a B cell coreceptor complex, and CR2 engagement with C3d enhances signaling through antigen-encountered B cell receptors and decreases the activation threshold of B cells (12, 15). The interaction between CR2 on follicular dendritic cells and C3d on antigens is important for antigen presentation to naive and primed B cells in the germinal center of lymph nodes, in the maturation of B cells, and in the generation of memory B cells. The role of complement proteins in the cognate interaction between antigen-presenting cells (APCs) and T cells is important in the T cell immune response (16). In addition to systemic production in the liver, complement proteins are also produced locally by T cells and APCs (17–20). The effects of complement proteins on activation, proliferation, and differentiation of T cells are mediated by the local complement activation, by production of C3a and C5a at the interface of T cells and APCs, and through anaphylatoxin receptors on T cells and APCs (17–19, 21, 22). Reducing the number of C3a and C5a receptors (C3aR and C5aR, respectively) on T cells or APCs impairs T cell immunity. Complement proteins and receptors are involved in different stages of the interaction between APCs and T cells. APCs produce C3 and express C3aR and C5aR, both of which are essential for their maturation and differentiation (19) and for effective antigen presentation to T cells (17, 23, 24). C3- or C3aR-deficient APCs are much less potent in inducing a T cell immune response compared with WT APCs (19, 25). After APCs present antigen to T cells, C5aR on the T cells is required for their proliferation. Binding of C5a to C5aR on T cells has both antiapoptotic and pro-proliferative effects (22). Nonimmune function of the complement system Cell-cell and stroma-cell interactions mediated by complement proteins regulate several physiologic processes, such as collective cell migration during embryogenesis (26), synaptic pruning during brain development (27–30), cell proliferation and differentiation during liver regeneration (31) and bone development (32, 33), and hematopoietic stem cell migration and engraftment during hematopoiesis (34). Complement and cancer The surge of interest in cancer immunotherapy is mainly focused on manipulating function or number of cytotoxic T cells. However, two important reasons justify studying the role of complement activation in cancer progression and the effect of complement manipulation in cancer therapy. First, the complement system is an important component of the inflammatory response, and inflammation is involved in various stages of tumorigenesis and cancer progression (35). Second, complement activation regulates adaptive immune response (15) and might have a role in regulating T cell response to tumors. Complement system in inflammation and tumorigenesis. Tumor-promoting inflammation has an important role in carcinogenesis and cancer progression (36–38). A series of elegant experiments established that activation of the complement system is an important component of tumor-promoting inflammation. Bonavita et al. showed that C3-deficient mice were protected against chemical carcinogenesis in mesenchymal and epithelial tissues (39), mainly because of reduced inflammation. Authors identified a humoral component of innate immunity, the long pentraxin PTX3, as an important negative regulator of inflammation and complement activation. PTX3-deficient mice were susceptible to chemical carcinogenesis, displaying an increased number of tumor-associated macrophages with M2 phenotype and increased concentration of CCL2 chemokine inside tumors. The tumor-promoting inflammation induced by PTX3 deficiency was complement-dependent and completely reversed after removal of C3, as manifested by a reduction in the susceptibility of Ptx3–/– C3–/– mice to chemical carcinogenesis. Similarly, treatment with the C5aR antagonist PMX-53 reversed the susceptible phenotype of Ptx3–/– mice without affecting the rate of tumorigenesis in Ptx3+/+ mice. Activation and regulation of complement pathways in tumors. Expression of complement and CRPs is increased in malignant tumors and cancer cell lines (summarized in Table 1). Complement proteins, C3 degradation products, and complement activation products (i.e., C5a, C3a, and C5b-9) are easily detectable in various types of cancer, consistent with complement activation inside these tumors. Table 1 Complement proteins in cancer The main pathway involved in activation of complement inside tumors is unclear, and evidence supports activation of each complement pathway in malignant tumors (40). To make matters more complicated, cancer cell membrane-bound serine proteases can also cleave C5 and generate C5a without complement activation (41). Additionally, complement proteins expressed in tumors might also play a role in cancer progression independent of complement activation, as was shown for C1q in a syngeneic murine model of melanoma, where C1q expression affected angiogenesis, tumor progression, and metastasis (42). In this murine model, C1q was expressed in endothelial cells, spindle-shaped fibroblasts, and tumor-infiltrating myeloid cells independently of C4. Lack of C4 coexpression in C1q-expressing tumors hints at a role for C1q in tumor progression independent of the classical pathway. Expression of CRPs, including both membrane proteins (CD55, CD59, MCP, or CD46) and soluble proteins (factor H and factor H–like proteins), is increased in cancer cells (43), although the overexpression of CRPs is heterogeneous among different cancer types and even between different tumor specimens of the same type of cancer (44). One interpretation of the presence of both complement activation products and CRPs in tumors is that complement activation is a host defense mechanism against cancer, and cancer cells resist complement attack by overexpressing CRPs. However, as discussed later in this Review, several recent studies do not support this interpretation and suggest another scenario in which local complement activation inside tumors enhances tumor growth. Complement activation: antitumor or protumor? Evidence for the effects of complement on malignant transformation of epithelial cells and progression of cancer has evolved based on several recent studies showing complex and sometimes contradictory findings. This complexity is similar to the complex role of inflammation in cancer (45). Although inflammatory cells and cytokines are important in immune surveillance, exemplified by the benefit of bacillus Calmette-Guérin therapy in early stages of bladder cancer, chronic inflammation promotes carcinogenesis and tumor growth. Even immune cells, such as macrophages, can have both pro- and antitumor phenotypes. Despite this multifaceted picture, most evidence points toward a protumor effect of chronic inflammation (45). The long-held view of complement activation as an antitumor defense mechanism is based on two main concepts: first, the complement system’s participation in immune surveillance against malignant cells, and second, complement-dependent cytotoxicity of therapeutic monoclonal antibodies. I will discuss these concepts below, and summarize new information pointing toward a protumor effect of complement activation inside tumors. Complement and immune surveillance. The complement system’s ability to distinguish self from non-self makes it an important part of the innate immune response to invading pathogens (46). Expression of non-self antigens and lack of CRPs on microbes make them optimal targets for complement detection and, later on, complement-mediated elimination. Similarly, expression of danger signals and neoantigens by apoptotic cells and cellular debris optimizes their detection and removal by the complement system. Cancer cells, on the other hand, mostly express the same proteins as their normal epithelial cell counterparts, albeit occasionally with a different density. Furthermore, overexpression of CRPs by cancer cells limits immune surveillance by the complement system (3, 43, 46, 47). Putting these findings together, one can conclude that cell-mediated immunity plays a more important role than humoral immunity in immune surveillance against cancer cells (48, 49), and effectiveness of complement in early detection and elimination of cancer cells is uncertain (50). Complement-dependent cytotoxicity. Complement activation was considered detrimental to cancer cells via complement-dependent cytotoxicity, which causes cancer cell lysis via MAC accumulation or phagocytosis of opsonized cancer cells by macrophages and neutrophils. Complement-dependent cytotoxicity is considered to be the main mechanism for the effectiveness of antitumor monoclonal antibodies. Rituximab, an anti-CD20 antibody against malignant B cells, is among the oldest and most widely used therapeutic monoclonal antibodies. Although in vitro and in vivo studies show that rituximab activates the classical complement pathway (51, 52), the notion that its therapeutic benefits are mainly mediated by induction of complement attack on malignant B cells is questionable. In fact, the antitumor effect of rituximab was inhibited by deposited complement proteins on B cells (53), and was enhanced in complement-deficient mice (54). Therefore, the extent to which complement-dependent cytotoxicity contributes to other immunologic effects of rituximab, i.e., antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis, is unknown. Other studies on the therapeutic mechanism of rituximab also showed a complement-independent, proapoptotic effect mediated by cross-linking of CD20 (55), as well as antiproliferative and antisurvival effects that were mediated by inhibition of B cell receptors (56). Furthermore, many in vitro antitumor effects of complement-fixing antibodies on cancer cell lines were not reproduced in vivo (57). Complement activation promotes tumor growth. Considering that complement is not efficient in immune surveillance against cancer cells and that the main antitumor effect of monoclonal antibodies might not arise from complement activation, the data supporting an antitumor role for complement activation are scant. The question remains: If complement does not attack cancer cells, how does local complement activation and deposition of complement proteins affect tumors? To understand the consequence of complement activation inside tumors, it is helpful to reexamine the biological functions of complement activation products. C3b and its degradation products binding to CR1, CR2, and CR3 provide ligands and receptors for cell-cell and stroma-cell interactions in many physiologic and pathologic conditions. Complement activation generates C3a and C5a and MAC. The anaphylatoxin receptors C3aR and C5aR are G protein–coupled receptors present on many cell types, including lymphocytes, monocytes/macrophages, myeloid cells, hematopoietic stem cells, mesenchymal cells, and epithelial cells, including cancer cells. Anaphylatoxin receptor signaling has been studied extensively (58). Activation of C5aR promotes a range of responses depending on the cell type. Relevant to its role in cancer, C5aR activation generates prosurvival and antiapoptotic responses. For example, C5a binding to C5aR decreases apoptosis in neutrophils (59) and T cells (22), and increases cell proliferation in endothelial (60) and colon cancer cell lines (61). Activation of C3aR plays an important role in guiding collective cell migration (26) and epithelial-mesenchymal transition (62, 63), both important mechanisms in metastasis. In a sublytic density, MAC accumulation on the cell membrane promotes cell proliferation (64) and differentiation, inhibits apoptosis (10, 65), and protects cells against complement-mediated lysis (66). Markiewski et al. showed that the activation of the classical complement pathway inside implanted orthotopic tumors in mice enhanced tumor growth (67). Complement’s progrowth effect on tumors was C5a-dependent and was eliminated in C5aR-deficient mice and in WT mice treated with a C5aR antagonist. C5a modulates the immune response to tumors by acting as a chemotactic factor, increasing infiltration of myeloid-derived suppressor cells (MDSCs) and reducing the number of CD8+ cytotoxic T cells inside tumors. MDSCs are immature myeloid cells that increase in blood, bone marrow, and spleen of tumor-bearing mice and cancer patients (68, 69) and assist tumor cells in evading the antitumor immune response. MDSCs reduce proliferation and increase apoptosis in CD8+ T cells by generating ROS and reactive nitrogen species (70). Depletion of CD8+ T cells in mice eliminated the protective effect of complement deficiency against tumor growth. In summary, this study showed that the immunomodulatory effect of activated classical complement pathway inside tumors enhances tumor growth. The origin of complement proteins was the host, but activation of complement occurred inside the tumor microenvironment, and the final effect on the tumor was an indirect immunomodulatory effect mediated by MDSCs (Figure 2). Figure 2 Effect of complement activation in the tumor microenvironment. Activation of the complement system inside tumors releases C5a and C3a into the tumor microenvironment and promotes tumor growth. C5a attracts myeloid cell, including MDSCs, into the tumor. MDSCs then reduce cytotoxic T cell responses to the tumor by inducing apoptosis and inhibiting CD8+ TILs via generation of ROS and reactive nitrogen species and depletion of arginine. In melanoma, secretion of C3 by CD8+ TILs and complement activation in the vicinity of these cells reduce IL-10 production by TILs and inhibit their function. Some cancer cell types secrete complement proteins into the tumor microenvironment and initiate an autocrine loop that increases cell proliferation and promotes metastasis. The effect of complement activation on MDSCs, TILs, and cancer cells is mediated by the C5a and C3a receptors (C5aR and C3aR) on these cells. In a follow-up study, Nunez-Cruz et al. investigated complement’s role in tumorigenesis in a murine model of spontaneous ovarian cancer (71, 72). C3 or C5aR deficiency in these mice prevented the development of ovarian tumors, permitting no tumors or only small and poorly vascularized tumor formation (71). C3 deficiency was associated with a change in the immune profile of leukocytes infiltrating into the tumors, but C5aR deficiency reduced ovarian tumor size without altering the immune profile of infiltrating leukocytes. This result suggested the existence of a protumor effect of complement that was independent of its immunomodulatory effect. We investigated the effect of complement in murine models of ovarian cancer and confirmed activation of complement in the tumor microenvironment (73). However, complement proteins detected inside ovarian tumors originated not from the host, but from tumor cells themselves. Complement activation products were present even inside tumors implanted in C3-deficient mice lacking a functional complement system. Although orthotopic ovarian cancer tumors in C3-deficient mice reached to the same size as those in WT mice, reducing C3 or C5 production in cancer cells significantly reduced the tumor growth independent of the host’s complement sufficiency status. C3 synthesis can be detected in malignant epithelial cells originating from several different organs, particularly lung and ovary. Inhibiting synthesis of complement proteins in cancer cells altered the immune profile of leukocytes infiltrating into tumors, manifested by an increase in the number of CD8+ T cells and reduction in myeloid cells. However, immunomodulatory effect of complement inhibition was not the main mechanism responsible for the observed reduction in tumor growth. Inhibiting complement protein synthesis in cancer cells implanted in CD8+ T cell–deficient mice reduced tumor growth to the same magnitude as in WT mice. We investigated the possibility of an autocrine stimulation of cancer cells as a result of complement activation. Anaphylatoxin receptors are present on ovarian cancer cells, and stimulation of these receptors by C3a or C5a agonist peptides increased proliferation and invasiveness of ovarian cancer cells in vitro. Furthermore, knockdown of these receptors on cancer cells reduced growth of orthotopic ovarian tumors in mice. Our studies showed that local complement activation inside the tumor microenvironment enhances tumor growth via a direct autocrine effect on ovarian cancer cells increasing cell proliferation (Figure 2). In a murine model, Wang et al. reported another mechanism for the progrowth effect of complement activation in melanoma, showing that production of IL-10 by CD8+ tumor-infiltrating lymphocytes (TILs) is constitutively inhibited in an autocrine fashion by C3 originating from CD8+ TILs themselves, acting through C5aR and C3aR on the surface of these lymphocytes (74). C3aR and C5aR antagonists increased IL-10 production and activated CD8+ TILs that in turn reduce tumor growth. The IL-10–dependent antitumor activity of complement inhibitors in melanoma was independent of the PD-1/PD-L1 axis or MDSCs. This study provides evidence that local complement activation in the tumor microenvironment results in suppression of the immune response to melanoma by inhibiting CD8+ TIL function (Figure 2). The studies above describe different mechanisms by which complement activation in the tumor microenvironment can enhance tumor growth: (a) by altering the immune profile of tumor-infiltrating leukocytes, (b) by increasing cancer cell proliferation, and (c) by directly suppressing CD8+ TIL function. It is possible that different cancer types use different mechanisms to take advantage of ectopic complement activation inside tumors. For example, ovarian cancer cells synthesize a significant amount of complement proteins and initiate an autocrine loop resulting in increased cell proliferation by a direct effect of anaphylatoxins on cancer cells. Conversely, melanoma cells do not secrete complement proteins, and complement proteins produced by CD8+ TILs reduce their IL-10 production and antitumor activity. An important question remains whether complement activation has any role in malignant transformation of normal cells or only affects the expansion of already established malignant clones. Most available data are based on orthotopic murine models of ovarian cancer or mice genetically engineered to develop ovarian cancer by overexpression of oncogenes. These studies showed that complement promotes growth and expansion of malignant tumors. Bonavita et al. showed that complement promotes malignant transformation of cells exposed to chronic inflammation induced by chemical carcinogens (39). However, additional studies are required to dissect the effect of early versus late stages of complement activation on various stages of oncogenesis. Overexpression of CRPs on cancer cells If complement activation promotes tumor growth and oncogenesis, why are CRPs overexpressed on cancer cells? One would expect that cancer cells, under selective pressure, downregulate expression of CRPs to benefit from complement activation. To reconcile these seemingly counterintuitive observations, we put forward the following hypothesis: Anaphylatoxins and sublytic concentrations of MAC promote tumor growth, but higher concentrations of MAC have a tumoricidal effect. As a result, cancer cells benefit from early stages of complement activation and production of anaphylatoxins, but actively inhibit generation of MAC. Cancer cells reduce MAC concentration by overexpressing CD59, the most consistently overexpressed CRP on different cancer cells (75) and the most effective membrane regulatory protein against complement-mediated lysis (43, 76, 77), eliminating MAC from the cell surface through membrane vesiculation. Thus, from a therapeutic point of view, interventions that reduce complement activation or promote the generation of MAC inside tumors can be considered as logical options to counter the progrowth effect of complement on cancer. Complement activation in epithelial-mesenchymal transition Epithelial-mesenchymal transition (EMT) occurs in physiologic processes such as embryogenesis and organ development, and in pathologic conditions including tissue fibrosis and metastasis (78, 79). Complement participates in EMT in murine models of renal injury and fibrosis (62, 80, 81). We showed that complement activation inside tumors not only increases tumor growth but also enhances metastasis by promoting EMT in cancer cells. In ovarian cancer cells, the transcription factor TWIST1 upregulates C3 gene expression, generating C3a in the tumor microenvironment, which binds to C3aR on ovarian cancer cells. We further showed that C3aR signaling increases EMT and decreases E-cadherin expression in ovarian cancer cells via a Krüppel-like factor 5–dependent mechanism and promotes EMT and metastasis (63). In addition to promoting metastasis, EMT also induces resistance to complement-dependent cytotoxicity in lung cancer cells by increasing expression of CD59 (82). Inhibition or knockdown of CD59 restored sensitivity of cancer cells to complement-dependent lysis without altering the morphologic features or protein markers of EMT in these cells. Therapeutic potential of targeting complement activation in cancer Our understanding of the role of the complement system in cancer biology is evolving, changing our approach to the therapeutic use of reagents modifying the complement system. Traditional methods targeting cancer cells using antitumor antibodies to promote lysis of cancer cells by MACs require identification of tumor-specific antigens that either are expressed with a higher density on cancer cells than normal epithelial cells, or are only expressed on cancer cells. Antibodies against EGFR and CD20 are among the most successful therapeutic antibodies. Development of therapeutic antibodies was initially complicated by induction of an immune response to polyclonal antibodies developed in nonhuman hosts. Development of monoclonal murine antibodies; later, chimeric human-mouse antibodies; and recently, humanized antibodies (83) helps overcome this problem. However, a more important problem in harvesting complement-dependent cytotoxicity induced by therapeutic antibodies is overexpression of membrane CRPs by cancer cells that let cancer cells evade MAC-mediated cytolysis (43). As a result, blockade of membrane CRPs on cancer cells, alone or in conjunction with use of therapeutic antibodies, has been tried as another potential therapeutic strategy. Blocking CRPs reduces cancer cell proliferation in vitro (43) and tumor growth in mice (76). CD59 blocking antibodies or CD59 siRNA enhanced complement-mediated cytolysis induced by anti-EGFR monoclonal antibodies (trastuzumab and cetuximab) in human lung cancer cell lines (84). rILYd4, a recombinant protein inhibitor of CD59, increased sensitivity of malignant B cells to rituximab in vitro and in orthotopic murine models (85). Membrane CRPs are universally expressed, and an important theoretical complication of blocking CRPs is exposing normal cells to complement-dependent cytotoxicity. For example, CD59 and CD55 protect red blood cells against complement-induced hemolysis, and blocking CD59 might cause hemolysis. Interestingly, administration of rILYd4 in mice was not associated with significant increases in hemolysis (85). More recent studies showed a protumor effect of complement, and inhibition of complement activation in vitro or in murine models of cancer was investigated as a novel way to treat cancer. Understanding of the autocrine and paracrine effects of complement production and activation inside tumors versus its systemic immunomodulatory effect is not complete, but blocking complement activation or inhibiting C5aR and C3aR signaling inside tumors seems a reasonable approach. However, several questions and concerns regarding the therapeutic use of anticomplement reagents have not been addressed and require additional studies: Does systemic complement inhibition affect local production and activation of complement in the tumor microenvironment? Pharmacokinetic studies based on measurements of tissue concentration of various anticomplement reagents may resolve this issue, although the leakiness of tumor vasculature likely provides adequate tissue penetrance of these reagents. Does complement have a protumor effect in many or only in a few types of cancer? Recent studies showed that complement activation enhances growth of ovarian, cervical, and non–small-cell lung cancer and sarcoma, and deposition of complement proteins is detectable in more cancer cell types (Table 1). Which complement pathways are activated in cancer? Evidence supporting activation of classical, lectin, and alternative pathways in cancer exists. It is possible that in different types of cancer different pathways are functional. Therefore, it is more reasonable to target common complement proteins or receptors in antitumor therapies. What are the effects of early (C3a and C5a) versus late (MAC) complement activation end products on cancer cells? If higher concentrations of C3a or C5a promote, and denser MAC deposit reduces, tumor growth, developing bispecific inhibitory antibodies targeting C5aR (or C3aR) and CD59 simultaneously may increase the potency of the antitumor effect of complement therapy. Complement-dependent cytotoxicity has been considered an important component of the therapeutic benefit of monoclonal antibodies in malignant B cell disorders; however, the effects of complement activation on white blood cell dyscrasia have not been studied. A few reports point to a prognostic significance of expression of complement genes in leukemic blasts (86, 87). More comprehensive studies on the role of complement activation in leukemia and lymphoproliferative disorders might reveal possible therapeutic benefits of anticomplement reagents in these disorders. Eculizumab, a humanized anti-C5 monoclonal antibody, is currently available on the market and is used to treat paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. A single i.v. infusion of eculizumab blocks complement activation in plasma for 2–3 weeks (88, 89), but its potency and half-life in the interstitial tissue are unknown. Eculizumab blocks generation of C5a and MAC, but would not affect synthesis and secretion of complement proteins by cancer cells or C3a generation in the tumor microenvironment. Currently, no ongoing clinical trials are evaluating eculizumab in cancer patients; however, because of the clinical use of this reagent for other indications, we have a relatively clear picture of its side effect profile. Patients on eculizumab are at risk for developing infections with encapsulated microorganisms and should receive meningococcal vaccination before initiation of therapy. Lack of bone marrow suppression with eculizumab is a therapeutic advantage that can be used in designing clinical trials combining this reagent with chemotherapeutic reagents in cancer patients. In a few animal studies, C5aR antagonists, including PMX-53, have been shown to be effective in reducing tumor size in mice (67, 71), but this or similar reagents have not entered into clinical practice yet. Targeting C5aR rather than C5 or C3 might have the potential benefit of leaving opsonization and MAC generation intact. Intact opsonization of bacteria would reduce the risk of infectious complications in individuals undergoing treatment, and the generation of lytic concentrations of MAC might have a tumoricidal effect. On the other hand, targeting C5aR has the disadvantage of leaving other complement effector molecules, such as C3a, uninhibited. A potential advantage of using anticomplement reagents in cancer treatment is that they can be combined with traditional chemotherapies without increasing myelosuppression associated with chemotherapies; and combined with immune checkpoint inhibitors, because they have different targets. While checkpoint inhibitors increase proliferation of cytotoxic T cells, complement inhibitors decrease MDSCs infiltrating into the tumor microenvironment, reduce MDSC-induced T cell suppression, and enhance T cell function. Based on experiences collected with the clinical use of eculizumab, another advantage of complement inhibitors is their relatively few side effects. Any therapeutic use of anticomplement therapies in solid or liquid tumors should be carefully balanced with possible interference of complement inhibition with the efficacy of other antitumor reagents: (a) The outcome of combining anticomplement reagents with monoclonal antibodies (such as cetuximab, rituximab, or trastuzumab) may depend on the importance of complement-dependent cytotoxicity in the function of these antibodies. (b) Chimeric antigen receptor (CAR) T cell therapies depend on in vitro expansion and in vivo proliferation of T cells. Complement inhibition may decrease the proliferation of CAR T cells in vivo and may reduce their efficacy. Conclusions By mediating cell-cell and cell-stroma interactions, complement proteins have several immune and nonimmune functions in both plasma and the extravascular interstitial tissue. Activation of the complement system in the tumor microenvironment enhances tumor growth via different mechanisms. Anticomplement reagents might have a place in the therapeutic armamentarium against cancer and, because of their limited non-myelosuppressive side effects and nonoverlapping pharmacodynamics, could be combined with traditional chemotherapies or immunotherapies. Acknowledgments This work is supported in part by NIH grant CA177909 (to VAK). The author thanks Michael Kroll for his valuable comments. Footnotes Conflict of interest: The author has declared that no conflict of interest exists. Reference information: J Clin Invest. 2017;127(3):780–789. https://doi.org/10.1172/JCI90962. References Krem MM, Di Cera E. Evolution of enzyme cascades from embryonic development to blood coagulation. Trends Biochem Sci. 2002;27(2):67–74. View this article via: PubMed CrossRef Google Scholar Sjöberg AP, Trouw LA, Blom AM. Complement activation and inhibition: a delicate balance. Trends Immunol. 2009;30(2):83–90. View this article via: PubMed CrossRef Google Scholar Zipfel PF, Skerka C. 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