AUTOIMMUNITY

BCR-independent memory B cell reactivation via TLR7 or TLR8 activation, type I interferon production, immune complex formation and T helper cell signalling is central in SLE pathogenesis. Dörner and Lipsky discuss the potential of targeting these pathways to eliminate autoreactive memory B cells...

Aberrant immune responses to viral pathogens contribute to pathogenesis, but our understanding of pathological immune responses caused by viruses within the human virome, especially at a population scale, remains limited. We analyzed whole-genome sequencing datasets of 6,321 Japanese individuals, including patients with autoimmune diseases (psoriasis vulgaris, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), pulmonary alveolar proteinosis (PAP) or multiple sclerosis) and coronavirus disease 2019 (COVID-19), or healthy controls. We systematically quantified two constituents of the blood DNA virome, endogenous HHV-6 (eHHV-6) and anellovirus. Participants with eHHV-6B had higher risks of SLE and PAP; the former was validated in All of Us. eHHV-6B-positivity and high SLE disease activity index scores had strong correlations.

Genome-wide association study and long-read sequencing mapped the integration of the HHV-6B genome to a locus on chromosome 22q. Epitope mapping and single-cell RNA sequencing revealed distinctive immune induction by eHHV-6B in patients with SLE. In addition, high anellovirus load correlated strongly with SLE, RA and COVID-19 status. Our analyses unveil relationships between the human virome and autoimmune and infectious diseases. Analysis of the blood DNA virome in patients with COVID-19 and autoimmune disease associates endogenous HHV-6 (eHHV-6) and high anellovirus load with increased disease risk, most notably for systemic lupus erythematosus. eHHV-6 carriers show a distinct immune response.

Published in NAt. Genetics (Jan. 3, 2025):

https://doi.org/10.1038/s41588-024-02022-z 

WEDNESDAY, Dec. 4, 2024 (HealthDay News) -- Inflammatory bowel disease (IBD) in older adults has become a global public health burden, according to a study published in the Jan. 31, 2025, issue of Autoimmunity Reviews.

As a pivotal cytokine, IL-22 plays a crucial role in the pathogenesis of autoimmune diseases. In this comprehensive review, we initially provide an overvie

Sixteen presentations of preclinical studies funded by the Lupus Research Alliance (LRA) to advance understanding of lupus and potential treatment pathways,...

Identification of HSP60 as an antigenic target of a subset of synovial B cell–derived anti-FLS RA-rmAbs with partial cross-reactivity to other stromal-related antigens. Of 71 rmAbs generated from 4 ELS+ RA synovial tissues, we previously showed that 10 RA-rmAbs display anti-FLS immunoreactivity with variable immunofluorescence patterns (Figure 1A and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI169754DS1). Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of a 60 kDa band of total protein detected via Western blotting by several RA-rmAbs targeting RA-FLSs identified HSP60 as one of the most abundant proteins (among >100 detected by the LC-MS/MS analysis), with a high amount of sequence coverage (68%) across the full length of HSP60 (Figure 1B). HSP60 is a molecular chaperone expressed in different intracellular compartments (e.g., cytosol) as well as extracellularly that migrates to an approximately 58 kDa position in SDS-PAGE (Figure 1C). Figure 1Synovial RA-rmAbs display immunoreactivity to RA-FLS. (A) Schematic summarizing the origin and reactivity characterization of the RA-rmAbs (n = 71) against NETs/cit-histones and RA-FLS. Schematic in A was created using BioRender.com. (B) RA-FLS protein extract was probed with an anti-FLS RA-rmAb. A protein of approximately 60 kDa was excised and analyzed by MS. The analysis detected a high amount of sequence coverage (68%) across the full length of the HSP60 protein in the RA-FLS protein extract. (C) rhHSP60 and RA-FLS protein extracts were subjected to Western blotting and probed with an anti–human HSP60 antibody. (D) Representative immunofluorescence images of RA-FLS incubated with the RA-rmAbs, showing immunoreactivity of 3 RA-rmAbs to FLS-derived antigens (green). RA056/11.95.2 was used as a negative control. Nuclei were stained with DAPI (blue). Original magnification, ×20. (E) Representative immunofluorescence image of RA-FLS showing expression of HSP60 (red). Original magnification, ×40. Therefore, we next performed immunofluorescence studies to determine whether HSP60 and/or other stromal-related antigens were the target of the subset of RA-rmAbs reactive to RA-FLSs (Figure 1D and Supplemental Figure 1). We confirmed the expression of HSP60 in RA-FLSs by Western blotting of the RA-FLS total protein extract and by immunofluorescence staining (Figure 1, C and E). We then quantitatively assessed in an ELISA the binding of the anti-FLS RA-rmAbs to a recombinant human HSP60 (rhHSP60) protein. We identified 3 RA-rmAbs (RA057/11.35.1, RA056/11.76.1, RA056/11.48.2) that showed binding to the rhHSP60 in a dose-dependent manner (Figure 2A). The RA057/11.35.1 rmAb showed the strongest reactivity to rhHSP60. In contrast, 1 RA-rmAb, RA056/11.95.2, which did not bind to RA-FLS (Figure 1D) and was thus used as a negative control, failed to show any binding to rhHSP60 (Figure 2A). The observed immunoreactivity against HSP60 was not due to polyreactivity, as none of the 3 RA-rmAbs displayed polyreactivity to multiple structurally unrelated antigens (Supplemental Figure 2) (2, 20, 21). We also investigated whether the 3 RA-rmAbs with anti-HSP60 immunoreactivity displayed enhanced binding to an in vitro citrullinated form of HSP60 (cit-rHSP60) (Supplemental Figure 3A). As shown in Supplemental Figure 3B, only RA056/11.76.1 rmAbs displayed increased binding to cit-rHSP60. Next, the 3 RA-rmAbs were tested for their reactivity in ELISAs to other RA-associated antigens including anti–cyclic citrullinated peptide (anti-CCP) ELISA, citrullinated fibrinogen, and the FLS-associated antigens calreticulin and vimentin, which are known RA autoantigens and migrate to a similar 60 kDa position in RA-FLS protein extract electrophoresis. As shown in Supplemental Figure 4, we also observed a variable degree of reactivity of the anti-HSP60 RA-rmAb to calreticulin and vimentin, whereas only anti-RA056/11.76.1 rmAb displayed low citrullinated fibrinogen–binding activity. We next conducted a competitive binding assay to further confirm whether HSP60 was recognized by the RA-rmAbs. As shown in Figure 2B, incubation of the RA-rmAbs with soluble rhHSP60 significantly reduced, but did not completely abrogate, the binding to HSP60 in the ELISA. In order to support the HSP60/RA-rmAb binding results, IP assays were performed. As shown in Figure 2C, IP of rhHSP60 with 2 RA-rmAbs (RA057/11.35.1 and RA056/11.76.1) showed a band of approximately 60 kDa, confirming the ELISA data. The absence of signal for the RA-rmAb RA057/11.48.2 might be explained by the low level of binding to HSP60 observed in the ELISA. Figure 2RA-rmAbs with anti-FLS reactivity recognize HSP60. (A) Binding of RA057/11.35.1, RA056/11.76.1, and RA056/11.48.2 rmAbs and a negative RA-rmAb (RA056/11/95.2) to rhHSP60. All RA-rmAbs were tested at a concentration of 100 mg/mL followed by 6 serial dilutions (1:2). (B) Competitive binding of the 3 RA-rmAbs to rhHSP60 preincubated with or without soluble HSP60 (competitor). Results are expressed as a percentage of HSP60 binding. (C) IP results of rhHSP60 and the RA-rmAbs. As a control, rhHSP60 alone was loaded. (D) Representative immunofluorescence image showing staining for HSP60 (red) and RA-rmAbs (green). Nuclei were stained with DAPI (blue). Original magnification, ×63. The data are the results of 3 independent experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with multiple comparisons. We next sought to confirm that the RA-rmAbs target FLS-derived HSP60 by immunofluorescence staining. As shown in Figure 2D, double-immunofluorescence staining with the 3 RA-rmAbs in combination with an anti–human HSP60 antibody demonstrated a partial cellular colocalization with the HSP60. We quantified the degree of colocalization using ImageJ (NIH), with a Pearson’s correlation coefficient of r = 0.4–0.5, which suggested a positive correlation (22, 23). Since the staining morphology of our anti-FLS rmAbs suggested partial binding to cytoskeletal proteins, either through direct biding or interaction with HSP60 (24), we performed double-immunofluorescence staining of the RA-rmAbs with an anti–human F-actin antibody showing variable but largely incomplete colocalization for the 3 RA-rmAbs with a structural FLS antigen (Supplemental Figure 5). Anti-FLS antibodies accelerate arthritis in the CIA mouse model. We explored whether a cocktail of anti-FLS antibodies containing the RA057/11.35.1 and RA056/11.76.1 anti-HSP60 antibodies could have a functional role in the CIA mouse model. CIA was induced by intradermal injection of 2 mg/mL bovine collagen type II on day 0, followed by a boost on day 21. Anti-FLS RA-rmAbs were administered i.p. at 4 time points. As a control, we used both PBS and a mix of RA-rmAbs targeting histone proteins, which were used as a negative control (2, 25). We found that the anti-FLS RA-rmAbs were able to enhance the arthritis score and increase inflammation in the paws compared with the control-treated group (Figure 3A). In contrast, the mice treated with anti-histone RA-rmAbs (2) showed a reduction in the arthritis score (Figure 3A), similar to previous data (25). Histological evaluation of the hind paws showed a large influx of inflammatory cells and signs of cartilage damage in the anti-FLS RA-rmAbs group versus the PBS- and anti-histone RA-rmAb–treated groups (Figure 3B and Supplemental Figure 6). Moreover, micro-CT (μCT) analysis confirmed the increased bone erosion induced by the anti-FLS RA-rmAbs compared with both the control- and anti–histone RA-rmAb–treated group (Figure 3, C–E). Figure 3Effect of anti-FLS RA-rmAbs in CIA. (A) Graph shows the arthritis scores for CIA DBA/1 mice treated i.p. with anti-FLS rmAbs (n = 20), PBS (controls, n = 40), and anti-histone rmAbs (n = 20). Anti-FLS rmAbs were administered on days 17, 28, 38, and 49. The anti-FLS rmAbs cocktail consisted of RA056/11.76.1, RA057/11.35.1, and RA057/11.89.1 antibodies. The anti-histone rmAbs cocktail consisted of RA015/11.58, RA015/11.88, and RA015/11.91 antibodies. Results are from 2 independent experiments. The scores are reported as the mean of the sum of the score ± SEM assessed for each paw of the mouse. The P values correspond to the treatment × time element from the ANOVA linear model between groups. (B) Representative IHC (top) and safranin-O (SO) (bottom) images of control- and anti-FLS–treated mice. Scale bars: 2.5 mm. Original magnification, ×1.5 (enlarged insets, control) and ×23 (anti-FLS). (C) Representative μCT scan of a hind paw from a CIA mouse and the scoring system used to evaluate bone erosion (erosion score). The score was given for each digit (color-coded area), with a maximum score of 24. 0 = normal; 1 = signs of pathological bone changes; 2 = heavy pathological bone changes. (D) Representative μCT scan images of control-treated, anti-FLS–treated (n = 5) and anti-histone–treated (n = 5) mice. (E) Graph shows the μCT erosion score for CIA DBA/1 mice treated with PBS, anti-FLS RA-rmAbs, or anti-histone RA-rmAbs. Five representative hind paws were used for each group. The scoring was done blindly by 3 independent evaluators and is reported as the mean of the sum ± SEM. *P < 0.05 and **P < 0.01, by 1-way ANOVA with multiple comparisons. Antigenic targets of anti-FLS rmAbs such as HSP60 are preferentially expressed in the lympho-myeloid pathotype, colocalize with synovial ELSs, and are upregulated in FLSs derived from leukocyte-rich RA joints. The distribution of HSP60 within RA synovial tissue and in matched synovial fluid (SF) and serum samples from patients with RA was examined by bulk RNA-Seq, immunofluorescence staining, and ELISA, respectively. RNA-Seq expression for HSPD1 (gene name for HSP60) within synovial tissue was compared between the lympho-myeloid, diffuse-myeloid, and pauci-immune fibroid histological groups in patients with early or established RA using bulk RNA-Seq data generated by us from synovial biopsies obtained as part of the Pathobiology of Early Arthritis Cohort (PEAC) study and the R4RA randomized clinical trial (26–28). HSPD1 was elevated in the lympho-myeloid versus the pauci-immune fibroid and diffuse-myeloid in both early (P = 0.0012) and established RA (P = 0.0015) patients (Figure 4, A and B). Moreover, at the protein level, HSP60 showed a preferential distribution around lymphocyte aggregates and periaggregates within the RA synovial tissue (Figure 4, C and D). In addition, we reanalyzed GeoMx Digital Spatial Profiler (DSP) data from the R4RA trial to characterize the spatial positioning of the HSPD1 transcript in association with the different RA histopathotypes (27). Consistent with the bulk RNA-Seq data, HSPD1 expression was significantly higher in the lympho-myeloid group versus the diffuse-myeloid group (P < 0.0001; Figure 4E). Like the HSP60 distribution within the RA synovial tissue determined by immunofluorescence, the spatial positioning of HSPD1 was observed mainly around lymphocyte aggregates independently of the sublining and lining areas (Figure 4, F and G). Moreover, we detected similar levels of HSP60 protein expression in the synovial sublining and lining fibroblasts, with HSP60 protein preferentially observed around lymphocyte aggregates, as previously mentioned (Figure 4C). This observation was supported by scRNA-Seq data from the Accelerating Medicines Partnership (AMP) RA database, in which HSPD1 was found to be highly expressed in fibroblasts from the patients with leukocyte-rich RA (29) (Figure 4H). However, HSPD1 expression was also detected in other cell types, like monocytes (Figure 4H). Finally, we observed higher levels of HSP60 protein in the SF of patients with RA compared with HSP60 levels at the systemic level (Figure 4I). Accumulation of HSP60 in the SF was preferentially observed in patients with RA (n = 20) versus patients with osteoarthritis (OA) (n = 11) (Figure 4J). Figure 4HSP60 expression in synovial tissue and SF of patients with RA. Synovium HSPD1 gene evaluation compared across histological pathotype in patients with early (A) and established (B) RA. Unit y axis: regularized log-normalized read. (C) Representative immunofluorescence and IHC images of synovial tissue from patients with RA showing staining for HSP60 (yellow)/CD90+ (green)/CD55+ (red) synovial fibroblasts and for CD20 (B cells), CD3 (T cells), and CD138 (plasma cells). Nuclei were stained with DAPI (blue). Original magnification, ×2 and ×10 (enlarged insets). (D) Quantification of HSP60 differential expression in RA synovial tissue (RA patients, n = 3) by QuPath analysis. Statistical analysis was done by Friedman’s test. (E) HSPD1 was differentially expressed in the lympho-myeloid (lymphoid) versus diffuse-myeloid (myeloid) pathotypes using GeoMx DSP data. (F) HSPD1 differential expression in the sublining, lining, and aggregated area using GeoMx DSP data. In E and F, statistical analysis was performed using the Mann-Whitney U test. (G) HSPD1 differential expression in the sublining and lining in diffuse-myeloid (myeloid) versus lympho-myeloid (lymphoid) pathotypes. Scatterplot shows individual regions of interest (ROIs)/. Statistical analysis was done using the Kruskal-Wallis test. (H) scRNA-Seq profiling of HSPD1 in synovial fibroblasts in leukocyte-rich/leukocyte-poor RA and OA patients (https://immunogenomics.io/ampra/). (I) Graph shows HSP60 levels (pg/mL) in matched sera and SF from patients with RA (n = 14). Statistical analysis was done with the Wilcoxon test. (J) Box plot displays SF HSP60 levels in RA (n = 20) versus OA (n = 11) patients. Statistical analysis was done using the Mann-Whitney U test. Each data point represents an individual patient. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Similar to HSP60, calreticulin (CALR) gene expression was significantly higher in the lympho-myeloid pathotype in bulk synovial RNA-Seq results and in spatial association with lymphoid aggregates using GeoMx analysis of patients with established RA (Supplemental Figure 7, A–D), supporting the conclusion that stromal-derived autoantigens involved in the cellular stress response were upregulated in RA patients with the lympho-myeloid pathotype and contributed to the generation of pathogenic autoantibodies. Anti-HSP60 antibodies are elevated in the SF of patients with RA. We then investigated the prevalence of anti-HSP60 antibodies in matched SF and serum samples from patients with RA. Serum and SF anti-HSP60 antibodies were measured by ELISA using rhHSP60. As shown in Figure 5A, binding of IgG, IgM, and IgA to HSP60 in SF was positively correlated to RA serum levels. We detected a significant difference of SF IgG, IgM, and IgA anti-HSP60 antibodies between patients with OA or RA, with an increase of IgM levels observed in the SF of patients with RA (Figure 5B and Supplemental Figure 8, A and B). Only 10% of the RA SF tested did not have anti-HSP60 antibodies. Ten percent of RA SF was characterized by the presence of IgG, IgM, and IgA binding to HSP60 (triple-positive), with 25% double-positive and 55% single-positive binding (Supplemental Figure 8C). Finally, patients with RA were divided into ACPA+ (serum, n = 11; SF, n = 10) and ACPA– (serum, n = 9; SF, n = 4) groups using a conventional anti-CCP2 test. As shown in Figure 5C, the levels of antibodies against HSP60 in ACPA+ RA patients were significantly increased for IgA antibodies, with only a trend for IgG and IgM anti-HSP60 antibodies for both serum and SF samples. Figure 5Expression of anti-HSP60 antibodies in serum and SF of patients with RA or OA. (A) Graphs show the correlation of matching RA SF IgG, IgM, and IgA binding to HSP60 with RA sera IgG, IgM, and IgA binding to HSP60. (B) Box plots display the levels of IgG, IgM, and IgA anti-HSP60 antibodies in SF of RA (n = 20) versus OA (n = 11) patients. (C) Scattered dot plots show IgG, IgM, and IgA binding to HSP60 in SF and sera of ACPA+ versus ACPA– RA patients. For B and C, the results are expressed as absorbance at 450 nm, and each data point represents an individual patient. *P < 0.05, **P < 0.01, and ****P < 0.0001, by Mann-Whitney U test. Synovial HSP60 expression is predictive of the clinical response to rituximab and is modulated by B cell depletion in good clinical responders. Given the high correlation of HSP60 with the presence of ELSs in the RA joints, we assessed the longitudinal effect of rituximab therapy on synovial HSPD1 expression before and after B cell depletion in the R4RA biopsy-driven randomized trial (Figure 6A) (27, 28). Baseline HSPD1 gene expression was significantly higher in the B cell–rich subset of patients with RA and was associated with good clinical responders, as defined by the R4RA trial’s clinical primary endpoint, the Clinical Disease Activity Index (CDAI), and by the secondary endpoint, the Disease Activity Score 28–C-reactive protein (DAS28-CRP) (Figure 6, B and C). Hence, we next explored the longitudinal effect of rituximab on HSPD1 within synovial tissue before and after B cell depletion. Using mixed-effects models applied to paired synovial samples (n = 29 individuals, n = 58 samples), we first observed a significant downregulation of HSPD1 expression 16 weeks after treatment with rituximab, but not following tocilizumab therapy (Figure 6D). For tocilizumab, baseline HSPD1 gene expression was not associated with good clinical responders, as defined by the R4RA trial clinical primary endpoint CDAI and by the secondary endpoint DAS28-CRP (Supplemental Figure 9). Interestingly, a significant downregulation of synovial HSPD1 expression upon B cell depletion was selectively observed in good responders compared with nonresponders and moderate responders (Figure 6, E and F) using mixed-effects models applied to paired synovial samples only, with similar results if all samples were included in the models (Supplemental Figure 10A). We observed similar results also for CALR expression (Supplemental Figure 7, E–G), suggesting a common gene expression profile linked to cellular stress response mechanisms in RA-FLS with local release of autoantigens. Figure 6HSP60 analysis of paired pre- and post-rituximab treatment synovial biopsies. (A) Schematic representation of the R4RA clinical trial (27, 28). At baseline, patients with RA underwent a synovial biopsy of a clinically active joint for histological evaluation. Following the synovial biopsy, patients were randomized 1:1 to either the rituximab or tocilizumab treatment group. An optional repeated synovial biopsy of the same joint previously sampled was performed at 16 weeks, followed by histological and transcriptomic evaluation. A CDAI of 50% was used as a primary endpoint to define responders and nonresponders to treatment at 16 weeks. Patients were initially followed up every 4 weeks, with the end of the trial at 48 weeks. Numbers in parentheses represent patients with available RNA-Seq data. (B) Synovium HSPD1 variance-stabilizing transformation (VST) gene expression in patients stratified by histology into B cell–poor (B poor) and B cell–rich (B rich) categories. (C) HSPD1-normalized synovial gene expression levels assessed at baseline in CDAI nonresponders versus responders and DAS28-CRP moderate/nonresponders versus responders to rituximab treatment. (D) HSPD1 RNA-Seq counts were assessed at baseline and 16 weeks following rituximab and tocilizumab treatment in paired synovial biopsies (rituximab, n = 29 individuals, n = 58 samples; tocilizumab, n = 15 individuals, n = 30 samples). (E and F) HSPD1 counts were assessed at baseline and 16 weeks following rituximab treatment in paired synovial biopsies. In D–F, gray data points represent RNA-Seq counts between paired samples for individuals. Statistical analysis was performed using a negative binomial mixed-effects model on RNA-Seq counts. Overlaid green/purple data points show estimated marginal means of the fitted mixed-effects model, with error bars showing 95% CIs for the fixed effects of the mixed-effects model. To assess the clinical response, the following parameters were used: CDAI 50% improvement and DAS28-CRP European Alliance of Associations for Rheumatology (EULAR) response. RTX, rituximab. Finally, in the R4RA cohort we observed a positive correlation between serum anti-HSP60 IgG with CDAI, tender joint count, and DAS28-ESR at baseline (Supplemental Figure 10B), even though IgG anti-HSP60 antibody levels did not change over time after rituximab treatment (Supplemental Figure 11).