The Role of TLR7 and TLR9 in the Pathogenesis of Systemic Sclerosis.

The bleomycin-induced scleroderma model is a well-established and dependable method for creating a mouse model of SSc (systemic sclerosis). In the field of skin connective tissue diseases, increasing evidence from clinical and animal experiments suggests that TLRs (Toll-like receptors) play an important role in several diseases. This study aimed to determine the role of TLR7 (Toll-like receptor 7) and TLR9 (Toll-like receptor 9) in the mechanisms of immune abnormalities and fibrosis in SSc. This study used TLR7-KO mice (TLR7-knockout mice with a balb/c background) and TLR9-KO mice (TLR9-knockout mice with a balb/c background) as well as WT mice (wild-type balb/c mice). All three kinds of mice were induced by BLM (bleomycin) in a scleroderma model as the experimental group; meanwhile, WT mice treated with PBS (phosphate-buffered saline) were used as the control group. We analyzed the fibrotic phenotype and the immunological abnormality phenotype of TLR7-deficient and TLR9-deficient mice in the SSc disease model using flow cytometry, RT-PCR (reverse transcription-polymerase chain reaction), a histological examination, and IHC (immunohistochemical staining). In a mouse model of SSc disease, the deletion of TLR7 attenuated skin and lung fibrosis, while the deletion of TLR9 exacerbated skin and lung fibrosis. The deletion of TLR7 resulted in a relative decrease in the infiltration and expression of various pro-inflammatory and fibrotic cells and cytokines in the skin. On the other hand, the deletion of TLR9 resulted in a relative increase in the infiltration and expression of various pro-inflammatory and cytokine-inhibiting cells and cytokines in the skin. Under the influence of pDCs (plasmacytoid dendritic cells), the balances of Beff/Breg (IL-6 + CD19 + B cell/IL-10 + CD19 + B cell), Th17/Treg (IL-17A + CD4 + T cell/Foxp3 + CD25 + CD4 + T cell), M1/M2 (CD86 + macrophage/CD206 + macrophage), and Th1/Th2 (TNFα + CD3 + CD4 + T cell/IL-4 + CD3 + CD4 + T cell) were biased towards the suppression of inflammation and fibrosis as a result of the TLR7 deletion. Comparatively, the balance was biased towards promoting inflammation and fibrosis due to the TLR9 deletion. In the SSc model, TLR7 promoted inflammation and fibrosis progression, while TLR9 played a protective role. These results suggest that TLR7 and TLR9 play opposite roles in triggering SSc to produce immune system abnormalities and skin fibrosis.

Clinical features of patients with connective tissue disease with anti-human upstream binding factor antibodies: A single-center retrospective study.

Anti-human upstream-binding factor (anti-hUBF) antibodies have been reported predominantly in patients with connective tissue diseases (CTDs); these have also been reported in patients without CTDs such as hepatocellular carcinoma. Because of the low frequency of expression and few case reports, there is no consensus on the clinical significance of these antibodies. Thus, we aimed to examine the clinical features of patients with anti-hUBF antibodies and analyzed 1042 patients with clinically suspected CTDs. The presence of anti-hUBF antibodies was screened using immunoprecipitation assays. Of the 1042 patients, 19 (1.82%) tested positive for anti-hUBF antibodies; among them, 10 (56%) were diagnosed with undifferentiated CTD (UCTD), six with systemic sclerosis (SSc) and three with other diseases. Five of the 10 patients with UCTD were referred to our hospital with suspected SSc. None of the five patients fulfilled the 2013 American College of Rheumatology/European League Against Rheumatism classification criteria, but three scored seven points, a relatively high score. Six anti-hUBF-positive patients with SSc had a significantly lower modified Rodnan skin score (mRSS) than that of anti-hUBF-negative patients with SSc (2 [0-2] vs 7 [0-49], p < 0.01). Compared with anti-topoisomerase I-positive patients, anti-hUBF-positive patients had a significantly lower mRSS (2 [0-2] vs 13 [0-42], p < 0.01) and lower incidence of scleroderma renal crisis (0 of 6 vs 8 of 184, p < 0.01). Compared with anti-centromere-positive patients, anti-hUBF-positive patients had a higher incidence of interstitial lung disease (ILD), but the difference was not statistically significant (4 of 6 vs 19 of 239). In conclusion, anti-hUBF antibodies were predominantly detected in patients with CTDs and UCTD. In patients with CTDs, SSc exhibited a high ratio, displaying a lower mRSS and higher incidence of ILD. In patients with UCTD, careful follow-up is recommended as they may develop CTDs in the future.

Anifrolumab for systemic lupus erythematosus: A clinical study of Japanese patients in Kanazawa University Hospital.

We investigated the effectiveness of anifrolumab in treating systemic lupus erythematosus (SLE). We treated seven patients with SLE (age range, 31-68 years; median age, 48 years); one male and six females) with anifrolumab between January 2022 and February 2023 at Kanazawa University Hospital. The period between the onset and initiation of anifrolumab treatment was 60-276 months (median, 234 months), and the SLE disease activity index-2000 (SLEDAI-2 K) before treatment was 2-6 months (median, 3 months). Five patients experienced skin rashes or alopecia, and their cutaneous lupus erythematosus disease area and severity index (CLASI) activity scores were 2-9 (median, 4). Six patients continued treatment with anifrolumab, but one did not because of uncontrolled pleurisy and pericarditis. Our results demonstrated that anifrolumab was effective in treating SLE and reducing both SLEDAI-2 K and CLASI activity scores (median decrease, 100%). Furthermore, the oral corticosteroid dosage could be reduced in all patients who were able to continue treatment. Our findings indicate that anifrolumab is effective not only for reducing disease and eruption activities, but also facilitates tapering of corticosteroid dosage.

Absence of interstitial lung disease at initial visit may predict a favorable outcome for Japanese patients with systemic sclerosis with anti-topoisomerase I antibody.

Interstitial lung disease (ILD) is recognized a prognostic factor and leading cause of death in patients with systemic sclerosis (SSc). The aim of the present study is to clarify factors at an initial visit that are associated with the deterioration of ILD in SSc patients with anti-topoisomerase I (anti-topo I) antibodies. This was a single-center, retrospective, observational study. Fifty-three consecutive SSc patients with anti-topo I antibodies were included in this study. Of the 53 patients, 43 had ILD at their initial visit, whereas 10 did not. We examined the clinical and immunological factors at an initial visit that were associated with the deterioration of ILD. The deterioration of ILD was defined as the administration of intravenous cyclophosphamide (IVCY) therapy. In this cohort, 45 (85%) patients had ILD at the time of the final observation, and only two who did not have ILD at their initial visit developed ILD during the follow-up period. Until the final observation, 26 (49%) patients received IVCY therapy for the progression of ILD. The age at onset, disease duration, SSc subtype, and skin score were similar between patients with and those without IVCY therapy. Approximately 60% (26 of 43) of patients with ILD at their initial visit received IVCY therapy. On the other hand, none of the 10 patients without ILD at their initial visit received IVCY therapy. Our multivariate analyses using Cox proportional hazards regression model revealed that the presence of ILD at an initial visit was an independent factor associated with the introduction of IVCY therapy (odds ratio, 2.8e+7 [95% confidence interval, 1.8e+17-uncalculated], p = 0.0048). Although anti-topo I antibodies are strongly associated with ILD, it was unlikely for SSc patients with anti-topo I antibodies to receive IVCY therapy when they did not have ILD at an initial visit.

Comprehensive autoantibody profiling in systemic autoimmunity by a highly-sensitive multiplex protein array.

Comprehensive autoantibody evaluation is essential for the management of autoimmune disorders. However, conventional methods suffer from poor sensitivity, low throughput, or limited availability. Here, using a proteome-wide human cDNA library, we developed a novel multiplex protein assay (autoantibody array assay; A-Cube) covering 65 antigens of 43 autoantibodies that are associated with systemic sclerosis (SSc) and polymyositis/dermatomyositis (PM/DM). The performance of A-Cube was validated against immunoprecipitation and established enzyme-linked immunosorbent assay. Further, through an evaluation of serum samples from 357 SSc and 172 PM/DM patients, A-Cube meticulously illustrated a diverse autoantibody landscape in these diseases. The wide coverage and high sensitivity of A-Cube also allowed the overlap and correlation analysis between multiple autoantibodies. Lastly, reviewing the cases with distinct autoantibody profiles by A-Cube underscored the importance of thorough autoantibody detection. Together, these data highlighted the utility of A-Cube as well as the clinical relevance of autoantibody profiles in SSc and PM/DM.

Longitudinal changes in nailfold videocapillaroscopy findings differ by myositis-specific autoantibody in idiopathic inflammatory myopathy.

OBJECTIVE: To assess the longitudinal changes in nailfold videocapillaroscopy (NVC) in patients expressing myositis-specific autoantibodies [anti-aminoacyl-tRNA synthetase (ARS), anti-transcriptional intermediary factor 1 (TIF1), and anti-melanoma differentiation-associated gene 5 (MDA5)]. METHODS: This study was performed retrospectively, at a single site, on an observational cohort. Seventy-one idiopathic inflammatory myopathy patients were included (25 patients expressed anti-MDA5 Abs, 24 patients expressed anti-TIF1 Abs, and 22 patients expressed anti-ARS Abs). NVC findings included giant, enlarged, and reduced capillaries, haemorrhages, capillary ramification, disorganization of the vascular array, and capillary loss. NVC findings were compared from baseline to after disease activity stabilization. RESULTS: The frequency of enlarged capillaries at baseline was different among the three groups, and was significantly higher in patients with anti-TIF1 Abs compared with those with anti-ARS Abs (88% vs 55%, P < 0.05). Reduced capillaries were significantly increased in patients with anti-TIF1 Abs compared with those with anti-MDA5 (96% vs 44%, P < 0.0001) or anti-ARS Abs (96% vs 50%, P < 0.0005). Both enlarged and reduced capillaries improved after stabilization in patients with anti-MDA5 Abs (P < 0.0001 and P < 0.05, respectively). These improvements were not observed in patients expressing anti-TIF1 and anti-ARS Abs. However, a significant reduction in haemorrhages was observed in all three groups (P < 0.0001 for each group). CONCLUSIONS: The results of this study demonstrate that longitudinal changes in NVC findings may vary depending on myositis-specific Ab expression. Therefore, it is crucial to assess individual NVC findings separately, as each finding may impact disease activity in a different manner.

FcγRIIB inhibits inflammation in a murine model of psoriasis.

BACKGROUND: Psoriasis is a chronic, inflammatory cutaneous disease. FcγRIIB is a low-affinity receptor for the IgG Fc fragment that provides a negative feedback pathway to down-regulate B-cell antigen receptor signaling. OBJECTIVE: The aim of this study was to investigate the role of FcγRIIB in the development of murine imiquimod (IMQ)-induced, psoriasis-like skin inflammation. METHODS: The experimental psoriasis-like skin inflammation was induced by the topical application of IMQ to the ears of FcγRIIB deficient (FcγRIIB-/-) and wild-type (WT) mice. After 6 days, epidermal thickness and inflammatory cell infiltration of the skin were histopathologically assessed and cytokine and chemokine expression levels were measured with RT-PCR. RESULTS: Skin inflammation was significantly worse in FcγRIIB-/- mice than WT mice. In the skin, the numbers of Gr-1+ neutrophils, CD11c+ dendritic cells, and Foxp3+ T cells were significantly higher in FcγRIIB-/- mice than WT mice. In the spleen, the numbers of CD25+Foxp3+ T cells and CD19+IL-10+ B cells were also significantly higher in FcγRIIB-/-mice than WT mice. The mRNA expression of Il-6, Il-17a, and Il-23a was significantly enhanced in FcγRIIB-/- mice. An adoptive transfer of splenic leukocytes from FcγRIIB-/- mice into WT mice also exacerbated skin inflammation compared to WT mice that received splenic leukocytes from WT mice. Intravenous immunoglobulin significantly reduced skin inflammation in WT mice, but this improvement was not observed in FcγRIIB-/- mice. CONCLUSION: These results indicate that FcγRIIB likely plays a suppressive role in IMQ-induced, psoriasis-like skin inflammation. Furthermore, signal modulation via FcγRIIB is a potential therapeutic target for psoriasis.

Cytokine-producing B-cell balance associates with skin fibrosis in patients with systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin and lung fibrosis. Over 90% of patients with SSc are positive for autoantibodies. In addition, the serum levels of B-cell activating factor, a potent B-cell stimulator, are correlated with SSc severity and activity. Thus, B cells play an important role in SSc pathogenesis. However, two opposing B-cell subsets exist: effector B cells (Beff) and regulatory B cells (Breg). Interleukin (IL)-6-producing Beff have been shown to promote scleroderma in a mouse model, whereas IL-10-producing Breg inhibit scleroderma development. In the present study, we investigated the clinical association of effector and regulatory B cells in patients with SSc. The blood levels of IL-6-producing Beff and IL-10-producing Breg were measured in 30 patients with SSc and 21 healthy subjects by flow cytometry. The frequency of IL-6-producing Beff in the blood was significantly (p < 0.0001) elevated in patients with SSc (median, 56.2%; range, 35.3-81.3%) compared with that in healthy controls (median, 41.3%; range, 21.0-61.3%). In contrast, the frequency of IL-10-producing Breg in the blood was significantly (p < 0.05) decreased in patients with SSc (median, 1.4%; range, 0.5-2.8%) compared with that in healthy controls (median, 2.0%; range, 1.1-3.8%). The Beff/Breg ratio was significantly increased in patients with SSc. In addition, the Beff/Breg ratio was positively correlated with the skin score and extent of interstitial lung disease. These results suggest that dysregulation of effector and regulatory B-cell balance contributes to SSc pathogenesis.

Suppression of IL-23-mediated psoriasis-like inflammation by regulatory B cells.

Psoriasis is an inflammatory cutaneous disease mediated by T-cell dependent immune responses; however, B cells are also considered to play an important role its development. Regulatory B cells (Bregs) regulate immune responses negatively through interleukin-10 (IL-10) production. This study aimed to investigate the role of Bregs in IL-23-mediated psoriasis-like inflammation in mice. Psoriasis-like inflammation was induced in B cell-specific phosphatase and tensin homolog (PTEN)-deficient mice, in which Bregs were significantly expanded, and in their controls, by intradermal injection of 20 µL phosphate-buffered saline (PBS) containing 0.5 µg rmIL-23 into one ear, every other day for 16 days. IL-23-mediated psoriasis-like inflammation was suppressed in B cell-specific PTEN-deficient mice along with decreased ear thickness and epidermal thickness on day 15. Moreover, adoptive transfer of B1 B cells suppressed IL-23-mediated psoriasis-like inflammation. rmIL-23-injected B cell-specific PTEN-deficient mice showed expanded regulatory T cells (Tregs) in the spleen and draining lymph nodes along with increased Bregs. Further, T helper (Th) 17 differentiation in the rmIL-23-injected ear was suppressed in B cell-specific PTEN-deficient mice. Overall, these results indicate that increased Bregs suppress IL-23-mediated psoriasis-like inflammation through Treg expansion and inhibition of Th17 differentiation. Thus, targeting Bregs may be a feasible treatment strategy for psoriasis.

Immunomodulating role of the JAKs inhibitor tofacitinib in a mouse model of bleomycin-induced scleroderma.

BACKGROUND: Janus kinase (JAK)-signal transducer and activator of transcription (STAT) was hyperactivated in biopsies from patients with systemic sclerosis (SSc) and in several autoimmune disease models. Tofacitinib, a pan-JAK inhibitor, blocks the downstream signaling of multiple cytokines and has exhibited therapeutic efficacy in various autoimmune diseases, although its immunomodulating property in scleroderma is unclear. OBJECTIVE: To evaluate the effect of tofacitinib on the modulation of cytokine-producing T and B cells, and proinflammatory cells in a mouse model of SSc. METHODS: Bleomycin (BLM)-induced SSc was generated by intradermal injection of BLM or PBS for control. Mice received intraperitoneal tofacitinib (20 mg/kg) or vehicle 3 times per week from day 0-28. Mice were sacrificed at day 28 after the last BLM/PBS injection. RESULTS: Tofacitinib administration significantly alleviated fibrosis of the skin and lungs in scleroderma mouse model. Furthermore, tofacitinib suppressed adaptive and innate immune responses by reducing splenocytes, total lymphocytes, CD4+ T helper cells (especially Th2 and Th17 subtypes), IL-6-producing effector B cells, PDCA-1+ dendritic cells in the spleen, and infiltration of F4/80+, CD206+ and CD163+ macrophages in the skin and lungs. Conversely, tofacitinib increased the proportions of splenic regulatory T and B cells. The mRNA expression of extracellular matrix proteins and fibrogenic cytokines was downregulated by tofacitinib in both the skin and lungs. CONCLUSION: These observations suggest JAK inhibition as a therapeutic approach for the treatment of inflammatory and fibrotic diseases, and highlight the potential of tofacitinib as a promising candidate for treating patients with scleroderma.

Regulatory B cells and T cell Regulation in Cancer.

Recent researches shed light on B cell role on various autoimmune diseases, including autoantibody-mediated diseases as well as T cell-mediated autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. B cells play a critical role in the immune response beyond the production of antibodies through mechanisms such as antigen presentation and cytokine production. Furthermore, B cells have recently been recognized to play a role in promoting tumor immunity against cancer. However, not all B cells positively regulate immune responses. Regulatory B cells negatively regulate immune responses by the production of anti-inflammatory cytokines such as interleukin (IL)-10, IL-35, and transforming growth factor-beta. Thus, a balance between effector and regulatory B cells regulates the immune response through the release of cytokines. In this review, we highlight the main emerging roles of B cells in tumor immunity with a focus on the T cell response. These findings can guide a protocol for selectively depleting regulatory B cells as a potential therapeutic strategy for patients with cancer.