A novel selective spleen tyrosine kinase inhibitor SKI-O-703 (cevidoplenib) ameliorates lupus nephritis and serum-induced arthritis in murine models.

Spleen tyrosine kinase (Syk) plays a pivotal role in the activation of B cells and innate inflammatory cells by transducing immune receptor-triggered signals. Dysregulated activity of Syk is implicated in the development of antibody-mediated autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the effect of Syk inhibition on such diseases remains to be fully evaluated. We have developed a novel selective Syk inhibitor, SKI-O-592, and its orally bioavailable salt form, SKI-O-703 (cevidoplenib). To examine the efficacy of SKI-O-703 on the progression of SLE, New Zealand black/white mice at the autoimmunity-established phase were administrated orally with SKI-O-703 for 16 weeks. Levels of IgG autoantibody, proteinuria, and glomerulonephritis fell significantly, and this was associated with hypoactivation of follicular B cells via the germinal center. In a model of serum-transferred arthritis, SKI-O-703 significantly ameliorated synovitis, with fewer neutrophils and macrophages infiltrated into the synovial tissue. This effect was recapitulated when mice otherwise refractory to anti-TNF therapy were treated by TNF blockade combined with a suboptimal dose of SKI-O-703. These results demonstrate that the novel selective Syk inhibitor SKI-O-703 attenuates the progression of autoantibody-mediated autoimmune diseases by inhibiting both autoantibody-producing and autoantibody-sensing cells.

DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1ß. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.

Splenic Long-Lived Plasma Cells Promote the Development of Follicular Helper T Cells during Autoimmune Responses.

Long-lived plasma cells (LLPCs) develop under the help of follicular helper T (Tfh) cells and reside mainly in the bone marrow. However, these cells are unusually abundant in the spleen of several autoimmune models including K/BxNsf mice, yet their pathogenic impact remains unknown. To investigate a previously unappreciated role of splenic LLPCs, we sorted splenic plasma cells (PCs) from K/BxNsf and K/BxN mice, corresponding to LLPCs and conventional short-lived PCs, respectively, and compared their phenotypes and ability to prime and induce the differentiation of naive CD4(+) T cells into effector cells in vitro and in vivo. We found that K/BxNsf PCs had lower levels of the Ag presentation machinery and costimulators than K/BxN PCs, and also a lower CD4(+) T cell priming capacity. Autoantigen-pulsed K/BxNsf PCs selectively polarized cognate CD4(+) T cells toward the expression of molecules necessary for Tfh development and function. As a result, the K/BxNsf PC-primed CD4(+) T cells were more effective in stimulating B cells to produce autoantigen-specific IgGs than K/BxN PCs or even dendritic cells. Adoptive transfer of K/BxNsf PCs, but not K/BxN PCs, to K/BxN mice increased numbers of Tfh cells in draining lymph nodes. These results propose that abnormal accumulation of LLPCs in the spleen of autoimmune models drives the differentiation of autoantigen-primed CD4(+) T cells to Tfh cells. This positive feedback loop between splenic LLPCs and Tfh cells may contribute to the persistence of humoral autoimmunity.

Foxp3+ regulatory T cells ensure B lymphopoiesis by inhibiting the granulopoietic activity of effector T cells in mouse bone marrow.

Foxp3(+) Treg cells are crucial for maintaining T-cell homeostasis, but their role in B-cell homeostasis remains unclear. Here, we found that Foxp3 mutant scurfy mice had fewer B-lineage cells and progenitors, including common lymphoid progenitors and lymphoid-primed multipotent progenitors, but higher myeloid-lineage cell numbers in BM compared with WT littermates. Homeostasis within the HSC compartment was also compromised with apparent expansion of long- and short-term HSCs. This abnormality was due to the lack of Treg cells, but not to the Treg-cell extrinsic functions of Foxp3 or cell-autonomous defects. Among cytokines enriched in the BM of scurfy mice, IFN-γ affected only B lymphopoiesis, but GM-CSF, TNF, and IL-6 collectively promoted granulopoiesis at the expense of B lymphopoiesis. Neutralization of these three cytokines reversed the hematopoietic defects on early B-cell progenitors in scurfy mice. Treg cells ensured B lymphopoiesis by reducing the production of these cytokines by effector T cells, but not by directly affecting B lymphopoiesis. These results suggest that Treg cells occupy an important niche in the BM to protect B-lineage progenitor cells from excessive exposure to a lymphopoiesis-regulating milieu.

Swimming exercise alleviates pathological bone features in curdlan-injected SKG mice by inducing irisin expression.

This study assessed the therapeutic potential of swimming exercise in the curdlan-injected SKG mouse model and investigated the modulatory effects of irisin on inflammation. Curdlan-injected SKG were randomly assigned to either a home-cage group or a swimming group for 6 weeks. Changes in clinical arthritis scores and ankle thickness were measured weekly. Post-swimming program, mice were anesthetized for collection of vastus lateralis muscle and blood, which was followed by histological analysis, micro-CT imaging of the ankle joints, and the measurement of pro-inflammatory cytokines and irisin levels. Additionally, curdlan-injected SKG mice were intravenously injected with recombinant irisin protein and observed. Finally, serum levels of irisin in healthy control and ankylosing spondylitis (AS) patient groups were measured by ELISA. The swimming group of curdlan-injected SKG mice exhibited significant improvements in arthritis and enthesitis compared to the home-cage group. In particular, micro-CT and histological analyses revealed a notable reduction in pathological bone features in the swimming group compared to the home-cage group. Muscle endurance was also enhanced in the swimming group compared to the home-cage group, as determined by the wire-hanging test. Intriguingly, irisin levels not only were statistically increased in the swimming group but, also, TNF-α, IL-1ß, and IL-6 levels were decreased. Additionally, injection of irisin protein slightly attenuated both arthritis and enthesitis in curdlan-injected SKG mice. Meanwhile, irisin serum levels were declined in AS patients. Overall, we found that swimming exercise attenuated pathological bone features in an AS animal model, potentially mediated by increased irisin serum levels with associated anti-inflammatory effects.

The complement factor H-related protein-5 (CFHR5) exacerbates pathological bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. KEY MESSAGES: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS.

Interleukin-21 promotes osteoclastogenesis in humans with rheumatoid arthritis and in mice with collagen-induced arthritis.

OBJECTIVE: Bone destruction is a critical pathology involved in the functional disability caused by rheumatoid arthritis (RA). Osteoclasts, which are specialized bone-resorbing cells regulated by cytokines such as RANKL, are implicated in bone destruction in RA. The aim of this study was to determine whether interleukin-21 (IL-21), a potent immunomodulatory 4-α-helical bundle type 1 cytokine, has osteoclastogenic activity in patients with RA and in mice with collagen-induced arthritis (CIA). METHODS: The expression of IL-21 in synovial tissue was examined using immunohistochemistry. The concentrations of IL-21 in serum and synovial fluid were determined by enzyme-linked immunosorbent assay. The levels of RANKL and osteoclastogenic markers were measured using real-time polymerase chain reaction. CD14+ monocytes from patients with RA or mouse bone marrow cells were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA or CD4+ T cells from mice with CIA in the presence of IL-21 and subsequently stained for tartrate-resistant acid phosphatase activity to determine osteoclast formation. RESULTS: IL-21 was up-regulated in the synovium, synovial fluid, and serum of patients with RA and in the synovium and serum of mice with CIA. IL-21 induced RANKL expression in mixed joint cells and CD4+ T cells from mice with CIA and in CD4+ T cells and FLS from patients with RA. Moreover, IL-21 enhanced in vitro osteoclastogenesis without the presence of RANKL-providing cells and by inducing RANKL expression in CD4+ T cells and FLS. CONCLUSION: Our data suggest that IL-21 promotes osteoclastogenesis in RA. We believe that therapeutic strategies targeting IL-21 might be effective for the treatment of patients with RA, especially in preventing bone destruction.

Foxp3+ regulatory T cells control humoral autoimmunity by suppressing the development of long-lived plasma cells.

Foxp3(+) regulatory T cells (Tregs) are crucial for maintaining T cell tolerance, but their role in humoral autoimmunity remains unclear. To address this, we combined a model of autoantibody-dependent arthritis (K/BxN) with Foxp3 mutant scurfy mice to generate Treg-deficient K/BxN mice, referred to as K/BxNsf mice. The disease symptoms of K/BxNsf mice were exacerbated, and this coincided with increases in extrafollicular Th cells, follicular Th cells, and germinal centers. Surprisingly, the K/BxNsf mice exhibited an abnormal accumulation of mature plasma cells in their spleens and a corresponding loss of bone marrow plasma cells. The plasma cells were unresponsive to the bone marrow homing chemokine CXCL12, despite normal expression of the chemokine receptor CXCR4. Importantly, they were long-lived and less susceptible to the cytotoxic action of cyclophosphamide. They also expressed less FcγRIIb and were less apoptotic in response to autoantigen-autoantibody immune complexes. This suggests that Tregs control plasma cell susceptibility to cell death induced by engagement of FcγRIIb with immune complexes. Direct cytotoxic effects of Tregs also contribute to the death of plasma cells. Thus, our results reveal that Tregs suppress the emergence of long-lived splenic plasma cells by affecting plasma cell-autonomous mechanisms as well as T cell help, thereby avoiding the persistence of humoral autoimmunity.

A novel NLRP3 inhibitor as a therapeutic agent against monosodium urate-induced gout.

Background: Since NEK7 is critical for NLRP3 inflammasome activation, NEK7 inhibitors could be employed as therapeutic agents against gout, a representative disease caused by NLRP3 inflammasome. Methods: We designed NEK7 inhibitors based on biochemical kinome profiling of 2,7-substituted thieno[3,2-d]pyrimidine derivatives (SLC3031~3035 and SLC3037). Inflammasome activation was assessed by ELISA of IL-1b and immunoblotting of IL-1b maturation after treatment of bone marrow-derived macrophages with LPS+monosodium urate (MSU). NLPR3 binding to NEK7 and oligomerization were examined using immunoprecipitation and Blue Native gel electrophoresis, respectively. In vivo effect was investigated by studying gross and histopathological changes of food pad tissue of MSU-injected mice, together with assays of maturation of IL-1b and ASC speck in the tissue. Results: SLC3037 inhibited inflammasome by MSU and other inflammasome activators through blockade of NLRP3 binding to NEK7 or oligomerization, and subsequent ASC oligomerization/phosphorylation. SLC3037 significantly reduced foot pad thickness and inflammation by MSU, which was superior to the effects of colchicine. SLC3037 significantly reduced content or maturation of IL-1b and ASC speck in the food pad. The number and height of intestinal villi were decreased by colchicine but not by SLC3037. Conclusion: SLC3037, a NLRP3 inhibitor blocking NEK7 binding to NLRP3, could be a novel agent against diseases associated with NLRP3 inflammasome activation such as gout, cardiovascular diseases, metabolic syndrome or neurodegenerative diseases.

The Expression of the Alpha7 Nicotinic Acetylcholine Receptor and the Effect of Smoking in Curdlan-Administered SKG Mice.

Nicotine, an abundant molecule in tobacco, has immunomodulatory effects on inflammatory diseases, primarily due to the activation of alpha7 nicotinic acetylcholine receptor (α7 nAChR). We aim to evaluate the expression of the α7 nAChR+ cells in joint tissue and the effect of smoking on immune cells and peripheral arthritis in curdlan-administered SKG mice, a murine model of spondyloarthropathy (SpA). The SKG mice were injected with curdlan two times at 2-week intervals and were divided into two groups; one exposed to cigarette smoke and the other not exposed. We found that the α7 nAChR+ cells increased in the joint tissue of curdlan-administered SKG mice compared to in the wild type. Furthermore, the peripheral arthritis scores and histological scores for synovial inflammation were lower in smoke-exposed curdlan-administered SKG mice than in mice not exposed to smoke. Immunofluorescence staining of the α7 nAChR+ and IL-17A+ cells was lower in the synovia of smoke-exposed mice than the control mice. The proportions of α7 nAChR+IL-17A+ and α7 nAChR+IL-17A+FOXP3+ cells also decreased in the synovia of smoke-exposed mice compared with the controls. We observed an increase in the α7 nAChR+ cells within the joint tissue of curdlan-administered SKG mice and that cigarette smoke had an influence on both peripheral arthritis and immune cell population, especially α7 nAChR+ cells. Thus, exposure to cigarette smoke after arthritogenic stimuli may have an anti-arthritogenic effect in curdlan-administered SKG mice.

Platelet-Derived Growth Factor B Is a Key Element in the Pathological Bone Formation of Ankylosing Spondylitis.

Enthesophyte formation plays a crucial role in the development of spinal ankylosis in ankylosing spondylitis (AS). We aimed to investigate the role of platelet-derived growth factor B (PDGFB) in enthesophyte formation of AS using in vitro and in vivo models and to determine the association between PDGFB and spinal progression in AS. Serum PDGFB levels were measured in AS patients and healthy controls (HC). Human entheseal tissues attached to facet joints or spinous processes were harvested at the time of surgery and investigated for bone-forming activity. The impact of a pharmacological agonist and antagonist of platelet-derived growth factor B receptor (PDGFRB) were investigated respectively in curdlan-treated SKG mice. PDGFB levels were elevated in AS sera and correlated with radiographic progression of AS in the spine. Mature osteoclasts secreting PDGFB proteins were increased in the AS group compared with HC and were observed in bony ankylosis tissues of AS. Expression of PDGFRB was significantly elevated in the spinous enthesis and facet joints of AS compared with controls. Moreover, recombinant PDGFB treatment accelerated bone mineralization of enthesis cells, which was pronounced in AS, whereas PDGFRB inhibition efficiently reduced the PDGFB-induced bone mineralization. Also, PDGFRB inhibition attenuated the severity of arthritis and enthesophyte formation at the joints of curdlan-treated SKG mice. This study suggests that regulating PDGFB/PDGFRB signaling could be a novel therapeutic strategy to block key pathophysiological processes of AS. © 2022 American Society for Bone and Mineral Research (ASBMR).

Role of IL-17A in the development of colitis-associated cancer.

A close relationship between inflammation and colon cancer has been widely accepted, and interleukin (IL)-17A plays an important role in controlling colonic inflammation. However, the role of IL-17A has not yet been validated in colitis-associated cancer (CAC). This study aims to identify the effects of IL-17A in tumorigenesis utilizing IL-17A-deficient mice in an experimental CAC model. CAC was induced in both the IL-17A-deficient and the C57BL/6 (wild-type, WT) mice by injection of 12.5 mg/kg azoxymethane followed by three rounds of 1.7% dextran sodium sulfate exposure to elicit colitis. On day 63 after the start of the study, mice were sacrificed. Colonic inflammation, proliferation and tumorigenesis were evaluated. Tumor numbers per mouse (1.43 versus 5.80; P = 0.02) and mean tumor size (1.17 versus 3.58 mm; P = 0.01) were significantly decreased in IL-17A-deficient mice compared with WT mice. Furthermore, the inflammation and the proliferation scores of IL-17A-deficient mice were significantly lower than WT mice. In the analysis of inflammatory mediators, IL-6, interferon-γ, tumor necrosis factor-α and IL-17A were markedly decreased in IL-17A-deficient mice compared with WT mice. In the western blot analysis, p-STAT3, cyclin D1, cyclin-dependent kinase 2, cyclin E, Glycogen synthase kinase 3-ß and p-Akt were downregulated in IL-17A-deficient mice. Immunohistochemical staining with p-STAT3, Ki-67 and ß-catenin revealed lower number of stained cells in IL-17A-deficient mice compared with WT mice. IL-17A ablation significantly decreases CAC tumorigenesis and thus may play an important role associated with chronic colitis.

5,7-Dihydroxy-3,4,6-trimethoxyflavone inhibits intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 via the Akt and nuclear factor-κB-dependent pathway, leading to suppression of adhesion of monocytes and eosinophils to bronchial epithelial cells.

5,7-Dihydroxy-3',4',6'-trimethoxyflavone (eupatilin), the active pharmacological ingredient from Artemisia asiatica Nakai (Asteraceae), is reported to have a variety of anti-inflammatory properties in intestinal epithelial cells. However, little information is known about the molecular mechanism of eupatilin-induced attenuation of bronchial epithelial inflammation. This study investigates the role of eupatilin in the adhesion of inflammatory cells such as monocytes and eosinophils to bronchial epithelial cells. Stimulation of a human bronchial epithelial cell line (BEAS-2B) with tumour necrosis factor-α (TNF-α) increased the expression of surface adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in which eupatilin significantly inhibited the expression of those adhesion molecules in a dose-dependent manner. Eupatilin suppressed the TNF-α-induced activation of IκBα and nuclear factor-κB (NF-κB) signals in BEAS-2B cells. The IκB kinase (IKK) activation was also significantly reduced in eupatilin-pre-treated BEAS-2B and primary normal human bronchial epithelial (NHBE) cells. However, eupatilin did not influence AP-1 activity in TNF-α-stimulated cells. Suppression of NF-κB signalling induced by eupatilin resulted in the inhibition of the expression of adhesion molecules and the adhesion of monocytes and eosinophils to BEAS-2B cells. Furthermore, eupatilin suppressed the phosphorylation of Akt in TNF-α-stimulated BEAS-2B and NHBE cells, leading to down-regulation of NF-κB activation and adhesion molecule expression and finally to suppression of the inflammatory cell adhesion to epithelial cells. These results suggest that eupatilin can inhibit the adhesion of inflammatory cells to bronchial epithelial cells via a signalling pathway, including activation of Akt and NF-κB, as well as expression of adhesion molecules.

B Cell-Specific Deletion of CR6-Interacting Factor 1 Drives Lupus-like Autoimmunity by Activation of Interleukin-17, Interleukin-6, and Pathogenic Follicular Helper T Cells in a Mouse Model.

OBJECTIVE: CR6-interacting factor 1 (CRIF1) is a nuclear transcriptional regulator and a mitochondrial inner membrane protein; however, its functions in B lymphocytes have been poorly defined. This study was undertaken to investigate the effects of CRIF1 on B cell metabolic regulation, cell function, and autoimmune diseases. METHODS: Using mice with B cell-specific deletion of CRIF1 (Crif1ΔCD19 mice), we assessed the relevance of CRIF1 function for lupus disease parameters, including anti-double-stranded DNA (anti-dsDNA), cytokines, and kidney pathology. RNA sequencing was performed on B cells from Crif1ΔCD19 mice. The phenotypic and metabolic changes in immune cells were evaluated in Crif1ΔCD19 mice. Roquinsan/+ mice crossed with Crif1ΔCD19 mice were monitored to assess the functionality of CRIF1-deficient B cells in lupus development. RESULTS: Crif1ΔCD19 mice showed an autoimmune lupus-like phenotype, including high levels of autoantibodies to dsDNA and severe lupus nephritis with increased mesangial hypercellularity. While loss of CRIF1 in B cells showed impaired mitochondrial oxidative function, CRIF1-deficient B cells promoted the production of interleukin-17 (IL-17) and IL-6 and was more potent in helping T cells develop into follicular helper T cells. In a mouse model of autoimmune lupus, depletion of CRIF1 in B cells exacerbated lupus severity, and CRIF1 overexpression prevented lupus development in roquinsan/san mice. CONCLUSION: These results demonstrated that CRIF1 negatively correlates with disease severity and that overexpression of CRIF1 ameliorates disease development. Our findings suggest that CRIF1 is essential for preventing lupus development by maintaining B cell self tolerance.

Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil apoptosis in early and late periods of stimulation.

Although Helicobacter pylori infections of the gastric mucosa are characterized by the infiltration of inflammatory cells such as eosinophils, the responses of eosinophils to H. pylori vacuolating cytotoxin (VacA) have not been fully elucidated. This study investigates the role of VacA in the apoptosis of human eosinophils. We treated human eosinophils with purified H. pylori VacA and observed that induction of apoptosis is a relatively late event. Expression of cellular inhibitor of apoptosis protein (c-IAP)-2 was upregulated during the early period of VacA stimulation, and transfection with c-IAP2 siRNA augmented apoptotic cell death. VacA caused the translocation of cytoplasmic Bax to the mitochondria and increased cytochrome c release from mitochondria in eosinophils. Transfection of an EoL-1 eosinophil cell line with Bax siRNA decreased the release of cytochrome c and DNA fragmentation. Furthermore, apoptosis facilitated by Bax and cytochrome c was primarily regulated by p38 MAPK in VacA-treated eosinophils. These results suggest that the exposure of human eosinophils to H. pylori VacA induces the early upregulation of c-IAP2 and a relatively late apoptotic response, with the apoptosis progressing through a sequential pathway that includes p38 MAPK activation, Bax translocation, and cytochrome c release.

A positive feedback loop of IL-21 signaling provoked by homeostatic CD4+CD25- T cell expansion is essential for the development of arthritis in autoimmune K/BxN mice.

Rheumatoid arthritis is a joint-specific autoimmune inflammatory disease of unknown etiology. The K/BxN mouse is a model of rheumatoid arthritis that is thought to be mainly due to autoantibody-mediated inflammatory responses. We showed previously that homeostatic proliferation of autoreactive CD4(+) T cells is required for disease initiation in the K/BxN mice. In this study, we show that the homeostatically proliferating CD4(+)CD25(-) T cells produce IL-21. We generated IL-21R-deficient (IL-21R(-/-)) K/BxN mice and found that these mice were completely refractory to the development of spontaneous arthritis. They contained fewer CD4(+) T cells with a reduced proportion of homeostatically proliferating cells, fewer follicular Th cells, and, surprisingly, more Th17 cells than their control counterparts. They also failed to develop IgG1(+) memory B cells and autoantigen-specific IgG1 Ab-secreting cells. IL-21 induced expression of receptor activator of NF-kappaB ligand (RANKL) a regulator of osteoclastogenesis, and few RANKL-expressing infiltrates were found in the synovia of IL-21R(-/-) K/BxN mice. Thus, our results demonstrate that IL-21 forms a positive feedback autocrine loop involving homeostatically activated CD4(+) cells and that it plays an essential role in the development of autoimmune arthritis by mechanisms dependent on follicular Th cell development, autoreactive B cell maturation, and RANKL induction but independent of Th17 cell function. Consistent with this, in vivo administration of soluble the IL-21R-Fc fusion protein delayed the onset and progression of arthritis. Our findings suggest that effective targeting of IL-21-mediated processes may be useful in treating autoimmune arthritis.

An Inflammatory Loop Between Spleen-Derived Myeloid Cells and CD4+ T Cells Leads to Accumulation of Long-Lived Plasma Cells That Exacerbates Lupus Autoimmunity.

Splenic long-lived plasma cells are abnormally numerous and deleterious in systemic autoimmune diseases, yet how they accumulate remains poorly understood. We demonstrate here that a pathological role of spleen-derived CD11b+Gr-1+ myeloid cells (SDMCs) underpins the accumulation of splenic long-lived plasma cells in a lupus-prone model named sanroque. We found that SDMCs were progressively accumulated in sanroque mice from the early clinical phase. Transcriptome profiles revealed that SDMCs have a predominant shift toward an inflammatory phenotype relative to the bone marrow-derived counterparts and are distinct from neutrophils and monocytes. SDMCs were expanded in situ via splenic extramedullary myelopoiesis under the proinflammatory cytokine milieu during lupus progression. SDMCs promoted the development of IFN-γ-secreting Th1 and follicular helper T cells, thereby licensing CD4+ T cells to be pathologic activators of SDMCs and plasma cells. SDMCs also directly promoted the survival of plasma cells by providing B-cell activating factor of the TNF family. The frequency of SDMCs correlated with that of splenic long-lived plasma cells. Selective depletion of CD11b+Gr-1+ cells reduced autoantibody production in sanroque mice. Thus, our findings suggest that SDMCs expanded in situ establish a positive feedback loop with CD4+ T cells, leading to accumulation of long-lived plasma cells which exacerbates lupus autoimmunity.

Acetylated Diacylglycerol 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol in Autoimmune Arthritis and Interstitial Lung Disease in SKG Mice.

Acetylated diacylglycerol 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) is a lipid molecule from the antlers of sika deer that might reduce inflammation by effectively controlling neutrophil infiltration, endothelial permeability and inflammatory chemokine production. Therefore, we evaluated the modulatory effect of PLAG on arthritis and interstitial lung disease (ILD) in an autoimmune arthritis model. We injected curdlan into SKG mice and PLAG was orally administered every day from 3 weeks to 20 weeks after the curdlan injection. The arthritis score was measured every week after the curdlan injection. At 20 weeks post-injection, the lung specimens were evaluated with H&E, Masson's trichrome and multiplexed immunofluorescent staining. Serum cytokines were also analyzed using a Luminex multiple cytokine assay. PLAG administration decreased the arthritis score until 8 weeks after the curdlan injection. However, the effect was not sustained thereafter. A lung histology revealed severe inflammation and fibrosis in the curdlan-induced SKG mice, which was attenuated in the PLAG-treated mice. Furthermore, immunofluorescent staining of the lung tissue showed a GM-CSF+ neutrophil accumulation and a decreased citrullinated histone 3 expression after PLAG treatment. PLAG also downregulated the levels of IL-6 and TNF-α and upregulated the level of sIL-7Rα, an anti-fibrotic molecule. Our results indicate that PLAG might have a preventative effect on ILD development through the resolution of NETosis in the lung.

Bacillus subtilis-specific poly-gamma-glutamic acid regulates development pathways of naive CD4(+) T cells through antigen-presenting cell-dependent and -independent mechanisms.

Peripheral naive CD4(+) T cells selectively differentiate to type 1 T(h), type 2 T(h) and IL-17-producing T(h) (T(h)17) cells, depending on the priming conditions. Since these subsets develop antagonistically to each other to elicit subset-specific adaptive immune responses, balance between these subsets can regulate the susceptibility to diverse immune diseases. The present study was undertaken to determine whether poly-gamma-glutamic acid (gamma-PGA), an edible and safe exopolymer that is generated by microorganisms such as Bacillus subtilis, could modulate the development pathways of T(h) subsets. The presence of gamma-PGA during priming promoted the development of T(h)1 and T(h)17 cells but inhibited development of T(h)2 cells. gamma-PGA up-regulated the expression of T-bet and ROR-gammat, the master genes of T(h)1 and T(h)17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of T(h)2 cells. gamma-PGA induced the expression of IL-12p40, CD80 and CD86 in dendritic cells (DC) and macrophages in a Toll-like receptor-4-dependent manner, and the effect of gamma-PGA on T(h)1/T(h)2 development was dependent on the presence of antigen-presenting cells (APC). Furthermore, gamma-PGA-stimulated DC favored the polarization of naive CD4(+) T cells toward T(h)1 cells rather than T(h)2 cells. In contrast, gamma-PGA affected T(h)17 cell development, regardless of the presence or absence of APC. Thus, these data demonstrate that gamma-PGA has the potential to regulate the development pathways of naive CD4(+) T cells through APC-dependent and -independent mechanisms and to be applicable to treating T(h)2-dominated diseases.

CCAAT/enhancer binding protein ß Induces Post-Switched B Cells to Produce Blimp1 and Differentiate into Plasma Cells.

Long-lasting post-switched plasma cells (PCs) arise mainly from germinal center (GC) reactions, but little is known about the mechanism by which GC B cells differentiate into PCs. Based on our observation that the expression of the transcription factor CCAAT/enhancer binding protein ß (C/EPBß) is associated with the emergence of post-switched PCs, we enquired whether a cell-autonomous function of C/EPBß is involved in the program for PC development. To address this, we generated C/EPBß-deficient mice in which the Cebpb locus was specifically deleted in B cells after transcription of the Ig γ1 constant gene segment (Cγ1). In response to in vitro stimulation, B cells from these Cebpbfl/flCγ1Cre/+ mice had defects in the induction of B lymphocyte-induced maturation protein 1 (Blimp1) and the formation of IgG1+ PCs, but not in proliferation and survival. At steady state, the Cebpbfl/flCγ1Cre/+ mice had reduced serum IgG1 titers but normal IgG2c and IgM titers. Moreover, upon immunization with T-dependent Ag, the mice produced reduced levels of Ag-specific IgG1 Ab, and were defective in the production of Ag-specific IgG1 Ab-secreting cells. These results suggest that a cell-autonomous function of C/EPBß is crucial for differentiation of post-switched GC B cells into PCs through a Blimp1-dependent pathway.