CD11b regulates the Treg/Th17 balance in murine arthritis via IL‐6

Th17 cells are often associated with autoimmunity and been shown to be increased in CD11b^?/? mice. Here, we examined the role of CD11b in murine collagen‐induced arthritis (CIA). C57BL/6 and CD11b^?/? resistant mice were immunized with type II collagen. CD11b^?/? mice developed arthritis with early onset, high incidence, and sustained severity compared with C57BL/6 mice. We observed a marked leukocyte infiltration, and histological examinations of the arthritic paws from CD11b^?/? mice revealed that the cartilage was destroyed in association with strong lymphocytic infiltration. The CD11b deficiency led to enhanced Th17‐cell differentiation. CD11b^?/? dendritic cells (DCs) induced much stronger IL‐6 production and hence Th17‐cell differentiation than wild‐type DCs. Treatment of CD11b^?/? mice after establishment of the Treg/Th17 balance with an anti‐IL‐6 receptor mAb significantly suppressed the induction of Th17 cells and reduced arthritis severity. Finally, the severe phenotype of arthritis in CD11b^?/? mice was rescued by adoptive transfer of CD11b⁺ DCs. Taken together, our results indicate that the resistance to CIA in C57BL/6 mice is regulated by CD11b via suppression of IL‐6 production leading to reduced Th17‐cell differentiation. Therefore, CD11b may represent a susceptibility factor for autoimmunity and could be a target for future therapy.     

Treatment of collagen‐induced arthritis by Natura‐α via regulation of Th‐1/Th‐17 responses

Cytokines and CD4⁺ Th cells play a crucial role in the pathogenesis of rheumatoid arthritis. Among the Th populations, Th‐1 and Th‐17 have been described as pathogenic in collagen‐induced arthritis (CIA) whereas Th‐2 and Treg were found to have protective effects. The objective of this study was to examine the affect of Natura‐α, a newly developed cytokine regulator, on CIA and on Th cell development. Natura‐α treatment was administered before or during arthritis induction. Anti‐type II collagen antibodies and cytokine expression were evaluated by ELISA. Emergence of CD4⁺CD25⁺Foxp3⁺ T cells was assessed by flow cytometry. Th‐17 differentiation of naive CD4 T cells was assessed in cultures with anti‐CD3 and anti‐CD28. We showed that Natura‐α both prevented and treated CIA. We further demonstrated that in vivo treatment with Natura‐α inhibited IL‐17 production and anti‐type II collagen IgG development. We showed in vitro, using an APC‐free system, that Natura‐α acted directly on differentiating T cells and inhibiting the formation of Th‐1 and Th‐17 cells but did not affect Th‐2 cells. Since Natura‐α inhibits a large spectrum of important pathogenic factors in CIA, it may provide a new and powerful approach to the treatment of rheumatoid arthritis and other inflammatory diseases.     

A bone‐protective role for IL‐17 receptor signaling in ovariectomy‐induced bone loss

Post‐menopausal osteoporosis is considered to be an inflammatory process, in which numerous pro‐inflammatory and T‐cell‐derived cytokines play a bone‐destructive role. IL‐17A is the signature cytokine of the pro‐inflammatory Th17 population and plays dichotomous roles in diseases that affect bone turnover. Although IL‐17A promotes bone loss in rheumatoid arthritis, it is protective against pathogen‐induced bone destruction in a periodontal disease model. We used a model of ovariectomy‐induced osteoporosis (OVX) in IL‐17 receptor (IL‐17RA)^?/? mice to evaluate the role of the IL‐17A in bone loss caused by estrogen deficiency. Unexpectedly, IL‐17RA^?/? mice were consistently and markedly more susceptible to OVX‐induced bone loss than controls. There were no changes in prototypical Th1, Th2 or Th17 cytokines in serum that could account for increased bone loss. However, IL‐17RA^?/? mice exhibited constitutively elevated leptin, which further increased following OVX. Consistently, IL‐17A and IL‐17F treatment of 3T3‐L1 pre‐adipocytes inhibited adipogenesis, leading to reduced production of leptin. In addition to its role in regulating metabolism and satiety, leptin can regulate bone turnover. Accordingly, these data show that IL‐17A negatively regulates adipogenesis and subsequent leptin expression, which correlates with increased bone destruction during OVX.     

Protection against collagen‐induced arthritis in mice afforded by the parasitic worm product,ES‐62, is associated with restoration of the levels of interleukin‐10‐producing B cells and reduced plasma cell infiltration of the joints

We have previously reported that ES‐62, a molecule secreted by the parasitic filarial nematode Acanthocheilonema viteae, protects mice from developing collagen‐induced arthritis (CIA). Together with increasing evidence that worm infection may protect against autoimmune conditions, this raises the possibility that ES‐62 may have therapeutic potential in rheumatoid arthritis and hence, it is important to fully understand its mechanism of action. To this end, we have established to date that ES‐62 protection in CIA is associated with suppressed T helper type 1 (Th1)/Th17 responses, reduced collagen‐specific IgG2a antibodies and increased interleukin‐10 (IL‐10) production by splenocytes. IL‐10‐producing regulatory B cells have been proposed to suppress pathogenic Th1/Th17 responses in CIA: interestingly therefore, although the levels of IL‐10‐producing B cells were decreased in the spleens of mice with CIA, ES‐62 was found to restore these to the levels found in naive mice. In addition, exposure to ES‐62 decreased effector B‐cell, particularly plasma cell, infiltration of the joints, and such infiltrating B cells showed dramatically reduced levels of Toll‐like receptor 4 and the activation markers, CD80 and CD86. Collectively, this induction of hyporesponsiveness of effector B‐cell responses, in the context of the resetting of the levels of IL‐10‐producing B cells, is suggestive of a modulation of the balance between effector and regulatory B‐cell responses that may contribute to ES‐62‐mediated suppression of CIA‐associated inflammation and inhibition of production of pathogenic collagen‐specific IgG2a antibodies.     

TACI‐dependent APRIL signaling maintains autoreactive B cells in a mouse model of systemic lupus erythematosus

Autoantibodies contribute to the development of systemic lupus erythematosus (SLE). APRIL (a proliferation‐inducing ligand), a member of the TNF superfamily, regulates plasma‐cell survival and binds to TACI (transmembrane activator CAML interactor) and BCMA (B‐cell maturation antigen). We previously showed that APRIL blockade delayed disease onset in lupus‐prone mice. In order to evaluate the role of APRIL receptors in the development of SLE, APRIL, TACI, BCMA , or double TACI.BCMA null mutations were introduced into the Nba2.Yaa (Y‐linked autoimmune acceleration) spontaneous lupus mouse model. Mortality as a consequence of glomerulonephritis (GN) was reduced in Nba2.APRIL^?/?.Yaa , Nba2.TACI^?/?.Yaa and double‐KO mice compared with Nba2.Yaa mice and correlated with lower levels of circulating antibodies, while splenic populations remained unchanged. In contrast, the appearance of symptoms was accelerated in BCMA‐deficient mice, in which TACI signaling was increased. Finally, lupus‐prone mice deficient for the APRIL‐TACI axis produced less pathogenic antibodies and developed less GN. Disease reduction was attributed to impaired T‐independent type 2 responses when the APRIL‐TACI signaling axis was disrupted. Collectively, our results have identified and confirmed APRIL as a new target involved in B‐cell activation, in the maintenance of plasma cell survival and subsequent increased autoantibody production that sustains lupus development in mice.     

T‐bet protects against exacerbation of schistosome egg‐induced immunopathology by regulating Th17‐mediated inflammation

C57BL/6 mice infected with Schistosoma mansoni naturally develop mild CD4⁺ T‐cell‐mediated immunopathology characterized by small hepatic granulomas around parasite eggs. However, immunization with soluble egg Ag in CFA markedly exacerbates the lesions by inducing a potent proinflammatory environment with high levels of IFN‐γ and IL‐17, which are signature cytokines of distinct Th1‐ versus Th17‐cell lineages. To determine the relative role of these subsets in disease exacerbation, we examined mice deficient in T‐bet (T‐bet^?/?), which is required for Th1 differentiation and IFN‐γ production. We now report that immunization with soluble egg Ag in CFA caused a significantly greater enhancement of egg‐induced hepatic immunopathology in T‐bet^?/? mice compared with WT controls, and analysis of their granulomas disclosed a higher proportion of activated DC and CD4⁺ T cells, as well as a marked influx of neutrophils. The absence of IFN‐γ in the T‐bet^?/? mice correlated with a marked increase in IL‐23p19, IL‐17 and TNF‐α in granulomas and MLN. In contrast, T‐bet^?/? mice had lower levels of IL‐4, IL‐5 and IL‐10 and a reduction in FIZZ1 and FoxP3 expression, suggesting diminished regulatory activity, respectively, by alternatively activated macrophages and Treg. These findings demonstrate that T‐bet‐dependent signaling negatively regulates Th17‐mediated immunopathology in severe schistosomiasis.     

DRB1*0402 may influence arthritis by promoting naive CD4+ T‐cell differentiation in to regulatory T cells

HLA‐DRB1*0401 expression in humans has been associated with a predisposition to developing rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA), while HLA‐DRB1*0402 is not associated with susceptibility. Here, we determined if mice transgenic (Tg) for human *0401 have a CD4⁺ T‐cell repertoire that predetermines proinflammatory cytokine production. The data show that both *0401 and *0402 Tg mice can produce TH1/TH17 cytokines, although the kinetics of response may be different. However, in the context of antigen‐specific responses in a CIA model, *0402 Tg mice generate a TH2 response that may explain their resistance to developing arthritis. In addition, a significant subset of naïve CD4⁺ T cells from *0402 Tg mice can be activated in polarizing conditions to differentiate into Treg cells that produce IFN‐γ. *0401 Tg mice harbor memory CD4⁺ T cells that differentiate into IL‐17⁺ cells in various polarizing conditions. Our data suggest that *0401 Tg mice generate a strong immune response to lipopolysaccharide and may be efficient in clearing infection, and may *0401 have been evolutionarily selected for this ability. Autoimmunity, such as RA, could likely be a bystander effect of the cytokine storm that, along with the presence of low Treg‐cell numbers in *0401 Tg mice, causes immune dysregulation.     

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease

Act1 is a negative regulator of B‐cell activation factor of the TNF family (BAFF) and CD40L‐induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti‐nuclear IgG autoantibody (ANA‐IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T‐cell dependent or T‐cell independent manner, we investigated the role of T‐cell help during Act1‐mediated autoimmunity. Act1‐deficiency was bred onto C57Bl/6 (B6.Act1^?/?) mice and B6.TCRβ^?/?TCRδ^?/?Act1^?/? (TKO) mice were generated. While TCRβ/δ‐sufficient B6.Act1^?/? mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA‐IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1^?/? nor TKO mice developed SjS‐like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1^?/? mice. Interestingly, BAFF‐driven transitional B‐cell abnormalities, previously reported in BALB/C.Act1^?/? mice, were intact in B6.Act1^?/? mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE‐like disease in B6.Act1^?/? mice, but not BAFF‐driven transitional B‐cell differentiation.     

Gadd45b and Gadd45g are important for anti‐tumor immune responses

An effective Th1 type cell‐mediated immune response against cancer cells is critical in limiting cancer progression. Gadd45b, a signaling molecule highly up‐regulated during Th1 type responses, is studied for its role in limiting tumor growth. Mouse B16 melanoma cells implanted into Gadd45b^?/? mice grew faster than those in WT or Gadd45b^+/? littermate controls. The defect of Gadd45b^?/? mice in tumor immunosurveillance was attributed to the reduced expression of IFN‐γ, granzyme B, and CCR5 in Gadd45b^?/? CD8⁺ T cells at the tumor site. Activation of p38 MAP kinase, but not ERK or JNK, by either TCR‐stimuli or IL‐12 and IL‐18 is diminished in Gadd45b^?/? CD8⁺ T cells, resulting in reduced production of IFN‐γ. In addition, mRNA of T‐bet and Eomes were reduced in Gadd45b^?/? CD8⁺ T cells, supporting a critical role of Gadd45b in shaping the Th1 fate. More importantly, the tumor vaccination, which is effective in WT mice, failed in Gadd45b/Gadd45g doubly deficient mice. Collectively, these data demonstrate that members of the Gadd45 gene family are important for anti‐tumor immune responses.     

IL‐2 is positively involved in the development of colitogenic CD4+ IL‐7Rαhigh memory T cells in chronic colitis

IL‐2 and IL‐7 share a common γ‐chain receptor and are critical for T‐cell homeostasis. We aimed to clarify the reciprocal roles of IL‐2 and IL‐7 in the development and persistence of chronic colitis. We performed a series of adoptive transfers of IL‐2^?/? CD4⁺CD45RB^high T cells into RAG‐2^?/? mice and assessed the role of IL‐2 in the induction of IL‐7Rα on colitogenic CD4⁺ T cells and the development of chronic colitis. RAG‐2^?/? mice transferred with WT but not with IL‐2^?/? CD4⁺CD45RB^high T cells developed Th1/Th17‐mediated colitis. Consistently, re‐expression of IL‐7Rα was severely impaired on IL‐2^?/? but not on WT CD4⁺ T cells from the transferred mice. To exclude a contribution of the preclinical autoimmunity of IL‐2^?/?mice, WT Ly5.1⁺ or IL‐2^?/? Ly5.2⁺ CD4⁺CD45RB^high T cells from GFP mice previously transplanted with the same number of WT and IL‐2^?/? BM cells were transferred into RAG‐2^?/? mice. RAG‐2^?/? mice transferred with IL‐2^?/?‐derived CD4⁺CD45RB^high T cells did not develop colitis, but their splenic CD4⁺ T cells changed from effector‐memory to central‐memory type. These results show that IL‐2 is critically involved in the establishment and maintenance of IL‐7‐dependent colitogenic memory CD4⁺IL‐7Rα^high T cells.     

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose‐dependent regulation of IgE formation. Methods Wild‐type mice as well as T helper (Th)1‐deficient IL‐12p40^?/? and IFN‐γ^?/? mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen‐dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT‐PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL‐4, IL‐5 and IL‐13, but no induction of IFN‐γ production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen‐ and isotype‐specific manner. Neither CD4⁺ nor CD8⁺ T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4^?CD8^? double‐negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4^?CD8^? dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.     

IL‐25 exhibits disparate roles during Th2‐cell differentiation versus effector function

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL‐25 has been shown to enhance Th2‐type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL‐25^?/? C57BL/6 mice with G4 IL‐4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL‐4‐independent Th2‐cell differentiation. These assays were used to determine whether IL‐25 was critical for the formation of Th2 cells. We found there was no physiological role for IL‐25 in either the differentiation of Th2 cells or their development to effector or memory Th2‐cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL‐25 and its proposed role in promoting Th2‐cell responses.     

Flk‐1+ mesenchymal stem cells aggravate collagen‐induced arthritis by up‐regulating interleukin‐6

The immunomodulatory ability of mesenchymal stem cells (MSCs) may be used to develop therapies for autoimmune diseases. Flk‐1⁺ MSCs are a population of MSCs with defined phenotype and their safety has been evaluated in Phase 1 clinical trials. We designed this study to evaluate whether Flk‐1⁺ MSCs conferred a therapeutic effect on collagen‐induced arthritis (CIA), an animal model of rheumatic arthritis, and to explore the underlying mechanisms. Flk‐1⁺ MSCs, 1–2 × 10⁶, were injected into CIA mice on either day 0 or day 21. The clinical course of arthritis was monitored. Serum cytokine profile was determined by cytometric bead array kit or enzyme‐linked immunosorbent assay. Flk‐1⁺ MSCs and splenocytes co‐culture was conducted to explore the underlying mechanisms. Flk‐1⁺ MSCs did not confer therapeutic benefits. Clinical symptom scores and histological evaluation suggested aggravation of arthritis in mice treated with MSCs at day 21. Serum cytokine profile analysis showed marked interleukin (IL)‐6 secretion immediately after MSC administration. Results of in vitro culture of splenocytes confirmed that the addition of Flk‐1⁺ MSCs promoted splenocyte proliferation and increased IL‐6 and IL‐17 secretion. Moreover, splenocyte proliferation was also enhanced in mice treated with MSCs at day 21. Accordingly, MSCs at low concentrations were found to promote lipopolysaccharide‐primed splenocytes proliferation in an in vitro co‐culture system. We propose that Flk‐1⁺ MSCs aggravate arthritis in CIA model by at least up‐regulating secretion of IL‐6, which favours Th17 differentiation. When Flk‐1⁺ MSCs are used for patients, we should be cautious about subjects with rheumatoid arthritis.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

T‐bet is essential for Th1‐mediated,but not Th17‐mediated,CNS autoimmune disease

T cells that produce both IL‐17 and IFN‐γ, and co‐express ROR‐γt and T‐bet, are often found at sites of autoimmune inflammation. However, it is unknown whether this co‐expression of T‐bet with ROR‐γt is a prerequisite for immunopathology. We show here that T‐bet is not required for the development of Th17‐driven experimental autoimmune encephalomyelitis (EAE). The disease was not impaired in T‐bet^?/? mice and was associated with low IFN‐γ production and elevated IL‐17 production among central nervous system (CNS) infiltrating CD4⁺ T cells. T‐bet^?/? Th17 cells generated in the presence of IL‐6/TGF‐β/IL‐1 and IL‐23 produced GM‐CSF and high levels of IL‐17 and induced disease upon transfer to naïve mice. Unlike their WT counterparts, these T‐bet^?/– Th17 cells did not exhibit an IL‐17→IFN‐γ switch upon reencounter with antigen in the CNS, indicating that this functional change is not critical to disease development. In contrast, T‐bet was absolutely required for the pathogenicity of myelin‐responsive Th1 cells. T‐bet‐deficient Th1 cells failed to accumulate in the CNS upon transfer, despite being able to produce GM‐CSF. Therefore, T‐bet is essential for establishing Th1‐mediated inflammation but is not required to drive IL‐23‐induced GM‐CSF production, or Th17‐mediated autoimmune inflammation.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

MicroRNA‐155 Deficiency Suppresses Th17 Cell Differentiation and Improves Locomotor Recovery after Spinal Cord Injury

Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Mir‐155 has been reported to be involved in both innate and adaptive immune responses. But the role of Mir‐155 in spinal cord injury is still unknown. In our current study, Mir‐155 deficiency displays increased myelin sparring and enhanced SC repair process. The number of T cells, B cells and neutrophils are all significantly lower in Mir‐155^?/? group than that in WT group after SCI. IL‐17A‐producing cells and the expression of IL‐17A are markedly lower in Mir‐155^?/? mice than that in WT mice. We also found higher production of IL‐17 by WT CD4⁺ T cells than Mir‐155^?/? CD4⁺ T cells in vitro. In our further DC‐T cell coculture system, Mir‐155 deficiency in DCs results in significantly less IL‐17 production from T cells. Furthermore, the inhibited Th17 differentiation induced by Mir‐155 deficiency is partly dependent on increased expression of SOCS1. In conclusion, our present work provides evidence to support the concept that Mir‐155 deficiency suppresses Th17 cell differentiation and improves locomotor recovery after SCI.