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Emergency myelopoiesis (EM) is a hematopoietic response against systemic infections that quickly supplies innate immune cells. As lymphopoiesis is strongly suppressed during EM, the role of lymphocytes in that process has not received much attention. Here, we found that myeloid-like B cells (M-B cells), which express myeloid markers, emerge in the bone marrow (BM) after the induction of EM. M-B cells were mainly derived from pre-B cells and preferentially expressed IL-10, which directly stimulates hematopoietic progenitors to enhance their survival and myeloid-biased differentiation. Indeed, lacking IL-10 in B cells, blocking IL-10 in the BM with a neutralizing antibody, and deleting the IL-10 receptor in hematopoietic progenitors significantly suppressed EM, which failed to clear microbes in a cecal ligation and puncture model. Thus, a distinct B cell subset generated during infection plays a pivotal role in boosting EM, which suggests the on-demand reinforcement of EM by adaptive immune cells.

Acquired immune reaction is initiated by dendritic cells (DCs), which present Ags to a few naive Ag-specific T cells. Deregulation of gene expression in DCs may alter the outcome of the immune response toward immunodeficiency and/or autoimmune diseases. Expression of TRIM28, a nuclear protein that mediates gene silencing through heterochromatin, decreased in DCs from old mice, suggesting alteration of gene regulation. Mice specifically lacking TRIM28 in DCs show increased DC population in the spleen and enhanced T cell priming toward inflammatory effector T cells, leading to acceleration and exacerbation in experimental autoimmune encephalomyelitis. TRIM28-deficient DCs were found to ectopically transcribe endogenous retrovirus (ERV) elements. Combined genome-wide analysis revealed a strong colocalization among the decreased repressive histone mark H3K9me3-transcribed ERV elements and the derepressed host genes that were related to inflammation in TRIM28-deficient DCs. This suggests that TRIM28 occupancy of ERV elements critically represses expression of proximal inflammatory genes on the genome. We propose that gene silencing through repressive histone modification by TRIM28 plays a role in maintaining the integrity of precise gene regulation in DCs, which prevents aberrant T cell priming to inflammatory effector T cells.

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.

Hematopoiesis is a system that provides red blood cells (RBCs), leukocytes, and platelets, which are essential for oxygen transport, biodefense, and hemostasis; its balance thus affects the outcome of various disorders. Here, we report that stem cell antigen-1 (Sca-1), a cell surface marker commonly used for the identification of multipotent hematopoietic progenitors (Lin−Sca-1+c-Kit+ cells; LSKs), is not suitable for the analysis of hematopoietic responses under biological stresses with interferon production. Lin−Sca-1−c-Kit+ cells (LKs), downstream progenitors of LSKs, acquire Sca-1 expression upon inflammation, which makes it impossible to distinguish between LSKs and LKs. As an alternative and stable marker even under such stresses, we identified CD86 by screening 180 surface markers. The analysis of infection/inflammation-triggered hematopoiesis on the basis of CD86 expression newly revealed urgent erythropoiesis producing stress-resistant RBCs and intact reconstitution capacity of LSKs, which could not be detected by conventional Sca-1–based analysis.

The balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell-mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.

Osteopontin (OPN) is a protein, generally considered to play a pro-tumorigenic role, whereas several reports have demonstrated the anti-tumorigenic function of OPN during tumor development. These opposing anti- and pro-tumorigenic functions are not fully understood. Here, we report that host-derived OPN plays an anti-tumorigenic role in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model and a TRAMP tumor transplant model. Tumor suppression mediated by OPN in Rag2-/- mice suggests that OPN is dispensable in the adaptive immune response. We found that host-derived OPN enhanced infiltration of natural killer (NK) cells into TRAMP tumors. The requirement of OPN in NK cell migration towards TRAMP cells was confirmed by an ex vivo cell migration assay. In contrast to TRAMP cells, in vivo B16 tumor development was not inhibited by OPN, and B16 tumors did not show OPN-mediated cell recruitment. It is possible that low levels of chemokine expression by B16 cells do not allow OPN to enhance immune cell recruitment. In addition to demonstrating the anti-tumorigenic role of OPN in TRAMP tumor development, this study also suggests that the contribution of OPN to tumor development depends on the type of tumor as well as the source and isoform of OPN.

Objective Syndecan 4 has been implicated as a critical mediator of inflammatory responses because of its functions as a coreceptor and reservoir for growth factors and chemokines. Although syndecan 4 is known to be expressed on B cells, its role in immune responses remains unclear. The purpose of this study was to investigate the contribution of syndecan 4 to the development of immune arthritis in murine models. Methods The clinical severity of 3 immunopathologically distinct models, collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and collagen antibody–induced arthritis (CAIA), was evaluated in wild-type (WT) mice and in syndecan 4–deficient (Sdc4–/–) mice. Germinal center (GC) formation, B cell profiles, humoral immune responses, and B cell migration were analyzed during the course of CIA. Results Sdc4–/– mice were resistant to the induction of CIA, which is T cell and B cell dependent, but AIA and CAIA, which are T cell dependent and T cell independent, respectively, occurred with equal frequency in WT mice and Sdc4–/– mice. Furthermore, Sdc4–/– mice had reduced numbers of B cells and deficient GC formation in draining lymph nodes (DLNs) during the course of CIA, resulting in reduced production of collagen-specific autoantibodies. Compared with B cells from WT mice, B cells from Sdc4–/– mice showed reduced chemotactic migration toward stromal cell–derived factor 1 (SDF-1) and reduced SDF-1–mediated Akt phosphorylation. Consistent with these findings, in vivo transfer experiments showed that fewer Sdc4–/– B cells than WT B cells were found in and around the follicles in the DLNs. Conclusion Our findings suggest that syndecan 4 contributes to the development of CIA by promoting GC formation and autoantibody production through its regulation of SDF-1–mediated B cell migration.

The lung is constantly exposed to the outer environment; thus, it must maintain a state of immune ignorance or tolerance not to overrespond to harmless environmental stimuli. How cells in the lung control immune responses under nonpathogenic condition is not fully understood. In this study, we found that autophagy plays a critical role in the lung-specific immune regulation that prevents spontaneous inflammation. Autophagy in pulmonary myeloid cells plays a role in maintaining low burdens of environmental microbes in the lung, as well as in lowering mitochondrial reactive oxygen species production and preventing overresponse to TLR4 ligands in alveolar macrophages. Based on these mechanisms, we also found that intranasal instillation of antibiotics or an inhibitor of reactive oxygen species was efficient in preventing spontaneous pulmonary inflammation. Thus, autophagy in myeloid cells, particularly alveolar macrophages, is critical for inhibiting spontaneous pulmonary inflammation, and pulmonary inflammation caused by dysfunctional autophagy is pharmacologically prevented.

Tumor-derived matricellular proteins such as osteopontin (OPN) and tenascin-C (TN-C) have been implicated in tumor growth and metastasis. However, the molecular basis of how these proteins contribute to tumor progression remains to be elucidated. Importantly, these matricellular proteins are known to interact with α9β1 integrin. Therefore, we hypothesized that tumor-derived α9β1 integrin may contribute to tumor progression. To clarify the roles of α9β1 integrin in tumor growth and lymphatic metastasis, we used an inhibitory anti-human α9β1 integrin antibody (anti-hα9β1 antibody) and a α9β1 integrin-positive human breast cancer cell line, MDA-MB-231 luc-D3H2LN (D3H2LN), in vitro functional assays, and an in vivo orthotopic xenotransplantation model. In this study, we demonstrated that tumor, but not host α9β1 integrin, contributes to tumor growth, lymphatic metastasis, recruitment of cancer-associated fibroblasts (CAFs), and host-derived OPN production. We also found that CAFs contributed to tumor growth, lymphatic metastasis, and host-derived OPN levels. Consistent with those findings, tumor volume was well-correlated with numbers of CAFs and levels of host-derived OPN. Furthermore, it was shown that the inoculation of D3H2LN cells into mammary fat pads with mouse embryonic fibroblasts (MEFs), obtained from wild type, but not OPN knock-out mice, resulted in enhancement of tumor growth, thus indicating that CAF-derived OPN enhanced tumor growth. These results suggested that tumor α9β1-mediated signaling plays a pivotal role in generating unique primary tumor tissue microenvironments, which favor lymphatic metastasis and tumor growth.Tumor α9β1 integrin promotes lymphatic metastasis through enhancing invasion. Tumor α9β1 integrin promotes tumor growth through CAFs. Tumor α9β1 integrin enhances the recruitment of CAFs into the primary tumor. Tumor cells induce the production of OPN by CAFs in the primary tumor. CAF-derived OPN promotes tumor growth.

Recruiting pathogenic T cells to the central nervous system (CNS) is a critical step during the development of experimental autoimmune encephalomyelitis (EAE). Here, we report that the absence of autophagy and microtubule-associated protein 1A/1B-light chain 3-associated phagocytosis significantly delayed the onset of EAE in Atg7 conditional knockout (Atg7 CKO) mice in myeloid cells. T-helper cell-cell priming appeared to be normal in the Atg7 CKO mice, but the mice showed significant accumulation of Th17 cells in the lung. The data suggested that the stalling of Th17 cells in the lung en route to the CNS caused the delay. The lung of Atg7 CKO mice, in which we previously demonstrated spontaneous mild inflammation, showed high expression of CCL20, a chemokine that attracts Th17 cells. We have also shown that LPS intranasal instillation delayed EAE onset, suggesting that pulmonary inflammation has an impact on EAE development. Based on our data, therapeutic immunomodulation targeted to the lung, rather than systemically, might be a possible future option to treat multiple sclerosis.

Osteopontin is critically involved in rheumatoid arthritis; however, the molecular cross-talk between osteopontin and joint cell components that leads to the inflammatory joint destruction is largely unknown. We found that not only osteopontin but also tenascin-C and their common receptor, α9 integrin, are expressed at arthritic joints. The local production of osteopontin and tenascin-C is mainly due to synovial fibroblasts and, to a lesser extent, synovial macrophages. Synovial fibroblasts and macrophages express α9 integrin, and autocrine and paracrine interactions of α9 integrin on synovial fibroblasts and macrophages and its ligands contribute differently to the production of proinflammatory cytokines and chemokines. α9 integrin is also involved in the recruitment and accumulation of inflammatory cells. Inhibition of α9 integrin function with an anti-α9 integrin Ab significantly reduces the production of arthrogenic cytokines and chemokines and ameliorates ongoing arthritis. Thus, we identified α9 integrin as a critical intrinsic regulator that controls the development of autoimmune arthritis.

Osteopontin (OPN) contains at least two major integrin recognition domains, Arg159-Gly-Asp161 (RGD) and Ser162-Val-Val-Tyr-Gly-Leu-Arg168 (SVVYGLR), recognized by alphavbeta3 and alpha5beta1 and alpha4 and alpha9 integrins, respectively. OPN is specifically cleaved by thrombin and matrix metalloproteinase (MMP)-3 or MMP-7 at a position of Arg168/Ser169 (R/S) and Gly166/Leu167 (G/L), respectively. We in this study examined the requirement of residues within SVVYGLR for the alpha4 and alpha9 integrin recognition and how MMP-cleavage influences the integrin recognition. The residues, Val164, Tyr165, and Leu167 are critical for alpha4 and alpha9 integrin recognition in both cell adhesion and cell migration. The residue Arg168 is additionally required for alpha9 integrin recognition in cell adhesion and this explains why alpha9 integrin binds to only thrombin cleaved form of OPN. alpha4 integrin is able to bind to SVVYG (MMP-cleaved form of RAA OPN-N half), while alpha9 integrin is not, supporting the above notion that Arg168 is additionally required for alpha9 integrin-mediated cell adhesion. The residue Val163 is important for alpha4, but not for alpha9 integrin recognition in cell migration. Importantly, we found that the replacement of Arg168 by Ala (R168A mutant) induces the augmentation of cell migration via alpha4 and alpha9 integrins.

Osteopontin (OPN) has been implicated in various helper T cell type 1 immunity-mediated diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), Crohn's disease, and fulminant hepatitis. Increased expression of OPN has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-OPN antibody in mice. Antibody treatment may elicit side effects including allergic reactions against heterologous antibody proteins, thus necessitating humanization of antibody. To provide alternative means to neutralize OPN function, in this study we explored the possibility of using OPN small interfering RNA (siRNA) to silence OPN gene expression. In vitro, OPN siRNA efficiently silenced the expression of both exogenous and endogenous OPN gene. After hydrodynamic intravenous injection of OPN siRNA, OPN siRNA was efficiently delivered to the liver, which resulted in the efficient silencing of OPN gene expression in liver. In a murine model of concanavalin A (ConA)-induced fulminant hepatitis, OPN expression was elevated in liver and severe hepatic necrosis was induced. Importantly, after OPN siRNA treatment, the OPN expression level in liver was significantly reduced and liver tissue injury was ameliorated, as reflected by the significant reduction of serum alanine aminotransferase levels and almost normal liver histology. Thus, this study indicates that OPN siRNA delivery has therapeutic potential in various inflammatory diseases in which OPN play a critical role by silencing OPN gene expression in vivo.

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert anti-microbial responses. Here, we report a role of autophagy in an early host anti-fungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80hi macrophages in the spleen, peritoneum, and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80hi macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.

Osteopontin (OPN) is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7). Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to alpha 9b1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA). This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA.

Syndecan 4 has been implicated as a critical mediator of inflammatory responses because of its functions as a coreceptor and reservoir for growth factors and chemokines. Although syndecan 4 is known to be expressed on B cells, its role in immune responses remains unclear. The purpose of this study was to investigate the contribution of syndecan 4 to the development of immune arthritis in murine models.The clinical severity of 3 immunopathologically distinct models, collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and collagen antibody-induced arthritis (CAIA), was evaluated in wild-type (WT) mice and in syndecan 4-deficient (Sdc4(-/-) ) mice. Germinal center (GC) formation, B cell profiles, humoral immune responses, and B cell migration were analyzed during the course of CIA.Sdc4(-/-) mice were resistant to the induction of CIA, which is T cell and B cell dependent, but AIA and CAIA, which are T cell dependent and T cell independent, respectively, occurred with equal frequency in WT mice and Sdc4(-/-) mice. Furthermore, Sdc4(-/-) mice had reduced numbers of B cells and deficient GC formation in draining lymph nodes (DLNs) during the course of CIA, resulting in reduced production of collagen-specific autoantibodies. Compared with B cells from WT mice, B cells from Sdc4(-/-) mice showed reduced chemotactic migration toward stromal cell-derived factor 1 (SDF-1) and reduced SDF-1-mediated Akt phosphorylation. Consistent with these findings, in vivo transfer experiments showed that fewer Sdc4(-/-) B cells than WT B cells were found in and around the follicles in the DLNs.Our findings suggest that syndecan 4 contributes to the development of CIA by promoting GC formation and autoantibody production through its regulation of SDF-1-mediated B cell migration.

Sphingosine 1-phosphate (S1P) plays a role in lymphocyte egress from lymphoid organs. However, it remains unclear how S1P production and secretion are regulated. We show that under inflammatory conditions, α9 integrin, which is closely associated with activated β1 integrin, and its ligand, tenascin-C, colocalize on medullary and cortical sinuses of draining lymph nodes (dLNs), which is a gate for lymphocyte exit, and that inhibition of lymphocyte egress is evident by blockade of α9 integrin-mediated signaling at dLNs. Based on in vitro analysis using lymphatic endothelial cells obtained from mice embryos, we suggested the possibility that stimulation of lymphatic endothelial cells by tenascin-C enhances S1P secretion in an α9 integrin-dependent manner without affecting S1P synthesis and/or degradation. Blockade of α9 integrin-mediated signaling reduced lymphocyte egress from dLNs in several models, including experimental autoimmune encephalomyelitis, where it improved clinical scores and pathology. Therefore, manipulating α9 integrin function may offer a therapeutic strategy for treating various inflammatory disorders.

Objective The role of the joint tissue microenvironment in the pathogenesis of human RA has recently attracted much attention. The present study investigated the roles of α9β1 integrin and its ligands in synovial specimens of human RA patients in generating the unique human arthritic tissue microenvironment. Methods Synovial fibroblasts and macrophages were isolated from the synovial tissue of patients with RA or OA. The expression of α9β1 integrin was analysed using FACS with multicolour staining. The production of MMPs and proinflammatory cytokines was analysed in cultures of synovial fibroblasts and macrophages with α9β1 integrin ligands. Results Synovial fibroblasts and macrophages derived from arthritic joints spontaneously secreted tenascin-C and osteopontin. Synovial fibroblasts and macrophages obtained from patients with RA expressed α9β1 integrins, a common receptor for osteopontin and tenascin-C. In the synovial fibroblasts of RA, the amount of tenascin-C protein produced was much greater than that of osteopontin in synovial fibroblasts of RA. Importantly, autocrine and paracrine interactions of α9β1 integrin and tenascin-C induced the expression of MMPs and IL-6 in synovial fibroblasts, as well as TNF-α and IL-1β in synovial macrophages. Conclusion These findings indicate that autocrine and paracrine interaction of α9β1 integrin and tenascin-C in the joint tissue microenvironment contributes to the pathogenesis of RA. Therefore α9β1 integrin may become a potential therapeutic target for RA.

Rheumatoid arthritis (RA), a chronic systemic inflammatory disorder that principally attacks synovial joints, afflicts over 2 million people in the United States. Interleukin (IL)-17 is considered to be a master cytokine in chronic, destructive arthritis. Levels of the ganglioside GM3, one of the most primitive glycosphingolipids containing a sialic acid in the structure, are remarkably decreased in the synovium of patients with RA. Based on the increased cytokine secretions observed in in vitro experiments, GM3 might have an immunologic role. Here, to clarify the association between RA and GM3, we established a collagen-induced arthritis mouse model using the null mutation of the ganglioside GM3 synthase gene. GM3 deficiency exacerbated inflammatory arthritis in the mouse model of RA. In addition, disrupting GM3 induced T cell activation in vivo and promoted overproduction of the cytokines involved in RA. In contrast, the amount of the GM3 synthase gene transcript in the synovium was higher in patients with RA than in those with osteoarthritis. These findings indicate a crucial role for GM3 in the pathogenesis and progression of RA. Control of glycosphingolipids such as GM3 might therefore provide a novel therapeutic strategy for RA.

Objective— Interleukin(IL)-17A, an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results— To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE −/− IL-17A −/− ) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6 to 8 weeks. We found that splenic CD4 + T-cells produced high amounts of IL-17A in ApoE −/− mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE −/− IL-17A −/− mice compared with ApoE −/− mice. Splenic CD4 + T-cells of ApoE −/− IL-17A −/− mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased interferon gamma and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE −/− IL-17A −/− mice after HFD feeding for 8 weeks. Conclusion— These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating interferon gamma and IL-5 production from CD4 + T-cells.

The interaction between matricellular proteins such as tenascin-C (TN-C) and osteopontin (OPN) and integrins has been implicated in the pathology of rheumatoid arthritis in which Th17 cells are recognized as primary pathogenic cells. The differentiation of Th17 cells is tightly regulated by cytokines derived from APCs, receiving various signals including TLR stimuli. In this study, we used a collagen-induced arthritis model and found that increased numbers of α9 integrin-positive conventional dendritic cells and macrophage were detectable in the draining lymph node (dLN) shortly following first immunization, and these cells produced both TN-C and OPN, ligands for α9 integrin. α9 integrin-mediated signaling, induced by TN-C and OPN, promoted the production of Th17-related cytokines by conventional dendritic cells and macrophages in synergy with TLR2 and 4 signaling. This led to the Th17 cell differentiation and arthritis development. Moreover, Th17 cells generated under blocking of α9 integrin-mediated signaling showed low level of CCR6 expression and impaired migration ability toward CCL20. Thus, we have identified α9 integrin-mediated signaling by TN-C and OPN as a novel intrinsic regulator of pathogenic Th17 cell generation that contributes to the development of rheumatoid arthritis.

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MΦs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MΦs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MΦ (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80hiMac-1low MΦs residing in the red pulp showed different expression patterns of surface markers compared with F4/80+Mac-1hi monocytes/MΦs. Studies with purified cell populations demonstrated that F4/80hiMac-1low MΦs regulated CD4+ T cell responses by producing soluble suppressive factors, including TGF-β and IL-10. Moreover, F4/80hiMac-1low MΦs induced the differentiation of naive CD4+ T cells into functional Foxp3+ regulatory T cells. Additionally, we found that the differentiation of F4/80hiMac-1low MΦs was critically regulated by CSF-1, and in vitro-generated bone marrow-derived MΦs induced by CSF-1 suppressed CD4+ T cell responses and induced the generation of Foxp3+ regulatory T cells in vivo. These results suggested that splenic CSF-1–dependent F4/80hiMac-1low MΦs are a subpopulation of RPMs and regulate peripheral immune homeostasis.

To avoid excessive inflammation, molecular signaling that triggers immune responses has to be well restrained by various cellular and molecular regulatory mechanisms. However, in infections, such restraints must be removed to exert quick and efficient anti-microbial responses. Here, we report a novel role of autophagy in systemic Candida infection by enhancing early immune responses through increased nuclear factor kappa B (NFκB) activity. First, we found that Atg7 conditional knockout (CKO) mice with LysM-Cre were susceptible to Candida infection. Our ex vivo experiments showed that autophagy did not affect Candida-phagocytosis and inhibition of Candida expansion by macrophages and neutrophils. We also found that phagocytosed conidia were not contained in LC3-positive vesicles. The data suggested that it is unlikely that autophagy played a role in “direct” Candida killing by myeloid cells. Unexpectedly, instead, enhancement of NFκB activation was observed in a certain macrophage subset. Enhancement of NFκB activation was achieved by autophagy-mediated sequestration of an NFκB inhibitor. This allows the macrophages to release chemokines to effectively attract neutrophils to infected sites, resulting in better resistance to Candida in wild-type mice compared to Atg7 CKO mice. Thus, autophagy protects mice from systemic Candida infection by a mechanism distinct from those that protect mice from bacteria and viruses.

Syndecan-4 (Syn4), a cell-surface heparan sulfate proteoglycan has been implicated as a critical mediator for inflammatory responses. Recent report has demonstrated that dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL) binds to Syn4 on activated T cells, resulting in the inhibition of T cell responses. Here, we have investigated the role of Syn4 in the development of Rheumatoid Arthritis (RA). We have used two RA models; T cell-dependent collagen-induced arthritis (CIA) model and T cell-independent collagen antibodies-induced arthritis (CAIA) model. In CAIA model, Syn4-wild-type and Syn4-deficient (Syn4 KO) mice showed similar levels of clinical severity. In contrast, in CIA model, Syn4 KO mice revealed attenuated RA development. Syn4 expression was detectable on activated CD4+ T cells and B cells in the draining LN (dLN). Syn4 KO mice showed a tendency to generate reduced numbers of Th1 and Th17 cells in the dLN after immunization, and helper T cells generated in Syn4 KO mice revealed different expression patterns of chemokine receptors. Although both groups showed comparable numbers of Tfh and GC (germinal center) B cells, Syn4 KO mice produced lower level of type-II collagen-specific antibody, as compared with WT mice. These results suggest that Syn4 may regulate the development of RA by modulating T and B cell responses. We are currently investigating if Syn4-deficiency affects the functions of Tfh and GC B cells.

Th17 cells are essential regulator of autoimmune diseases. It is known that antigen presenting cells including conventional dendritic cells (cDCs) play a critical role in the differentiation of Th17 cells as the supplier of several Th17-polarizing cytokines such as IL-6 and IL-23. We have previously shown that the interaction between α9 integrin (α9) and its ligands such as tenascin-C (TN-C) enhance the production of a variety of cytokines including IL-6 by fibroblasts and macrophages. To examine the in vivo significance of α9 in Th17 development, we used murine collagen-induced arthritis (CIA). We demonstrated that α9 and TN-C were expressed by cDCs in the regional lymph nodes and the interaction between α9 and TN-C promoted the production of IL-6 from cDCs. The administration of anti-α9 antibody (55A2C) before and 2 days after first immunization with typeII collagen significantly suppressed the gene expression of IL-6 and IL-23 and the generation of Th17, but not Th1 cells in regional lymph nodes at day 7. Surprisingly at day 24, Th17, but not Th1 cell was significantly accumulated in regional lymph nodes of 55A2C-treated mice due to the down-regulation of CCR6 on Th17 cells. Nevertheless, arthritis was significantly attenuated in 55A2C-treated mice. These data suggest that α9-mediated signaling contributes to the generation of functional Th17 cells through the activation of cDCs in regional lymph node.

The balance between immune activation and tolerance must be regulated to maintain immune homeostasis. It has been well demonstrated that dendritic cells are critical regulators to maintain such balance. Tissue macrophages (Mφs) constitute the major cellular subsets of antigen presenting cells within body, however, it is poorly understood what kind of tissue resident macrophages is involved in the regulation of immune homeostasis in the peripheral lymphoid tissues. Here, we found that splenic Mφs could be divided into two subsets due to α9 integrin expression (F4/80intMac-1hiα9- and F4/80hiMac-1lowα9+). F4/80intMac-1hiα9- Mφ spontaneously secreted IL-6 and IL-12p40 and induced robust proliferation and differentiation of naive CD4+ T cells. These characteristics were met with the features of conventional splenic Mφ (cMφ). Mφs expressing α9 integrin (α9Mφs) were mainly localized in the red pulp. Unlike cMφs, α9Mφs expressed TGF-β and inhibited T cell proliferation by a mechanism partially dependent on TGF-β. Furthermore, α9Mφs were capable of inducing the differentiation of naive CD4+ T cells into Foxp3+ regulatory T cells via TGF-β. These results suggest that these distinct two Mφ subsets reciprocally control T cell immune responses in the spleen.

It has been shown that osteopontin (OPN) is critically involved in various Th1 type immune-mediated disorders such as rheumatoid arthritis (RA) and multiple sclerosis. In this study, we demonstrated that the expression of both OPN and tenascin-C (TN-C), and its common receptor, α9 integrin are up-regulated at the pathological foci of autoimmune arthritis. The local production of OPN and TN-C in arthritic joints is due to mainly synovial fibroblsts and to the lesser extent synovial macrophages. Majority of synovial fibroblasts and a subpopulation of synovial macrophages express α9 integrin. Importantly, autocrine and paracrine interactions of α9 integrin on synovial macrophages and fibroblasts and its ligands differently contribute to the production of pro-inflammatory cytokines and chemokines. α9 integrin is also involved in the recruitment and accumulation of inflammatory cells, through interaction with vascular cell adhesion molecule-1 (VCAM-1). Inhibition of α9 integrin function with an inhibitory anti-α9 integrin antibody significantly reduces production of arthrogenic cytokines and chemokines and ameliorates on-going arthritis. Thus, we identified α9 integrin as a critical intrinsic regulator that control the development of autoimmune arthritis. These results suggest that α9 integrin serves as a novel therapeutic target for the treatment of autoimmune disorders including RA.