Search

Early T-cell development from CD4

As a multisystem autoimmune disorder disease, systemic sclerosis (SSc) is characterized by inflammation and fibrosis in the skin and other internal organs. However, mechanisms underlying the inflammatory response that drives the development of SSc remain largely unknown.ADAR1 heterozygous knockout (AD1+/-) mice and myeloid-specific ADAR1 knockout mice were used to determine the function of ADAR1 in SSc. Histopathological analyses and western blot confirmed the role of ADAR1 in bleomycin-induced increased skin and lung fibrosis.In this study, we discover that adenosine deaminase acting on RNA (ADAR1), a deaminase converting adenosine to inosine (i.e., RNA editing) in RNA, is abundantly expressed in macrophages in the early stage of bleomycin-induced SSc. Importantly, ADAR1 is essential for SSc formation and indispensable for classical macrophage activation because ADAR1 deficiency in macrophages significantly ameliorates skin and lung sclerosis and inhibits the expression of inflammation mediator inducible NO synthase (iNOS) and IL-1β in macrophages. Mechanistically, deletion of ADAR1 blocks macrophage activation through diminishing NF-κB signaling.Our studies reveal that ADAR1 promotes macrophage activation in the onset of SSc. Thus, targeting ADAR1 could be a potential novel therapeutic strategy for treating sclerosis formation.

Upon virus infection, CD8

T-cell development in the thymus undergoes the process of differentiation, selective proliferation, and survival from CD4−CD8−double negative (DN) stage to CD4+CD8+double positive (DP) stage prior to the formation of CD4+helper and CD8+cytolytic T cells ready for circulation. Each developmental stage is tightly regulated by sequentially operating molecular networks, of which only limited numbers of transcription regulators have been deciphered. Here, we identified Zfp335 transcription factor as a new player in the regulatory network controlling thymocyte development in mice. We demonstrate thatZfp335intrinsically controls DN to DP transition, as T-cell-specific deficiency inZfp335leads to a substantial accumulation of DN3 along with reduction of DP, CD4+, and CD8+thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ (iTCRβ) expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factorsBcl6andRorcby Zfp335. Importantly, enhanced expression of TCRβ andBcl6/Rorcrestores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role ofZfp335in controlling T-cell development by maintaining iTCRβ expression-mediated β-selection and independently activating cell survival signaling.

Effector CD8

Memory CD8

Rationale: Abdominal aortic aneurysm (AAA) is a permanent and localized dilatation of abdominal aorta with potentially fatal consequence of aortic rupture. No effective pharmacological approach has been identified to limit AAA progression and rupture. AAA is characterized by extensive aortic wall matrix degradation that contributes to arterial wall remodeling and eventual rupture, in which smooth muscle cell (SMC) phenotypic transition and MMPs (matrix metalloproteinases), especially MMP2 (matrix metalloproteinase-2) and MMP9, play critical roles. Objective: Our previous study showed that ADAR1 (adenosine deaminases acting on RNA 1) regulates SMC phenotype, which prompted us to study if ADAR1 is involved in AAA development. Methods and Results: We used Ang II (angiotensin II) infusion ApoE −/− mouse model combined with ADAR1 global and SMC-specific knockout to study the role of ADAR1 in AAA formation/dissection. Aortic transplantation was conducted to determine the importance of vascular cell ADAR1 in AAA development/dissection. Primary cultured SMC were used to study how ADAR1 regulates the inflammatory SMC phenotype and MMP production/activity. Patient specimens were obtained to investigate the relevance of ADAR1 expression to human AAA disease. ADAR1 was induced in abdominal aortic SMC in both mouse and human AAA tissues. Heterozygous knockout of ADAR1 diminished the Ang II–induced AAA/dissection in ApoE −/− mice. Mouse aortic transplantation showed that ADAR1 in vascular cells was essential for AAA formation. SMC-specific ADAR1 knockout reduced experimental AAA formation/dissection. Mechanistically, ADAR1 interacted with HuR (human antigen R) to increase the stability of MMP2 and MMP9 mRNA, leading to increased MMP levels and activities. Conclusions: ADAR1 is novel regulator of AAA development/dissection, and thus may represent a potential new therapeutic target to hinder AAA growth and rupture.

Under steady‐state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL‐7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell‐specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild‐type (WT) and Med1‐deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1‐deficient CD8+ T cells compared with WT counterparts. Finally, Med1‐deficient CD8+ T cells exhibited a decreased expression of interleukin‐7 receptor α (IL‐7Rα), down‐regulation of phosphorylated‐STAT5 (pSTAT5) and Bim up‐regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL‐7Rα/STAT5 pathway‐mediated cell survival.

Systemic sclerosis (SSc) is a multisystem autoimmune disorder that is characterized by inflammation and fibrosis in the skin and internal organs. Previous studies indicate that inflammatory cells and cytokines play essential roles in the pathogenesis of SSc; however, the mechanisms that underlie the inflammation-driven development of SSc are not fully understood. In this study, we show that response gene to complement 32 (RGC32) is abundantly expressed in mouse macrophages in the early stage of bleomycin-induced SSc. Importantly, RGC32 is required to induce the inflammatory response during the onset of SSc, because RGC32 deficiency in mice significantly ameliorates skin and lung sclerosis and inhibits the expression of inflammatory mediators inducible NO synthase (iNOS) and IL-1β in macrophages. RGC32 appears to be a novel regulator for the differentiation of classically activated macrophages (M1 macrophages). IFN-γ and LPS stimulation induces RGC32 expression in primary peritoneal macrophages and bone marrow–derived macrophages. RGC32 deficiency impairs the polarization of M1 macrophages and attenuates iNOS and IL-1β production. Mechanistically, RGC32 interacts with NF-κB proteins and promotes iNOS and IL-1β expression by binding to their promoters. Collectively, our data reveal that RGC32 promotes the onset of SSc by regulating the inflammatory response of M1 macrophages, and it may serve as a promising therapeutic target for treating SSc.

Proper development of taste organs including the tongue and taste papillae requires interactions with the underlying mesenchyme through multiple molecular signaling pathways. The effects of bone morphogenetic proteins (BMPs) and antagonists are profound, however, the tissue-specific roles of distinct receptors are largely unknown. Here, we report that constitutive activation (ca) of ALK2-BMP signaling in the tongue mesenchyme (marked by Wnt1-Cre) caused microglossia-a dramatically smaller and misshapen tongue with a progressively severe reduction in size along the anteroposterior axis and absence of a pharyngeal region. At E10.5, the tongue primordia (branchial arches 1-4) formed in Wnt1-Cre/caAlk2 mutants while each branchial arch responded to elevated BMP signaling distinctly in gene expression of BMP targets (Id1, Snai1, Snai2, and Runx2), proliferation (Cyclin-D1) and apoptosis (p53). Moreover, elevated ALK2-BMP signaling in the mesenchyme resulted in apparent defects of lingual epithelium, muscles, and nerves. In Wnt1-Cre/caAlk2 mutants, a circumvallate papilla was missing and further development of formed fungiform papillae was arrested in late embryos. Our data collectively demonstrate that ALK2-BMP signaling in the mesenchyme plays essential roles in orchestrating various tissues for proper development of the tongue and its appendages in a region-specific manner.

Monocytes and macrophages have long been associated with acute and chronic allograft rejection; this is mediated by their abilities to promote inflammation, kill target cells via antibody-dependent cytotoxicity and modulate adaptive immunity. Our present study showed that allogeneic antigen-primed macrophages acutely rejected skin grafts with specificity after adoptive transfer into MHC-matched immunodeficient mice. The ability of primed macrophages to reject allografts essentially requires the help of CD4(+) T cells and does not require the help of CD8(+) T cells. Moreover, the primed, perforin-deficient macrophages rejected the skin grafts in a significantly delayed pattern compared with WT macrophages, indicating that the perforin pathway of the primed macrophages is likely involved in the rejection process. Thus, primed macrophages are endowed with adaptive immunity-like features, such as specificity, with the help of CD4(+) T cells during the immune response to allografts. The present study challenges our traditional views of macrophage functions and highlights the biological functions of macrophages beyond innate immunity in mammals.

Thymic epithelial cells (TECs) are a key cell type in the thymic microenvironment essential for T cell development. However, intrinsic molecular mechanisms controlling TEC differentiation and activities are poorly defined. In this study, we found that deficiency of p53-induced phosphatase 1 (Wip1) in mice selectively caused severe medullary TEC (mTEC) maturation defects in an intrinsic manner. Wip1 knockout (KO) mice had decreased mature epithelial cell adhesion molecule+Ulex europaeus agglutinin-1 (UEA-1)+ mTECs, including UEA-1+MHC class IIhigh, UEA-1+CD80+, UEA-1+CD40+, and UEA-1+Aire+ cells, but not decreased numbers of cortical epithelial cell adhesion molecule+BP-1+ TECs, in the postnatal stage but not in the fetal stage. Wip1-deficient mTECs express fewer tissue-restricted Ags and UEA-1+involucrin+ terminal-differentiated cells. Animal models, including grafting fetal Wip1-deficient thymic tissue into T cell–deficient nude mice and reconstitution of lethally irradiated Wip1KO mouse recipients with wild-type bone marrow cells, also showed the impaired mTEC components in Wip1KO thymi, indicating the intrinsic regulatory role of Wip1 in mTEC maturation. Furthermore, thymus regeneration was significantly less efficient in adult Wip1KO mice than in wild-type mice after cyclophosphamide treatment. Wip1 deficiency resulted in elevated p38 MAPK activity in mTECs. Activated p38 MAPK has the ability to suppress CD40 expression on mTECs. Wip1-deficient thymi displayed poor response to CD40L in the fetal thymus organ culture system. Thus, Wip1 positively controls mTEC maturation, homeostasis, and regeneration through limiting the p38 MAPK pathway.

P0-Cre and Wnt1-Cre mouse lines have been widely used in combination with loxP-flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1-Cre has been regarded as the gold standard and there have been concerns about the specificity of P0-Cre because it is not clear about the timing and spatial distribution of the P0-Cre transgene in labeling NC cells at early embryonic stages. We re-visited P0-Cre and Wnt1-Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26-lacZ Cre reporter responded to Cre activity more reliably than CAAG-lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0-Cre and reporter (lacZ and RFP) activity in P0-Cre/R26-lacZ and P0-Cre/R26-RFP embryos was detected in the cranial NC and notochord regions in E8.0-9.5 (4-19 somites) embryos. P0-Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0-Cre and Wnt1-Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1-Cre and in the hindbrain of P0-Cre embryos. The difference between P0-Cre and Wnt1-Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre-driven genetic modifications.

Immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells and TGF-β have been shown to negatively regulate host immunity against allografts. Our results demonstrated that Smad3-deficient mice or mice reconstituted with Smad3-deficient hematopoietic cells rejected allogeneic skin or heart grafts in a significantly slower manner compared with littermates or wild-type (WT) control mice. Transplanted Smad3−/− recipients produced markedly less anti-donor IgG Abs, especially IgG1 and IgG2b subclasses. T cells in alloskin-grafted Smad3-deficient mice were more likely to participate in a Th2-type immune response, as evidenced by more Th2-specific transcription factor, GATA3 expression, and increased IL-4 and IL-10 production, as well as less Th1-specific transcription factor, T-bet expression, and decreased IL-2 and IFN-γ production. More CD11b+Gr1+ neutrophil infiltration and less monocyte/macrophage and T cell infiltration in allografts were observed in Smad3−/− recipients compared with WT recipients. Increased CXCL1 and CXCL2 as well as decreased CCL3, MCP-1, and RANTES chemokines in allografts of Smad3−/− recipients were consistently detected by real-time PCR. Further studies indicated that the increased CD11b+Gr1+ myeloid cells in Smad3-deficient mice were immunosuppressive and responsible for the delayed allograft rejection mainly via an NO-dependent pathway. Thus, this study identifies Smad3 as an intrinsic negative regulator that critically inhibits the differentiation and function of immunosuppressive CD11b+Gr1+ myeloid-derived suppressor cells.

A variety of immunosuppressive drugs are currently used in patients with allo-grafts or autoimmune diseases. Though the effects of rapamycin (RPM) and other immunosuppressant on the CD4 + CD25 + Foxp3 + T regulatory cells (Tregs) were studied, their impact on Ag-specific Tregs during immune response was not well defined. In our studies, we adoptively transferred TCR-transgenic CD4 + KJ1-26 + T cells, CD4 + KJ1-26 + CD25 − naive T cells or CD4 + KJ1-26 + CD25 + Tregs into syngeneic BALB/c mice. 24 h later, we treated the recipients with OVA immunization and immunosuppressant including rapamycin (RPM), fingolimod (FTY720), cyclosporin A (CsA), mycophenolate mofetil (MMF), leflunomide (LEF), cyclophosphamide (Cy) or none, respectively. The levels and function of CD4 + KJ1-26 + CD25 + Foxp3 + Tregs in draining lymph nodes (dLNs) and spleens were determined at different time points. Significantly higher percentage and cell number of Ag-specific CD4 + KJ1-26 + CD25 + Foxp3 + Tregs were observed in OVA immunized mice treated with RPM or FTY720 compared with mice that received OVA immunization alone. Furthermore, RPM augmented the population of functional iTregs in dLNs and spleens whereas inhibited nTregs during immune response. In contrast to RPM and FTY720, MMF, LEF, CsA, and Cy markedly decreased the levels of Ag-specific CD4 + KJ1-26 + CD25 + Foxp3 + Tregs during immune response. Thus, different immunosuppressive drugs have distinct effects on the Ag-specific CD4 + CD25 + Foxp3 + Tregs during immune response. The stronger inhibiting effects of MMF, LEF, CsA and Cy on CD4 + CD25 + Foxp3 + Tregs than on T effectors may block the host immune tolerance potentiality.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, produced in the thymus or periphery as a functionally mature T cell subpopulation, play pivotal roles in maintenance of self-tolerance and negative regulation of immune responses. Aspirin (ASA) is widely used to reduce pain, the risk of cardiovascular diseases and allo-graft rejection. However, the effect of ASA on CD4(+)CD25(+)Foxp3(+) Treg cells has yet to be determined. The frequency, phenotype and immunosuppressive function of CD4(+)CD25(+)Foxp3(+) Treg cells were detected in BALB/c mice treated with low or high doses of ASA for 4 weeks. ASA significantly decreased the percentage and number of CD4(+) T cells in the periphery, while ASA remarkably increased the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells in CD4(+)T cells. The total cell numbers of thymocytes were significantly decreased in ASA-treated mice, but the number of CD4(+) CD25(+)Fxop3(+) cells and its ratio in CD4(+)CD8(-) thymocytes were markedly enhanced in the thymi of ASA-treated mice. The phenotype of CD4(+)CD25(+) Treg cells, including the expressions of CD44, CD45RB, CD62L, CD69, GITR and CTLA-4, did not show detectable changes in ASA-treated mice. CD4(+)CD25(+) Treg cells in ASA-treated mice exhibited unimpaired immunosuppressive function on CD4(+)CD25(-) T effector cells. ASA significantly enhanced the frequency of functional CD4(+)CD25(+)Foxp3(+) Treg cells in mice in a therapeutic dose range. The different effects of ASA on CD4(+)CD25(+)Foxp3(+) Treg cells and CD4(+)CD25(-) T cells may potentially make hosts susceptible to tolerance induction which would be beneficial for tolerance induction in patients with autoimmune diseases or allo-grafts. This study may have potential impacts in the clinical application of ASA.

Thymic epithelial cells (TECs) form a 3-dimentional network supporting thymocyte development and maturation. Besides epithelium and thymocytes, heterogeneous fibroblasts are essential components in maintaining thymic microenvironments. However, thymic fibroblast characteristics, development and function remain to be determined. We herein found that thymic non-hematopoietic CD45-FSP1+ cells represent a unique Fibroblast specific protein 1 (FSP1)—fibroblast-derived cell subset. Deletion of these cells in FSP1-TK transgenic mice caused thymus atrophy due to the loss of TECs, especially mature medullary TECs (MHCIIhigh, CD80+ and Aire+). In a cyclophosphamide-induced thymus injury and regeneration model, lack of non-hematopoietic CD45-FSP1+ fibroblast subpopulation significantly delayed thymus regeneration. In fact, thymic FSP1+ fibroblasts released more IL-6, FGF7 and FSP1 in the culture medium than their FSP1- counterparts. Further experiments showed that the FSP1 protein could directly enhance the proliferation and maturation of TECs in the in vitro culture systems. FSP1 knockout mice had significantly smaller thymus size and less TECs than their control. Collectively, our studies reveal that thymic CD45-FSP1+ cells are a subpopulation of fibroblasts, which is crucial for the maintenance and regeneration of TECs especially medullary TECs through providing IL-6, FGF7 and FSP1.

Bacterial infection often follows virus infection due to pulmonary interferon-γ (IFN-γ) production during virus infection, which down-regulates macrophage phagocytosis. The molecular mechanisms for this process are still poorly understood. In the present study, IFN-γ treatment significantly inhibited the ability of mouse macrophages to phagocytize nonopsonized chicken red blood cells (cRBCs), bacteria and beads in vitro, while it enhanced IgG- and complement-opsonized phagocytosis. IFN-γ treatment decreased the expression of MARCO (macrophage receptor with collagenous structure) in macrophages. Macrophages showed lower binding to and phagocytic ability of cRBCs when MARCO was blocked with antibody. In addition, IFN-γ induced high activity of mTOR (mammalian target of rapamycin) and decreased the expression of c/EBPβ (CCAAT enhancer-binding protein β) in macrophages. Rapamycin, a specific mTOR inhibitor, significantly reversed the inhibitory effect of IFN-γ on nonopsonized phagocytosis of macrophages and restored c/EBPβ and MARCO expression. Biochemical assays showed that c/EBPβ directly bound to the MARCO gene promoter. Rapamycin significantly hampered the viral-bacterial synergy and protected influenza-infected mice from subsequent bacterial infection. Thus, IFN-γ inhibited the nonopsonized phagocytosis of macrophages through the mTOR-c/EBPβ-MARCO pathway. The present study offered evidence indicating that mTOR may be one of the key target molecules for the prevention of secondary bacterial infection caused by primary virus infection.

Regulatory T cells, especially CD4(+)CD25(+) regulatory T cells are critical regulators of immune tolerance in humans and mice. Mice with humanized immunity have been developed by various transplantation strategies of human tissues or cells related to immunity, which are being extensively applied in biomedical research. However, it is unclear whether human CD4(+)CD25(+) regulatory T cells can normally develop in human thymic grafts and efficiently populate in the periphery in NOD/SCID mouse recipients. In human thymic grafts, high percentage of mature human CD4(+)CD25(high) regulatory T cells was detected. Human CD4(+)CD25(+) regulatory T cells maturing in fetal human thymus grafts could subsequently output to the periphery of NOD/SCID mouse recipients. Importantly, these cells exhibited Foxp3(+)CD45RO(+)CTLA4(+)CD127(-) phenotype, similarly to those in healthy individuals. In addition, human CD4(+)CD25(+) regulatory T cells maturing in human thymic grafts suppressed proliferative response of CD4(+)CD25(-) T cells to allogeneic antigens, though the peripheral CD4(+)CD25(+) regulatory T cells in fetal human thymus-grafted NOD/SCID mice showed somewhat decreased immunosuppressive ability compared with normal CD4(+)CD25(+) regulatory T cells. Thus, this humanized animal model is suitable for examining development and function of human CD4(+)CD25(+) regulatory T cells in vivo.

Xenogeneic thymus transplantation can efficiently induce specific immune tolerance to donor antigens in athymic recipients. However, many nude mice suffer from autoimmune diseases (AID) for over 10 weeks after xenogeneic thymus transplantation. CD4+CD25+Foxp3+ regulatory T (Treg) cells were recently determined to play a pivotal role in keeping immune tolerance in humans and mice. Thus, we investigated this subpopulation of Treg cells in the periphery of pig thymus-grafted nude mice suffering from AID. Our results showed that the expression of Foxp3, CTLA-4 and GITR on mouse CD4+CD25+ T cells and the ratio of CD4+CD25+Foxp3+ Treg cells to CD4+ T cells were significantly decreased in the periphery of pig thymus-grafted nude mice suffering from AID, compared with healthy pig or mouse thymus-grafted nude mice. Furthermore, mouse CD4+CD25+ T cells in pig thymus-grafted nude mice suffering from AID showed more severe deficiency in immunosuppressive function compared with the counterpart in xenogeneic pig or syngeneic thymus-grafted nude mice without AID. Thus, the decreased frequency, altered phenotype and functional deficiency of mouse CD4+CD25+ Treg cells in pig thymus-grafted nude mice may contribute to the development of AID in this model.