Jianxia Peng

The phenotype and functional alterations of macrophages in mice with hyperglycemia for long term

Abnormal immunity and its related complications are the major causes of mortality and morbidity in diabetes patients. Macrophages, as one of the important innate cells, play pivotal roles in controlling immune homeostasis, immunity, and tolerance. The effects of hyperglycemia on the function of macrophages in hosts remain to be determined. Here we used mice with streptozotocin (STZ)‐induced diabetes for long term to study the changes of macrophages. We found that F4/80+ peritoneal exudate macrophages (PEMs) from mice with diabetes for 4 months displayed significantly reduced CD86 and CD54 expression and tumor necrosis factor (TNF)‐α and IL‐6 production but enhanced nitric oxide (NO) secretion compared with control mice when treated with interferon (IFN)‐γ and lipopolysaccharide (LPS), while the activity of arginase in PEMs from diabetic mice was significantly higher than control mice when stimulating with IL‐4. These dysfunctions of macrophages could be efficiently reversed by insulin treatment. Importantly, in vitro bone marrow‐derived macrophages showed similar functional changes, indicating the epigenetic alteration of macrophage precursors in these mice. In an in vitro culture system, high glucose and insulin significantly altered TNF‐α, IL‐6, and NO production and arginase activity of macrophages, which was reversed by the treatment with AKT and ERK inhibitors. Therefore, hyperglycemia and insulin deficiency can modify macrophage function through AKT‐mTOR and ERK pathways and through epigenetic effects on macrophage precursors. To further identify different components of diabetes on the dysfunction of macrophages is important for efficient prevention of diabetic complications. J. Cell. Physiol. 227: 1670–1679, 2012.

Smad3-Deficient CD11b+Gr1+ Myeloid-Derived Suppressor Cells Prevent Allograft Rejection via the Nitric Oxide Pathway

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.

Immunosuppressive drugs on inducing Ag-specific CD4+CD25+Foxp3+ Treg cells during immune response in vivo

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(-) naïve T cells or CD4(+)KJ1-26(+)CD25(+) Tregs into syngeneic BALB/c mice. 24h 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.

Induction of M2-like macrophages in recipient NOD- scid mice by allogeneic donor CD4 + CD25 + regulatory T cells

CD4+CD25+ regulatory T cells (Tregs) play an important role in maintaining host immune tolerance via regulation of the phenotype and function of the innate and adaptive immune cells. Whether allogeneic CD4+CD25+ Tregs can regulate recipient mouse macrophages is unknown. The effect of allogeneic donor CD4+CD25+ Tregs on recipient mouse resident F4/80+macrophages was investigated using a mouse model in which allogeneic donor CD4+CD25+ Tregs were adoptively transferred into the peritoneal cavity of host NOD-scid mice. The phenotype and function of the recipient macrophages were then assayed. The peritoneal F4/80+ macrophages in the recipient mice that received the allogeneic CD4+CD25+ Tregs expressed significantly higher levels of CD23 and programmed cell death-ligand 1(PD-L1) and lower levels of CD80, CD86, CD40 and MHC II molecules compared to the mice that received either allogeneic CD4+CD25− T cells (Teffs) or no cells. The resident F4/80+ macrophages of the recipient mice injected with the allogeneic donor CD4+CD25+ Tregs displayed significantly increased phagocytosis of chicken red blood cells (cRBCs) and arginase activity together with increased IL-10 production, whereas these macrophages also showed decreased immunogenicity and nitric oxide (NO) production. Blocking arginase partially but significantly reversed the effects of CD4+CD25+ Tregs with regard to the induction of the M2 macrophages in vivo. Therefore, the allogeneic donor CD4+CD25+ Tregs can induce the M2 macrophages in recipient mice at least in part via an arginase pathway. We have provided in vivo evidence to support the unknown pathways by which allogeneic donor CD4+CD25+ Tregs regulate innate immunity in recipient mice by promoting the differentiation of M2 macrophages.

The different effects of indirubin on effector and CD4+CD25+ regulatory T cells in mice : potential implication for the treatment of autoimmune diseases

CD4+CD25+ regulatory T (Treg) cells play an essential role in the induction and maintenance of peripheral self-tolerance. Indirubin, a traditional Chinese medicine, was clinically used in the treatment of chronic myelocytic leukemia as well as some autoimmune diseases, including Alzheimer’s disease, diabetes, and so on. The effects of indirubin on CD4+CD25+Treg cells, which play a critical role in controlling autoimmunity, have not been addressed. In the present study, we observed the cell levels, phenotypes, and immunoregulatory function of CD4+CD25+Treg cells in indirubin-treated mice. Treatment with indirubin significantly enhanced the ratios of CD4+CD25+Treg cells or CD4+CD25+Foxp3+Treg cells to CD4+T cells in peripheral blood, lymph nodes, and spleens (P < 0.01 compared with control mice). CD4+CD25+Foxp3+Treg cells to CD4 single positive cells in the thymi of indirubin-treated mice were significantly higher than those in control mice. Furthermore, splenic CD4+CD25+Treg cells in indirubin-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4+CD25+Treg cells in vitro. The present studies indicate that CD4+CD25+Treg cells are more resistant to indirubin than effector T cells in vivo. The selectively enhanced CD4+CD25+Treg cell levels by indirubin made host to be more favorable for immune tolerance induction, which opened one possibility for indirubin to treat autoimmune diseases.

Alterations of peripheral CD4 + CD25 + Foxp3 + T regulatory cells in mice with STZ-induced diabetes

Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+ T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+ Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+ Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+ Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+ Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25− T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25− T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios−Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase in CD4+CD25+ Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.

The immunity of splenic and peritoneal F4/80 + resident macrophages in mouse mixed allogeneic chimeras

Mixed allogeneic chimeras are emerging as a prospective approach to induce immune tolerance in clinics. However, the immunological function of macrophages in mixed chimeras has not been evaluated. Using a B6→BALB/c mixed chimera model, we investigated the phenotype and function of F4/80+ resident peritoneal exudate macrophage (PEMs) and splenic macrophages (SPMs) in vitro and in vivo. Recipient F4/80+PEMs and SPMs in mixed chimeras expressed significantly lower levels of MHC-II, CD54, and CD23 than those in non-chimeric mice before lipopolysaccharide stimulation. Recipient F4/80+PEMs and SPMs in mixed chimeras induced normal cell proliferation and delayed-type hypersensitivity of allo-T cells, but they induced more IFN-γ and IL-2 products and less IL-10 and TGF-β products of allo-T cells compared with those of non-chimeras. Furthermore, recipient F4/80+PEMs and SPMs had significantly higher phagocytotic capacity against chicken red blood cells or allo-T cells than those of controls while they had normal phagocytosis to Escherichia coli. Although some slight but significant alterations of recipient macrophages have been detected, these results provide direct evidences for the efficient immunity of recipient macrophages in mixed allogeneic chimeras. The present study also, for the first time, offered basic information for macrophages maturing in heterogeneous environments.

An instructive role of donor macrophages in mixed chimeras in the induction of recipient CD4 + Foxp3 + Treg cells

The immune regulatory function of macrophages (M⊘s) in mixed chimeras has not been determined. In the present study, with a multi‐lineage B6‐to‐BALB/c mixed chimeric model, we examined the ability of donor‐derived splenic M⊘s in the induction of regulatory T cells (Treg). B6 splenic M⊘s from mixed chimeras induced significantly less cell proliferation, more IL‐10 and TGF‐β, and less IL‐2 and IFN‐γ productions of CD4+ T cells from BALB/c mice than naive B6 M⊘s did, whereas they showed similar stimulatory activity to the third part C3H CD4+ T cells. Importantly, highly purified donor F4/80+CD11c− M⊘s efficiently induced recipient CD4+Foxp3+ Treg cells from CD4+CD25−Foxp3− T cells. Furthermore, donor M⊘s of mixed chimeras produced more IL‐10 and less IFN‐γ than those of naive mice when cultured with BALB/c but not the third party C3H CD4+ T cells. Induction of recipient CD4+ Treg cells by donor M⊘s was significantly blocked by anti‐IL‐10, but not by anti‐TGF‐β mAb. Therefore, donor M⊘s have the ability to induce recipient CD4+Foxp3+ Treg cells in a donor antigen‐specific manner, at least partially, via an IL‐10‐dependent pathway. This study for the first time showed that, in mixed allogeneic chimeras, donor M⊘s could be specifically tolerant to recipients and gained the ability to induce recipient but not the third party Foxp3+ Treg cells. Whether this approach is involved in transplant immune tolerance needs to be determined.

Induction of regulatory T cells from mature T cells by allogeneic thymic epithelial cells in vitro

The ability of thymic epithelial cells (TEC) to re‐educate mature T cells to be regulatory T cells has not been addressed. In the present study, this issue was directly investigated by co‐culturing of mature T cells and allo‐TECs. B6 macrophage cell line 1C21‐cultured BALB/c splenocytes responded to B6 antigens in vitro. However, BALB/c splenocytes precultured with B6‐derived TECs 1‐4C18 or 1C6 did not proliferate to B6 antigens, but responded to rat antigens. Exogenous interleukin‐2 (IL‐2) failed to revise the unresponsiveness of these T cells. Allo‐TEC‐cultured T cells predominantly expressed Th2 cytokines (IL‐4 and IL‐10). B6 TEC‐cultured BALB/c splenocytes markedly inhibited the immune responses of naïve BALB/c splenocytes to B6 antigens, but not to rat or the third‐party mouse antigens. BALB/c nude mice that received naïve syngeneic splenocytes rejected B6 or rat skin grafts by 17 days postskin grafting; however, co‐injection of B6 TEC‐cultured BALB/c splenocytes significantly delayed B6 skin graft rejection (P < 0.01), with the unchanged rejection of rat skin grafts. These studies demonstrate that allo‐TECs are able to ‘educate’ mature T cells to be regulatory cells, and suggest that regulatory cells derived from mature T cells by TECs may play an important role in T cell tolerance to allo‐ and auto‐antigens.

Interferon-γ Inhibits Nonopsonized Phagocytosis of Macrophages via an mTORC1-c/EBPβ Pathway

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.