TSC1 controls IL-1ß expression in macrophages via mTORC1-dependent C/EBPß pathway.

The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR), which serves as a key regulator of inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Previous studies have shown that TSC1 knockout (KO) macrophages produced increased inflammatory responses including tumor necrosis factor-α (TNF-α) and IL-12 to pro-inflammatory stimuli, but whether and how TSC1 regulates pro-IL-1ß expression remains unclear. Here using a mouse model in which myeloid lineage-specific deletion of TSC1 leads to constitutive mTORC1 activation, we found that TSC1 deficiency resulted in impaired expression of pro-IL-1ß in macrophages following lipopolysaccharide stimulation. Such decreased pro-IL-1ß expression in TSC1 KO macrophages was rescued by reducing mTORC1 activity with rapamycin or deletion of mTOR. Rictor deficiency has no detectable effect on pro-IL-1ß synthesis, suggesting that TSC1 positively controls pro-IL-1ß expression through mTORC1 pathway. Moreover, mechanism studies suggest that mTORC1-mediated downregulation of the CCAAT enhancer-binding protein (C/EBPß) critically contributes to the defective pro-IL-1ß expression. Overall, these findings highlight a critical role of TSC1 in regulating innate immunity by control of the mTOR1-C/EBPß pathway.

Interferon-γ inhibits nonopsonized phagocytosis of macrophages via an mTORC1-c/EBPß 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/EBPß (CCAAT enhancer-binding protein ß) in macrophages. Rapamycin, a specific mTOR inhibitor, significantly reversed the inhibitory effect of IFN-γ on nonopsonized phagocytosis of macrophages and restored c/EBPß and MARCO expression. Biochemical assays showed that c/EBPß 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/EBPß-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.

TSC1 controls macrophage polarization to prevent inflammatory disease.

Macrophages acquire distinct phenotypes during tissue stress and inflammatory responses, but the mechanisms that regulate the macrophage polarization are poorly defined. Here we show that tuberous sclerosis complex 1 (TSC1) is a critical regulator of M1 and M2 phenotypes of macrophages. Mice with myeloid-specific deletion of TSC1 exhibit enhanced M1 response and spontaneously develop M1-related inflammatory disorders. However, TSC1-deficient mice are highly resistant to M2-polarized allergic asthma. Inhibition of the mammalian target of rapamycin (mTOR) fails to reverse the hypersensitive M1 response of TSC1-deficient macrophages, but efficiently rescues the defective M2 polarization. Deletion of mTOR also fails to reverse the enhanced inflammatory response of TSC1-deficient macrophages. Molecular studies indicate that TSC1 inhibits M1 polarization by suppressing the Ras GTPase-Raf1-MEK-ERK pathway in mTOR-independent manner, whereas TSC1 promotes M2 properties by mTOR-dependent CCAAT/enhancer-binding protein-ß pathways. Overall, these findings define a key role for TSC1 in orchestrating macrophage polarization via mTOR-dependent and independent pathways.

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.

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-ß 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.

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)-ß, 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 iCD4+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 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.

The effects of leflunomide on CD4(+)CD25 (+)Foxp3 (+) T regulatory cells in mice receiving allogeneic bone marrow transplantation.

OBJECTIVE AND DESIGN: Leflunomide (LEF) is effective not only in different animal models of autoimmune diseases and the therapy of patients with rheumatoid arthritisbut also in graft rejection. The effect of LEF on CD4(+)CD25(+)T regulatory cells (Treg) was determined in a mouse model of allogeneic bone marrow transplantation. MATERIAL OR SUBJECTS: BALB/c and C57BL/6 mice were used as donors and recipients, respectively. TREATMENT: C57BL/6 mice were given 2 Gy total-body irradiation, followed by an intravenous injection of 2 × 10(7) BALB/c bone marrow cells (BMCs). Mice were treated with LEF daily at a dose of 30 mg/kg/day for 2 weeks. RESULTS: In naïve mice, LEF significantly decreased the percentage of CD4(+)CD25(+) Treg cells in spleens (P < 0.05), but not in lymph nodes, though LEF enhanced the percentages of CD4(+)CD25(+) Treg cells in CD4 single positive cells in the thymocytes and blood (P < 0.05). Furthermore, LEF significantly decreased the percentage of CD4(+)CD25(+) Treg cells in the spleens of mice that received allogeneic BMCs. CONCLUSIONS: LEF decreases peripheral CD4(+)CD25(+) Treg cells in un-immunizing and immunizing recipients, indicating that LEF might not be an ideal candidate for the treatment of autoimmune diseases or graft rejection with respect to induction of immune tolerance.

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

The immune regulatory function of macrophages (Møs) 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 Møs in the induction of regulatory T cells (Treg). B6 splenic Møs from mixed chimeras induced significantly less cell proliferation, more IL-10 and TGF-ß, and less IL-2 and IFN-γ productions of CD4(+) T cells from BALB/c mice than naive B6 Møs did, whereas they showed similar stimulatory activity to the third part C3H CD4(+) T cells. Importantly, highly purified donor F4/80(+)CD11c(-) Møs efficiently induced recipient CD4(+)Foxp3(+) Treg cells from CD4(+)CD25(-)Foxp3(-) T cells. Furthermore, donor Møs 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 Møs was significantly blocked by anti-IL-10, but not by anti-TGF-ß mAb. Therefore, donor Møs 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 Møs 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.

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.

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-gamma and IL-2 products and less IL-10 and TGF-beta 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.

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.