Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kappaB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kappaB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to na?ve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kappaB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.     

Controlling NF-kappaB activation in T cells by costimulatory receptors

Full and productive activation of T lymphocytes relies on the simultaneous delivery of T cell receptor (TCR)- and coreceptor-derived signals. In na?ve T cells engagement of the TCR alone causes anergy, while TCR triggering of preactivated T cells results in activation-induced cell death. Costimulatory signals are prominently mirrored by the activation of NF-kappaB, which needs input from the TCR as well as from coreceptors in order to be fully activated and to fulfil its crucial function in the immune response. Coreceptor-generated signals tightly control the duration and amplitude of the NF-kappaB response. The activation of IkappaB kinase (IKK) complex at the contact zone between a T cell and an antigen-presenting cell offers the unique opportunity to study the spatial organization of IKK activation. Recent studies indicate that coreceptor pathways influence the threshold activities of many signalling mediators and thus act on multiple layers of the NF-kappaB pathway.     

The role of antigen in the activation of regulatory T cells by immune B cells

The transfer of B cells from mice immunized with Type III pneumococcal polysaccharide (SSS-III) results in the activation of suppressor and amplifier T cells that control the magnitude of the antibody response in recipient mice, immunized subsequently with SSS-III. Prior treatment of transferred B cells with an excess of enzyme (polysaccharide depolymerase) capable of hydrolyzing SSS-III, does not alter the capacity of these cells to activate regulatory T cells. These findings indicate that the activation of regulatory T cells by immune B cells is not mediated by residual antigen on the surface of transferred cells.     

IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.     

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.     

Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes

CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-kappaB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-kappaB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.     

The pro-metastatic role of bone marrow-derived cells: a focus on MSCs and regulatory T cells

Several bone marrow-derived cells have been shown to promote tumour growth and progression. These cells can home to the primary tumour and become active components of the tumour microenvironment. Recent studies have also identified bone marrow-derived cells—such as mesenchymal stem cells and regulatory T cells—as contributors to cancer metastasis. The innate versatility of these cells provides diverse functional aid to promote malignancy, ranging from structural support to signal-mediated suppression of the host immune response. Here, we review the role of mesenchymal stem cells and regulatory T cells in cancer metastasis. A better understanding of the bipolar nature of these bone marrow-derived cells in physiological and malignant contexts could pave the way for new therapeutics against metastatic disease.     

A limited role for regulatory T cells in post-ischemic neovascularization

Recently, it was demonstrated that arteriogenesis is enhanced in mice deficient in regulatory T cells (CD4(+) CD25(+) FoxP3(+) T cell), which can suppress effector T cell responses. The present study investigates the effects of these regulatory T cells on arteriogenesis in more detail by either specific expanding or depleting regulatory T cells. Hind limb ischemia was induced by electro-coagulation of the femoral artery in mice. Regulatory T cells were either expanded by injecting mice with a complex of interleukin (IL)-2 with the IL-2 monoclonal antibody JES6-1, or depleted by anti-CD25 antibody or diphtheria toxin injections in DEREG mice (depletion of regulatory T cells). Blood flow restoration was monitored using laser Doppler perfusion imaging. Collateral arteries were visualized by immunohistochemistry. Regulatory T cell expansion led to a moderate though significant suppression of blood flow restoration after ischemia induction. Surprisingly, depletion of regulatory T cells resulted in minor increase on blood flow recovery. However, collateral and capillary densities in the post-ischemic skeletal muscle were significantly increased in DEREG mice depleted for regulatory T cells. The presence of regulatory T cells after ischemia induction when analysed in non-depleted DEREG mice could be demonstrated by green fluorescent protein staining only in lymph nodes in the ischemic area, and not in the ischemic muscle tissue. The current study demonstrates that, even under conditions of major changes in regulatory T cell content, the contribution of regulatory T cells to the regulation of the arteriogenic response is only moderate.     

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.     

The role of suppressors of cytokine signalling in thymopoiesis and T cell activation

Cytokines play an essential role in mediating interactions between cells of the immune system. Suppressors of cytokine signalling proteins act to negatively regulate these cytokine signals, thereby exerting control over the expression of cytokine responsive genes. Various lines of experimental evidence suggest that two closely related members of the this family, suppressor of cytokine signalling 1 and 3, are important in the processes of T cell development, activation and homeostasis. This review outlines the principles underlying these processes and relates these to the potentially important roles played by suppressor of cytokine signalling 1 and 3.     

Resveratrol attenuates TNF-alpha-induced activation of coronary arterial endothelial cells: role of NF-kappaB inhibition

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its cardioprotective effects are not completely understood. Because TNF-alpha-induced endothelial activation and vascular inflammation play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits TNF-alpha-induced signal transduction in human coronary arterial endothelial cells (HCAECs). We found that TNF-alpha significantly increased adhesiveness of the monocytic THP-1 cells to HCAECs, an effect that could be inhibited by pretreatment with resveratrol and the NF-kappaB inhibitor pyrrolidine dithiocarbamate. Previously, we found that TNF-alpha activates NAD(P)H oxidases, and our recent data showed that TNF-alpha-induced endothelial activation was prevented by the NAD(P)H oxidase inhibitor apocynin or catalase plus SOD. Resveratrol also inhibited H(2)O(2)-induced monocyte adhesiveness. Using a reporter gene assay, we found that, in HCAECs, TNF-alpha significantly increased NF-kappaB activity, which could be inhibited by resveratrol (>50% inhibition at 10(-6) mol/l) and pyrrolidine dithiocarbamate. Resveratrol also inhibited TNF-alpha-induced, NF-kappaB-driven luciferase expression in rat aortas electroporated with the reporter gene construct. In TNF-alpha-treated HCAECs, resveratrol (in the submicromolar range) significantly attenuated expression of NF-kappaB-dependent inflammatory markers inducible nitric oxide synthase, IL-6, bone morphogenetic protein-2, ICAM-1, and VCAM. Thus resveratrol at nutritionally relevant concentrations inhibits TNF-alpha-induced NF-kappaB activation and inflammatory gene expression and attenuates monocyte adhesiveness to HCAECs. We propose that these anti-inflammatory actions of resveratrol are responsible, at least in part, for its cardioprotective effects.     

A regulatory role for the CD4 and CD8 molecules in T cell activation

The role of the CD4 and CD8 molecules in T cell activation is presently a matter of controversy. Although their role as associative binding elements to MHC class II or class I is well documented, their influence on the triggering process in unclear. Because antibodies to CD4 or CD8 block T cell activation in the absence of their respective ligands, a negative signaling by these molecules has been suggested. However, recent experimental evidence argues against a negative regulatory effect of these molecules, since, e.g., simultaneous cross-linking of TCR and CD4 leads to enhanced T cell activation. Therefore, a current model suggests that the association of TCR and CD4 in the membrane gives a positive signal essential for triggering. In this report we present evidence that this model is likely to be too simple. Anti-CD4 and CD8 antibodies inhibit alternative, nonreceptor pathways of T cell triggering via Tp103 and Tp44 in the absence of class II positive accessory or target cells. These antibodies also inhibit bypass activation of T cells by phorbol ester and calcium ionophore in an accessory cell-free system. Furthermore, if the CD4 or CD8 molecules are removed from the cell surface by antibody-induced modulation, the proliferative and cytotoxic response of T cell clones is enhanced. This enhancement is also observed if resting peripheral blood T cells are used as responder cells. These data show that the CD4 or CD8 molecules have a complex regulatory function in T cell activation beyond the requirement for co-cross-linking with the TCR.     

Plasmacytoid dendritic cells are inefficient in activation of human regulatory T cells

Background

Dendritic cells (DC) play a key role in initiation and regulation of immune responses. Plasmacytoid DC (pDC), a small subset of DC, characterized as type-I interferon producing cells, are critically involved in anti-viral immune responses, but also mediate tolerance by induction of regulatory T cells (Treg). In this study, we compared the capacity of human pDC and conventional DC (cDC) to modulate T cell activity in presence of Foxp3⁺ Treg.

Principal Findings

In coculture of T effector cells (Teff) and Treg, activated cDC overcome Treg anergy, abrogate their suppressive function and induce Teff proliferation. In contrast, pDC do not break Treg anergy but induce Teff proliferation even in coculture with Treg. Lack of Treg-mediated suppression is independent of proinflammatory cytokines like IFN-α, IL-1, IL-6 and TNF-α. Phenotyping of pDC-stimulated Treg reveals a reduced expression of Treg activation markers GARP and CTLA-4. Additional stimulation by anti-CD3 antibodies enhances surface expression of GARP and CTLA-4 on Treg and consequently reconstitutes their suppressive function, while increased costimulation with anti-CD28 antibodies is ineffective.

Conclusions/Significance

Our data show that activated pDC induce Teff proliferation, but are insufficient for functional Treg activation and, therefore, allow expansion of Teff also in presence of Treg.     

Chloroplast-to-nucleus signalling: a role for Mg-protoporphyrin

Signalling from chloroplasts to the nucleus is an important feature of the coordination of nuclear and chloroplast gene expression required for the assembly of functional chloroplasts. Recent studies have indicated that accumulation of Mg-protoporphyrin, the first committed precursor of chlorophyll, is both necessary and sufficient for the regulation of nuclear genes by chloroplasts.     

IDO and regulatory T cell support are critical for cytotoxic T lymphocyte-associated Ag-4 Ig-mediated long-term solid organ allograft survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.