Absence Of Inflammatory Signals Research Articles

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked tothe induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for theregulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to theunderstanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Stiffening of the extracellular matrix is a sufficient condition for airway hyperreactivity.

The current therapeutic approach to asthma focuses exclusively on targeting inflammation and reducing airway smooth muscle force to prevent the recurrence of symptoms. However, even when inflammation is brought under control, airways in an asthmatic can still hyperconstrict when exposed to a low dose of agonist. This suggests that there are mechanisms at play that are likely triggered by inflammation and eventually become self-sustaining so that even when airway inflammation is brought back under control, these alternative mechanisms continue to drive airway hyperreactivity in asthmatics. In this study, we hypothesized that stiffening of the airway extracellular matrix is a core pathological change sufficient to support excessive bronchoconstriction even in the absence of inflammation. To test this hypothesis, we increased the stiffness of the airway extracellular matrix by photo-crosslinking collagen fibers within the airway wall of freshly dissected bovine rings using riboflavin (vitamin B2) and Ultraviolet-A radiation. In our experiments, collagen crosslinking led to a twofold increase in the stiffness of the airway extracellular matrix. This change was sufficient to cause airways to constrict to a greater degree, and at a faster rate when they were exposed to 10-5 M acetylcholine for 5 min. Our results show that stiffening of the extracellular matrix is sufficient to drive excessive airway constriction even in the absence of inflammatory signals.NEW & NOTEWORTHY Targeting inflammation is the central dogma on which current asthma therapy is based. Here, we show that a healthy airway can be made to constrict excessively and at a faster rate in response to the same stimulus by increasing the stiffness of the extracellular matrix, without the use of inflammatory agents. Our results provide an independent mechanism by which airway remodeling in asthma can sustain airway hyperreactivity even in the absence of inflammatory signals.

REVIEW ARTICLE: THE ROLE OF DENDRITIC CELLS IN THE CONTROL ON INNATE IMMUNITY

Dendritic cells play an important role in host immunity by inducing innate inflammatory responses to pathogens, efficiently priming immature T cells, activating memory T cells and promoting B cell activation. Dendritic cells are also integral in maintaining steady- state immune homeostasis by continually presenting tissue-derived selfantigens to CD4+ and CD8+ T cells in the absence of inflammatory signals, leading to tolerance against those self-antigens. The important of innate immunity lies not only in directly confronting pathogenic and non-pathogenic insults but also in instructing the development of an efficient adaptive immune response.

Osteoclast Activated FoxP3+ CD8+ T-Cells Suppress Bone Resorption in vitro

BackgroundOsteoclasts are the body’s sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (TEFF) increase bone resorption by osteoclasts. Prolonged exposure to the TEFF produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (TcREG). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption.Principal FindingsTo determine the net effect of TcREG on osteoclast activity we used a number of in vitro assays. We found that TcREG can potently and directly suppress bone resorption by osteoclasts. TcREG could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that TcREG suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of TcREG by osteoclasts is antigen-dependent, suppression of osteoclasts by TcREG does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the TcREG and osteoclasts.SignificanceWe have determined that osteoclast-induced TcREG can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology.

Commensal bacteria trigger a full dendritic cell maturation program that promotes the expansion of non-Tr1 suppressor T cells

Dendritic cells (DCs) orchestrate the immune response establishing immunity versus tolerance. These two opposite functions may be dictated by DC maturation status with maturity linked to immunogenicity. DCs directly interact with trillions of noninvasive intestinal bacteria in vivo, a process that contributes to gut homeostasis. We here evaluated the maturation program elicited in human DCs by direct exposure to commensal-related bacteria (CB) in the absence of inflammatory signals. We showed that eight gram(+) and gram(-) CB strains up-regulated costimulatory molecule expression in DCs and provoked a chemokine receptor switch similar to that activated by gram(+) pathogens. CB strains may be classified into three groups according to DC cytokine release: high IL-12 and low IL-10; low IL-12 and high IL-10; and low IL-12 and IL-10. All CB-treated DCs produced IL-1beta and IL-6 and almost no TGF-beta. Yet, CB instructed DCs to convert naive CD4+ T cells into hyporesponsive T cells that secreted low or no IFN-gamma, IL-10, and IL-17 and instead, displayed suppressor function. These data demonstrate that phenotypic DC maturation combined to an appropriate cytokine profile is insufficient to warrant Th1, IL-10-secreting T regulatory Type 1 (Tr1), or Th17 polarization. We propose that commensal flora and as such, probiotics manipulate DCs by a yet-unidentified pathway to enforce gut tolerance.

N‐terminal trimer extension of nominal CD8 T cell epitopes is sufficient to promote cross‐presentation to cognate CD8 T cells in vivo

Cross‐presentation pathway is essential for generating effective CD8 T cell responses against cell‐associated antigens derived from either infectious pathogens or malignant cells. In this study, we investigated the regulation of cross‐presentation pathway through manipulation of the CD8 T cell epitopes using TCR transgenic mice as model system. Previous studies have shown that nominal peptide is inefficiently cross‐presented and that proteins and large polypeptides that require proteasomal processing are the main source of naturally cross‐presented antigens. Here we show that N‐terminal extension of nominal peptide by as few as three residues is sufficient to produce a substrate for TAP dependent cross‐presentation that is highly efficient in cross‐priming murine CD8 T cells in vivo. On a molar basis, cross‐priming with trimer extended peptide is 20 fold more efficient than priming with intact protein. Depending on the presence or absence of inflammatory signals, this method can lead to immunity or tolerance. Moreover, cross‐presentation of N‐terminal modified CD8 epitopes derived from insulin can significantly protect NOD mice from developing type I diabetes. Therefore, this method of peptide extension should prove of great value in facilitating in vivo studies of CD8 immunity and tolerance that rely on cross‐presentation.(This work was supported by NIH grants DK50824 and U19 AI050864)

Using gamma‐cytokine complexes to improve antigen specific CD8 T cell responses in tumor‐bearing mice

In the absence of inflammatory signals, the exposure of T cells to tumor antigen in the periphery results in tolerance. Remaining tumor specific T cells are usually of low avidity or anergic. Immunotherapy aims at reversing this tolerance to induce an efficient T cell response. STAT5 Cytokines of the interleukin‐2 family signal through STAT5 and are candidates to increase T cell anti‐tumor response. The effects of these cytokines are greatly increased when the cytokine is complexed with a specific antibody.We have studied the potential of cytokine complexes to serve as adjuvants for CD8 cells activation in a murine model, RIP‐Tag2‐HA, in which pancreatic insulomas expressing the influenza hemagglutinin (HA) as a surrogate antigen develop spontaneously. HA specific CD8 cells become effector after 3 days of treatment with IL‐2 complexes and antigen in vivo. This differentiation leads to the elimination of endogenous tumors by high avidity HA specific CD8 cells. Survival of CD8 cells is increased by using the TLR ligands polyIC or by extending the length of treatment with IL‐2 complexes. Under such conditions, even low avidity HA specific CD8 cells eliminate the tumors. We further show that cytokine complexes also increase survival of adoptively transferred, ex‐vivo activated HA specific CD8 cells. These results indicate that IL‐2 complexes are strong adjuvants that greatly enhance tumor vaccines or immunotherapy.

Antigen-Driven Interactions with Dendritic Cells and Expansion of Foxp3+ Regulatory T Cells Occur in the Absence of Inflammatory Signals

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.

Dendritic Cells Break Bonds to Tolerize

Typically, dendritic cells (DCs) induce peripheral tolerance under steady state but immunity during inflammation or infection. In this issue of Immunity, Jiang et al. (2007) identify disruption of E-cadherin interactions as a unique maturation pathway by which DCs are capable of mediating tolerance.

Balancing between immunity and tolerance: an interplay between dendritic cells, regulatory T cells, and effector T cells

Dendritic cells (DC), professional antigen-presenting cells of the immune system, exert important functions both in induction of T cell immunity, as well as tolerance. It is well established that the main function of immature DC (iDC) in their in vivo steady-state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. Previously, it was believed that T cell unresponsiveness induced after stimulation with iDC is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. Moreover, several reports indicate that traditional DC maturation can no longer be used to distinguish tolerogenic and immunogenic properties of DC. This review will focus on the complementary role of dendritic cells in inducing both tolerance and immunity, and we will discuss the clinical implications for dendritic cell-based therapies.

Immunosuppression induced by immature dendritic cells is mediated by TGF‐β/IL‐10 double‐positive CD4+ regulatory T cells

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-β and interleukin (IL)-10 double-positive CD4+ T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-β and IL-10 secreted by CD4+CD25−FOXP3− T cells. In addition, the suppressive capacity of CD4+ T cells conditioned by iDC was transferable to already primed antigen-specific CD8+ T cell cultures. In contrast, addition of CD4+ T cells conditioned by mDC to primed antigen-specific CD8+ T cells resulted in enhanced CD8+ T cell responses, notwithstanding the presence of TGF-β+/IL-10+ T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4+ T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4+ T cells. Furthermore, TGF-β+/IL-10+ T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.

Retinoids regulate survival and antigen presentation by immature dendritic cells.

Maturation of dendritic cells (DCs) is a critical step for the induction of an immune response. We have examined the role of retinoid nuclear receptor pathways in this process. Retinoids induce DC apoptosis, in the absence of inflammatory signals, through retinoic acid receptor (RAR)α/retinoic X receptor (RXR) heterodimers. In contrast, via a cross talk with inflammatory cytokines, retinoids increase DNA binding activity of nuclear factor κB in DCs, trigger membrane major histocompatibility complex class II and costimulatory molecule expression, induce the differentiation of immature DCs into mature DCs, and enhance antigen-specific T cell response. This maturation of DCs is mediated via a RXR-dependent/RAR-independent pathway and via an RARα/RXR pathway distinct from the one responsible for apoptosis. Apoptosis and activation, mediated through distinct nuclear retinoid receptor pathways, can be dissociated from each other with selective synthetic retinoids. We identify a novel cellular function for retinoids and suggest that selective retinoids might be of interest for controlling antigen presentation.

Porcine alveolar macrophages: poor accessory or effective suppressor cells for T-lymphocytes

Porcine Alv-Mø from bronchoalveolar lavages were tested for their function in an in vitro foot-and-mouth disease virus (FMDV)-specific lymphoproliferative recall response. The Alv-Mø were seen to be poor accessory cells when compared with peripheral blood monocytes. This poor capacity was evident despite an efficient expression of SLA-DR region antigens, and other co-stimulatory adhesion molecules. It was noted that Alv-Mø secrete relatively little interleukin 1 (IL-1β), with or without LPS induction, even though mRNA for the cytokine could be detected. In contrast, blood monocytes with their effective accessory activity were potent secretors of IL-1. Although this IL-1β would be important with respect to the accessory capacity of monocytic cells, it was noted that the absence of bioactive IL-1 from the Alv-Mø cultures was not solely responsible for their poor accessory function. In fact, the Alv-Mø produced factors which not only inhibited IL-1 bioactivity, but were also responsible for a clear suppression of lymphoproliferation. This suppressor activity was dependent on the type of monocytic cell present in the culture, being more prominent when “scavenger” phagocytes were present. Thus, the major role of Alv-Mø is not as an accessory cell akin to monocytes, but as both a scavenger cell, related to Mø derived from monocytes in the absence of inflammatory signals, and an immunoregulatory cell.

Modulating Dendritic Cells to Optimize Mucosal Immunization Protocols

Oral administration of soluble protein Ag induces tolerance, a phenomenon that has hampered mucosal vaccine design. To provoke active immunity, orally administered Ag must be fed together with a mucosal adjuvant such as cholera toxin (CT). Unfortunately, CT is not suitable for clinical use because of its associated toxicity. There is, therefore, a need to develop alternative mucosal immunization regimens. Here we have attempted to alter the intrinsically tolerogenic nature of the intestine and improve immunization potential by expanding and activating intestinal APC in vivo. Previous studies have indicated that intestinal dendritic cells (DC) present oral Ag, but do so in a tolerogenic manner. In the present study we investigated whether DC can be converted from tolerogenic into immunogenic APC by treating mice with Flt3 ligand (Flt3L), a DC growth factor, and then immunizing with CT. We observed increased local and systemic responses to CT in the presence of elevated numbers of intestinal DC. In parallel, CT induced up-regulation of CD80 and CD86 on these Flt3L-expanded DC. In an attempt to develop a toxin-free adjuvant system, we investigated whether IL-1 could be used as an alternative DC-activating stimulus. Using a combination of Flt3L and IL-1alpha, we observed a potent active response to fed soluble Ag, rather than the tolerogenic response normally observed. These data suggest that Flt3L-expanded DC are well positioned to regulate intestinal responses depending on the presence or the absence of inflammatory signals. Flt3L may therefore be a reagent useful for the design of mucosal immunization strategies.

Dendritic cells need T cell help to prime cytotoxic T cell responses to strong antigens

Peptide-pulsed mouse dendritic cells (DC) primed peptide-specific CD8+ cytotoxic T cell responses very effectively if they expressed minor histocompatibility antigens, which could stimulate a CD4+ T helper cell response. These DC could also prime most syngeneic mice, although there was no deliberate immunization for help (the DC were prepared in syngeneic mouse serum, to avoid any response to fetal calf serum antigens). In contrast, DC were unable to prime MHC class II-deficient mice for cytotoxic responses to the classical helper-dependent antigens Qa1a and HY. More strikingly, Balb.B DC failed to prime B6 MHC class II-deficient mice for cytotoxic responses to Balb minor antigens, even though these two strains differ at more than 40 minor histocompatibility loci. When peptide-pulsed DC were prepared without enzymes (used to release DC from lymphoid tissues), they failed to prime the majority of normal syngeneic mice, even though they expressed high levels of B7 and ICAM-1 co-stimulatory molecules, suggesting that help was provided by responses to antigens in the enzyme cocktail. The enzyme treatment itself did not provide signals that could substitute for help, since DC prepared with enzymes could not prime MHC class II-deficient mice. The observation that highly immunogenic minor-incompatible DC failed to prime MHC class II-deficient mice suggests that in the absence of inflammatory signals, even strong antigens cannot stimulate CD8+ T cell responses without help.