Astrid M. Westendorf

The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor best known for mediating the toxicity of dioxin. Environmental factors are believed to contribute to the increased prevalence of autoimmune diseases, many of which are due to the activity of TH17 T cells, a new helper T-cell subset characterized by the production of the cytokine IL-17. Here we show that in the CD4+ T-cell lineage of mice AHR expression is restricted to the TH17 cell subset and its ligation results in the production of the TH17 cytokine interleukin (IL)-22. AHR is also expressed in human TH17 cells. Activation of AHR by a high-affinity ligand during TH17 cell development markedly increases the proportion of TH17 T cells and their production of cytokines. CD4+ T cells from AHR-deficient mice can develop TH17 cell responses, but when confronted with AHR ligand fail to produce IL-22 and do not show enhanced TH17 cell development. AHR activation during induction of experimental autoimmune encephalomyelitis causes accelerated onset and increased pathology in wild-type mice, but not AHR-deficient mice. AHR ligands may therefore represent co-factors in the development of autoimmune diseases.

Frontline: Neuropilin-1: a surface marker of regulatory T cells

CD4+CD25+ regulatory T cells (Treg cells) control immune responsiveness to a large variety of antigens. The isolation and therapeutic manipulation of Treg cells requires the use of reliable surface receptors that are selectively up‐regulated in Treg cells. On the basis of global gene expression studies, we identified neuropilin‐1 (Nrp1) as a specific surface marker for CD4+CD25+ Treg cells. Nrp1, a receptor involved in axon guidance, angiogenesis, and the activation of T cells, is constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status. In contrast, Nrp1 expression is down‐regulated in naive CD4+CD25– T cells after TCR stimulation. Furthermore, CD4+Nrp1high T cells express high levels of Foxp3 and suppress CD4+CD25– T cells. Thus, Nrp1 constitutes a useful surface marker to distinguish Treg cells from both naive and recently activated CD4+CD25+ non‐regulatory T cells.

Purinergic Control of T Cell Activation by ATP Released Through Pannexin-1 Hemichannels

Pannexin hemichannel–mediated release of ATP provides an autocrine, costimulatory signal for T cell activation. ATP Signals T Cells to Activate Sustained influx of extracellular Ca2+ is a critical event in the activation of T cells. One consequence of increased cytosolic Ca2+ concentration is the uptake of Ca2+ by mitochondria, which leads to the synthesis of adenosine triphosphate (ATP). Activation of purinergic receptors upon T cells is known to affect the outcome of stimulation of the T cell receptor (TCR), but how extracellular ATP might affect T cell function in the context of inflammation is unclear. Schenk et al. now show that on TCR triggering, ATP is released from T cells through pannexin hemichannels and functions in an autocrine fashion as a costimulator of T cell activation. Blocking ATP signaling mediated by purinergic P2X receptors on T cells in the context of TCR stimulation led to decreased T cell activation and increased expression of anergy-associated genes. Moreover, administration of a P2X receptor antagonist to mouse models of type 1 diabetes and inflammatory bowel disease substantially inhibited the development of effector T cells and lessened tissue damage compared with that in untreated mice. Together, these data suggest that therapeutic intervention against ATP synthesis and release may be of benefit in the treatment of T cell–mediated inflammatory diseases. T cell receptor (TCR) stimulation results in the influx of Ca2+, which is buffered by mitochondria and promotes adenosine triphosphate (ATP) synthesis. We found that ATP released from activated T cells through pannexin-1 hemichannels activated purinergic P2X receptors (P2XRs) to sustain mitogen-activated protein kinase (MAPK) signaling. P2XR antagonists, such as oxidized ATP (oATP), blunted MAPK activation in stimulated T cells, but did not affect the nuclear translocation of the transcription factor nuclear factor of activated T cells, thus promoting T cell anergy. In vivo administration of oATP blocked the onset of diabetes mediated by anti-islet TCR transgenic T cells and impaired the development of colitogenic T cells in inflammatory bowel disease. Thus, pharmacological inhibition of ATP release and signaling could be beneficial in treating T cell–mediated inflammatory diseases.

Myeloid-Derived Suppressor Cells in Inflammatory Bowel Disease: A New Immunoregulatory Pathway

BACKGROUND & AIMS CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) have been shown to cause T-cell tolerance in tumor-bearing mice; however, little is known about the role of MDSCs in chronic inflammation. Here, for the first time, we have identified and analyzed their role in inflammatory bowel disease (IBD). METHODS Repetitive adoptive transfer of clone 4/T-cell receptor (CL4-TCR) transgenic CD8(+) T cells into VILLIN-hemagglutinin (HA) transgenic mice was performed on days 1, 12, and 27. Recipient mice were analyzed for immunopathology, HA-specific CD8(+) T-cell responses, and CD11b(+)Gr-1(+) MDSCs (frequency, phenotype, expression analysis, and in vitro as well as in vivo function). In addition, peripheral blood from patients with active Crohns disease and ulcerative colitis was examined for the presence and function of human MDSCs denoted as CD14(+)HLA-DR(-/low) cells. RESULTS Repetitive transfer of HA-specific CD8(+) T cells prevented VILLIN-HA recipient mice from development of severe enterocolitis, which is seen after a single transfer of T cells. Repeated transfer of antigen-specific T cells led to an increase in the frequency of nitric oxide synthase 2 and arginase-expressing CD11b(+)Gr-1(+) MDSCs in spleen and intestine of VILLIN-HA mice with immunosuppressive function. Cotransfer of MDSCs with HA-specific CD8(+) T cells into naive VILLIN-HA mice ameliorated enterocolitis, indicating a direct immune regulatory effect of MDSCs on induction of IBD by antigen-specific T cells. Finally, an increase in the frequency of human MDSCs with suppressor function was observed in peripheral blood from patients with IBD. CONCLUSIONS These results identify MDSCs as a new immune regulatory pathway in IBD.

Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of ∼100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.

ATP Inhibits the Generation and Function of Regulatory T Cells Through the Activation of Purinergic P2X Receptors

Purinergic signaling during inflammation converts immunosuppressive CD4+ T cells into proinflammatory ones. T Cells Lose Their Identity Regulatory T cells (Tregs) inhibit the actions of inflammatory T cells during immune responses and prevent autoimmunity. Schenk et al. showed that adenosine triphosphate (ATP) signaling through purinergic receptors on Tregs inhibited their immunosuppressive effects and exacerbated tissue inflammation in mice. Worse still, autocrine ATP signaling made the Tregs lose their identity, through the loss of their signature transcription factor Foxp3, and induced their conversion into proinflammatory, interleukin-17–secreting cells. These data suggest that ATP signaling through purinergic receptors might be an effective therapeutic target to shape immune responses, a suggestion supported by the maintenance of the identity and immunosuppressive function of Tregs through pretreatment with a purinergic receptor antagonist. Extracellular nucleotides are pleiotropic regulators of mammalian cell function. Adenosine triphosphate (ATP) released from CD4+ helper T cells upon stimulation of the T cell receptor (TCR) contributes in an autocrine manner to the activation of mitogen-activated protein kinase (MAPK) signaling through purinergic P2X receptors. Increased expression of p2rx7, which encodes the purinergic receptor P2X7, is part of the transcriptional signature of immunosuppressive CD4+CD25+ regulatory T cells (Tregs). Here, we show that the activation of P2X7 by ATP inhibits the suppressive potential and stability of Tregs. The inflammatory cytokine interleukin-6 (IL-6) increased ATP synthesis and P2X7-mediated signaling in Tregs, which induced their conversion to IL-17–secreting T helper 17 (TH17) effector cells in vivo. Moreover, pharmacological antagonism of P2X receptors promoted the cell-autonomous conversion of naïve CD4+ T cells into Tregs after TCR stimulation. Thus, ATP acts as an autocrine factor that integrates stimuli from the microenvironment and cellular energetics to tune the developmental and immunosuppressive program of the T cell in adaptive immune responses.

Neuropilin 1 deficiency on CD4+Foxp3+ regulatory T cells impairs mouse melanoma growth

Neuropilin 1 mediates anti-tumor control by promoting regulatory T cell infiltration.

The use of calcium phosphate nanoparticles encapsulating Toll-like receptor ligands and the antigen hemagglutinin to induce dendritic cell maturation and T cell activation

Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells. Antigen presentation by mature (activated) DCs is a prerequisite for the stimulation of antigen-specific T cells, whereas antigen presentation by immature DCs results in the generation of specific tolerance. Our aim was to develop calcium phosphate nanoparticles which can serve as carriers of immunoactive oligonucleotides into dendritic cells for their activation. We analyzed size, surface charge, and morphology of calcium phosphate nanoparticles loaded with the TLR ligands CpG and poly(I:C) and also with the antigen hemagglutinin (HA) by scanning electron microscopy, dynamic light scattering, Brownian motion analysis and ultracentrifugation. The uptake of fluorescence-labeled nanoparticles into dendritic cells was illustrated by confocal laser scanning microscopy. Immunostimulatory effects of these nanoparticles on DCs were studied, i.e., cytokine production and activation of the cells in terms of upregulation of surface molecules. We show that functionalized calcium phosphate nanoparticles are capable to induce both innate and adaptive immunity by activation of DCs.

CD4+ Foxp3+ regulatory T cell expansion induced by antigen-driven interaction with intestinal epithelial cells independent of local dendritic cells

Background: Regulatory T cells (Tregs) have potential anti-inflammatory effects and are likely to be important in the pathogenesis of chronic inflammatory bowel disease (IBD). However, the induction and expansion of Tregs at sites of mucosal inflammation are not yet fully understood and may involve antigen presentation by local dendritic cells (DCs) and/or intestinal epithelial cells (IECs). Methods: To determine the unique ways in which the gut induces or expands Tregs, a transgenic mouse model that is based on the specific expression of a model autoantigen (influenza haemagglutinin (HA)) in the intestinal epithelium (VILLIN-HA) was used. Gut-associated DCs and IECs isolated from these mice were phenotypically and functionally characterised for the potential to interact with HA-specific Tregs in vitro and in vivo. Results: Intestinal self-antigen expression leads to peripheral expansion of antigen-specific CD4+Foxp3+ Tregs. Although gut-associated DCs can induce antigen-specific CD4+Foxp3+ T cell proliferation, in vivo depletion of DCs did not preclude proliferation of these cells. Interestingly, antigen presentation by primary IECs is sufficient to expand antigen-specific CD4+Foxp3+ Tregs efficiently. This is dependent on major histocompatibility complex class II, but, in contrast to DCs, is unlikely to require transforming growth factor β and retinoic acid. Conclusion: This study provides experimental evidence for a new concept in mucosal immunity: in contrast to current thinking, expansion of Tregs can be achieved independently of local DCs through antigen-specific IEC–T cell interactions.

Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells

The uptake of calcium phosphate nanoparticles (diameter 120nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3h of incubation.