Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

CD4+ T cells differentiate into subsets that promote immunity or minimize damage to the host. T helper 17 cells (Th17) are effector cells that function in inflammatory responses. T regulatory cells (Tregs) maintain tolerance and prevent autoimmunity by secreting immunosuppressive cytokines and expressing check point receptors. While the functions of Th17 and Treg cells are different, both cell fate trajectories require T cell receptor (TCR) and TGF-β receptor (TGF-βR) signals, and Th17 polarization requires an additional IL-6 receptor (IL-6R) signal. Utilizing high-resolution phosphoproteomics, we identified that both synergistic and additive interactions between TCR, TGF-βR, and IL-6R shape kinase signaling networks to differentially regulate key pathways during the early phase of Treg versus Th17 induction. Quantitative biochemical analysis revealed that CD4+ T cells integrate receptor signals via SMAD3, which is a mediator of TGF-βR signaling. Treg induction potentiates the formation of the canonical SMAD3/4 trimer to activate a negative feedback loop through kinases PKA and CSK to suppress TCR signaling, phosphatidylinositol metabolism, and mTOR signaling. IL-6R signaling activates STAT3 to bind SMAD3 and block formation of the SMAD3/4 trimer during the early phase of Th17 induction, which leads to elevated TCR and PI3K signaling. These data provide a biochemical mechanism by which CD4+ T cells integrate TCR, TGF-β, and IL-6 signals via generation of alternate SMAD3 complexes that control the development of early signaling networks to potentiate the choice of Treg versus Th17 cell fate.     

Pharmacologic IL-6Rα inhibition in cholangiocarcinoma promotes cancer cell growth and survival

Biliary tract cancer (BTC) represents a malignant tumor of the biliary tract including cholangiocarcinoma (CCA) and the carcinoma of the gallbladder (GBC) with a 5-year survival rate between 5 and 18% due to late diagnosis and rapid disease progression. Chronic inflammation is one of the main risk factors for CCA and GBC in particular. IL-6, as a mediator of inflammation, can act through a membrane-bound receptor alpha-chain (mIL-6R, “IL-6 classic signaling”) or via soluble forms (sIL-6R, “IL-6 trans-signaling”). However, little is known about the impact on cellular responses of IL-6 trans-signaling on BTC. We analyzed primary tumors as whole sections and as tissue microarrays, and also searched The Cancer Genome Atlas database. Compared to non-neoplastic, non-inflamed gallbladder tissue, IL-6Rα was downregulated in GBC, and this correlated with the patients' overall survival. Furthermore, different CCA cell lines and compounds for activation (IL-6 and Hyper-IL-6) or inhibition (Tocilizumab and sgp130Fc) of IL-6 classic signaling and trans-signaling were used to determine their effects on cellular processes between the two modes of IL-6 signaling. Inhibition of IL-6 trans-signaling by sgp130Fc reduced CCA cell line viability and apoptosis, whereas migration and proliferation were increased. We conclude that IL-6Rα expression is a good prognostic marker for GBC, and that the blocking of IL-6 trans-signaling and activation of IL-6 classic signaling have tumor promoting activity. These findings warrant the exclusion of patients with GBC or other malignancies associated with bile metabolism from IL-6R inhibitor therapy.     

IL-9 Induces CCL11 Expression via STAT3 Signalling in Human Airway Smooth Muscle Cells

Background

Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood.

Methodology/Principal Findings

In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3β over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression.

Conclusion/Significance

Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.     

Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8^{- /-}) and wild-type (WT) mice. We found that CCR8^-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca²⁺ flux and CCL1-driven PMφ aggregation. Similar to CCR8^-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8^-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8^-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8^-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.     

The IL-2A receptor pathway and its role in lymphocyte differentiation and function

Murine myeloid cells are developed from hemopoietic stem/progenitor cells. Different types of progenitor cells have variable differentiation potentials. Among the ten main types of cells differentiated from lymphoid progenitor cells, regulatory T cells (Tregs), an important cell subpopulation regulating immune and inflammatory responses, arise from the hematopoietic stem cells in the bone marrow. Tregs then differentiate into T lymphocytes and migrate to the thymus and finally generate Treg subsets, which are subsequently activated and regulated by inflammatory cytokines in the peripheral blood. Tregs also have different phenotypes and immunomodulatory functions. The cytokine interleukin-2/interleukin-2 receptor (IL-2/IL-2R) pathway is an important regulatory signaling pathway of Tregs. Besides, different types of CD4⁺ and CD8⁺ cells have different immune effects in the absence of IL-2. IL-2R consists of three subunits, α chain (CD25), β chain (CD122), and γ chain (CD132). Different subunit combinations have different effects on the activation of immune cells. Multiple studies have shown that IL2RA deficiency has various effects on the immune function in mice. This article reviews the subunit composition and signaling pathway of IL-2R, the classification of Tregs in a murine myeloid cell line and the regulatory effect of IL-2/IL-2R on them, the regulatory impact and signaling mechanism of IL-2/IL-2R on CD4⁺/CD8⁺ lymphocyte differentiation, the primary manifestations and molecular mechanism of immune dysfunction in IL-2- and IL-2R-deficient mice, soluble IL-2Rα as a biomarker for diagnosis, prognosis and therapeutic efficacy of treatment in immune system disorders, and the development and clinical application of IL-2 mutants.     

All-trans retinoic acid ameliorates inflammatory response mediated by TLR4/NF-κB during initiation of diabetic nephropathy

Diabetic nephropathy (DN) is the leading cause of renal failure worldwide and its complications have become a public health problem. Inflammation, oxidative stress and fibrosis play central roles in the progression of DN that lead to renal failure. Potential deleterious effect of inflammation in early evolution of DN is not fully disclosed. Therefore, it is relevant to explore therapies that might modulate this process in order to reduce DN progression. We explored the beneficial effect of all-trans retinoic acid (ATRA) in early inflammation in glomeruli, proximal and distal tubules in streptozotocin (STZ)-induced diabetes. ATRA was administered (1 mg/kg daily by gavage) on days 3 to 21 after STZ administration. It was found that 21 days after STZ injection, diabetic rats exhibited proteinuria, increased natriuresis and loss of body weight. Besides, diabetes induced an increase in interleukins [IL-1β, IL-1α, IL-16, IL-13, IL-2; tumor necrosis factor alpha (TNF-α)] and transforming growth factor-beta 1 (TGF-β1), chemokines (CCL2, CCL20, CXCL5 and CXCL7), adhesion molecules (ICAM-1 and L-selectin) and growth factors (GM-CSF, VEGF, PDGF) in glomeruli and proximal tubules, whereas ATRA treatment remarkably ameliorated these alterations. To further explore the mechanisms through which ATRA decreased inflammatory response, the NF-κB/p65 signaling mediated by TLR4 was studied. We found that ATRA administration attenuates the TLR4/NF-κB inflammatory signaling and prevents NF-κB nuclear translocation in glomeruli and proximal tubules.     

The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN.     

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.     

myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience: A NEW MECHANISM RELEVANT TO THE PATHOGENESIS OF DIABETIC NEPHROPATHY*

Diabetic nephropathy (DN) is characterized by perturbations in metabolic/cellular signaling pathways with generation of reactive oxygen species (ROS). The ROS are regarded as a common denominator of various pathways, and they inflict injury on renal glomerular cells. Recent studies indicate that tubular pathobiology also plays a role in the progression of DN. However, the mechanism(s) for how high (25 mm) glucose (HG) ambience induces tubular damage remains enigmatic. myo-Inositol oxygenase (MIOX) is a tubular enzyme that catabolizes myo-inositol to d-glucuronate via the glucuronate-xylulose (G-X) pathway. In this study, we demonstrated that G-X pathway enzymes are expressed in the kidney, and MIOX expression/bioactivity was up-regulated under HG ambience in LLC-PK1 cells, a tubular cell line. We further investigated whether MIOX overexpression leads to accentuation of tubulo-interstitial injury, as gauged by some of the parameters relevant to the progression of DN. Under HG ambience, MIOX overexpression accentuated redox imbalance, perturbed NAD⁺/NADH ratios, increased ROS generation, depleted reduced glutathione, reduced GSH/GSSG ratio, and enhanced adaptive changes in the profile of the antioxidant defense system. These changes were also accompanied by mitochondrial dysfunctions, DNA damage and induction of apoptosis, accentuated activity of profibrogenic cytokine, and expression of fibronectin, the latter two being the major hallmarks of DN. These perturbations were largely blocked by various ROS inhibitors (Mito Q, diphenyleneiodonium chloride, and N-acetylcysteine) and MIOX/NOX4 siRNA. In conclusion, this study highlights a novel mechanism where MIOX under HG ambience exacerbates renal injury during the progression of diabetic nephropathy following the generation of excessive ROS via an unexplored G-X pathway.     

Paracrine IL-6 signaling mediates the effects of pancreatic stellate cells on epithelial-mesenchymal transition via Stat3/Nrf2 pathway in pancreatic cancer cells

BackgroundWe previously showed that pancreatic stellate cells (PSC) secreted interleukin (IL)-6 and promoted pancreatic ductal adenocarcinoma (PDAC) cell proliferation via nuclear factor erythroid 2 (Nrf2)-mediated metabolic reprogramming. Epithelial-mesenchymal transition (EMT) is a key process for the metastatic cascade. To study the mechanism of PDAC progression to metastasis, we investigated the role of PSC-secreted IL-6 in activating EMT and the involvement of Nrf2 in this process.MethodsGene expression of IL-6 and IL-6Rα in PSC and PDAC cells was measured with qRT-PCR. The role of PSC-secreted IL-6, JAK/Stat3 signaling, and Nrf2 mediation on EMT-related genes expression was also examined with qRT-PCR. EMT phenotypes were assessed with morphological change, wound healing, migration, and invasion.ResultsPSC expressed higher mRNA levels of IL-6 but lower IL-6Rα compared to PDAC cells. Neutralizing IL-6 in PSC secretion reduced mesenchymal-like morphology, migration and invasion capacity, and mesenchymal-like gene expression of N-cadherin, vimentin, fibronectin, collagen I, Sip1, Snail, Slug, and Twist2. Inhibition of JAK/Stat3 signaling induced by IL-6 repressed EMT and Nrf2 gene expression. Induction of Nrf2 activity by tert-butylhydroquinone (tBHQ) increased both EMT phenotypes and gene expression (N-cadherin, fibronectin, Twist2, Snail, and Slug) repressed by IL-6 neutralizing antibody. Simultaneous inhibition of Nrf2 expression with siRNA and Stat3 signaling further repressed EMT gene expression, indicating that Stat3/Nrf2 pathway mediates EMT induced by IL-6.ConclusionsIL-6 from PSC promotes EMT in PDAC cells via Stat3/Nrf2 pathway.General significanceTargeting Stat3/Nrf2 pathway activated by PSC-secreted IL-6 may provide a novel therapeutic option to improve the prognosis of PDAC.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the functions of membrane receptors in T cells and suppressed T cell-mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n−3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study concluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.     

CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200^−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.