Cell Functional Responses Research Articles

Weight-Independent Mechanisms of Glucose Control After Roux-en-Y Gastric Bypass.

Roux-en-Y gastric bypass results in large and sustained weight loss and resolution of type 2 diabetes in 60% of cases at 1–2 years. In addition to calorie restriction and weight loss, various gastro-intestinal mediated mechanisms, independent of weight loss, also contribute to glucose control. The anatomical re-arrangement of the small intestine after gastric bypass results in accelerated nutrient transit, enhances the release of post-prandial gut hormones incretins and of insulin, alters the metabolism and the entero-hepatic cycle of bile acids, modifies intestinal glucose uptake and metabolism, and alters the composition and function of the microbiome. The amelioration of beta cell function after gastric bypass in individuals with type 2 diabetes requires enteric stimulation. However, beta cell function in response to intravenous glucose stimulus remains severely impaired, even in individuals in full clinical diabetes remission. The permanent impairment of the beta cell may explain diabetes relapse years after surgery.

Role of gut peptide in diabetes remission after Roux-en-Y gastric bypass surgery

Roux-en-Y gastric bypass surgery results in the resolution of type 2 diabetes in ∼60% of cases. Pre-surgery beta-cell function, with the large and sustained weight loss resulting from the surgery, are the main predictors of diabetes remission. However, enteric mechanisms, independent of weight loss, have been identified as important players. They include, among others, changes in intestinal metabolism, the gut hormones, the bile acids and the microbiome. The enhanced release of glucagon-like peptide 1 (GLP-1) contribute to the improvement of the incretin effect on insulin secretion during meals. GLP-1 and other gut peptides such as peptide YY and oxyntomodulin, may also contribute to the increased satiety and the decreased reward power of food observed after bypass, both contributing to decreased food intake and sustained weight loss. Although enteric factors are key for the full recovery of beta cell function during meals, the recovery of beta cell function in response to an IV glucose stimulus is more modest in magnitude, even in individual in diabetes remission. This may explain, in part, the long-term diabetes relapse in some patients. With the improvement of gut-mediated improved insulin secretion, RYGB results in large increase in insulin sensitivity and clearance, both contributors to improved glucose metabolism.

Sex Hormones Regulate Innate Immune Cells and Promote Sex Differences in Respiratory Virus Infection.

Sex differences in the incidence and severity of respiratory virus infection are widely documented in humans and murine models and correlate with sex biases in numbers and/or functional responses of innate immune cells in homeostasis and lung infection. Similarly, changes in sex hormone levels upon puberty, pregnancy, and menopause/aging are associated with qualitative and quantitative differences in innate immunity. Immune cells express receptors for estrogens (ERα and ERβ), androgens (AR), and progesterone (PR), and experimental manipulation of sex hormone levels or receptors has revealed that sex hormone receptor activity often underlies sex differences in immune cell numbers and/or functional responses in the respiratory tract. While elegant studies have defined mechanistic roles for sex hormones and receptors in innate immune cells, much remains to be learned about the cellular and molecular mechanisms of action of ER, PR, and AR in myeloid cells and innate lymphocytes to promote the initiation and resolution of antiviral immunity in the lung. Here, we review the literature on sex differences and sex hormone regulation in innate immune cells in the lung in homeostasis and upon respiratory virus infection.

Abstract 1709: Enhanced proliferation and effector functions of human immune cells in response to immune checkpoint therapy of MDA-MB-231 tumor bearing huNOG mice engrafted with human CD34+ cells

Abstract Given the recent success of cancer immunotherapies, well characterized pre-clinical models are needed to enable drug discovery efforts. The recently developed humanized mouse models composed of various types of immune cells offer a unique tool to evaluate aspects of the human immune system in response to immunotherapy. MDA-MB231 cells express high levels of the T-inhibitory molecule PD-L1 making this breast cancer line an ideal candidate for testing the efficacy of the immune-checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the CD34+ huNOG humanized mouse strain. We analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in blood, spleen and tumor samples from MDA-MB231 tumor bearing huNOG humanized treated-mice via flow cytometry. We found that combination therapy enhanced the effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-gamma and Granzyme B. Supporting this data, we observed an increase in Ki-67+ cells which correlates with a decrease in PD-1 expression in response to pembrolizumab therapy associated with activation of T lymphocytes. In summary, these results demonstrate the potential of humanized mouse models to evaluate the anti-tumor activity of human immune cells in a preclinical setting in response to human specific immune checkpoint targeted therapies. Citation Format: Thi Bui, Karyn McArtan Shinn, Emily G. O'Koren, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Jason Davis, Aidan Synnott, Anya Avrutskaya, Paula L. Miliani De Marval. Enhanced proliferation and effector functions of human immune cells in response to immune checkpoint therapy of MDA-MB-231 tumor bearing huNOG mice engrafted with human CD34+ cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1709.

Abstract 3960: The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes

Abstract Introduction: In cancer patients, the development of metastases is associated with a poor clinical prognosis. Metastatic dissemination requires cancer cells to detach from the primary tumor and invade adjacent or distant tissue. This process involves epithelial-to-mesenchymal transition (EMT), whereby tumor cells lose cell-cell adhesion mediated by E-cadherin repression. The CD44 family of transmembrane glycoproteins comprises alternatively spliced variants that are involved in many cellular processes. The isoform CD44v6 has been shown to play a major role in tumor growth and metastasis. CD44v6 acts as a co-receptor for the receptor tyrosine kinases (RTKs) c-MET, RON and VEGFR-2, and is preferentially expressed in a variety of epithelial cancers. We have previously shown that the co-receptor function of CD44v6 for c-MET is the driving force for metastasis. c-Met and RON facilitate metastasis through MAPK signaling and VEGFR-2 plays a role in angiogenesis. AMC303, an allosteric and selective peptide inhibitor of CD44v6, prevents phosphorylation of the CD44v6 dependent RTKs and respective cellular processes. Methods: The effect of AMC303 on cancer cell function in response to the growth factors HGF, MSP and VEGF was studied in various assays. Cell migration and invasion was analyzed in scratch assays and the Boyden Chamber. Morphological changes of EMT were monitored using microscopy in scattering assays in the presence of growth factors. EMT markers were analyzed by Western blot and immunohistochemistry. Tube formation was microscopically analyzed. Results: AMC303 dose dependently and at low nanomolar concentrations (IC50 10 - 20nM) prevents HGF- or VEGF-induced activation of c-MET/VEGFR-2 in various cancer cells of pancreatic, lung and colonic origin or endothelial cells, which cannot be overcome by excess HGF and VEGF. HGF- and MSP-dependent cell migration, invasion and wound closure in scratch assays was prevented by AMC303 using different cancer cell lines. AMC303 had a strong inhibitory effect on HGF- and MSP- induced cell scattering, a morphological feature of EMT. Consistent with this, AMC303 inhibited the downregulation of HGF induced EMT markers (E-cadherin). In primary endothelial cells, AMC303 blocked activation of VEGFR-2 and VEGF-A induced formation of a tubular network. Conclusions: Phosphorylation of CD44v6 dependent RTKs is inhibited in primary endothelial cells and different epithelial tumor cells by binding of AMC303 to the extracellular domaine of CD44v6. This novel mode of action results in strong anti-tumor and anti-metastatic effects of AMC303 involving inhibition of migration and invasion of tumor cells and prevention of EMT. AMC303 is currently tested in a First-in-Human Phase I/Ib study in cancer patients with solid epithelial tumors. Citation Format: Thorsten Läufer, Martin Augsten, Vanessa Al-Rawi, Birgit Hotz, Yvonne Heneka, Oliver Coutelle, Alexandra Matzke-Ogi. The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3960.

Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system l-amino acid transport is blocked. We identify SLC7A5 as the predominant system l-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

Innate immune responses to toll-like receptor stimulation are altered during the course of pregnancy

During pregnancy the maternal immune system has to develop tolerance towards the developing fetus. These changes in maternal immunity can result in increased severity of certain infections, but also in amelioration of autoimmune diseases. Pregnancy-related hormones have been suggested to play a central role in the adaptation of the maternal immune system, but their specific effects on innate immune function is not well understood. In a longitudinal study of pregnant women, we investigated innate immune cell function in response to toll-like receptors (TLR) 4 and 7 stimulation, two TLR pathways playing a critical role in early innate immune recognition of bacteria and viruses. IFNα production by TLR7-stimulated pDCs was decreased in early pregnancy, and increased towards the end of pregnancy. In contrast, pro-inflammatory TLR4-induced TNFα production by monocytes was increased during early pregnancy, but declined after the first trimester. Changes in cytokine production were associated with changes in pregnancy-related hormones and monocyte subpopulations over the course of pregnancy. These data demonstrating a significant association between pregnancy-related hormones and modulation of innate immune responses mediated by TLRs provide novel insights into the immunological adaptations occurring during pregnancy.

Deficient mitochondrial biogenesis in IL-2 activated NK cells correlates with impaired PGC1-α upregulation in elderly humans.

Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000 U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000 U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence.

An Improved Multi-parametric Programming Algorithm for Flux Balance Analysis of Metabolic Networks

Flux balance analysis has proven an effective tool for analyzing metabolic networks. In flux balance analysis, reaction rates and optimal pathways are ascertained by solving a linear program, in which the growth rate is maximized subject to mass-balance constraints. A variety of cell functions in response to environmental stimuli can be quantified using flux balance analysis by parameterizing the linear program with respect to extracellular conditions. However, for most large, genome-scale metabolic networks of practical interest, the resulting parametric problem has multiple and highly degenerate optimal solutions, which are computationally challenging to handle. An improved multi-parametric programming algorithm based on active-set methods is introduced in this paper to overcome these computational difficulties. Degeneracy and multiplicity are handled, respectively, by introducing generalized inverses and auxiliary objective functions into the formulation of the optimality conditions. These improvements are especially effective for metabolic networks because their stoichiometry matrices are generally sparse; thus, fast and efficient algorithms from sparse linear algebra can be leveraged to compute generalized inverses and null-space bases. We illustrate the application of our algorithm to flux balance analysis of metabolic networks by studying a reduced metabolic model of Corynebacterium glutamicum and a genome-scale model of Escherichia coli. We then demonstrate how the critical regions resulting from these studies can be associated with optimal metabolic modes and discuss the physical relevance of optimal pathways arising from various auxiliary objective functions. Achieving more than fivefold improvement in computational speed over existing multi-parametric programming tools, the proposed algorithm proves promising in handling genome-scale metabolic models.

The adaptor molecule CD2AP in CD4 T cells modulates differentiation of follicular helper T cells during chronic LCMV infection.

CD4 T cell-mediated help to CD8 T cells and B cells is a critical arm of the adaptive immune system required for control of pathogen infection. CD4 T cells express cytokines and co-stimulatory molecules that support a sustained CD8 T cell response and also enhance generation of protective antibody by germinal center B cells. However, the molecular components that modulate CD4 T cell functions in response to viral infection or vaccine are incompletely understood. Here we demonstrate that inactivation of the signaling adaptor CD2-associated protein (CD2AP) promotes CD4 T cell differentiation towards the follicular helper lineage, leading to enhanced control of viral infection by augmented germinal center response in chronic lymphocytic choriomeningitis virus (LCMV) infection. The enhanced follicular helper differentiation is associated with extended duration of TCR signaling and enhanced cytokine production of CD2AP-deficient CD4 T cells specifically under TH1 conditions, while neither prolonged TCR signaling nor enhanced follicular helper differentiation was observed under conditions that induce other helper effector subsets. Despite the structural similarity between CD2AP and the closely related adaptor protein CIN85, we observed defective antibody-mediated control of chronic LCMV infection in mice lacking CIN85 in T cells, suggesting non-overlapping and potentially antagonistic roles for CD2AP and CIN85. These results suggest that tuning of TCR signaling by targeting CD2AP improves protective antibody responses in viral infection.

Regulation of TCR signaling by CD2AP in CD4 T cells modulates antibody responses to chronic LCMV infection

Abstract CD4 T cell-mediated help to CD8 T cells and B cells is a critical arm of the adaptive immune system required for control of pathogens. CD4 T cells express cytokines and co-stimulatory molecules that support a sustained CD8 T cell response and also enhance generation of protective antibody by germinal center B cells. However, the molecular components that regulate CD4 T cell functions in response to viral infection or vaccine is incompletely understood. Here we demonstrate that inactivation of the signaling adaptor CD2AP promotes CD4 T cell differentiation towards the follicular helper lineage, leading to enhanced control of viral infection by augmented germinal center response in chronic LCMV infection. The enhanced follicular helper differentiation is associated with extended duration of TCR signaling and enhanced cytokine production of CD2AP-deficient CD4 T cells specifically under TH1 conditions, while neither prolonged TCR signaling nor enhanced follicular helper differentiation was observed under conditions that induce other helper effector subsets. These results suggest that tuning of TCR signaling by targeting CD2AP improved protective antibody responses in viral infection.

Mast cell corticotropin releasing factor receptor CRF2 regulates mast cell function in response to psychological and immunological stress

Abstract Psychological stress is a significant risk factor in a number of inflammatory and allergic diseases. We have previously demonstrated that CRF1 receptors expressed on mast cells (MCs) can potentiate psychological stress-induced MC degranulation intestinal permeability, and IgE-mediated anaphylaxis. In this study, we investigated the role of the CRF2 receptors in regulating MC activation in response to psychological and immunological stress. CRF2-deficient (CRF2−/−) bone marrow derived MCs exhibited enhanced IgE-and c48/80-mediated mast cell degranulation, determined by β-hex and histamine release. Heightened degranulation in CRF2−/− MCs were mediated by enhanced intracellular Ca2+ mobilization from endoplasmic reticulum stores. Similar results were obtained with human LAD2 mast cells treated with the CRF2 antagonist Astressin 2B. In vitro findings translated to in vivo studies as MC-deficient KitW-sh/W-sh mice systemically engrafted with CRF2−/− BMMCs exhibited a 3-fold increase in serum histamine levels and intestinal permeability following acute restraint stress or passive systemic anaphylaxis. Contrasting effects of CRF2 were observed with de novo synthesized mediator release as IL-4, IL-6, MCP-1 and prostaglandin D2 release were reduced (by ~40–50%) in CRF2−/− BMMCs following stimulation with IgE-antigen, LPS and IL-33. In summary, these data show that CRF2 signaling inhibits MC degranulation and associated pathophysiology induced by acute psychological stress and IgE-mediated anaphylaxis, while enhancing the release of de novo synthesized mediators. Therefore, targeting CRF2 may represent a novel therapeutic approach for preventing stress-related allergic and inflammatory disease flares.

Loss of the von-Hippel Lindau gene in osteocytes alters B lymphocyte development and immune response

Abstract The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor (HIF) degradation. Conditional-deletion of VHL in osteoblasts and in hematopoietic progenitors have demonstrated a role for VHL in these cell types, but how changes in bone homeostasis affects immune development are not fully understood. Osteoblasts (OBs) support B cell development. OBs mature into osteocytes (OCYs) and to test if OCYs also support B cell development, we generated OCY-specific (Dmp-Cre) conditional VHL knockout (cKO) mice. cKO mice display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and overall decrease in BM cellularity compared to controls. In line with this, the frequencies and numbers of B cell precursors (B220+ IgM−), immature B (B220+ IgMint) and recirculating B (B220+ IgMhigh) cells in the bone marrow are significantly decreased. These data suggest that the change in bone homeostasis adversely affects B cell development in a cell-extrinsic manner. Although the absolute numbers of B cells in the spleens of cKOs were normal, we hypothesized that loss of VHL in OCYs could permanently and adversely affect B cell functional response. To test this, VHL cKO mice were immunized with a T-independent antigen (NP-Ficoll). IgM and IgG3 antigen-specific titers to NP-Ficoll in VHL cKOs were similar to WT mice. Testing of B cell responses to T-dependent antigens (NP-OVA) is ongoing. Taken together, our data suggest that OCY-specific VHL deletion might be helpful to increase bone density, but at the expense of B cell numbers. Further investigation is required to understand the molecular mechanisms by which VHL in OCYs contribute to its putative role as a B cell niche cell.

Tart cherry extract improves skeletal muscle recovery by increasing type II MHC expression, MCP-1 and HGF secretion, and attenuating ROS production by neutrophils

Abstract Ingestion of tart cherry extract (TCE), a rich source of polyphenolic anti-oxidant/anti-inflammatory compounds, improves skeletal muscle (SM) recovery following exercise-induced muscle damage. This response may be due in part to attenuated oxidative/pro-inflammatory activities of recruited neutrophils and macrophages during SM regeneration. How TCE promotes the molecular mechanisms regulating SM regeneration or attenuates those controlling myeloid cell pro-inflammatory responses is unknown. Herein, we describe how TCE affects two murine cell models optimized to recapitulate in vivo interactions between regenerating SM and recruited pro-inflammatory myeloid cells: 1) strained skeletal muscle tissue derived from C2C12 myoblasts, and 2) mature myeloid lineages derived from ex vivo-cultured primary bone marrow. We show that TCE treatment may improve SM regeneration by increasing MHC protein expression plus MCP-1 and HGF secretion. Importantly, TCE treatment modestly reduced myoblast proliferation but without affecting viability, and did not affect proliferation or differentiation-specific protein expression profiles (via imaging flow cytometry) of myeloblasts or derived progenitors. Moreover, TCE treatment reduced levels of reactive oxygen species (ROS) released by activated neutrophils. Our continued analyses will identify how TCE either independently affects damaged SM regeneration or myeloid cell functional responses, or dependently affects the interactions between these disparate cell types upon muscle injury. Our studies may reveal that this natural compound not only improves muscle recovery following damage but also attenuates pro-inflammatory responses of recruited myeloid cells.

Diacylglycerol kinase ζ (DGKζ) and Casitas b-lineage proto-oncogene b-deficient mice have similar functional outcomes in T cells but DGKζ-deficient mice have increased T cell activation and tumor clearance.

Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.

Effect of incubation temperature on neuropeptide Y and neuropeptide Y receptors in turkey and chicken satellite cells

Neuropeptide Y (NPY) is an appetite stimulating peptide released from the central nervous system and impacts the function of many different cell types. A recent transcriptome study showed that NPY expression was altered when turkey breast muscle satellite cells were incubated at low or high temperatures, suggesting NPY may mediate temperature effects on satellite cells. However, to date minimal information exists describing the expression and function of NPY in satellite cells. The objective of this study was to determine how temperature impacts NPY and NPY receptor gene expression in satellite cells isolated from turkeys and chickens with differing genetic lineages. Two broiler and two turkey breast muscle satellite cell lines were incubated at 35, 38 or 41 °C during proliferation and differentiation. In both turkey lines, NPY, and receptors NPY2R and NPY5R expression increased at elevated temperatures after 72 h of proliferation. During differentiation NPY and NPY5R expression increased in both turkey lines with higher temperatures, whereas NPY2R was minimally affected by temperature. In contrast, in both chicken cell lines there were few significant differences for NPY and NPY receptor expression across temperature during proliferation. During differentiation, the temperature effect was different in the two chicken cell lines. In the BPM8 chicken line, there were few differences in NPY and NPY receptors across temperature; whereas elevated temperatures increased NPY, NPY2R, and NPY5R expression in the 708 line. The differences between turkey and chicken lines suggest NPY has species specific satellite cell functions in response to heat stress.

Aryl Hydrocarbon Receptor Ligands Indoxyl 3-sulfate and Indole-3-carbinol Inhibit FMS-like Tyrosine Kinase 3 Ligand-induced Bone Marrow-derived plasmacytoid Dendritic Cell Differentiation.

Aryl hydrocarbon receptor (AhR) regulates both innate and adaptive immune responses by sensing a variety of small synthetic and natural chemicals, which act as its ligands. AhR, which is expressed in dendritic cells (DCs), regulates the differentiation of DCs. However, effects of AhR on the differentiation of DCs are variable due to the heterogeneity of DCs in cell surface marker expression, anatomical location, and functional responses. The plasmacytoid DCs (pDCs), one of DC subsets, not only induce innate as well as adaptive immune responses by secreting type I interferons and pro-inflammatory cytokines, but also induce IL-10 producing regulatory T cell or anergy or deletion of antigen-specific T cells. We showed here that AhR ligands indoxyl 3-sulfate (I3S) and indole-3-carbinol (I3C) inhibited the development of pDCs derived from bone marrow (BM) precursors induced by FMS-like tyrosine kinase 3 ligand (Flt3L). I3S and I3C downregulated the expression of signal transducer and activator of transcription 3 (STAT3) and E2-2 (Tcf4). In mice orally treated with I3S and I3C, oral tolerance to dinitrofluorobenzene was impaired and the proportion of CD11c+B220+ cells in mesenteric lymph nodes was reduced. These data demonstrate that AhR negatively regulates the development of pDCs from BM precursors induced by Flt3L, probably via repressing the expression of STAT3.

Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.

The effects of liquid crystal-based composite substrates on cell functional responses of human umbilical cord-derived mesenchymal stem cells by mechano-regulatory process.

Physical properties of extracellular matrix, including elasticity and microstructure, have been considered as important factors inducing stem cell differentiation. This study developed a novel type of liquid crystal-based matrix by combining the elastic property of polyurethane with viscoelastic liquid crystal to generate a soft elastic response resembling physical microenvironment of stem cell niche, and explored the mechano-driving cell behaviors. Addition of varying liquid crystal concentration (10 wt%, 30 wt% and 50 wt%) had great effects on surface morphology and elastic modulus of liquid crystal/ polyurethane composite substrates. Changes in microstructure and elastic modulus of the substrates could cause intense cell responses that influenced cell properties, including proliferation, adhesion, and osteogenic differentiation. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured on both liquid crystal-10/polyurethane and liquid crystal-30/polyurethane substrates exhibited higher viability, more actin filament, and larger spreading area while liquid crystal-50/polyurethane substrates seemed not to favor cell attachment and spreading. Alkaline phosphatase activity and calcium deposition were significantly improved with hUC-MSCs on both liquid crystal-10/ polyurethane and liquid crystal-30/ polyurethane substrates, and the maximal alkaline phosphatase activity was observed in liquid crystal-10/ polyurethane while the lowest in liquid crystal-50/ polyurethane. Osteopontin was upregulated to a high level in both liquid crystal-10/ polyurethane and liquid crystal-30/ polyurethane groups after 14 days culturing; the maximal expression of osteocalcin and related transcription factor 2 were found in liquid crystal-10/ polyurethane group on day 21. Our findings revealed that hUC-MSCs could intensely sense the bioactive patterns and soft-matter feature of liquid crystal domains and subsequently modulated cell behaviors, which may prove useful in the development of new class of biomaterials for applications in tissue engineering and regenerative medicine.

Srebp-controlled glucose metabolism is essential for NK cell functional responses.

Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.