Ligand Lipopolysaccharide Research Articles

Expression and function of macrophage-inducible C-type lectin (Mincle) in inflammation driven parturition in fetal membranes and myometrium.

The pivotal role of inflammatory processes in human parturition is well known, but not completely understood. We have performed a study to examine the role of macrophage-inducible C-type lectin (Mincle) in inflammation-associated parturition. Using human samples, we show that spontaneous labour is associated with up-regulated Mincle expression in the myometrium and fetal membranes. Mincle expression was also increased in fetal membranes and myometrium in the presence of pro-labour mediators, the proinflammatory cytokines interleukin (IL)-1B and tumour necrosis factor (TNF), and Toll-like receptor (TLR) ligands fsl-1, poly(I:C), lipopolysaccharide (LPS) and flagellin. These clinical studies are supported by mouse studies, where an inflammatory challenge in a mouse model of preterm birth increased Mincle expression in the uterus. Importantly, elimination of Mincle decreased the effectiveness of proinflammatory cytokines and TLR ligands to induce the expression of pro-labour mediators; namely, proinflammatory cytokines and chemokines, contraction-associated proteins and prostaglandins, and extracellular matrix remodelling enzymes, matrix metalloproteinases. The data presented in this study suggest that Mincle is required when inflammatory activation precipitates parturition.

Nicotinamide alleviates kidney injury and pregnancy outcomes in lupus-prone MRL/lpr mice treated with lipopolysaccharide

Systemic lupus erythematosus (SLE) increases the risk of preterm birth and preeclampsia (PE). The flares of SLE during pregnancy or after delivery are also problematic. We have previously demonstrated that nicotinamide (NAM), a non-teratogenic amide of vitamin B3, reduces inflammation and oxidative stress and improves PE-like phenotype and pregnancy outcomes in the mouse models of PE. The present study aimed to establish a model to investigate the pregnancy outcomes and flares of SLE in pregnant mice with SLE and to examine whether NAM is beneficial to pregnant mice with SLE. We used pregnant and non-pregnant lupus-prone MRL/lpr mice treated with or without a Toll-like receptor (TLR) ligand lipopolysaccharide (LPS) because TLR4 signaling reportedly exacerbates SLE and pregnancy; MRL/+ mice were used as controls. Blood pressure (BP) and urinary albumin excretion were increased only in the pregnant MRL/lpr-LPS mice. LPS together with pregnancy exacerbated glomerulonephritis, and the most severe inflammation was observed in the kidneys of the pregnant MRL/lpr-LPS mice. The shortening of pregnancy periods, increase in fetal demise percentage, and reduction in fetal weight were observed only in the pregnant MRL/lpr-LPS mice. NAM improved BP and kidney injury, prolonged pregnancy periods, and improved fetal growth in the pregnant MRL/lpr-LPS mice. The results suggest that SLE patients are prone to develop poor pregnancy outcome, and likely develop severe nephropathy and kidney inflammation. NAM may be a novel therapeutic option that improves kidney injury and pregnancy outcomes, thereby benefiting pregnant patients with SLE.

Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells.

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous member of the GRK family that restrains cellular activation by G protein-coupled receptor (GPCR) phosphorylation leading to receptor desensitization and internalization, but has been identified to regulate a variety of signaling molecules, among which may be associated with inflammation. In this study, we attempted to establish the regulatory role of GRK2 in the Toll-like receptor (TLR) signaling pathway for inducible nitric oxide synthase (iNOS) expression in microglial cells. When mouse MG6 cells were stimulated with the TLR4 ligands lipopolysaccharide (LPS) and paclitaxel, we found that interferon regulatory factor 1 (IRF1) protein expression and activation was upregulated, transcription of interferon-β (IFN-β) was accelerated, induction/activation of STAT1 and activation of STAT3 were promoted, and subsequently iNOS expression was upregulated. The ablation of GRK2 by small interfering RNAs (siRNAs) not only eliminated TLR4-mediated upregulation of IRF1 protein expression and nuclear translocation but also suppressed the activation of the STAT pathway, resulting in negating the iNOS upregulation. The TLR3-mediated changes in IRF1 and STAT1/3, leading to iNOS induction, were also abrogated by siRNA knockdown of GRK2. Furthermore, transfection of GRK2 siRNA blocked the exogenous IFN-β supplementation-induced increases in phosphorylation of STAT1 as well as STAT3 and abrogated the augmentation of iNOS expression in the presence of exogenous IFN-β. Taken together, our results show that GRK2 regulates the activation of IRF1 as well as the activation of the STAT pathway, leading to upregulated transcription of iNOS in activated microglial cells. Modulation of the TLR signaling pathway via GRK2 in microglia may be a novel therapeutic target for treatment of neuroinflammatory disorders.

Effects of ATP on expression of inflammatory factors in endothelial progenitor cells induced by LPS and the mechanisms

To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms. Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway. Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05). Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.

KDM6B overexpression activates innate immune signaling and impairs hematopoiesis in mice

AbstractKDM6B is an epigenetic regulator that mediates transcriptional activation during differentiation, including in bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Overexpression of KDM6B has been reported in BM HSPCs of patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Whether the overexpression of KDM6B contributes to the pathogenesis of these diseases remains to be elucidated. To study this, we generated a Vav-KDM6B mouse model, which overexpresses KDM6B in the hematopoietic compartment. KDM6B overexpression alone led to mild hematopoietic phenotype, and chronic innate immune stimulation of Vav-KDM6B mice with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS) resulted in significant hematopoietic defects. These defects recapitulated features of MDS and CMML, including leukopenia, dysplasia, and compromised repopulating function of BM HSPCs. Transcriptome studies indicated that KDM6B overexpression alone could lead to activation of disease-relevant genes such as S100a9 in BM HSPCs, and when combined with innate immune stimulation, KDM6B overexpression resulted in more profound overexpression of innate immune and disease-relevant genes, indicating that KDM6B was involved in the activation of innate immune signaling in BM HSPCs. Finally, pharmacologic inhibition of KDM6B with the small molecule inhibitor GSK-J4 ameliorated the ineffective hematopoiesis observed in Vav-KDM6B mice. This effect was also observed when GSK-J4 was applied to the primary BM HSPCs of patients with MDS by improving their repopulating function. These results indicate that overexpression of KDM6B mediates activation of innate immune signals and has a role in MDS and CMML pathogenesis, and that KDM6B targeting has therapeutic potential in these myeloid disorders.

Imaging the Vascular Bone Marrow Niche During Inflammatory Stress.

Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation. Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry and histology showed vascular changes to the bone marrow niche. Specifically, proliferating endothelial cells gave rise to new vasculature in the bone marrow during hypoxic conditions. We studied these vascular changes with complementary intravital microscopy and positron emission tomography/magnetic resonance imaging. Fluorescence and positron emission tomography integrin αVβ3 imaging signal increased during lipopolysaccharide-induced vascular remodeling. Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when neutrophils departed and hematopoietic stem and progenitor cells proliferated more vigorously. Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature may provide a key to unlock therapeutic targets regulating systemic inflammation.

Butyrate upregulates the TLR4 expression and the phosphorylation of MAPKs and NK-κB in colon cancer cell in vitro.

Microbiota and its induced inflammation in colorectal mucosa have been considered risk factors for the development of colorectal carcinogenesis. Previous studies demonstrated that the coexisting elements of microbiota in the gut, such as short chain fatty acids (SCFAs) and lipopolysaccharides (LPS), which exhibited regulatory effects on the intestinal epithelial cells individually. Unfortunately, the association between butyrate and the toll-like receptor (TLR) signaling pathway in the development of colon cancer is not fully elucidated. In the present study, by culturing human colon cancer SW480 cells or mouse colon cancer CT26 cells with butyrate and/or TLR4 ligand LPS in vitro, it was identified that butyrate suppressed the growth and promoted apoptosis of these cancer cells. Notably, the expression levels of TLR4 and CD14 were markedly increased on these butyrate-treated cells, but not on LPS-alone treated cells. Additionally, butyrate treatment induced the phosphorylation of extracellular signal-regulated kinase, tumor protein 38, c-Jun NH2-terminal kinase and nuclear factor-κB (NF-κB) p65, and then promoted the pro-inflammatory cytokine tumor necrosis factor-α, but not interleukin 6 secretion in SW480 and CT26 cells. Therefore, butyrate treatment regulates the expression of TLR4, mitogen-activated protein kinase and NF-κB signal pathway activation and pro-inflammatory response in vitro. Although the exact mechanisms have not been fully explored, these results suggested that butyrate and LPS-TLR4 signaling mediated innate immunity in colon cancer cells through two distinct but inter-regulated pathways. Thus, butyrate can further initiate innate immunity against tumor cells by upregulating the TLR4 expression and activation to preserve intestinal homeostasis.

Anti-inflammatory properties of cilostazol: Its interruption of DNA binding activity of NF-κB from the Toll-like receptor signaling pathways

Cilostazol, a selective inhibitor of phosphodiesterase type III with anti-platelet, anti-mitogenic, and vasodilating properties, is widely used to treat ischemic symptoms of peripheral vascular disease. Ample evidence has suggested that cilostazol also exhibits an anti-inflammatory effect, but its anti-inflammatory mechanism is not fully understood. Here, we showed that cilostazol specifically inhibited expression of cytokines, which are induced by nuclear factor-κB (NF-κB) activation, in RAW264.7 macrophage cells stimulated with different Toll-like receptor (TLR) ligands. Cilostazol was found to significantly reduce TLR-4 and TLR-3 ligands-stimulated NF-κB transcriptional activity, which was quantified by luciferase reporter assays. However, cilostazol was without effect on IκBα degradation and NF-κB p65 phosphorylation and nuclear translocation after challenge with the TLR-4 ligand lipopolysaccharide (LPS). Cilostazol did not also prevent the LPS-induced increase in phosphorylated levels of the mitogen-activated protein kinase (MAPK) family. On the other hand, using chromatin immunoprecipitation assays, we demonstrated that cilostazol reduced the LPS-induced transcriptional activities of interleukin-6 and tumor necrosis factor-α by preventing the recruitment of NF-κB p65 to these gene promoters. When cilostazol was given to mice by oral gavage daily for 7 days, LPS-induced aberrant pro-inflammatory cytokine production and end-organ tissue injury were significantly reduced. The results of this study suggest that cilostazol is capable of directly interrupting DNA binding activity of NF-κB proteins from the TLR signaling pathways. The therapy to specifically intervene in this pathway may be potentially beneficial for the prevention of different inflammatory disorders.

Early minor stimulation of microglial TLR2 and TLR4 receptors attenuates Alzheimer's disease-related cognitive deficit in rats: behavioral, molecular, and electrophysiological evidence.

At early stages of Alzheimer's disease (AD), soluble amyloid beta (Aβ) accumulates in brain while microglia are in resting state. Microglia can recognize Aβ long after formation of plaques and release neurotoxic mediators. We examined impact of early minor activation of microglia by Toll-like receptors (TLRs) 2 and 4 agonists on Alzheimer's disease-related disturbed synaptic function and spatial memory in rats. Microglial BV-2 cells were treated by 0.1, 1, and 10μg/mL of the TLRs ligands lipopolysaccharide, monophosphoryl lipid A (MPL), and Pam3Cys for 24 hours. Culture medium was then changed with media containing 1-μM Aβ. Tumour necrosis factor (TNF)-α and CCL3 levels were measured in the supernatant, 24 hours thereafter. One μg of TLRs ligands which was able to release low level of TNF-α and CCL3, was administered intracerebroventricularly (i.c.v) to adult male rats every 3days for 24days. At the half of the treatment period, Aβ1-42 was infused i.c.v (0.075μg/hour) for 2 weeks. Finally, the following factors were measured: memory performance by Morris water maze, postsynaptic potentials of dentate gyrus following perforant pathway stimulation, hippocampal inflammatory cytokines interleukin 1 (IL-1)βand TNF-α, anti-inflammatory cytokines IL-10 and TGF-1β, microglia marker arginase 1, Aβ deposits, and the receptor involved in Aβ clearance, formyl peptide receptor 2 (FPR2). TLRs ligands caused dose-dependent release of TNF-α and CCL3 by BV-2 cells. Aβ-treated cells did not release TNF-α and CCL3, whereas those pretreated with MPL and Pam3Cys significantly released these cytokines in response to Aβ. Low-dose TLRs ligands improved the disturbance in spatial and working memory; restored the impaired long-term potentiation induced by Aβ; decreased TNF-α, and Aβ deposits; enhanced TGF-1β, IL-10, and arginase 1 in the hippocampus of Aβ-treated rats; and increased polarization of hippocampal microglia to the anti-inflammatory phenotype. The ligands increased formyl peptide receptor 2 in both BV-2 cells and hippocampus/cortex of Aβ-treated rats. Microglia can sense/clear soluble Aβ by early low-dose MPL and Pam3Cys and safeguard synaptic function and memory in rats.

The role of TLR4/P38/JNK signaling pathway in apoptosis of hippocampal neurons

To investigate the effects of the Toll like receptor 4 (TLR4) -P38-JNK signaling pathway in the apoptosis of hippocampal neurons in rats and its mechanisms. And to provide new experimental evidences for the pathogenesis research, prevention and treatment of neurodegenerative diseases (ND). The hippocampal neurons derived from newborn rat were cultured for 7 d in vitro. The purity of hippocampal neurons was identified by immunofluorescence method. In order to activate or block the action of TLR4, the hippocampal neurons were pretreated with TLR4 ligand lipopolysaccharide (LPS) or TLR4 antibody. In the experiment 1, the hippocampal neurons were divided into normal control group, LPS group and TLR4 antibody+LPS group. The expressions of P-P38 and P-JNK were deteced by immunofluorescence. In the experiment 2, the hippocampal neurons were divided into 6 groups:normal control group, LPS group, TLR4 antibody +LPS group, SB202190(inhibitor P38)+LPS group, SP600125(inhibitor JNK)+LPS group, PD98059(inhibitor ERK)+LPS group. The cells in above mentioned groups were pretreated with TLR4 antibody, the inhibitors of P38, JNK or ERK for 2 h respectively. Then, all the six groups were stimulated by LPS for 24 h. The expressions of Bcl-2, Bax and Active-caspase-3 were detected by Western blot. The hippocampal neuronal apoptosis rate were tested with flow cytometry. The expressions of P-P38 and P-JNK of hippocampal neurons in LPS group were higher than those in normal control group (P<0.01). Compared with LPS group, the expressions of P-P38 and P-JNK were decreased significantly in TLR4 antibody +LPS group (P<0.01). Compared with the normal control group, the expressions of Bcl-2/Bax were decreased, while the expression of Active-caspase-3 was increased in the hippocampal neurons after LPS stimulation (P<0.01). The apoptotic rate of hippocampal neurons was higher in LPS group than that in the control group (P<0.01). Compared with LPS group, the expressions of Bcl-2/Bax were increasd and the expression of Active-caspase-3 was decreased in TLR4 antibody+LPS group, SB202190+LPS group, and SP600125+LPS group. The apoptotic rate of hippocampal neurons was significantly lower than that in the LPS group (P<0.05, P<0.01). The cell apoptosis rate had no significant differences between PD98059+LPS group and LPS group. ①TLR4-mediated P38/JNK signaling pathway exists in hippocampal neurons. ②After TLR4 activation in hippocampal neurons, the expressions of P-P38 and P-JNK are up-regulated, the expressions of Bcl-2/Bax are decreased and the expression of Active-caspase-3 is increased, which promote apoptosis of hippocampal neurons. TLR4/P38/JNK signaling pathway is involved in apoptosis of hippocampal neurons.

Toll-Like Receptors Drive Specific Patterns of Tolerance and Training on Restimulation of Macrophages.

Tolerance is a long-recognized property of macrophages that leads to an altered response to repeated or chronic exposure to endotoxin. The physiological role of tolerance is to limit the potential damage to host tissue that may otherwise result from prolonged production of pro-inflammatory cytokines. Tolerance is induced by all toll-like receptor (TLR) ligands tested to date, however, tolerance induced by the TLR4 ligand lipopolysaccharide (LPS) is by far the best studied. LPS tolerance involves a global transcriptional shift from a pro-inflammatory response toward one characterized by the expression of anti-inflammatory and pro-resolution factors. Although largely reversible, LPS-tolerance leads to a hybrid macrophage activation state that is pro-inflammatory in nature, but possesses distinct regulatory anti-inflammatory features. Remarkably, a comparative transcriptomic analysis of tolerance induced by different TLR ligands has not previously been reported. Here, we describe the transcriptomic profiles of mouse macrophages tolerized with ligands for TLR2, TLR3, TLR4 and TLR 9. While we identified TLR-specific transcriptional profiles in macrophages tolerized with each ligand, tolerance induced by TLR4 represented an archetype pattern, such that each gene tolerized by any of the TLRs tested was also found to be tolerized by TLR4. Pro-inflammatory cytokines are not universally suppressed in all tolerant cells, but distinct patterns of cytokine expression distinguished TLR-specific tolerance. Analysis of gene regulatory regions revealed specific DNA sequence motifs associated with distinct states of TLR tolerance, implicating previously identified as well as novel transcriptional regulators of tolerance in macrophages. These data provide a basis for the future exploitation of TLR-specific tolerant states to achieve therapeutic re-programming of the innate immune response.

Differential surface phenotype and context-dependent reactivity of functionally diverse NKT cells

Abstract Natural Killer T (NKT) cells are a functionally diverse population that recognize lipid-based antigens in association with the antigen-presenting molecule CD1d. Here we define a technique to separate the functionally distinct NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD279 (ICOS) and the activation-associated glycoform of CD43, enabling the investigation of subset-specific effector-functions. We report that all three subsets express the transcription factor GATA-3 and the potential to produce IL-4 and IL-10 following activation. This questions the notion that NKT2 cells are the predominant source of IL-4 within the NKT cell pool, and suggests that IL-10-production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4+ and are biased towards Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll-like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell-biased response, even in the absence of exogenous antigen, and that combining LPS with α-GalCer resulted in enhanced IL-17A-production, and reduced levels of the immunosuppressive cytokine IL-10. This study provides a novel means to examine the context-dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.

Bone marrow lympho-myeloid malfunction in obesity requires precursor cell-autonomous TLR4

Abstract Obesity, a prevalent condition in adults and children, impairs bone marrow (BM) function. However, the underlying mechanisms are unclear. Here we show that obese mice exhibit poor emergency immune responses in a toll-like receptor 4 (TLR4)-dependent manner. Canonical myeloid genes (c-fms, pu.1, runx) are enhanced, and lymphoid genes (flt3, e47, ebf) are reduced. Using adoptive transfer and mixed BM chimera approaches we demonstrate that myeloid&amp;gt;lymphoid bias arises after only six weeks of high fat diet and depends on precursor cell-autonomous TLR4. Further, lean mice exposed to the TLR4 ligand lipopolysaccharide (LPS) at doses similar to that detectable in obese serum recapitulates BM lympho-myeloid alterations. Together, these results establish a mechanistic contribution of BM cell-intrinsic TLR4 in obesity-driven BM malfunction and demonstrate the importance of LPS. Our findings raise important questions about the impact of maternal obesity and endotoxemia to fetal hematopoiesis since fetal immune precursors are also sensitive to TLR4 signals.

Aerobic Exercise and Environmental Heat Stress as Adjuvants to Seasonal Influenza Vaccine

Influenza strain diversity and variability and individual immune health contribute to reduced vaccine efficacy. Among adjuvants that enhance vaccine effectiveness, several studies have examined the relationship between acute exercise stress and influenza vaccination immune responses in humans. It is proposed that acute bouts of stress enhance immune response, but optimal intervention, timing of intervention, and mechanisms have yet to be identified. As Toll‐like receptor (TLR) agonists enhance innate immune responses and support development of chronic humoral responses, exercise induced release of the TLR4 ligand lipopolysaccharide (LPS) into circulation may be a mechanism for benefit of pre‐vaccine exercise. This study aimed to assess the effects of exercise and exercise‐heat stress completed before seasonal influenza vaccination (IV) on short‐ and long‐term immune responses. Thirty‐four males (body mass, 77.41±12.48kg; age, 21.5±4.4y; maximal aerobic capacity (VO2max), 43.53±8.24mL/kg/min) were randomized into 1 of 3 groups: no exercise (NE, n=12) or 60 minutes of 60%VO2max exercise with (MEH, n=11) or without (ME, n=11) environmental heat stress. Exercise and baseline blood samples were completed 25.11±5.92h before IV, and subsequent blood samples were collected 23.45±6.13h, 13.06±2.96d, and 29.54±3.73d following IV. Exercise environmental conditions were different between ME and MEH, respectively (average wet bulb globe temperature, 32.91±2.67°C, 41.21±2.88°C; p&lt;0.001), but physiological stress was similar (average rectal temperature, 37.30±0.48°C, 27.61±1.36°C, p=0.513; heart rate, 114±105bpm, 118±105bpm, p=0.103) due to controlled exercise intensity. On‐going research will utilize hemagglutinin inhibition, cortisol, and LPS assays to quantify virus‐specific antibody responses, stress, and TLR4 activation respectively. Understanding innate and humoral responses will elucidate the potential of exercise and exercise‐heat stressors as adjuvants for IV.Support or Funding InformationNew investigator start‐up, InCHIP Interdisciplinary Seed GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential surface phenotype and context-dependent reactivity of functionally diverse NKT cells.

Natural Killer T (NKT) cells are a functionally diverse population that recognizes lipid-based antigens in association with the antigen-presenting molecule CD1d. Here, we define a technique to separate the functionally distinct thymic NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD278 (ICOS) and the activation-associated glycoform of CD43, enabling the investigation of subset-specific effector-functions. We report that all three subsets express the transcription factor GATA-3 and the potential to produce IL-4 and IL-10 following activation. This questions the notion that NKT2 cells are the predominant source of IL-4 within the NKT cell pool, and suggests that IL-10-production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4+ and are biased toward Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll-like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell-biased response, even in the absence of exogenous antigen, and that combining LPS with α-GalCer resulted in enhanced IL-17A-production, and reduced levels of the immunosuppressive cytokine IL-10. This study provides a novel means to examine the context-dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.

Chondroitin sulfate proteoglycans as novel drivers of leucocyte infiltration in multiple sclerosis

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with leucocytes in inflammatory perivascular cuffs in all four patients. We conclude that the accumulation of chondroitin sulfate proteoglycans in the perivascular cuff in multiple sclerosis and experimental autoimmune encephalomyelitis boosts the activity and migration of leucocytes across the glia limitans into the central nervous system parenchyma. Thus, chondroitin sulfate proteoglycans represent a new class of molecules to overcome in order to reduce the inflammatory cascades and clinical severity of multiple sclerosis.

Bone marrow lympho-myeloid malfunction in obesity requires precursor cell-autonomous TLR4

Obesity, a prevalent condition in adults and children, impairs bone marrow (BM) function. However, the underlying mechanisms are unclear. Here, we show that obese mice exhibit poor emergency immune responses in a toll-like receptor 4 (TLR4)-dependent manner. Canonical myeloid genes (Csf1r, Spi1, Runx1) are enhanced, and lymphoid genes (Flt3, Tcf3, Ebf1) are reduced. Using adoptive transfer and mixed BM chimera approaches we demonstrate that myeloid>lymphoid bias arises after 6 weeks of high-fat diet and depends on precursor cell-autonomous TLR4. Further, lean mice exposed to the TLR4 ligand lipopolysaccharide (LPS) at doses similar to that detectable in obese serum recapitulates BM lympho-myeloid alterations. Together, these results establish a mechanistic contribution of BM cell-intrinsic TLR4 to obesity-driven BM malfunction and demonstrate the importance of LPS. Our findings raises important questions about the impact of maternal obesity and endotoxemia to fetal hematopoiesis, as fetal immune precursors are also sensitive to TLR4 signals.

Induction of Ocular Complement Activation by Inflammatory Stimuli and Intraocular Inhibition of Complement Factor D in Animal Models.

Genome-wide association studies suggest a role for the complement system in age-related macular degeneration (AMD). We characterized ocular complement activation and evaluated a complement factor D (FD) neutralizing antibody. Mice were treated with toll-like receptor (TLR) ligands, intravitreal injection (IVT), or corneal debridement. Levels of complement proteins and mRNA were measured. A FD neutralizing antibody was administered IVT into eyes of rabbits that were challenged with LPS (lipopolysaccharide) administered intravenously. Levels of C3 and factor B (FB) mRNA and protein in the eye were increased following intraperitoneal injection of TLR4 ligand LPS. Increased levels of C3 and FB breakdown products were observed in both eye tissues and plasma. Complement activation products were markedly reduced in C3-/- and Cfb-/- mice challenged with LPS. Ocular complement levels were also elevated in mice treated systemically with TLR2 and -3 ligands, injured by IVT injection or corneal debridement, or even in normal aging. IVT administration of a complement FD neutralizing antibody in rabbits inhibited LPS-induced complement activation in the posterior segment of the eye, but not in the anterior segment of the eye or in plasma. Systemic TLR stimulation and eye tissue injury induced time-dependent alternative complement pathway activation in the eye. Ocular complement levels were also gradually elevated during aging. An anti-FD antibody IVT potently inhibited LPS-induced complement activation in the posterior segment of the eye. This study provides insights into the dynamic profile of ocular complement activation, which is valuable for complement research in eye diseases and for developing complement therapeutics for AMD.

Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4.

Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional conditions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho-PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein synthesis in skeletal muscle in part by induction of ER stress.

Transcriptional and translational-uncoupling in regulation of the CXCL12 and its receptors CXCR4, 7 in THP-1 monocytes and macrophages.

IntroductionThe chemokine CXCL12 and its receptors CXCR4 and 7 play crucial roles in the immune system. In the present study, regulation of this pathway was further examined using the in‐vitro model of undifferentiated human THP‐1 monocytes (u‐THP‐1) and phorbol 12‐myristate 13‐acetate (PMA)‐differentiated THP‐1 macrophages (d‐THP‐1), to assess the effects of differentiation and the TLR4 ligand lipopolysaccharide (LPS) on the pathway.Methods/ResultsDifferentiation did not affect the CXCR4, 7 mRNA levels. Interestingly, the CXCL12 and CXCR7 proteins but not CXCR4 were found to be up‐regulated during differentiation. LPS, through CD14‐dependent pathway, induced CXCL12 and CXCR4, 7 mRNA levels to a greater magnitude in d‐ than u‐THP‐1. The induction effect on CXCL12 stimulated by LPS was confirmed using ELISA. Increased migration of u‐THP‐1 was observed using conditioned medium from LPS‐treated d‐THP‐1. Additionally, d‐THP‐1, although expressed higher CXCR7 protein levels, failed to migrate toward CXCL12. In contrast, LPS did not affect CXCR4, 7 protein levels.ConclusionHence, this study indicated that CXCL12, CXCR4, and CXCR7 were differentially expressed and regulated in u‐THP‐1 and d‐THP‐1 cells in response to external stimuli. Importantly, we reported here a novel observation that uncoupling exists between transcriptional and translational regulation of CXCR4, 7 expressions by differentiation and TLR stimuli.