CD14+ monocytes repress gamma globin expression at early stages of erythropoiesis.

In ß-hemoglobinopathies, reactivation of gamma- at the expense of beta-globin is a prominent therapeutic option. Expression of the globin genes is not strictly intrinsically regulated during erythropoiesis, supported by the observation that fetal erythroid cells switch to adult hemoglobin expression when injected in mice. We show cultured erythroblasts are a mix of HbA restrictive and HbA/HbF expressing cells and that the proportion of cells in the latter population depends on the starting material. Cultures started from CD34+ cells contain more HbA/HbF expressing cells compared to erythroblasts cultured from total peripheral blood mononuclear cells (PBMC). Depletion of CD14+ cells from PBMC resulted in higher HbF/HbA percentages. Conversely, CD34+ co-culture with CD14+ cells reduced the HbF/HbA population through cell-cell proximity, indicating that CD14+ actively repressed HbF expression in adult erythroid cultures. RNA-sequencing showed that HbA and HbA/HbF populations contain a limited number of differentially expressed genes, aside from HBG1/2. Co-culture of CD14+ cells with sorted uncommitted hematopoietic progenitors and CD34-CD36+ erythroblasts showed that hematopoietic progenitors prior to the hemoglobinized erythroid stages are more readily influenced by CD14+ cells to downregulate expression of HBG1/2, suggesting temporal regulation of these genes. This possibly provides a novel therapeutic avenue to develop ß-hemoglobinopathies treatments.

Sexual Dimorphic Role of CD14 (Cluster of Differentiation 14) in Salt-Sensitive Hypertension and Renal Injury.

Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)-a cell surface protein involved in innate immune system activation-is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), we created a targeted mutation in the CD14 gene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14-/- animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14-/- females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14+/+ or CD14-/- bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.

The prognostic value of Presepsin for postoperative complications following pancreatic resection: A prospective study.

BACKGROUND: Presepsin is involved in binding lipopolysaccharides and previous studies have confirmed its value as a marker for early diagnosis and prediction of severity in sepsis. Comparable studies assessing the predictive potential regarding postoperative complications and mortality following pancreatic resection are lacking. METHODS: This prospective study included 70 patients undergoing pancreatic resection from December 2017 until May 2019. Presepsin was measured preoperatively, on postoperative day 1, 3 and 8 (POD1/3/8) and correlated with the clinical course and mortality. RESULTS: Severe complications (Clavien-Dindo ≥3a) occurred in 28 patients (40%), postoperative pancreatic fistula (POPF) grade B/C occurred in 20 patients (28.6%), infectious complications in 28 (40%), and four patients (5.7%) died during hospital stay. Presepsin levels at any timepoint did not correlate with further development of postoperative complications or in-hospital mortality whereas CRP levels on postoperative day (POD) 3 were significantly associated with clinically relevant POPF (AUC 0.664, 95%CI 0.528-0.800; p = 0.033). Preoperative Presepsin levels as well as Presepsin on POD1 were significantly elevated in patients with malignant compared to benign underlying disease (299pg/ml vs. 174pg/ml and 693.5pg/ml vs. 294pg/ml; p = 0.009 and 0.013, respectively). CONCLUSION: In our cohort, Presepsin was not eligible to predict the postoperative course following pancreatic resection. However, Presepsin levels were significantly elevated in patients with malignant disease, this finding warrants further investigation.

Pharmacological inhibitory profile of TAK-828F, a potent and selective orally available RORγt inverse agonist.

Retinoic acid-related orphan receptor γt (RORγt) is a key master regulator of the differentiation and activation of IL-17 producing CD4+ Th17, CD8+ Tc17 and IL-17/IFN-γ co-producing cells (Th1/17 cells). These cells play critical roles in the pathogenesis of autoimmune diseases such as inflammatory bowel disease and multiple sclerosis. Thus, RORγt is an attractive target for the treatment of these diseases. We discovered TAK-828F, an orally available potent and selective RORγt inverse agonist. The inhibitory effect on the activation and differentiation of Th17 cells by TAK-828F was evaluated in mouse and human primary cells. TAK-828F inhibited IL-17 production from mouse splenocytes and human peripheral blood mononuclear cells dose-dependently at concentrations of 0.01-10 µM without affecting the production of IFN-γ. Additionally, TAK-828F strongly inhibited Th17, Tc17 and Th1/17 cells' differentiation from naive T cells and memory CD4+ T cells at 100 nM without affecting Th1 cells' differentiation. In addition, TAK-828F improved Th17/Treg cells' population ratio by inhibiting Th17 cells' differentiation and up-regulating Treg cells. Furthermore, TAK-828F, at 100 nM, reduced the production of Th17-related cytokines (IL-17, IL-17F and IL-22) without affecting IFN-γ production in whole blood. These results demonstrate that TAK-828F has the potent and selective inhibitory activity against RORγt both in mouse and human cells. Additionally, oral administration of TAK-828F showed promising efficacy in naive T cell transfer mouse colitis model. TAK-828F may provide a novel therapeutic option to treat immune diseases by inhibiting Th17 and Th1/17 cells' differentiation and improving imbalance between Th17 and Treg cells.

CD14 Plays a Protective Role in Experimental Inflammatory Bowel Disease by Enhancing Intestinal Barrier Function.

Intestinal homeostasis disturbance through intestinal barrier disruption presumably plays a key role in inflammatory bowel disease (IBD) development. Genetic and candidate gene analyses in an Il10-deficient IBD mouse model system identified Cd14 as a potentially protective candidate gene. The role of Cd14 in colitis development was determined using dextran sulfate sodium (DSS)-induced acute and an Il10-deficiency-induced chronic model of intestinal inflammation. Intestinal permeability was investigated by fluorescein isothiocyanate-dextran uptake assay, quantitative RT-PCR analysis of tight junction proteins, myosin light chain kinase, and proinflammatory cytokine expression. Immunohistological staining of occludin, Ki-67, NF-κB-p65, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was performed, and intestinal inflammation severity was evaluated histologically. Untreated B6-Cd14-/- mice and wild-type controls did not differ in intestinal barrier function. However, DSS-treated Cd14-deficient and B6-Il10-/-Cd14-/- mice exhibited more severe intestinal barrier disruption, with increased histological scores and proinflammatory cytokine expression, compared to controls. Therefore, Cd14 deficiency did not influence epithelial integrity under steady-state conditions but caused intestinal barrier dysfunction under inflammation. As expected, CD14 overexpression increased barrier integrity. No difference in intestinal epithelial NF-κB translocation was observed between the investigated groups. Intestinal myosin light chain kinase expression decreased in Cd14-deficient mice under steady-state conditions and in the chronic model, whereas no difference was detected in the DSS models. Thus, CD14 plays a protective role in IBD development by enhancing intestinal barrier function.

Kinetics of plasma procalcitonin, soluble CD14, CCL2 and IL-10 after a sublethal infusion of lipopolysaccharide in horses.

Endotoxemia represents a significant clinical and economic problem for the equine industry. This study assesses the kinetics of soluble CD14 (sCD14), chemokine (CC motif) ligand 2 (CCL2), interleukin 10 (IL-10) and plasma procalcitonin (PCT) in healthy horses after the intravenous infusion of lipopolysaccharide (LPS). The aim was to contribute to the basic understanding of the equine species-specific kinetics of these molecules in response to LPS exposure, which could support further findings in clinical studies and identify valuable inflammatory biomarkers for equine practice. Eleven healthy horses were involved in this experimental in vivo study. Horses were classified as healthy before the LPS infusion. After the pre-infusion blood collection (T0), all horses received an infusion of E. coli endotoxin (30ng/kg over 30min). Data and samples were collected 1h (T1), 2 (T2), 3 (T3) and 24h (T24) after infusion. Plasma sCD14, CCL2 and IL-10 were evaluated with a fluorescent bead-based assay, while PCT was evaluated with an equine PCT ELISA assay. A one-way ANOVA test was performed between each blood-sampling time for PCT, sCD14 and IL-10, and a Friedman test was performed for CCL2. Plasma PCT, IL-10 and CCL2 concentrations increased statistically significantly at T1, T2 and T3 compared to T0. No statistically significant differences were found between plasma IL-10 and CCL2 concentrations between T0 vs T24, although plasma PCT values remained high 24h after LPS infusion. Plasma sCD14 concentration showed no statistically significant differences for any of sampling times. Our results demonstrate that LPS injection into healthy horses results in PCT, CCL2 and IL-10 increases in plasma without an increase in sCD14. The increases in PCT, CCL2 and IL-10 are related to the inflammatory response induced by circulating lipopolysaccharide.

Dynamic lipopolysaccharide transfer cascade to TLR4/MD2 complex via LBP and CD14.

Toll-like receptor 4 (TLR4) together with MD2, one of the key pattern recognition receptors for a pathogen-associated molecular pattern, activates innate immunity by recognizing lipopolysaccharide (LPS) of Gram-negative bacteria. Although LBP and CD14 catalyze LPS transfer to the TLR4/MD2 complex, the detail mechanisms underlying this dynamic LPS transfer remain elusive. Using negative-stain electron microscopy, we visualized the dynamic intermediate complexes during LPS transfer-LBP/LPS micelles and ternary CD14/LBP/LPS micelle complexes. We also reconstituted the entire cascade of LPS transfer to TLR4/MD2 in a total internal reflection fluorescence (TIRF) microscope for a single molecule fluorescence analysis. These analyses reveal longitudinal LBP binding to the surface of LPS micelles and multi-round binding/unbinding of CD14 to single LBP/LPS micelles via key charged residues on LBP and CD14. Finally, we reveal that a single LPS molecule bound to CD14 is transferred to TLR4/MD2 in a TLR4-dependent manner. These discoveries, which clarify the molecular mechanism of dynamic LPS transfer to TLR4/MD2 via LBP and CD14, provide novel insights into the initiation of innate immune responses. [BMB Reports 2017; 50(2): 55-57].

Contribution of CD14 and TLR4 to changes of the PI(4,5)P2 level in LPS-stimulated cells.

LPS binds sequentially to CD14 and TLR4/MD2 receptor triggering production of proinflammatory mediators. The LPS-induced signaling is controlled by a plasma membrane lipid PI(4,5)P2 and its derivatives. Here, we show that stimulation of murine peritoneal macrophages with LPS induces biphasic accumulation of PI(4,5)P2 with peaks at 10 and 60-90 min that were still seen after silencing of TLR4 expression. In contrast, the PI(4,5)P2 elevation was abrogated when CD14 was removed from the cell surface. To assess the contribution of CD14 and TLR4 to the LPS-induced PI(4,5)P2 changes, we used HEK293 transfectants expressing various amounts of CD14 and TLR4. In cells with a low content of CD14 and high of TLR4, no accumulation of PI(4,5)P2 occurred. With an increasing amount of CD14 and concomitant decrease of TLR4, 2 peaks of PI(4,5)P2 accumulation appeared, eventually approaching those found in LPS-stimulated cells expressing CD14 alone. Mutation of the signaling domain of TLR4 let us conclude that the receptor activity can modulate PI(4,5)P2 accumulation in cells when expressed in high amounts compared with CD14. Among the factors limiting PI(4,5)P2 accumulation are its hydrolysis, phosphorylation, and availability of its precursor, PI(4)P. Inhibition of PLC and PI3K or overexpression of PI4K IIα that produces PI(4)P promoted PI(4,5)P2 elevation in LPS-stimulated cells. The elevation of PI(4,5)P2 was dispensable for TLR4 signaling yet enhanced its magnitude. Taken together, these data suggest that LPS-induced accumulation of PI(4,5)P2 that maximizes TLR4 signaling is controlled by CD14, whereas TLR4 can fine tune the process by affecting the PI(4,5)P2 turnover.

Critical residues involved in Toll-like receptor 4 activation by cationic lipid nanocarriers are not located at the lipopolysaccharide-binding interface.

DiC14-amidine is a cationic lipid that was originally designed as a lipid nanocarrier for nucleic acid transport, and turned out to be a Toll-like receptor 4 (TLR4) agonist as well. We found that while E. coli lipopolysaccharide (LPS) is a TLR4 agonist in all species, diC14-amidine nanoliposomes are full agonists for human, mouse and cat receptors but weak horse agonists. Taking advantage of this unusual species specificity, we used chimeric constructs based on the human and horse sequences and identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine. Interestingly, these regions lie outside the known LPS-binding domain. Competition experiments also support our hypothesis that diC14-amidine interacts primarily with TLR4 hydrophobic crevices located at the edges of the TLR4/TLR4* dimerization interface. We have characterized potential binding modes using molecular docking analysis and suggest that diC14-amidine nanoliposomes activate TLR4 by facilitating its dimerization in a process that is myeloid differentiation 2 (MD-2)-dependent and cluster of differentiation 14 (CD14)-independent. Our data suggest that TLR4 may be activated through binding at different anchoring points, expanding the repertoire of TLR4 ligands to non-MD-2-binding lipids.

Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response.

Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1ß (IL1ß) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1ß. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.

Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

Human skin dendritic cells in health and disease.

Dendritic cells (DCs) are specialized antigen presenting cells abundant in peripheral tissues such as skin where they function as immune sentinels. Skin DCs migrate to draining lymph node where they interact with naïve T cells to induce immune responses to microorganisms, vaccines, tumours and self-antigens. In this review, we present the key historical developments and recent advances in human skin DC research. We also integrate the current understanding on the origin and functional specializations of DC subsets in healthy skin with findings in inflammatory skin diseases focusing on psoriasis and atopic eczema. A comprehensive understanding of the dynamic changes in DC subsets in health and disease will form a strong foundation to facilitate the clinical translation of DC-based therapeutic and vaccination strategies.

Normal mesenteric lymph ameliorates acute kidney injury following lipopolysaccharide challenge in mice.

BACKGROUND: The kidney is one of the prior damaged organs subjected to severe infection and sepsis shock. Our previous studies have shown that the normal mesenteric lymph (NML) obtained from healthy dogs could alleviate multiple organ injuries following endotoxic shock. In the current study, we further investigated the beneficial effect of NML from healthy mice on acute kidney injury (AKI) induced by lipopolysaccharide (LPS) in mice. METHODS: The mice in LPS and LPS + NML groups received an intraperitoneal injection of LPS (35 mg/kg). One hour later, the treatment of NML was performed and kept for 6 h. Then, the renal function indices, renal morphology, the levels of phosphorylation mitogen-activated protein kinases (MAPKs), markers of sensitization to LPS, as well as pro-inflammatory mediators in renal tissue were observed. RESULTS: Intraperitoneal injection of LPS induced an increased level of urea in plasma, lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), but no obvious changes in the MAPKs in renal tissue. NML treatment decreased the levels of urea, CD14, TNF-α and IL-6 in mice after LPS injection. CONCLUSION: The current results indicate that NML alleviates LPS-induced AKI through its attenuation of sensitization to LPS.

Novel synthetic toll-like receptor 4/MD2 ligands attenuate sterile inflammation.

Toll-like receptor (TLR) stimulation has been implicated as a major contributor to chronic inflammation. Among these receptors, TLR4 has been described as a key regulator of endogenous inflammation and has been proposed as a therapeutic target. Previously, we discovered by high-throughput screening a group of substituted pyrimido[5,4-b]indoles that activated a nuclear factor-κB reporter in THP-1 human monocytic cells. A biologically active hit compound was resynthesized, and derivatives were prepared to assess structure-activity relationships. The derived compounds activated cells in a TLR4/myeloid differentiation protein 2 (MD2)-dependent and CD14-independent manner, using the myeloid differentiation primary response 88 and Toll/IL-1 receptor domain-containing adapter-inducing interferon-ß pathways. Two lead compounds, 1Z105 and 1Z88, were selected for further analysis based on favorable biologic properties and lack of toxicity. In vivo pharmacokinetics indicated that 1Z105 was orally bioavailable, whereas 1Z88 was not. Oral or parenteral doses of 1Z105 and 1Z88 induced undetectable or negligible levels of circulating cytokines and did not induce hepatotoxicity when administered to galactosamine-conditioned mice, indicating good safety profiles. Both compounds were very effective in preventing lethal liver damage in lipopolysaccharide treated galatosamine-conditioned mice. Orally administered 1Z105 and parenteral 1Z88 prevented arthritis in an autoantibody-driven murine model. Hence, these low molecular weight molecules that target TLR4/MD2 were well tolerated and effective in reducing target organ damage in two different mouse models of sterile inflammation.

LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14.

Treatment of cultures with toll-like receptor (TLR) ligands or cytokines has become a popular approach to investigate astrocyte neuroinflammatory responses and to simulate the neural environment in various CNS disorders. However, despite much effort, the mechanism of astrocyte activation such as their responses to the TLR ligands and IL-1 remain highly debated. We compared highly pure primary mouse and human astrocyte cultures in their ability to produce proinflammatory mediators (termed "A1") and immunoregulatory mediators (termed "A2") in response to LPS, poly IC, and IL-1 stimulation. In human astrocytes, IL-1 induced both A1 and A2 responses, poly IC induced mostly A2, and LPS induced neither. In mouse astrocytes, LPS induced mostly an A1-predominant response, poly IC induced both A1 and A2, and IL-1 neither. In addition, mouse astrocytes produce abundant IL-1 protein, whereas human astrocytes did not, despite robust IL-1 mRNA expression. Of the TLR4 receptor complex proteins, human astrocytes expressed TLR4 and MD2 but not CD14, whereas mouse astrocytes expressed all three. Mouse astrocyte CD14 (cell-associated and soluble) was potently upregulated by LPS. Silencing TLR4 or CD14 by siRNA suppressed LPS responses in mouse astrocytes. In vivo, astrocytes in LPS-injected mouse brains also expressed CD14. Our results show striking differences between human and mouse astrocytes in the use of TLR/IL-1R and subsequent downstream signaling and immune activation. IL-1 translational block in human astrocytes may be a built-in mechanism to prevent autocrine and paracrine cell activation and neuroinflammation. These results have important implications for translational research of human CNS diseases.

TLR2 & Co: a critical analysis of the complex interactions between TLR2 and coreceptors.

TLRs play a major role in microbe-host interactions and innate immunity. Of the 10 functional TLRs described in humans, TLR2 is unique in its requirement to form heterodimers with TLR1 or TLR6 for the initiation of signaling and cellular activation. The ligand specificity of TLR2 heterodimers has been studied extensively, using specific bacterial and synthetic lipoproteins to gain insight into the structure-function relationship, the minimal active motifs, and the critical dependence on TLR1 or TLR6 for activation. Different from that for specific well-defined TLR2 agonists, recognition of more complex ligands like intact microbes or molecules from endogenous origin requires TLR2 to interact with additional coreceptors. A breadth of data has been published on ligand-induced interactions of TLR2 with additional pattern recognition receptors such as CD14, scavenger receptors, integrins, and a range of other receptors, all of them important factors in TLR2 function. This review summarizes the roles of TLR2 in vivo and in specific immune cell types and integrates this information with a detailed review of our current understanding of the roles of specific coreceptors and ligands in regulating TLR2 functions. Understanding how these processes affect intracellular signaling and drive functional immune responses will lead to a better understanding of host-microbe interactions and will aid in the design of new agents to target TLR2 function in health and disease.

Selective expansion of pro-inflammatory chemokine CCL2-loaded CD14+CD16+ monocytes subset in HIV-infected therapy naïve individuals.

We have previously demonstrated the critical role of C-C chemokine CCL2 in HIV-1 pathogenesis, and circulating monocytes as the major source of CCL2. Since the functional aspect of monocyte subsets in context to CCL2 production is unclear, we investigated the frequency and production of CCL2 by circulating monocyte subsets in a cohort of HIV- therapy naïve patients. A cohort of HIV-infected therapy naïve patients (n=9) and healthy controls (n=6) were recruited for this study. To examine monocyte subset frequency and CCL2 production, we performed surface and intra-cellular staining of freshly isolated peripheral blood mononuclear cells (PBMC) and subjected to flow cytometry. A preferential expansion of CD14(+)CD16(+) monocyte subset, coupled with increased intracellular production of CCL2 was observed in HIV-1 patients compared to healthy controls. Interestingly this phenotype was mostly restricted to CD14(+)CD16(+) monocyte subsets. This study identifies pro-inflammatory CCL2 producing CD14(+)CD16(+) monocyte subset that expands selectively in HIV-1 infection and could potentially participate in causing immuno-pathology.

Mucosal CXCR4+ IgG plasma cells contribute to the pathogenesis of human ulcerative colitis through FcγR-mediated CD14 macrophage activation.

BACKGROUND: Chronic inflammation characterised by IgG-producing plasma cell infiltration of colonic mucosa is a histological hallmark of ulcerative colitis (UC); however, whether its function is pathogenic or protective remains unclear. OBJECTIVE: To explore the contribution of intestinal IgG plasma cells to UC pathogenesis. METHODS: We isolated lamina propria mononuclear cells (LPMCs) from intestinal mucosa of UC patients and analysed the characteristics of intestinal plasma cells (expression profiles of differentiation molecules and chemokine receptors). We investigated the involvement of IgG-immune complex (IC)-Fc gamma receptor (FcγR) signalling in intestinal inflammation by examining the cytokine production by LPMCs in response to IgG-IC stimulation. RESULTS: IgG plasma cells that were markedly increased in number in the inflamed mucosa of UC patients showed a distinct expression profile (CD19(+)CD27(low), CCR10(low)CXCR4(high)) compared with IgA plasma cells (CD19(+/-)CD27(high), CCR10(high)CXCR4(-/low)). In vitro IgG-IC stimulation activated intestinal CD14 macrophages that were increased in number in the inflamed mucosa of UC patients via FcγRI and FcγRII, and induced the extensive production of pro-inflammatory cytokines such as tumour necrosis factor (TNF) and interleukin-1ß (IL-1ß), comparable to the effect of commensal bacteria stimulation. Co-stimulation with IgG-IC and commensal bacteria increased TNF and IL-1ß production more than stimulation with the latter alone. Furthermore, IgG-IC notably up-regulated the expression of TL1A, whereas commensal bacteria specifically induced IL-23. CONCLUSIONS: Collectively, these results demonstrate a novel aspect of UC pathogenesis in which unique IgG plasma cells infiltrate the inflamed mucosa via CXCR4, and critically influence UC pathogenesis by exacerbating mucosal inflammation through the activation of 'pathogenic' intestinal CD14 macrophages via IgG-IC-FcγR signalling.

Differential expression of CD14-dependent and independent pathways for chemokine induction regulates neutrophil trafficking in infection.

Previous studies have shown that CD14(-/-) mice are resistant to peritoneal infection with some clinical isolates of Escherichia coli and that this resistance is accompanied by an enhanced ability to clear the bacteria; in contrast, normal mice expressing CD14 fail to clear the bacteria, causing severe sepsis and death. The enhanced clearance in CD14(-/-) mice is dependent on early neutrophil recruitment to the local foci of infection in the PC. The studies described show that neutrophil recruitment in CD14(-/-) mice occurs as a result of the local induction of the CXCL1 and CXCL2 chemokines, KC and MIP-2. Although local induction of these chemokines also occurs in normal mice, their effects on neutrophil recruitment to the PC appear to be counterbalanced by very high levels of these chemokines in the blood of normal, but not CD14(-/-), mice. Neutrophil recruitment to the PC is also inhibited in normal mice in response to LPS, which also induces high chemokine levels in the blood of normal, but not CD14(-/-), mice. However, MPLA, a monophosphorylated derivative of LPS, is able to induce early neutrophil recruitment in normal mice; this is because MPLA, unlike LPS or E. coli, induces MIP-2 and KC in the PC but not in the blood of normal mice. The pretreatment of normal mice with MPLA is able to protect them from a lethal E. coli infection. Thus, stimulation of a local CD14-independent chemokine induction pathway without triggering a systemic CD14-dependent chemokine pathway can protect against severe E. coli infections.