Kinetics of Toll-like receptor-4 splice variants expression in lipopolysaccharide-stimulated antigen presenting cells of healthy donors and patients with cystic fibrosis

Toll-like receptors (TLR) are key components of innate immune system. As TLR activation could induce potentially harmful inflammatory response, activation of TLR signaling pathways has to be under tight control. Besides other control mechanisms, an inhibitory function of murine TLR4 splice variants was recently demonstrated. In this study we investigated expression of four TLR4 splice variants in human antigen presenting cells (APC). Furthermore, we studied modification in TLR4 splice variants expression in APC in cystic fibrosis (CF) patients chronically infected by Gram-negative bacteria. We developed a novel reliable real-time PCR detection system that allowed monitoring of individual TLR4 splice variants expression. In APC from healthy donors we detected a characteristic transient increase of two out of four splice variants after lipopolysaccharide (LPS) stimulation. Similarly to murine TLR4, one of these variants, NM 003266, might translate to a potentially inhibitory protein. In contrast to controls, CF monocytes had significantly changed LPS-induced expression of TLR4 gene and its variants including reduced ability to up-regulate the expression of the potentially inhibitory variant upon stimulation. In accordance with this observation, monocytes from CF patients produced significantly more tumor necrosis factor after LPS stimulation than healthy controls. Our results thus describe the kinetics of TLR4 splicing variants expression after LPS stimulation and indicate a possible alteration of its regulation in CF patients.     

CFTR localization in native airway cells and cell lines expressing wild-type or F508del-CFTR by a panel of different antibodies.

The intracellular localization of cystic fibrosis transmembrane conductance regulator (CFTR) in native tissues is a major issue in the study of mutation, processing, and trafficking effects in CFTR and in the evaluation of therapeutic strategies in cystic fibrosis (CF). This work evaluated the applicability of ten different antibodies (Abs) under various fixation techniques for CFTR localization in fresh-brushed nasal epithelial cells collected from CF patients homozygous for F508del and control individuals. In parallel, the same Ab panel was also tested on BHK cell lines overexpressing wild-type or F508del CFTR. The Abs MATG1061, 169, Lis1, MP-CT1, CC24-R, MAB25031, and MAB1660 gave the best detection of CFTR in the apical region (AR) of nasal tall columnar epithelial (TCE) cells. The labeling pattern of these Abs was consistent with the postulated processing defect of F508del CFTR because only a minority of CF TCE cells present CFTR in the AR. In contrast, M3A7, MM13-4, and L12B4 weakly react with the AR and stain almost exclusively a cis-Golgi-like structure in the majority of CF and non-CF airway cells. In BHK cells, all the Abs enabled distinction between wild-type CFTR localization in cell membrane from F508del CFTR, which in these cells is exclusively located in the endoplasmic reticulum.     

Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.     

Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.     

Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-alpha production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-alpha in response to LPS. LPS-induced activation of both NF-kappaB and c-Jun NH(2)-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.     

Calcium homeostasis is abnormal in cystic fibrosis airway epithelial cells but is normalized after rescue of F508del-CFTR

Retention of F508del-CFTR proteins in the endoplasmic reticulum (ER) is dependent upon chaperone proteins, many of which require Ca(2+) for optimal activity. Here, we show in human tracheal gland CF-KM4 cells, that after correction of F508del-CFTR trafficking by miglustat (N-butyldeoxynojirimycin) or low temperature (27 degrees C), the Ca(2+) mobilization is decreased compared to uncorrected cells and becomes identical to the Ca(2+) response observed in non-CF MM39 cells. In CF-KM4 and human nasal epithelial CF15 cells, we also show that inhibiting vesicular trafficking by nocodazole prevents not only the rescue of F508del-CFTR but also the Ca(2+) mobilization decrease. Finally, experiments using the CFTR inhibitor CFTR(inh)-172 showed that the presence but not the channel activity of F508del-CFTR at the plasma membrane is required to decrease the Ca(2+) mobilization in corrected CF cells. These findings show that correction of the abnormal trafficking of F508del-CFTR proteins might have profound consequences on cellular homeostasis such as the control of intracellular Ca(2+) level.     

Cystic fibrosis transmembrane conductance regulator trafficking is mediated by the COPI coat in epithelial cells

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted beta-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. Beta-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of beta-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the alpha-, beta-, and gamma-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.     

Enteroendocrine cells express functional Toll-like receptors

Intestinal epithelial cells (IECs) provide a physical and immunological barrier against enteric microbial flora. Toll-like receptors (TLRs), through interactions with conserved microbial patterns, activate inflammatory gene expression in cells of the innate immune system. Previous studies of the expression and function of TLRs in IECs have reported varying results. Therefore, TLR expression was characterized in human and murine intestinal sections, and TLR function was tested in an IEC line. TLR1, TLR2, and TLR4 are coexpressed on a subpopulation of human and murine IECs that reside predominantly in the intestinal crypt and belong to the enteroendocrine lineage. An enteroendocrine cell (EEC) line demonstrated a similar expression pattern of TLRs as primary cells. The murine EEC line STC-1 was activated with specific TLR ligands: LPS or synthetic bacterial lipoprotein. In STC-1 cells stimulated with bacterial ligands, NF-kappaB and MAPK activation was demonstrated. Furthermore, the expression of TNF and macrophage inhibitory protein-2 were induced. Additionally, bacterial ligands induced the expression of the anti-inflammatory gene transforming growth factor-beta. LPS triggered a calcium flux in STC-1 cells, resulting in a rapid increase in CCK secretion. Finally, conditioned media from STC-1 cells inhibited the production of nitric oxide and IL-12 p40 by activated macrophages. In conclusion, human and murine IECs that express TLRs belong to the enteroendocrine lineage. Using a murine EEC model, a broad range of functional effects of TLR activation was demonstrated. This study suggests a potential role for EECs in innate immune responses.     

Penta-acylated lipopolisaccharide binds to murine MD-2 but does not induce the oligomerization of TLR4 required for signal transduction

A mutant lipopolysaccharide (LPS) lacking a myristate chain in lipid A was shown to be non-pathogenic both in humans and mice. The mutant penta-acylated LPS from the lpxM-strain did not induce TNF-alpha production in murine peritoneal macrophages, or activation of NF-kappaB in transfected cells expressing murine TLR4/MD-2. We prepared a recombinant murine MD-2 in Escherichia coli (E. coli), and examined the binding function. Unexpectedly, specific binding was detected to both wild type and mutant LPS. However, the mutant LPS did not induce conformation changes or oligomerization of TLR4, which have been shown to be required for signal transduction. Mutant LPS appears to fail to induce appropriate conformational changes, resulting in oligomerization of the murine complex for triggering cell responses.     

Dysregulation of TIM-3-galectin-9 pathway in the cystic fibrosis airways

The T-cell Ig and mucin domain-containing molecules (TIMs) have emerged as promising therapeutic targets to correct abnormal immune function in several autoimmune and chronic inflammatory conditions. It has been reported that proinflammatory cytokine dysregulation and neutrophil-dominated inflammation are the main causes of morbidity in cystic fibrosis (CF). However, the role of TIM receptors in CF has not been investigated. In this study, we demonstrated that TIM-3 is constitutively overexpressed in the human CF airway, suggesting a link between CF transmembrane conductance regulator (CFTR) function and TIM-3 expression. Blockade of CFTR function with the CFTR inhibitor-172 induced an upregulation of TIM-3 and its ligand galectin-9 in normal bronchial epithelial cells. We also established that TIM-3 serves as a functional receptor in bronchial epithelial cells, and physiologically relevant concentrations of galectin-9 induced TIM-3 phosphorylation, resulting in increased IL-8 production. In addition, we have demonstrated that both TIM-3 and galectin-9 undergo rapid proteolytic degradation in the CF lung, primarily because of neutrophil elastase and proteinase-3 activity. Our results suggest a novel intrinsic defect that may contribute to the neutrophil-dominated immune response in the CF airways.     

The IFN-inducible GTPase LRG47 (Irgm1) negatively regulates TLR4-triggered proinflammatory cytokine production and prevents endotoxemia

LRG47/Irgm1, a 47-kDa IFN-inducible GTPase, plays a major role in regulating host resistance as well as the hemopoietic response to intracellular pathogens. LRG47 expression in macrophages has been shown previously to be stimulated in vitro by bacterial LPS, a TLR4 ligand. In this study, we demonstrate that induction of LRG47 by LPS is not dependent on MyD88 signaling, but rather, requires STAT-1 and IFN-beta. In addition, LRG47-deficient mice are highly susceptible to LPS, but not TLR2 ligand-induced shock, an outcome that correlates with enhanced proinflammatory cytokine production in vitro and in vivo. Further analysis revealed that LPS-stimulated LRG47-deficient macrophages display enhanced phosphorylation of p38, a downstream response associated with TLR4/MyD88 rather than IFN-beta/STAT-1 signaling. In contrast, LPS-induced phosphorylation of IFN regulatory factor-3 and expression of IFN-beta or the type I IFN-regulated genes, CCL5 and CCL10, were unaltered in LRG47(-/-) cells. Together, these observations indicate that in LPS-stimulated murine macrophages LRG47 is induced by IFN-beta and negatively regulates TLR4 signaling to prevent excess proinflammatory cytokine production and shock. Thus, our findings reveal a new host-protective function for this GTPase in the response to pathogenic encounter.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

Bruton's tyrosine kinase is involved in p65-mediated transactivation and phosphorylation of p65 on serine 536 during NFkappaB activation by lipopolysaccharide

Bruton's tyrosine kinase (Btk) has recently been shown to participate in the induction of nuclear factor kappaB (NFkappaB)-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). In this study we have examined the mechanism whereby Btk participates in this response. Treatment of the murine monocytic cell line Raw264.7 with LFM-A13, a specific Btk inhibitor, blocked LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk had no effect on NFkappaB-dependent reporter gene expression but strongly promoted transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS was intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibited LPS-driven p65 mediated transactivation. Additionally LFM-A13 impaired phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response was impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4, which culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB.     

Unique properties of the chicken TLR4/MD-2 complex: selective lipopolysaccharide activation of the MyD88-dependent pathway

During evolution, mammals have evolved a powerful innate immune response to LPS. Chickens are much more resistant to LPS-induced septic shock. Herein we report that chickens sense LPS via orthologs of mammalian TLR4 and myeloid differentiation protein-2 (MD-2) rather than the previously implicated chicken TLR2 isoform type 2 (chTLR2t2) receptor. Cloning and expression of recombinant chTLR4 and chMD-2 in HeLa 57A cells activated NF-kappaB at concentrations of LPS as low as 100 pg/ml. Differential pairing of chicken and mammalian TLR4 and MD-2 indicated that the protein interaction was species-specific in contrast to the formation of functional human and murine chimeric complexes. The chicken LPS receptor responded to a wide variety of LPS derivatives and to the synthetic lipid A compounds 406 and 506. The LPS specificity resembled the functionality of the murine rather than the human TLR4/MD-2 complex. Polymorphism in chTLR4 (Tyr(383)His and Gln(611)Arg) did not influence the LPS response. Interestingly, LPS consistently failed to activate the MyD88-independent induction of IFN-beta in chicken cells, in contrast to the TLR3 agonist poly(I:C) that yielded a potent IFN-beta response. These results suggest that chicken lack a functional LPS-specific TRAM-TRIF (TRIF-related adapter molecule/TIR-domain-containing adapter-inducing IFN-beta) signaling pathway, which may explain their aberrant response to LPS compared with the mammalian species.     

CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-alpha signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-alpha to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-alpha signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-DeltaTRL), TNF-alpha increased the amount of WtCFTR but not CFTR-DeltaTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-DeltaTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.     

Localization of cystic fibrosis transmembrane conductance regulator to lipid rafts of epithelial cells is required for Pseudomonas aeruginosa-induced cellular activation

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein is an epithelial cell receptor for the outer core oligosaccharide of the Pseudomonas aeruginosa LPS. Bacterial binding leads to CFTR-dependent bacterial internalization, initiation of NF-kappaB nuclear translocation, cellular desquamation, and eventual apoptosis of the infected cells, all of which are critical for innate immune resistance to infection with this pathogen. Lack of this reaction in CF patients underlies their hypersusceptibility to chronic P. aeruginosa infection. In this study we tested whether these epithelial cell responses are dependent upon the localization of CFTR to lipid rafts. Confocal microscopy showed that green fluorescent protein-tagged CFTR (GFP-CFTR) and the lipid raft marker ganglioside GM1 colocalized at sites of P. aeruginosa contact and internalization. GFP-CFTR localized to low density Triton X-100-insoluble fractions in lysates of Madin-Darby canine kidney GFP-CFTR cells, and P. aeruginosa infection increased the levels of GFP-CFTR in these fractions as determined by Western blot. Cells expressing GFP-DeltaF508-CFTR did not have rafts with detectable CFTR protein. Extraction of cell surface cholesterol via cyclodextrin treatment of the cells inhibited CFTR entry into rafts. In addition, cyclodextrin treatment of both human and canine epithelial cells inhibited cellular ingestion of P. aeruginosa, NF-kappaB nuclear translocation, and apoptosis. These results indicate that lipid raft localization of CFTR is required for signaling in response to P. aeruginosa infection. Such signaling is needed for the coordination of innate immunity to P. aeruginosa lung infection, a process that is defective in CF.     

Inhibition of high-mobility group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas Aeruginosa pneumonia in cystic fibrosis

Pulmonary infection with Pseudomonas aeruginosa and neutrophilic lung inflammation significantly contribute to morbidity and mortality in cystic fibrosis (CF). High-mobility group box 1 protein (HMGB1), a ubiquitous DNA binding protein that promotes inflammatory tissue injury, is significantly elevated in CF sputum. However, its mechanistic and potential therapeutic implications in CF were previously unknown. We found that HMGB1 levels were significantly elevated in bronchoalveolar lavage fluids (BALs) of CF patients and cystic fibrosis transmembrane conductance regulator (CFTR )(-/-) mice. Neutralizing anti-HMGB1 monoclonal antibody (mAb) conferred significant protection against P. aeruginosa-induced neutrophil recruitment, lung injury and bacterial infection in both CFTR(-/-) and wild-type mice. Alveolar macrophages isolated from mice treated with anti-HMGB1 mAb had improved phagocytic activity, which was suppressed by direct exposure to HMGB1. In addition, BAL from CF patients significantly impaired macrophage phagocytotic function, and this impairment was attenuated by HMGB1-neutralizing antibodies. The HMGB1-mediated suppression of bacterial phagocytosis was attenuated in macrophages lacking toll-like receptor (TLR)-4, suggesting a critical role for TLR4 in signaling HMGB1-mediated macrophage dysfunction. These studies demonstrate that the elevated levels of HMGB1 in CF airways are critical for neutrophil recruitment and persistent presence of P. aeruginosa in the lung. Thus, HMGB1 may provide a therapeutic target for reducing bacterial infection and lung inflammation in CF.     

Folimycin (concanamycin A) inhibits LPS-induced nitric oxide production and reduces surface localization of TLR4 in murine macrophages

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and signals through a receptor complex which consists of TLR4, MD-2 and CD14. LPS signaling in macrophages induces the production of many pro-inflammatory molecules, including nitric oxide (NO). In this study, we have shown that folimycin, a macrolide antibiotic and a specific inhibitor of vacuolar ATPase (V-ATPase), inhibits LPS-induced NO production, but not TNFalpha production, in murine elicited peritoneal macrophages. However, folimycin did not affect interferon-gamma induced NO production. LPS-induced iNOS mRNA and protein expression and NF-kappaB activation were also inhibited by folimycin. Interestingly, folimycin-treated cells showed reduced surface expression of TLR4 molecules and dilated Golgi apparatus. These findings suggest that folimycin, by inhibiting V-ATPases, alters intra-Golgi pH, which in turn causes defective processing and reduced surface expression of TLR4 reducing the strength of LPS signaling in murine macrophages.     

Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1alpha (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.