A high number of PD-L1+ CD14+ monocytes in peripheral blood is correlated with shorter survival in patients receiving immune checkpoint inhibitors.

PURPOSE: Targeting of anti-programmed cell death protein-1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) is a standard therapeutic strategy for various cancers. The aim of the present study was to investigate the prognostic effect of pretreatment PD-L1 expression levels in peripheral blood mononuclear cell (PBMC) subsets for patients with several cancer types receiving anti-PD-1 blockade therapies. PATIENTS AND METHODS: Thirty-two patients undergoing anti-PD-L1 blockade therapy, including 15 with non-small cell lung cancer, 14 with gastric cancer, 1 with melanoma, 1 with parotid cancer, and 1 with bladder cancer, were recruited for the present study. PD-L1 expression levels in CD3+, CD4+, CD8+, CD45RA+ and CCR7+ T cells; CD20+ B cells; CD14+ and CD16+ monocytes were measured via flow cytometry before treatment. The percentages of PD-L1+ cells in respective PBMC subsets were compared with respect to different clinicopathological conditions and the association with overall survival (OS) was assessed. RESULTS: The percentages of PD-L1+ with CD3+, CD4+ and CD8+ T cells including naïve and memory T cell subsets, or CD20+ B cells during pretreatment were not markedly correlated with the OS of patients (p > 0.05); however, the percentage of the PD-L1+ CD14+ monocyte subset was significantly correlated with OS (p = 0.0426). CONCLUSION: Increase in pretreatment expression levels of PD-L1 on CD14+ monocytes is associated with the OS of patients treated with immune checkpoint inhibitors. Further evaluation of large sample size and each specific cancer type might clarify the predictive role of PBMC in patients.

Involvement of Pattern Recognition Receptors in the Direct Influence of Bacterial Components and Standard Antiacne Compounds on Human Sebaceous Gland Cells.

INTRODUCTION: Pattern recognition receptors are involved in innate and adaptive immunity by detecting microbial components. Bacteria have been accused to play a role in inflammatory acne. We investigated the potential involvement of Toll-like receptor (TLR)2, TLR4, TLR6, and CD14 in the direct influence of bacterial components and standard antiacne compounds on human sebocytes. METHODS: mRNA and protein expression of TLR2, TLR4, TLR6, and CD14 in SZ95 sebocytes was evaluated by real-time qRT-PCR and immunocytochemistry. The effects of lipopolysaccharides (LPS) and lipoteichoic acid on TLR2, TLR4, and CD14 expression and of cytokine/chemokine secretion by 13-cis-retinoic acid, all-trans-retinoic acid, retinol, and hydrocortisone at the mRNA and protein levels were assessed by real-time qRT-PCR and ELISA and verified by cocultivation with neutralizing antibodies. RESULTS: The constitutive expression of TLR2, TLR4, and CD14 in SZ95 sebocytes was augmented by exposure to LPS. Hydrocortisone induced TLR2, but markedly reduced TLR4 expression. 13-cis-retinoic acid and all-trans-retinoic acid regulated IL-6 release. LPS enhanced and hydrocortisone reduced cytokine and chemokine release. Anti-TLR4 and anti-CD14 mAb blocked LPS-induced IL-8 and IL-6 release. CONCLUSIONS: Microbial components use pattern recognition receptors to directly activate sebocytes to express a wide range of proinflammatory molecules and especially IL-8 and IL-6 in a TLR4- and CD14-specific manner. Retinoids, but mostly corticosteroids, also use this pathway to exhibit anti-inflammatory effects.

Chronic ethanol consumption: role of TLR3/TRIF-dependent signaling.

Chronic ethanol consumption stimulates neuroimmune signaling in the brain, and Toll-like receptor (TLR) activation plays a key role in ethanol-induced inflammation. However, it is unknown which of the TLR signaling pathways, the myeloid differentiation primary response gene 88 (MyD88) dependent or the TIR-domain-containing adapter-inducing interferon-ß (TRIF) dependent, is activated in response to chronic ethanol. We used voluntary (every-other-day) chronic ethanol consumption in adult C57BL/6J mice and measured expression of TLRs and their signaling molecules immediately following consumption and 24 hours after removing alcohol. We focused on the prefrontal cortex where neuroimmune changes are the most robust and also investigated the nucleus accumbens and amygdala. Tlr mRNA and components of the TRIF-dependent pathway (mRNA and protein) were increased in the prefrontal cortex 24 hours after ethanol and Cxcl10 expression increased 0 hour after ethanol. Expression of Tlr3 and TRIF-related components increased in the nucleus accumbens, but slightly decreased in the amygdala. In addition, we demonstrate that the IKKε/TBK1 inhibitor Amlexanox decreases immune activation of TRIF-dependent pathway in the brain and reduces ethanol consumption, suggesting the TRIF-dependent pathway regulates drinking. Our results support the importance of TLR3 and the TRIF-dependent pathway in ethanol-induced neuroimmune signaling and suggest that this pathway could be a target in the treatment of alcohol use disorders.

Epinecidin-1 protects mice from LPS-induced endotoxemia and cecal ligation and puncture-induced polymicrobial sepsis.

The antimicrobial peptide, epinecidin-1 (Epi), was identified from Epinephelus coioides and may have clinical application for treating sepsis. Epi has been shown to ameliorate antibiotic-resistant bacteria-induced sepsis in mice, but further evaluation in mixed-flora models and a description of the protective mechanisms are essential to establish this peptide as a potential therapeutic. Therefore, we first tested the protective effects of Epi against polymicrobial sepsis-induced bactericidal infection, inflammation and lung injury that result from cecal ligation and puncture in mice. Furthermore, since lipopolysaccharide (LPS) is a key inducer of inflammation during bacterial infection and sepsis, we also tested the LPS-antagonizing activity and related mechanisms of Epi-mediated protection in mice with LPS-induced endotoxemia and LPS-treated Raw264.7 mouse macrophage cells. Epi rescued mice from both polymicrobial sepsis and endotoxemia after delayed administration and suppressed both lung and systemic inflammatory responses, while attenuating lung injury and diminishing bacterial load. In vitro studies revealed that Epi suppressed LPS-induced inflammatory cytokine production. Mechanistically, Epi disrupted the interaction between LPS and LPS binding protein, competed with LPS for binding on the cell surface, and inhibited Toll-like receptor 4 endocytosis, resulting in inhibition of LPS-induced reactive oxygen species/p38/Akt/NF-κB signaling and subsequent cytokine production. Overall, our results demonstrate that Epi is a promising therapeutic agent for endotoxemia and polymicrobial sepsis.

Combined Inhibition of Complement and CD14 Attenuates Bacteria-Induced Inflammation in Human Whole Blood More Efficiently Than Antagonizing the Toll-like Receptor 4-MD2 Complex.

BACKGROUND: Single inhibition of the Toll-like receptor 4 (TLR4)-MD2 complex failed in treatment of sepsis. CD14 is a coreceptor for several TLRs, including TLR4 and TLR2. The aim of this study was to investigate the effect of single TLR4-MD2 inhibition by using eritoran, compared with the effect of CD14 inhibition alone and combined with the C3 complement inhibitor compstatin (Cp40), on the bacteria-induced inflammatory response in human whole blood. METHODS: Cytokines were measured by multiplex technology, and leukocyte activation markers CD11b and CD35 were measured by flow cytometry. RESULTS: Lipopolysaccharide (LPS)-induced inflammatory markers were efficiently abolished by both anti-CD14 and eritoran. Anti-CD14 was significantly more effective than eritoran in inhibiting LPS-binding to HEK-293E cells transfected with CD14 and Escherichia coli-induced upregulation of monocyte activation markers (P < .01). Combining Cp40 with anti-CD14 was significantly more effective than combining Cp40 with eritoran in reducing E. coli-induced interleukin 6 (P < .05) and monocyte activation markers induced by both E. coli (P < .001) and Staphylococcus aureus (P < .01). Combining CP40 with anti-CD14 was more efficient than eritoran alone for 18 of 20 bacteria-induced inflammatory responses (mean P < .0001). CONCLUSIONS: Whole bacteria-induced inflammation was inhibited more efficiently by anti-CD14 than by eritoran, particularly when combined with complement inhibition. Combined CD14 and complement inhibition may prove a promising treatment strategy for bacterial sepsis.

Anti-ApoA-1 IgG serum levels predict worse poststroke outcomes.

BACKGROUND: Autoantibodies to apolipoprotein A-1 (anti-ApoA-1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored. MATERIALS AND METHODS: We included n = 76 patients admitted for AIS and we investigated whether baseline serum anti-ApoA-1 IgG levels could predict (i) AIS-induced clinical disability [assessed by the modified Rankin Scale (mRS)], and (ii) AIS-related ischaemic lesion volume [assessed by Computed Tomography (CT)]. We also evaluated the possible pro-apoptotic and pro-necrotic effects of anti-ApoA-1 IgG on human astrocytoma cell line (U251) using flow cytometry. RESULTS: High levels of anti-ApoA-1 IgG were retrieved in 15·8% (12/76) of patients. Increased baseline levels of anti-ApoA-1 IgG were independently correlated with worse mRS [ß = 0·364; P = 0·002; adjusted odds ratio (OR): 1·05 (95% CI 1·01-1·09); P = 0·017] and CT-assessed ischaemic lesion volume [ß = 0·333; P < 0·001; adjusted OR: 1·06 (95% CI 1·01-1·12); P = 0·048] at 3 months. No difference in baseline clinical, biochemical and radiological characteristics was observed between patients with high vs. low levels of anti-ApoA-1 IgG. Incubating human astrocytoma cells with anti-ApoA-1 IgG dose dependently induced necrosis and apoptosis of U251 cells in vitro. CONCLUSION: Anti-ApoA-1 IgG serum levels at AIS onset are associated with poorer clinical recovery and worse brain lesion volume 3 months after AIS. These observations could be partly explained by the deleterious effect of anti-ApoA-1 IgG on human brain cell survival in vitro and may have clinical implication in the prediction of poor outcome in AIS.

Female sex hormones modulate Porphyromonas gingivalis lipopolysaccharide-induced Toll-like receptor signaling in primary human monocytes.

BACKGROUND AND OBJECTIVE: Female sex hormones are elevated and are potential host response modifiers during pregnancy. Modulation of immune responses by estrogen and progesterone may be responsible for periodontal inflammation. Therefore, we aimed to investigate the role of ß-estradiol and progesterone in human monocyte immune responses, at cellular and molecular levels, to identify their role as a possible immunological link between pregnancy and periodontal disease. MATERIAL AND METHODS: Primary human monocytes were purified from human peripheral blood mononuclear cells by adherent method. Expression of Toll-like receptor (TLR) 2, 4 and CD14 was analyzed by flow cytometry. TLR2, TLR4, cyclooxygenase-2 (COX2), nuclear factor-kappa B (NF-κB) and NF-κB inhibitor-alpha mRNA expressions were measured using real-time reverse transcriptase-polymerase chain reaction and prostaglandin E2 secretion was assayed by enzyme-linked immunosorbent assay. NF-κB expression was also examined by immunofluorescence. Western blotting was performed to determine the activation of mitogen-activated protein kinase pathway. RESULTS: We report herein that both ß-estradiol and progesterone significantly reduced TLR2 expression at both protein and mRNA levels but had less of an effect on TLR4 expression in primary human monocytes. We also found that the hormones decreased monocyte cell surface protein expression of CD14. Significantly, ß-estradiol and progesterone dose-dependently downregulated monocyte expression of COX2 mRNA. Pretreatment monocytes with ß-estradiol or progesterone reduced effects of Porphyromonas gingivalis lipopolysaccharide (LPS) on COX2 mRNA expression and decreased prostaglandin E2 secretion by the monocytes. Furthermore, we demonstrated that both ß-estradiol and progesterone inhibited P. gingivalis LPS-induced NF-κB signaling pathway through the upregulation of NF-κB inhibitor-alpha expression. However, neither ß-estradiol nor progesterone altered the phosphorylation of the p38, the extracellular signal-regulated kinase 1/2 and the c-Jun N-terminal activated kinase in P. gingivalis LPS-stimulated monocytes. Thus, the inhibitory effects of these hormones on the response of human monocytes to P. gingivalis LPS appear to be independent on mitogen-activated protein kinase signaling pathway. CONCLUSION: The results of the present study suggest that ß-estradiol and progesterone could influence the immune response of human monocytes to periodontal pathogens and this process may have a role in the clinical manifestations of periodontal disease associated with pregnancy.

Human leukocyte antigen DR surface expression on CD14+ monocytes during adverse events after hematopoietic stem cell transplantation.

The human leukocyte antigen DR surface expression on CD14+ monocytes reflects the degree to which these cells have been activated. Given the central role monocytes and macrophages play in the immune system, a decreased human leukocyte antigen DR expression on CD14+ monocytes results in a hallmark of altered immune status during systemic inflammatory response syndrome. We hypothesize that human leukocyte antigen DR expression might be similarly altered after hematopoietic stem cell transplantation and during post-transplant complications. Using flow cytometry, this study investigates the human leukocyte antigen DR surface expression of CD14+ monocytes in 30 pediatric and young adult patients up to 1 year after hematopoietic stem cell transplantation. Normal values were derived from a control group of healthy children, adolescents, and young adults. Human leukocyte antigen DR expression decreased significantly prior and during bacterial infection or sepsis. By contrast, human leukocyte antigen DR expression levels were elevated before and at the time of viremia. Human leukocyte antigen DR expression was also elevated during acute graft-versus-host disease. In contrast, the expression was reduced when patients had hepatic veno-occlusive disease. A significant decrease of human leukocyte antigen DR expression was associated with a relapse of the underlying disease and before death. Human leukocyte antigen DR expression on CD14+ monocytes appears to be a promising parameter that might allow identification of patients at risk after hematopoietic stem cell transplantation.

Soluble CD14 is essential for lipopolysaccharide-dependent activation of human intestinal mast cells from macroscopically normal as well as Crohn's disease tissue.

Mast cells are now considered sentinels in immunity. Given their location underneath the gastrointestinal barrier, mast cells are entrusted with the task of tolerating commensal microorganisms and eliminating potential pathogens in the gut microbiota. The aim of our study was to analyse the responsiveness of mast cells isolated from macroscopically normal and Crohn's disease-affected intestine to lipopolysaccharide (LPS). To determine the LPS-mediated signalling, human intestinal mast cells were treated with LPS alone or in combination with soluble CD14 due to their lack of surface CD14 expression. LPS alone failed to stimulate cytokine expression in human intestinal mast cells from both macroscopically normal and Crohn's disease tissue. Upon administration of LPS and soluble CD14, there was a dose- and time-dependent induction of cytokine and chemokine expression. Moreover, CXCL8 and interleukin-1ß protein expression was induced in response to activation with LPS plus soluble CD14. Expression of cytokines and chemokines was at similar levels in mast cells from macroscopically normal and Crohn's disease-affected intestine after LPS/soluble CD14 treatment. In conclusion, human intestinal mast cells appear to tolerate LPS per se. The LPS-mediated activation in mast cells may be provoked by soluble CD14 distributed by other LPS-triggered cells at the gastrointestinal barrier.

The effects of selective complement and CD14 inhibition on the E. coli-induced tissue factor mRNA upregulation, monocyte tissue factor expression, and tissue factor functional activity in human whole blood.

BACKGROUND: The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood. METHODS: Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')2 monoclonal antibody. RESULTS: The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1beta, IL-6, IL-8, and platelet-derived growth factor bb. CONCLUSION: Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

Olaparib attenuates sepsis-induced acute multiple organ injury via ERK-mediated CD14 expression.

Sepsis is characterized by persistent systemic inflammation, which can cause multi-organ dysfunction. The poly polymerase-1 inhibitor olaparib possesses anti-inflammatory properties. This study aimed to assess the effects of olaparib (pre- and post-treatments) on sepsis, and to investigate whether it could suppress CD14 expression via the ERK pathway in polymicrobial sepsis and peritoneal macrophages models. Sepsis was induced by cecal ligation and puncture in C57BL/6 male mice. Fifty mice were randomly divided into five groups: The sham group was treated with vehicle or olaparib, the cecal ligation and puncture group with vehicle or with olaparib (5 mg/kg i.p.) 1 h before or 2 h after surgery. Olaparib pretreatment significantly improved the survival of septic mice (P < 0.001). Pre- and post-treatment of mice with olaparib partly alleviated cecal ligation and puncture-induced organ injury by decreasing the amounts of the pro-inflammatory mediators TNF-α and IL-6 as well as bacterial burden in the serum, peritoneal lavage fluid, and organs (P < 0.05). The protective effect of olaparib was associated with CD14 suppression via inhibition of ERK activation. Olaparib facilitated negative regulation of ERK-mediated CD14 expression, which may contribute to multi-organ injury in sepsis.

sCD14 in bronchoalveolar lavage 18, 42 and 162 hours after segmental allergen provocation.

Lipopolysaccharides (LPS) have been associated with a protective role in the development of asthma while higher levels of endotoxin have been linked with more severe asthma. LPS recruit neutrophils and eosinophils and activate macrophages via the CD14 receptor. The soluble CD 14 receptor (sCD14) has been found in bronchoalveolar lavage fluid in different diseases including allergic asthma. To elucidate the kinetics and the regulation of sCD14 concentrations in BAL in asthma, 18 patients with allergic asthma underwent segmental allergen challenge at different time points (10 min, 18, 42 and 162 h). In addition, CD14(+) peripheral blood mononuclear cell (PBMC-CD14(+)) cultures from seven allergic and seven non-allergic subjects were stimulated with LPS, leukotrien D(4) (LTD(4)), a combination of LPS and LTD(4), IL-17 and LTD(4) in presence of the leukotriene-receptor antagonist (LTRA) Montelukast for 6, 12 and 24 h. sCD14 concentrations in BAL and the supernatants were measured by ELISA. sCD14 concentrations in BAL were significantly increased 18 h after allergen challenge and peaked at 42 h. At 162 h, concentrations had returned to baseline levels. In PBMC-CD14(+) cultures, sCD14 levels increased significantly 24 h after stimulation with LTD(4) and Montelukast was able to block LTD(4)-induced stimulation. Allergen challenge leads to a significant increase in sCD14 concentrations in BAL and might modulate the allergen-induced inflammation. In addition, LTD(4) might play a role in the release of sCD14, and it could be speculated that sCD14 reduction by LTRA might contribute to the mechanisms of LTRA in the treatment of allergic asthma.

Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo.

BACKGROUND AND OBJECTIVE: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

Effect of hyperbaric oxygen and ulinastatin on plasma endotoxin, soluble CD14, endotoxin-neutralizing capacity and cytokines in acute necrotizing pancreatitis.

BACKGROUND: We sought to study the effect of a combination therapy comprised of hyperbaric oxygen (HBO) and ulinastatin on the plasma levels of endotoxin, soluble CD14 (sCD14), endotoxin neutralizing capacity (ENC) and cytokines in acute necrotizing pancreatitis (ANP) in rats. METHODS: We randomly allocated 90 Sprague-Dawley rats into 6 groups: group 1 (ordinary control), group 2 (sham operation), group 3 (ANP), group 4 (ANP with HBO), group 5 (ANP with ulinastatin) and group 6 (ANP with HBO and ulinastatin). We induced ANP by retrograde injection of 3.5% sodium taurocholate (2.5 mL/kg) via the pancreatic duct. Five minutes after induction, animals in groups 5 and 6 were infused with ulinastatin (20 000 U/kg) via the portal vein. Thirty minutes after induction, animals in groups 4 and 6 received HBO therapy. We collected samples 3, 6 and 10 hours after induction of ANP. RESULTS: We found that the plasma level of endotoxin in group 3 was significantly higher than in group 4 (3, 6 h, both p < 0.001), group 5 (3 h, p < 0.001; 6 h, p = 0.014) and group 6 (both p < 0.001). The level of plasma sCD14 in group 3 was significantly higher than in group 4 (3, 6 h, both p < 0.001), group 5 (3, 6 h, both p = 0.001) and group 6 (3 h, p < 0.001; 6 h, p = 0.001). The plasma endotoxin and sCD14 levels in group 6 were significantly lower than in groups 4 and 5. The plasma ENC level in group 6 was significantly higher than in groups 3, 4 and 5 (p < 0.001). The ENC level in groups 4 and 5 were higher than in group 3, but there was no significant difference. The plasma level of tumour necrosis factor-alpha (TNF-alpha) and IL-6 in group 6 were significantly lower than in groups 3, 4 and 5 (p < 0.001). The TNF-alpha and IL-6 levels in groups 4 and 5 were lower than in group 3, but there was no significant difference. CONCLUSION: The use of an early combination therapy of HBO and ulinastatin was more effective than either therapy alone in the treatment of ANP.

mCD14 expression in human monocytes is downregulated by ouabain via transactivation of epithelial growth factor receptor and activation of p38 mitogen-activated protein kinase.

BACKGROUND AND AIMS: The steroid ouabain is found in plasma and in many mammalian tissues, and is now considered as a hormone. In the immune system, ouabain regulates a number of lymphocyte functions, but little is known about its effects on monocyte function. Monocytes are important for adequate immune responses. The aim of this work was to analyze the effect of ouabain on mCD14 expression, a surface molecule involved in the response against Gram-negative bacteria and phagocytosis. METHODS: Human peripheral blood mononuclear cells obtained from healthy donors were separated by density gradient centrifugation. Monocytes were separated by adherence and treated for 24 h with 100 nM ouabain. mCD14, CD1a and P-p38 expression was analyzed by flow cytometry. Inhibitors of cell-signaling pathways, i.e. SB202190, reduced glutathione, rottlerin, tyrphostin A23, genistein, chelerythrine chloride, PD98059, PP1 and Ly 294002, were used concomitantly with ouabain to observe their effect on mCD14 expression. RESULTS: Ouabain induced a significant decrease in mCD14 expression. This feature was not related to receptor endocytosis or cell death. Furthermore, mCD14 downregulation did not reflect a shift in differentiation into dendritic cells because this hormone failed to induce CD1a expression. Amongst several inhibitors of cell-signaling pathways triggered by ouabain, only epidermal growth factor receptor (EGFR) and p38 mitogen-activated protein kinase (MAPK) inhibitors (tyrphostin A23 and SB202109) significantly reverted the effect of ouabain on mCD14 expression. Accordingly, the levels of P-p38 were increased on monocytes after ouabain treatment. However, incubation with epidermal growth factor did not alter mCD14 expression. CONCLUSION: These findings suggest that ouabain downregulates mCD14 expression on monocytes through EGFR transactivation and p38 MAPK activation.

Absence of GDF5 does not interfere with LPS Toll-like receptor signaling.

OBJECTIVES: Growth and differentiation factor 5 (GDF5), member of TGFBeta superfamily, has been implicated in limb development, and is known to play an important role in joint formation. Its absence leads to brachypodism in mice and a number of skeletal malformation syndromes in humans. Recently, an association was shown between osteo-arthritis and a 5' UTR polymorphism in GDF5 gene. In addition, the role of GDF5 may reach beyond the musculoskeletal system. GDF5 appears present in a lipopolysaccharide (LPS) receptor cluster. Absence of GDF5 may limit the response to LPS. This may have consequences for immune responses and macrophage function in general, and for arthritis in particular. Here we compared the sensitivity of Gdf5(Bp-J/Bp-J) mice and wild type (WT) mice to LPS. METHODS: Peritoneal macrophages from Gdf5(Bp-J/Bp-J) mice and WT mice were stimulated for 18h with LPS (0, 10 or 100 ng/ml). The supernatant was collected and TNF release was measured by ELISA and by an indirect luciferase assay using LNF-luc C3 cells. Gdf5(Bp-J/Bp-J) mice and WT mice were injected with LPS i.p. (30 mg/kg) and LPS induced lethality was checked every 3 hours for 36 hours. RESULTS: Gdf5(Bp-J/Bp-J) macrophages showed no difference in TNF expression upon LPS stimulation measured by ELISA and by indirect luciferase assay. Gdf5(Bp-J/Bp-J) mice died upon a lethal dose of LPS, as is seen in WT controls. CONCLUSION: Absence of Gdf5 appears not to affect the LPS response. Mice with a reduced expression of Gdf5 can be used in disease models which are dependent on LPS boost.

Fenoterol, a beta(2)-adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through beta-arrestin-2 in THP-1 cell line.

AIM: To investigate the molecular mechanism and signaling pathway by which fenoterol, a beta(2)-adrenergic receptor (beta(2)-AR) agonist, produces anti-inflammatory effects. METHODS: THP-1, a monocytic cell line, was used to explore the mechanism of beta(2)-AR stimulation in LPS-induced secretion of inflammatory cytokines and changes of toll-like receptors (TLRs). We labeled TLR4 and CD14 using monoclonal anti-TLR4 PE-conjugated and anti-CD14 FITC-conjugated antibodies in THP-1 cells stimulated by beta(2)-AR in the presence or absence of lipopolysaccharide (LPS) and small, interfering RNA (siRNA)-mediated knockdown of beta-arrestin-2, and then analyzed their changes in distribution by flow cytometry, Western blotting and confocal analysis. RESULTS: LPS-induced membrane-bound CD14, TLR4/CD14 complex levels and elevation of inflammatory cytokines were all significantly reduced by pre-incubation of fenoterol (P<0.05). However, the total level of CD14 and TLR4 was not significantly changed. Interestingly, confocal microscopy revealed redistribution of CD14 and TLR4/CD14 complex under beta(2)-AR stimulation. Furthermore, siRNA-mediated knockdown of beta-arrestin-2 eliminated the anti-inflammatory effects and redistribution of CD14 and TLR4/CD14 complex stimulated by beta(2)-AR. CONCLUSION: beta(2)-AR agonist exerts its anti-inflammatory effects by down-regulating TLR signaling in THP-1 cells, potentially resulting from beta-arrestin-2 mediated redistribution of CD14 and TLR14/CD14 complex.

Bacterial lipopolysaccharide regulates nociceptin expression in sensory neurons.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-related peptide that is markedly up-regulated in sensory neurons in vivo following peripheral inflammation and plays a key role in pain physiology. To identify substances that up-regulate N/OFQ expression in sensory neurons, we carried out an in vitro screen using purified adult mouse dorsal root ganglion (DRG) neurons and identified the potent proinflammatory agent bacterial lipopolysaccharide (LPS) as a very effective inducer of N/OFQ. The robust response of these neurons to LPS enabled us to identify the components of a putative neuronal LPS receptor complex. In contrast to the immune system, where the functional LPS receptor complex is composed of CD-14 together with either MD-2 and TLR4 on myeloid cells or the homologous receptors MD-1 and RP105 on mature B cells, DRG neurons express the unusual combination of CD-14, TLR4, and MD-1. Blocking antibodies against TLR4 and MD-1 prevented induction of N/OFQ by LPS, and, in immunoprecipitation experiments, MD-1 coprecipitated with TLR4. Our findings suggest that LPS regulates N/OFN expression in sensory neurons via a novel combination of LPS receptor components and demonstrate for the first time a direct action of a key initiator of innate immune responses on neurons.

Down-regulation of epidermal growth factor receptor-dependent signaling by Porphyromonas gingivalis lipopolysaccharide in life-expanded human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Human gingival fibroblasts exhibit proliferative responses following epidermal growth factor exposure, which are thought to enhance periodontal regeneration in the absence of bacterial products such as lipopolysacharide. However, lipopolysaccharide challenge activates human gingival fibroblasts to release several inflammatory mediators that contribute to the immune response associated with periodontitis and attenuate wound repair. We tested the hypothesis that Porphyromonas gingivalis lipopolysaccharide-activated signaling pathways down-regulate epidermal growth factor receptor-dependent events. MATERIAL AND METHODS: To study lipopolysaccharide/epidermal growth factor interactions in human gingival fibroblasts, we introduced the catalytic subunit of human telomerase into human gingival fibroblasts, thereby generating a more long-lived cellular model. These cells were characterized and evaluated for lipopolysaccharide/epidermal growth factor responsiveness and regulation of epidermal growth factor-dependent pathways. RESULTS: Comparison of human telomerase-transduced gingival fibroblasts with human gingival fibroblasts revealed that both cell lines exhibit a spindle-like morphology and express similar levels of epidermal growth factor receptor, CD14 and Toll-like receptors 2 and 4. Importantly, human telomerase-transduced gingival fibroblasts proliferation rates are increased 5-9 fold over human gingival fibroblasts and exhibit a longer life span in culture. In addition, human telomerase-transduced gingival fibroblasts and human gingival fibroblasts exhibit comparable profiles of mitogen-activated protein kinase kinase (extracellular signal-regulated kinase 1/2) activation upon epidermal growth factor or P. gingivalis lipopolysaccharide administration. Interestingly, treatment with P. gingivalis lipopolysaccharide leads to a down-regulation of epidermal growth factor-dependent extracellular signal-regulated kinase 1/2, p38 and cyclic-AMP response element binding protein phosphorylation in both cell types. CONCLUSION: These studies demonstrate that human telomerase-transduced gingival fibroblasts exhibit an extended life span and recapitulate human gingival fibroblasts biology. Moreover, this system has allowed for the first demonstration of lipopolysaccharide down-regulation of epidermal growth factor activated pathways in human gingival fibroblasts and should facilitate the analysis of signaling events relevant to the pathogenesis and treatment of periodontitis.

Glutamine as a modulator of the immune system of critical care patients: effect on Toll-like receptor expression. A preliminary study.

OBJECTIVE: We evaluated the expression of Toll-like receptors 2 and 4 (TLR-2 and TLR-4) in circulating monocytes from peripheral blood of critical care patients treated with and without glutamine. Because no research has been published to date on the effect of glutamine on TLR receptors in critical patients, it was determined in an initial sample of 30 patients. METHODS: This was a prospective, randomized, single-blind study with 15 patients assigned to receive parenteral nutrition with a daily glutamine supplement of 0.35 g/kg. The control group received isocaloric-isonitrogenous parenteral nutrition. Blood samples were extracted before beginning the treatment and at 5 and 14 d. Expressions of CD14, TLR-2, and TLR-4 were determined by flow cytometry. Levels of TLRs were expressed as mean fluorescence intensity (mfi). RESULTS: Basal characteristics were similar in both groups. The expressions of TLR-2 in the treatment group with glutamine were 4.67 +/- 3.82 mfi before treatment, 3.91 +/- 2.04 mfi at 5 d, and 4.28 +/- 2.47 mfi at 14 d. The expressions of TLR-2 in the control group were 5.49 +/- 3.20 mfi before treatment, 4.48 +/- 2.15 mfi at 5 d, and 4.36 +/- 2.36 mfi at 14 d. The expressions of TLR-4 in the treatment group were 1.65 +/- 1.89 mfi before treatment, 1.23 +/- 1.10 mfi at 5 d, and 1.77 +/- 1.97 at 14 d. The expressions of TLR-4 in the control group were 1.51 +/- 1.76 mfi before treatment, 1.36 +/- 0.99 mfi at 5 d, and 1.26 +/- 0.59 mfi at 14 d. Infections were detected in 11 patients who received glutamine and 13 control patients (P = 0.51). CONCLUSION: In critical care patients, parenteral nutrition supplemented with glutamine does not increase the expression of TLR-2 or TLR-4 in peripheral blood monocytes.