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.

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.

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.

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.

Effects of dexmedetomidine on CD42a+/CD14+, HLADR+/CD14+ and inflammatory cytokine levels in patients undergoing multilevel spinal fusion.

OBJECTIVE: To assess the effects of dexmedetomidine (Dex) on CD42a+/CD14+，HLADR+/CD14+ and inflammatory cytokine levels in patients undergoing multilevel spinal fusion. Patients and methods Forty ASA I-II patients undergoing multilevel spinal fusion were randomly divided into Dex and control groups (n=20). A continuous intravenous infusion of Dex (0.5µg/kg/h) or normal saline was started 10min prior to induction and was stopped 15min before operation completion. Serum levels of CD42a+/CD14+, HLADR+/CD14+, WBC, PLT, CRP, IL-6, IL-10, and TNF-α were measured before induction (T1), 30min (T2) after operation initiation, and 60min (T3), 1d (T4), 3d (T5), and 5d (T6) post-operation. VAS values were obtained at T3, T4, T5 and T6, as well as hospital days. RESULTS: Treatment with Dex significantly decreased CD42a+/CD14+ at T2, T3, and T4, and markedly increased HLADR+/CD14+ at T4 and T5 when compared with controls. CRP and WBC were markedly decreased at T2, T3, T4 and T5 (P<0.01 or P<0.05). Serum IL-6 and TNF-α level in Dex group was significantly increased at T3 and T4 (P<0.05), and IL-6 and TNF-α level in control group was significantly increased at T2, T3, T4 and T5 (P<0.05) when compared with their respective preoperative levels (T1). IL-6 and TNF-α levels at T2, T3, T4 and T5 in Dex group were significantly lower than those in control group (P<0.05). There were no significant differences in operation time, hospital days or VAS values between the two groups (P>0.05). CONCLUSION: Dex can inhibit the inflammatory response and reduce immunosuppression in patients undergoing multilevel spinal fusion.

Classical and alternative macrophages have impaired function during acute and chronic HIV-1 infection

Abstract Objectives: Three decades after HIV recognition and its association with AIDS development, many advances have emerged – especially related to prevention and treatment. Undoubtedly, the development of Highly Active Antiretroviral Therapy (HAART) dramatically changed the future of the syndrome that we know today. In the present study, we evaluate the impact of Highly Active Antiretroviral Therapy on macrophage function and its relevance to HIV pathogenesis. Methods: PBMCs were isolated from blood samples and monocytes (CD14+ cells) were purified. Monocyte-Derived Macrophages (MDMs) were activated on classical (MGM-CSF+IFN-γ) or alternative (MIL-4+IL13) patterns using human recombinant cytokines for six days. After this period, Monocyte-Derived Macrophages were stimulated with TLR2/Dectin-1 or TLR4 agonists and we evaluated the influence of HIV-1 infection and Highly Active Antiretroviral Therapy on the release of cytokines/chemokines by macrophages. Results: The data were obtained using Monocyte-Derived Macrophages derived from HIV naïve or from patients on regular Highly Active Antiretroviral Therapy. Classically Monocyte-Derived Macrophages obtained from HIV-1 infected patients on Highly Active Antiretroviral Therapy released higher levels of IL-6 and IL-12 even without PAMPs stimuli when compared to control group. On the other hand, alternative Monocyte-Derived Macrophages derived from HIV-1 infected patients on Highly Active Antiretroviral Therapy released lower levels of IL-6, IL-10, TNF-α, IP-10 and RANTES after LPS stimuli when compared to control group. Furthermore, healthy individuals have a complex network of cytokines/chemokines released by Monocyte-Derived Macrophages after PAMP stimuli, which was deeply affected in MDMs obtained from naïve HIV-1 infected patients and only partially restored in MDMs derived from HIV-1 infected patients even on regular Highly Active Antiretroviral Therapy. Conclusion: Our therapy protocols were not effective in restoring the functional alterations induced by HIV, especially those found on macrophages. These findings indicate that we still need to develop new approaches and improve the current therapy protocols, focusing on the reestablishment of cellular functions and prevention/treatment of opportunistic infections.

Antiinflammatory and ROS Suppressive Effects of the Addition of Fiber to a High-Fat High-Calorie Meal.

Background: Fiber intake is associated with a reduction in the occurrence of cardiovascular events and diabetes. Objective: To investigate whether the addition of fiber to a high-fat, high-calorie (HFHC) meal prevents proinflammatory changes induced by the HFHC meal. Design: Ten normal fasting subjects consumed an HFHC meal with or without an additional 30 g of insoluble dietary fiber on 2 separate visits. Blood samples were collected over 5 hours, and mononuclear cells (MNCs) were isolated. Results: Fiber addition to the HFHC meal significantly lowered glucose excursion in the first 90 minutes and increased insulin and C-peptide secretion throughout the 5-hour follow-up period compared with the meal alone. The HFHC meal induced increases in lipopolysaccharide (LPS) concentrations, MNC reactive oxygen species generation, and the expression of interleukin (IL)-1ß, tumor necrosis factor α (TNF-α), Toll-like receptor (TLR)-4, and CD14. The addition of fiber prevented an increase in LPS and significantly reduced the increases in ROS generation and the expression of IL-1ß, TNF-α, TLR-4, and CD14. In addition, the meal increased Suppressor of cytokine signaling (SOCS)-3 and protein tyrosine phosphatase 1B (PTP-1B) messenger RNA and protein levels, which were inhibited when fiber was added. Conclusions: The addition of fiber to a proinflammatory HFHC meal had beneficial anti-inflammatory and metabolic effects. Thus, the fiber content of the American Heart Association meal may contribute to its noninflammatory nature. If these actions of dietary fiber are sustained following long-term intake, they may contribute to fiber's known benefits in the prevention of insulin resistance, type 2 diabetes, and atherosclerosis.

Classical and alternative macrophages have impaired function during acute and chronic HIV-1 infection.

OBJECTIVES: Three decades after HIV recognition and its association with AIDS development, many advances have emerged - especially related to prevention and treatment. Undoubtedly, the development of Highly Active Antiretroviral Therapy (HAART) dramatically changed the future of the syndrome that we know today. In the present study, we evaluate the impact of Highly Active Antiretroviral Therapy on macrophage function and its relevance to HIV pathogenesis. METHODS: PBMCs were isolated from blood samples and monocytes (CD14+ cells) were purified. Monocyte-Derived Macrophages (MDMs) were activated on classical (MGM-CSF+IFN-γ) or alternative (MIL-4+IL13) patterns using human recombinant cytokines for six days. After this period, Monocyte-Derived Macrophages were stimulated with TLR2/Dectin-1 or TLR4 agonists and we evaluated the influence of HIV-1 infection and Highly Active Antiretroviral Therapy on the release of cytokines/chemokines by macrophages. RESULTS: The data were obtained using Monocyte-Derived Macrophages derived from HIV naïve or from patients on regular Highly Active Antiretroviral Therapy. Classically Monocyte-Derived Macrophages obtained from HIV-1 infected patients on Highly Active Antiretroviral Therapy released higher levels of IL-6 and IL-12 even without PAMPs stimuli when compared to control group. On the other hand, alternative Monocyte-Derived Macrophages derived from HIV-1 infected patients on Highly Active Antiretroviral Therapy released lower levels of IL-6, IL-10, TNF-α, IP-10 and RANTES after LPS stimuli when compared to control group. Furthermore, healthy individuals have a complex network of cytokines/chemokines released by Monocyte-Derived Macrophages after PAMP stimuli, which was deeply affected in MDMs obtained from naïve HIV-1 infected patients and only partially restored in MDMs derived from HIV-1 infected patients even on regular Highly Active Antiretroviral Therapy. CONCLUSION: Our therapy protocols were not effective in restoring the functional alterations induced by HIV, especially those found on macrophages. These findings indicate that we still need to develop new approaches and improve the current therapy protocols, focusing on the reestablishment of cellular functions and prevention/treatment of opportunistic infections.

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.

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.

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.

CD14 as a Mediator of the Mineralocorticoid Receptor-Dependent Anti-apolipoprotein A-1 IgG Chronotropic Effect on Cardiomyocytes.

In vitro and animal studies point to autoantibodies against apolipoprotein A-1 (anti-apoA-1 IgG) as possible mediators of cardiovascular (CV) disease involving several mechanisms such as basal heart rate interference mediated by a mineralocorticoid receptor-dependent L-type calcium channel activation, and a direct pro-inflammatory effect through the engagement of the toll-like receptor (TLR) 2/CD14 complex. Nevertheless, the possible implication of these receptors in the pro-arrhythmogenic effect of anti-apoA-1 antibodies remains elusive. We aimed at determining whether CD14 and TLRs could mediate the anti-apoA-1 IgG chronotropic response in neonatal rat ventricular cardiomyocytes (NRVC). Blocking CD14 suppressed anti-apoA-1 IgG binding to NRVC and the related positive chronotropic response. Anti-apoA-1 IgG alone induced the formation of a TLR2/TLR4/CD14 complex, followed by the phosphorylation of Src, whereas aldosterone alone promoted the phosphorylation of Akt by phosphatidylinositol 3-kinase (PI3K), without affecting the chronotropic response. In the presence of both aldosterone and anti-apoA-1 IgG, the localization of TLR2/TLR4/CD14 was increased in membrane lipid rafts, followed by PI3K and Src activation, leading to an L-type calcium channel-dependent positive chronotropic response. Pharmacological inhibition of the Src pathway led to the decrease of L-type calcium channel activity and abrogated the NRVC chronotropic response. Activation of CD14 seems to be a key regulator of the mineralocorticoid receptor-dependent anti-apoA-1 IgG positive chronotropic effect on NRVCs, involving relocation of the CD14/TLR2/TLR4 complex into lipid rafts followed by PI3K and Src-dependent L-type calcium channel activation.

Polychlorinated Biphenyls (PCB 101, 153, and 180) Impair Murine Macrophage Responsiveness to Lipopolysaccharide: Involvement of NF-κB Pathway.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are persistent organic pollutants, associated with a range of adverse health effects, including interference with the immune system. In this study, we investigate the capability of NDL-PCBs 101, 153, and 180, 3 of the 6 NDL-PCBs defined as indicators, to impair the immune response in lipopolysaccharide (LPS)-activated J774A.1 and primary murine macrophages. Our results clearly demonstrate that the exposure of J774A.1 and primary macrophages to NDL-PCB 153 or 180 or all NDL-PCBs mixtures causes a significant reduction in LPS-induced cytokine/chemokine synthesis, such as tumor necrosis factor-α and interleukin-6, together with monocyte chemoattractant protein-1, involved in cell recruitment. Moreover, PCBs were found to suppress LPS-stimulated NO production, and to reduce cyclooxygenase-2 and inducible nitric oxide synthase expression in J774A.1 and primary macrophages. At mechanistic level, PCBs significantly counteract the LPS-driven toll-like receptor (TLR) 4 and CD14 upregulation, therefore inhibiting downstream nuclear factor-κB (NF-κB) activation in J774A.1. Furthermore, PCBs determine a significant loss of macrophage endocytic capacity, a prerequisite for efficient antigen presentation. Taken together, these data indicate that NDL-PCBs reduce macrophage responsiveness, particularly when they are combined at concentrations per se inactive, impairing the capability to orchestrate a proper immune response to an infectious stimulus, disrupting TLR4/NF-κB pathway.

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.

Porphyromonas gingivalis lipopolysaccharide upregulates insulin secretion from pancreatic ß cell line MIN6.

BACKGROUND: A close association between periodontitis and diabetes has been demonstrated in human cross-sectional studies, but an exact relationship between periodontitis and prediabetes has not been established. Previous studies using animal model systems consistently have shown that hyperinsulinemia occurs in animals with periodontitis compared to animals with healthy periodontium (while maintaining normoglycemia). Because bacterial lipopolysaccharide (LPS) plays an important role in the pathogenesis of periodontitis, we hypothesized that LPS may stimulate insulin secretion through a direct effect on ß cell function. To test this hypothesis, pancreatic ß cell line MIN6 cells were used to determine the effect of Porphyromonas gingivalis (Pg) LPS on insulin secretion. Furthermore, expression of genes altered by Pg LPS in innate immunity and insulin-signaling pathways was determined. METHODS: MIN6 cells were grown in medium with glucose concentration of normoglycemia (5.5 mM). Pg LPS was added to each well at final concentrations of 50, 200, and 500 ng/mL. Insulin secretion was measured using enzyme-linked immunosorbent assay. Gene expression levels altered by Pg LPS were determined by polymerase chain reaction (PCR) array for mouse innate and adaptive immunity response and mouse insulin-signaling pathways, and results were confirmed for specific genes of interest by quantitative PCR. RESULTS: Pg LPS stimulated insulin secretion in the normoglycemic condition by ≈1.5- to 3.0-fold depending on the concentration of LPS. Pg LPS treatment altered the expression of several genes involved in innate and adaptive immune response and insulin-signaling pathway. Pg LPS upregulated the expression of the immune response-related genes cluster of differentiation 8a (Cd8a), Cd14, and intercellular adhesion molecule-1 (Icam1) by about two-fold. LPS also increased the expression of two insulin signaling-related genes, glucose-6-phosphatase catalytic subunit (G6pc) and insulin-like 3 (Insl3), by three- to four-fold. CONCLUSIONS: We have demonstrated for the first time that Pg LPS stimulates insulin secretion by pancreatic ß cell line MIN cells. Pg LPS may have significant implications on the development of ß cell compensation and insulin resistance in prediabetes in individuals with periodontitis.

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects.

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell-wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA-binding proteins (Sm.LTA-BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA-BPs in the saliva of caries-free and caries-positive human subjects using Sm.LTA-conjugated beads and LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Sm.LTA-beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon-γ-inducible protein 10 in a murine macrophage cell line and activation of Toll-like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA-BPs were isolated from pooled saliva prepared from 10 caries-free or caries-positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high-resolution mass spectrometry. A total of 8 and 12 Sm.LTA-BPs were identified with statistical significance in the pooled saliva from the caries-free and caries-positive human subjects, respectively. Unique Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1 and neutrophil defensin-1, and those in caries-positive saliva included cystatin-C, cystatin-SN, cystatin-S, cystatin-D, lysozyme C, calmodulin-like protein 3 and ß-actin. The Sm.LTA-BPs found in both groups were hemoglobin subunits α and ß, prolactin-inducible protein, protein S100-A9, and SPLUNC2. Collectively, we identified Sm.LTA-BPs in the saliva of caries-free and caries-positive subjects, which exhibit differential protein profiles.

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 class A scavenger receptor SR-A/CD204 and the class B scavenger receptor CD36 regulate immune functions of macrophages differently.

SR-A/CD204 and CD36 are major receptors responsible for oxidized lipoproteins uptake by macrophages in atherosclerotic plaques. Both receptors also share the role as receptors for different pathogens, but studies on their signaling have been hampered by the lack of selective ligands. We report that, upon specific ligation by Ab, SR-A does not induce cytokine production, but mediates inhibition of LPS-stimulated production of IL-6 and IL-12/23p40, enhancement of IL-10 release, and has no effect on TNF-α and RANTES production in murine macrophages. In contrast, anti-CD36 Ab alone stimulated production of all these cytokines, with IL-10 production being exceptionally high. Effects of anti-CD36 Ab, except of IL-10 production, were mediated by CD14 and TLR2, whereas those of SR-A ligation by heterotrimeric Gi/o proteins and by phosphatidylinositol 3-kinases. Surprisingly, we found that LPS uptake by macrophages was mediated in part by CD36 cooperating with CD14, whereas SR-A was not involved in this process. Finely, during in vitro Ag presentation to naïve CD4(+) lymphocytes, pre-incubation of macrophages with anti-CD36 Ab enhanced IFN-γ production in the co-culture, but exerted the opposite effect under conditions enabling IL-10 accumulation. In contrast, anti-SR-A Ab was ineffective alone, but reversed the Th1-polarizing effect of LPS.

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.