Myeloid-related proteins 8 and 14 contribute to monosodium urate monohydrate crystal-induced inflammation in gout.

OBJECTIVE: Monosodium urate monohydrate (MSU) crystal-induced interleukin-1 46; (IL-1 46;) secretion is a critical factor in the pathogenesis of gout. However, without costimulation by a proIL-1 46;-inducing factor, MSU crystals alone are insufficient to induce IL-1 46; secretion. The responsible costimulatory factors that act as a priming endogenous signal in vivo are not yet known. We undertook this study to analyze the costimulatory properties of myeloid-related protein 8 (MRP-8) and MRP-14 (endogenous Toll-like receptor 4 [TLR-4] agonists) in MSU crystal-induced IL-1 46; secretion and their relevance in gout. METHODS: MRP-8/MRP-14 was measured in paired serum and synovial fluid samples by enzyme-linked immunosorbent assay (ELISA) and localized in synovial tissue from gout patients by immunohistochemistry. Serum levels were correlated with disease activity, and MSU crystal-induced release of MRPs from human phagocytes was measured. Costimulatory effects of MRP-8 and MRP-14 on MSU crystal-induced IL-1 46; secretion from phagocytes were analyzed in vitro by ELISA, Western blotting, and polymerase chain reaction. The impact of MRP was tested in vivo in a murine MSU crystal-induced peritonitis model. RESULTS: MRP-8/MRP-14 levels were elevated in the synovium, tophi, and serum of patients with gout and correlated with disease activity. MRP-8/MRP-14 was released by MSU crystal-activated phagocytes and increased MSU crystal-induced IL-1 46; secretion in a TLR-4-dependent manner. Targeted deletion of MRP-14 in mice led to a moderately reduced response of MSU crystal-induced inflammation in vivo. CONCLUSION: MRP-8 and MRP-14, which are highly expressed in gout, are enhancers of MSU crystal-induced IL-1 46; secretion in vitro and in vivo. These endogenous TLR-4 ligands released by activated phagocytes contribute to the maintenance of inflammation in gout.

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-_4;B, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Expression and function of macrophage migration inhibitory factor (MIF) in melioidosis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis. METHODOLOGY AND PRINCIPAL FINDINGS: MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei. CONCLUSIONS: MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients' outcomes. In experimental melioidosis MIF impaired antibacterial defense.

Proinflammatory activity of cell-wall constituents from gram-positive bacteria.

Innate immunity reacts to conserved bacterial molecules. The outermost lipopolysaccharide (LPS) of Gram-negative organisms is highly inflammatory. It activates responsive cells via specific CD14 and toll-like receptor-4 (TLR4) surface receptor and co-receptors. Gram-positive bacteria do not contain LPS, but carry surface teichoic acids, lipoteichoic acids and peptidoglycan instead. Among these, the thick peptidoglycan is the most conserved. It also triggers cytokine release via CD14, but uses the TLR2 co-receptor instead of TLR4 used by LPS. Moreover, whole peptidoglycan is 1000-fold less active than LPS in a weight-to-weight ratio. This suggests either that it is not important for inflammation, or that only part of it is reactive while the rest acts as ballast. Biochemical dissection of Staphylococcus aureus and Streptococcus pneumoniae cell walls indicates that the second assumption is correct. Long, soluble peptidoglycan chains (approximately 125 kDa) are poorly active. Hydrolysing these chains to their minimal unit (2 sugars and a stem peptide) completely abrogates inflammation. Enzymatic dissection of the pneumococcal wall generated a mixture of highly active fragments, constituted of trimeric stem peptides, and poorly active fragments, constituted of simple monomers and dimers or highly polymerized structures. Hence, the optimal constraint for activation might be 3 cross-linked stem peptides. The importance of structural constraint was demonstrated in additional studies. For example, replacing the first L-alanine in the stem peptide with a D-alanine totally abrogated inflammation in experimental meningitis. Likewise, modifying the D-alanine decorations of lipoteichoic acids with L-alanine, or deacylating them from their diacylglycerol lipid anchor also decreased the inflammatory response. Thus, although considered as a broad-spectrum pattern-recognizing system, innate immunity can detect very subtle differences in Gram-positive walls. This high specificity underlines the importance of using well-characterized microbial material in investigating the system.

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia.

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1 46;. This IL-1 46; production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1 46; production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Hydrogen sulfide in posthemorrhagic shock mesenteric lymph drainage alleviates kidney injury in rats

Posthemorrhagic shock mesenteric lymph (PHSML) is a key factor in multiple organ injury following hemorrhagic shock. We investigated the role of hydrogen sulfide (H2S) in PHSML drainage in alleviating acute kidney injury (AKI) by administering D,L-propargylglycine (PPG) and sodium hydrosulfide hydrate (NaHS) to 12 specific pathogen-free male Wistar rats with PHSML drainage. A hemorrhagic shock model was established in 4 experimental groups: shock, shock+drainage, shock+drainage+PPG (45 mg/kg, 0.5 h prehemorrhage), and shock+drainage+NaHS (28 µmol/kg, 0.5 h prehemorrhage). Fluid resuscitation was performed after 1 h of hypotension, and PHMSL was drained in the last three groups for 3 h after resuscitation. Renal function and histomorphology were assessed along with levels of H2S, cystathionine- 47;-lyase (CSE), Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)- 45; in renal tissue. Hemorrhagic shock induced AKI with increased urea and creatinine levels in plasma and higher H2S, CSE, TLR4, IL-10, IL-12, and TNF- 45; levels in renal tissue. PHSML drainage significantly reduced urea, creatinine, H2S, CSE, and TNF- 45; but not TLR4, IL-10, or IL-12. PPG decreased creatinine, H2S, IL-10, and TNF- 45; levels, but this effect was reversed by NaHS administration. In conclusion, PHSML drainage alleviated AKI following hemorrhagic shock by preventing increases in H2S and H2S-mediated inflammation.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Inflammation and adipose tissue: effects of progressive load training in rats

Introduction: Cytokines (IL-6, IL-10 and TNF-alpha) are increased after exhaustive exercise in the rat retroperitoneal (RPAT) and mesenteric adipose tissue (MEAT) pads. On the other hand, these cytokines show decreased expression in these depots in response to a chronic exercise protocol. However, the effect of exercise with overload combined with a short recovery period on pro-and anti-inflammatory cytokine expression is unknown. In the present study, we investigated the regulation of cytokine production in the adipose tissue of rats after an overtraining-inducing exercise protocol. Methods: Male Wistar rats were divided into four groups: Control (C), Trained (Tr), Overtrained (OT) and recovered overtrained (R). Cytokines (IL-6, TNF-alpha and IL-10) levels and Toll Like Receptor 4 (TLR4), Nuclear Factor kBBp65 (NF-kBp65), Hormone Sensitive Lipase (HSL) and, Perilipin protein expression were assessed in the adipose tissue. Furthermore, we analysed plasma lipid profile, insulin, testosterone, corticosterone and endotoxin levels, and liver triacylglycerol, cytokine content, as well as apolipoprotein B (apoB) and TLR4 expression in the liver. Results: OT and R groups exhibited reduced performance accompanied by lower testosterone and increased corticosterone and endotoxin levels when compared with the control and trained groups. IL-6 and IL-10 protein levels were increased in the adipose tissue of the group allowed to recover, in comparison with all the other studied groups. TLR-4 and NF-kBp65 were increased in this same group when compared with both control and trained groups. The protein expression of HSL was increased and that of Perilipin, decreased in the adipose in R in relation to the control. In addition, we found increased liver and serum TAG, along with reduced apoB protein expression and IL-6 and IL-10 levels in the of R in relation to the control and trained groups. Conclusion: In conclusion, we have shown that increases in pro-inflammatory cytokines in the adipose tissue after an overtraining protocol may be mediated via TLR-4 and NF-kBp65 signalling, leading to an inflammatory state in this tissue.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polymorphisms of the TLR2 and TLR4 genes are associated with risk of gastric cancer in a Brazilian population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cinnamic acid is partially involved in propolis immunomodulatory action on human monocytes

Propolis is a beehive product used in traditional medicine due to its biological properties. It shows a complex chemical composition including phenolics, such as cinnamic acid (Ci). The mechanisms of action of propolis have been the subject of research recently; however, the involvement of Ci on propolis activity was not investigated on immune cells. Ci effects were evaluated on human monocytes, assessing the expression of Toll-like receptors (TLRs), HLA-DR, and CD80. Cytokine production (TNF-α and IL-10) and the fungicidal activity of monocytes were evaluated as well. Data showed that Ci downregulated TLR-2, HLA-DR, and CD80 and upregulated TLR-4 expression by human monocytes. High concentrations of Ci inhibited both TNF-α and IL-10 production, whereas the same concentrations induced a higher fungicidal activity against Candida albicans. TNF-α and IL-10 production was decreased by blocking TLR-4, while the fungicidal activity of monocytes was not affected by blocking TLRs. These results suggest that Ci modulated antigen receptors, cytokine production, and the fungicidal activity of human monocytes depending on concentration, and TLR-4 may be involved in its mechanism of action. Ci seemed to be partially involved in propolis activities. © 2013 Bruno José Conti et al.

Signaling Pathways Activation by Primary Endodontic Infectious Contents and Production of Inflammatory Mediators

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)