Lipopolysaccharide from Coxiella burnetii is involved in bacterial phagocytosis, filamentous actin reorganization, and inflammatory responses through Toll-like receptor 4

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4(-/-) mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4(-/-) macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4(-/-) macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4(-/-) mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4(-/-) mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-gamma and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.     

Gene expressions of Toll-like receptor 2, but not Toll-like receptor 4, is induced by LPS and inflammatory cytokines in mouse macrophages

Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1beta, IFN-gamma, or TNF-alpha significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-kappaB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.     

The in vitro bactericidal activity of peritoneal and spleen cells from Listeria-resistant and -susceptible mouse strains

Two days after Listeria-resistant (LrR) C57BL/10 mice were infected intraperitoneally with Listeria, their peritoneal macrophages demonstrated enhanced bactericidal activity beyond that seen in susceptible (LrS) BALB/c or CBA mice. Intravenous infection had no effect on peritoneal cell activity. The induction, but not expression, of the enhanced activity was radiosensitive. There was no significant difference between the strains with respect to the number of cells or cellular composition of the exudates. No difference in the in vitro chemotactic response of cells from the two strains could be demonstrated. Therefore there seems to be recruitment to the infected peritoneal cavity of C57BL/10 mice of young, efficiently bactericidal monocytes/macrophages. On the other hand, spleen cell bactericidal activity was intrinsically superior in C57BL/10 mice compared with BALB/c mice, possibly because, as a haemopoietic organ, the C57BL/10 spleen already contains high numbers of these efficient monocytes.     

Reactive nitrogen and oxygen species, and iron sequestration contribute to macrophage-mediated control of Encephalitozoon cuniculi (Phylum Microsporidia) infection in vitro and in vivo

Encephalitozoon cuniculi (Phylum Microsporidia) infects a wide range of mammals, and replicates within resting macrophages. Activated macrophages, conversely, inhibit replication and destroy intracellular organisms. These studies were performed to assess mechanisms of innate immune responses expressed by macrophages to control E. cuniculi infection. Addition of reactive oxygen and nitrogen species inhibitors to activated murine peritoneal macrophages statistically significantly, rescued E. cuniculi infection ex vivo. Mice deficient in reactive oxygen species, reactive nitrogen species, or both survived ip inoculation of E. cuniculi, but carried significantly higher peritoneal parasite burdens than wild-type mice at 1 and 2 weeks post inoculation. Infected peritoneal macrophages could still be identified 4 weeks post inoculation in mice deficient in reactive nitrogen species. L-tryptophan supplementation of activated murine peritoneal macrophage cultures ex vivo failed to rescue microsporidia infection. Addition of ferric citrate to supplement iron, however, did significantly rescue E. cuniculi infection in activated macrophages and further increased parasite replication in non-activated macrophages over non-treated resting control macrophages. These results demonstrate the contribution of reactive oxygen and nitrogen species, as well as iron sequestration, to innate immune responses expressed by macrophages to control E. cuniculi infection.     

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.     

Spontaneously diabetic Ins2(+/Akita):apoE-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis

Type 2 diabetes (T2DM) is characterized by hyperglycemia, dyslipidemia, and increased inflammation. Previously, we showed that high glucose (HG) induces Toll-like receptor (TLR) expression, activity, and inflammation via NF-κB followed by cytokine release in vitro and in vivo. Here, we determined how HG-induced inflammation is affected by free fatty acids (FFA) in human monocytes. THP-1 monocytic cells, CD14(+) human monocytes, and transiently transfected HEK293 cells were exposed to various FFA (0-500 μM) and glucose (5-20 mM) for evaluation of TLR2, TLR4, NF-κB, IL-1β, monocyte chemoattractant protein-1 (MCP-1), and superoxide release. In THP-1 cells, palmitate increased cellular TLR2 and TLR4 expression, generated reactive oxygen species (ROS), and increased NF-κB activity, IL-1β, and MCP-1 release in a dose- and time-dependent manner. Similar data were observed with stearate and FFA mixture but not with oleate. Conversely, NADPH oxidase inhibitor treatment repressed glucose- and palmitate-stimulated ROS generation and NF-κB activity and decreased IL-1β and MCP-1 expression. Silencing TLR2, TLR4, and p47phox with small inhibitory RNAs (siRNAs) significantly reduced superoxide release, NF-κB activity, IL-1β, and MCP-1 secretion in HG and palmitate-treated THP-1 cells. Moreover, data from transient transfection experiments suggest that TLR6 is required for TLR2 and MD2 for TLR4 to augment inflammation in FFA- and glucose-exposed cells. These findings were confirmed with human monocytes. We conclude that FFA exacerbates HG-induced TLR expression and activity in monocytic cells with excess superoxide release, enhanced NF-κB activity, and induced proinflammatory factor release.     

Aging enhances the production of reactive oxygen species and bactericidal activity in peritoneal macrophages by upregulating classical activation pathways

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.     

Regulatory role of thioredoxin in homocysteine-induced monocyte chemoattractant protein-1 secretion in monocytes/macrophages

We have previously shown that homocysteine (Hcy) can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes. Here, we show that Hcy upregulates expression of an important antioxidative protein, thioredoxin (Trx), via NADPH oxidase in human monocytes in vitro. The increase of Trx expression and activity inhibited Hcy-induced ROS production and MCP-1 secretion. Of note, 2-week hyperhomocysteinemia (HHcy) ApoE^−/− mice showed accelerated lesion formation and parallel lower Trx expression in macrophages than ApoE^−/− mice, suggesting that HHcy-induced sustained oxidative stress in vivo might account for impaired Trx and hence increased ROS production and MCP-1 secretion from macrophages, and subsequently accelerated atherogenesis.     

Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase

We investigated the role of neutrophil elastase (NE) in interactions between murine inflammatory neutrophils and macrophages infected with the parasite Leishmania major. A blocker peptide specific for NE prevented the neutrophils from inducing microbicidal activity in macrophages. Inflammatory neutrophils from mutant pallid mice were defective in the spontaneous release of NE, failed to induce microbicidal activity in wild-type macrophages, and failed to reduce parasite loads upon transfer in vivo. Conversely, purified NE activated macrophages and induced microbicidal activity dependent on secretion of TNF-alpha. Induction of macrophage microbicidal activity by either neutrophils or purified NE required TLR4 expression by macrophages. Injection of purified NE shortly after infection in vivo reduced the burden of L. major in draining lymph nodes of TLR4-sufficient, but not TLR4-deficient mice. These results indicate that NE plays a previously unrecognized protective role in host responses to L. major infection.     

Toll-like receptor 2 participates in mediation of immune response in experimental pneumococcal meningitis

Heterologous expression of Toll-like receptor (TLR)2 and CD14 in Chinese hamster ovary fibroblasts was reported to confer responsiveness to pneumococcal peptidoglycan. The present study characterized the role of TLR2 in the host immune response and clinical course of pneumococcal meningitis. Pneumococcal infection of mice caused a significant increase in brain TLR2 mRNA expression at both 4 and 24 h postchallenge. Mice with a targeted disruption of the TLR2 gene (TLR2-/-) showed a moderate increase in disease severity, as evidenced by an aggravation of meningitis-induced intracranial complications, a more pronounced reduction in body weight and temperature, and a deterioration of motor impairment. These symptoms were associated with significantly higher cerebellar and blood bacterial titers. Brain expression of the complement inhibitor complement receptor-related protein y was significantly higher in infected TLR2-/- than in wild-type mice, while the expression of the meningitis-relevant inflammatory mediators IL-1beta, TNF-alpha, IL-6, macrophage-inflammatory protein (MIP)-2, inducible NO synthase, and C3 was similar in both genotypes. We first ectopically expressed single candidate receptors in HEK293 cells and then applied peritoneal macrophages from mice lacking TLR2 and/or functional TLR4 for further analysis. Overexpression of TLR2 and TLR4/MD-2 conferred activation of NF-kappaB in response to pneumococcal exposure. However, pneumococci-induced TNF-alpha release from peritoneal macrophages of wild-type and TLR2/functional TLR4/double-deficient mice did not differ. Thus, while TLR2 plays a significant role in vivo, yet undefined pattern recognition receptors contribute to the recognition of and initiation of the host immune defense toward Streptococcus pneumoniae infection.     

Role of redox factor-1 in hyperhomocysteinemia-accelerated atherosclerosis

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. We have previously shown that homocysteine can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes in vitro. In the present study, we investigated whether redox factor-1 (Ref-1) is involved in HHcy-accelerated atherosclerosis. We used a mild HHcy animal model, aortic roots and peritoneal macrophages were isolated for immunohistochemistry and Western blotting, from apoE-/- and C57BL/6J mice fed a high Hcy diet (1.8 g/L) for 4 or 12 weeks. Four-week HHcy apoE-/- mice showed more plaques and significantly increased immunostaining of Ref-1 and MCP-1 in foam cells, and HHcy mice showed enhanced Ref-1 expression in peritoneal macrophages. To explore the mediating mechanism, incubation with Hcy (100 microM) increased Ref-1 protein level and translocation in human monocytes in vitro. In addition, Hcy-induced NADPH oxidase activity mediated the upregulation of Ref-1. Furthermore, overexpressed Ref-1 upregulated NF-kappaB and MCP-1 promoter activity, and antisense Ref-1 reduced Hcy-induced NF-kappaB DNA-binding activity and MCP-1 secretion. These data indicate that Hcy-induced ROS upregulate the expression and translocation of Ref-1 via NADPH oxidase, and then Ref-1 increases NF-kappaB activity and MCP-1 secretion in human monocytes/macrophages, which may accelerate the development of atherosclerosis.     

Cutting edge: impaired Toll-like receptor expression and function in aging

Toll-like receptors (TLR) are pattern recognition receptors that recognize conserved molecular patterns on microbes and link innate and adaptive immune systems. We investigated whether the enhanced susceptibility to bacterial, yeast, and viral infections and poor adaptive immune responses in aging are a result of diminished expression and function of TLRs. We examined the expression and function of all murine TLRs on macrophages from young and aged mice. Both splenic and activated peritoneal macrophages from aged mice expressed significantly lower levels of all TLRs. Furthermore, macrophages from aged mice secreted significantly lower levels of IL-6 and TNF-alpha when stimulated with known ligands for TLR1 and 2, 2 and 6,TLR3, TLR4, TLR5, and TLR9 when compared with those from young mice. These results support the concept that increased susceptibility to infections and poor adaptive immune responses in aging may be due to the decline in TLR expression and function.     

Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction

Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.     

ASK1-p38 MAPK-p47phox activation is essential for inflammatory responses during tuberculosis via TLR2-ROS signalling

The roles of intracellular reactive oxygen species (ROS) and related signalling pathways in mycobacterial infection are largely unknown. Here we show that tuberculin purified protein derivative (PPD)/Toll-like receptor (TLR) 2/ROS signalling through activation of apoptosis-regulating signal kinase (ASK) 1 and p47phox pathways is responsible for the induction of proinflammatory responses during tuberculosis (TB) infection. Tuberculin PPD stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs) and an early burst of ROS in monocytes/macrophages in a TLR2-dependent manner. PPD-induced ROS production led to robust activation of ASK1 upstream of p38 MAPK, via TLR2. Interestingly, phosphorylation of the cytosolic NADPH oxidase subunit p47phox and ASK1 activation are mutually dependent on PPD/TLR2-mediated signalling. Furthermore, active pulmonary TB patients showed upregulated ROS generation, as well as enhanced activation of ASK1/p38/p47phox pathways in their primary monocytes compared with healthy controls, which suggests a systemic primed status during TB. Taken together, these results indicate that activation of the ASK1/p38 MAPK/p47phox cascade plays a central role in PPD/TLR2-induced ROS generation and suggests the existence of a 'ROS/ASK1' inflammatory amplification feedback loop in monocytes/macrophages. The altered regulation of this axis with an increasing free-radical burden may contribute to the immunopathogenesis of human TB.     

Chemiluminescence response of phagocytes in E. coli induced experimental ascending pyelonephritis.

The mechanism of tissue injury at the cellular level by following the chemiluminescence response of various phagocytes in E. coli induced experimental pyelonephritis in mice was investigated. There was a marked increase in the capacity of various phagocytic cells viz; renal neutrophils and macrophages peritoneal macrophages, blood monocytes and neutrophils to produce reactive oxygens species through the respiratory burst activity as monitored by the chemiluminescence response. The chemiluminescence response was observed to be increased significantly (p less than 0.001) with increasing days post infection in all phagocytic cells. However, the quantity of total reactive oxygen species produced per million cells was much more in the renal and peritoneal macrophages as compared to blood monocytes and neutrophils. The peak chemiluminescence response time was observed to be decreased from 4 to 2 minutes with the progression of the diseases. The implications of these findings have been discussed.     

House dust mite regulate the lung inflammation of asthmatic mice through TLR4 pathway in airway epithelial cells

Aberrant innate and adaptive immune responsed to allergens and environmental pollutants lead to respiratory allergic disease such as asthma. In this study, we focused on toll-like receptor-4 (TLR4) expressed on airway epithelium to identify house dust mite (HDM)-regulated allergic inflammation via TLR4 signaling pathway and the triggering to alveolar macrophages (AM)-driven adaptive immune response. The authors found that mouse exposed to HDM showed more eosinophils, neutrophils, monocytes, lymphocytes as well as total cells in bronchoalveolar lavage fluid (BALF) confirmed by flow cytometry. Besides, the expression of TLR4 in airway epithelial cells was significantly increased in both mRNA and protein levels in mice treated with HDM and the expression of CD40 and CD86 in AM was also increased in mice exposed to HDM. Tight correlation between TLR4 protein and CD40, CD86 in AM was identified. This study demonstrates that TLR4 expression on airway epithelium played an essential role in HDM-induced activation of AM in immune responses and allergic inflammation. The airway epithelial TLR4 signaling pathway revealed tight connection between endotoxin exposure and asthma prevalence in the clinic.     

Toll-like receptor-2 is essential in murine defenses against Candida albicans infections

In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity of i.p. infected TLR2-/- mice. In contrast, in vitro phagocytosis of yeasts and production of reactive oxygen intermediates (ROI) were not affected in macrophages from TLR2-/- animals. Our data indicate that TLR2 plays a major role in the response of macrophages to C. albicans, triggering cytokine and chemokine expression, and it is essential for in vivo protection against infection.     

Alteration of superoxide- and nitric oxide-mediated antimicrobial function of macrophages by in vivo cocaine exposure

Cocaine is a popular drug of abuse and despite impressive advances in the understanding of its physiological, pharmacological, and toxic effects, its mechanism of immunosuppression at the cellular level is not well understood. In this paper we report the role of effector molecules like superoxide and nitric oxide in the antibacterial function of macrophages exposed to acute and chronic doses of cocaine in vivo. Bacterial killing by acute cocaine-exposed macrophages (ACE-Mphis) increased significantly, with a concomitant rise in respiratory burst and generation of superoxide and nitric oxide, compared with control macrophages. In contrast, chronic cocaine-exposed macrophages (CCE-Mphis) exhibited limited antimicrobial activity, which correlated closely with diminished respiratory burst and reduced production of superoxide and nitric oxide. Further, a killing assay was carried out in the presence of N(G)-methyl-L-arginine acetate, an inhibitor of iNOS, to evaluate the role of nitric oxide in the killing process. The results obtained indicate that while about 30% killing of input bacteria by control and ACE-Mphis was attributable to NO-mediated killing, only about 6% killing from NO was found with CCE-Mphis. The findings indicate that acute exposure to cocaine possibly caused upregulation of enzymes responsible for the generation of ROI (reactive oxygen intermediates) and RNI (reactive nitrogen intermediates), leading to enhanced antimicrobial function. On the other hand, chronic exposure to cocaine impaired the oxygen-dependent microbicidal capacity of macrophages, possibly through impaired expression of enzymes responsible for ROI and RNI formation. Proinflammatory cytokines may play a key role in cocaine-mediated immunosuppression, since exposure of macrophages to cocaine impairs the ability of the cells to produce these cytokines.