Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein-/- mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.     

Effects of Coxiella burnetii on MAPKinases phosphorylation

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.     

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.     

Visualization of the molecular dynamics of lipopolysaccharide on the plasma membrane of murine macrophages by total internal reflection fluorescence microscopy

The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4(-/-), MD2(-/-), MyD88(-/-), and TRIF(-/-) macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively.     

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

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

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages

There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27(p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. In this article, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (polyinosinic-polycytidylic acid) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LBP completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28)-producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)(+)F4/80(+)C5aR(+) phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor rather than the C5L2 receptor. After endotoxemia, C5aR(-/-) mice displayed higher plasma levels of IL-27(p28) compared with C57BL/6J mice. C5a did not affect the release of IL-27(p28) or the frequency of IL-27(p28)(+)F4/80(+) macrophages after engagement of TLR3. Mechanistically, LPS activated both the NF-κB and the PI3K/Akt pathways, whereas C5a activated only the PI3K/Akt pathway. Engagement of PI3K/Akt was inhibitory for IL-27(p28) production, because PI3K/Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K/Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, the PI3K/Akt pathway was not involved in TLR3-mediated release of IL-27(p28). These data provide new evidence about how complement activation may selectively interfere with production of T cell regulatory cytokines by APCs in the varying contexts of either bacterial (TLR4 pathway) or viral (TLR3 pathway) infection.     

Innate immune responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.     

Mouse resident peritoneal macrophages partially control in vitro infection with Coxiella burnetii phase II

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is a small, dimorphic, obligate intracellular bacterium, sheltered within large, acidified, and hydrolase-rich phagosomes. Although several primary and established cell lines, macrophage-like cells, and primary macrophages from other species have been infected with C. burnetii, the infection of mouse primary macrophages has not been sufficiently characterized. In this report quantification of DAPI (4', 6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy, and transmission electron microscopy were used to compare the infection of three mouse-derived cells, L929 fibroblasts, J774 macrophage-like cells, and resident peritoneal macrophages, with a phase II clone of C. burnetii known to be non-virulent for mammals. Infected peritoneal phagocytes differed from L929 or J774 cells in that: (a) large vacuoles took longer to appear (3-5 d instead of 2), and were only found in a subset (20-30%) of macrophages, as opposed to in more than 70% of the other cells; (b) total and vacuole-associated relative bacterial loads in L929 and J774 cells were several-fold higher than in peritoneal macrophages; (c) estimated doubling times of the bacteria were about 68 h in the primary macrophages, 18 h in J774 and 22 h in L929 cells. Thus, mouse resident peritoneal macrophages control both the formation of the large vacuoles and the intracellular proliferation of C. burnetii phase II.     

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A(-/-)), TLR4(-/-) and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4(-/-) or SR-A(-/-) macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.     

The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung

TLRs are important for the recognition of conserved motifs expressed by invading bacteria. TLR4 is the signaling receptor for LPS, the major proinflammatory component of the Gram-negative cell wall, whereas CD14 serves as the ligand-binding part of the LPS receptor complex. Triggering of TLR4 results in the activation of two distinct intracellular pathways, one that relies on the common TLR adaptor MyD88 and one that is mediated by Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF). Nontypeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen that expresses both TLR4 (LPS and lipooligosaccharide) and TLR2 (lipoproteins) ligands. To determine the roles of CD14, TLR4, and TLR2 during NTHi pneumonia, the following studies were performed: 1) Alveolar macrophages from CD14 and TLR4 knockout (KO) mice were virtually unresponsive to NTHi in vitro, whereas TLR2 KO macrophages displayed a reduced NTHi responsiveness. 2) After intranasal infection with NTHi, CD14 and TLR4 KO mice showed an attenuated early inflammatory response in their lungs, which was associated with a strongly reduced clearance of NTHi from the respiratory tract; in contrast, in TLR2 KO mice, lung inflammation was unchanged, and the number of NTHi CFU was only modestly increased at the end of the 10-day observation period. 3) MyD88 KO, but not TRIF mutant mice showed an increased bacterial load in their lungs upon infection with NTHi. These data suggest that the MyD88-dependent pathway of TLR4 is important for an effective innate immune response to respiratory tract infection caused by NTHi.     

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.     

Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-alpha, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-gamma. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-alpha production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.     

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.     

Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb

Previously we demonstrated that basolateral LPS inhibits HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through TLR4-dependent ERK activation. Here we report that the response of the MTAL to basolateral LPS requires TLR2 in addition to TLR4. The basolateral addition of LPS (ultrapure Escherichia coli K12) decreased HCO(3)(-) absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2(-/-) mice. In contrast, inhibition of HCO(3)(-) absorption by lumen LPS was preserved in TLR2(-/-) MTALs, indicating that TLR2 is involved specifically in mediating the basolateral LPS response. LPS also did not increase ERK phosphorylation in MTALs from TLR2(-/-) mice. TLR2 deficiency had no effect on expression of TLR4, MD-2, or MyD88. However, LPS-induced recruitment of MyD88 to the basolateral membrane was impaired in TLR2(-/-) MTALs. Inhibition of HCO(3)(-) absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO(3)(-) absorption by TLR2-specific ligands was preserved in MTALs from TLR4(-/-) mice. These results indicate that the effect of basolateral LPS to inhibit HCO(3)(-) absorption in the MTAL through MyD88-dependent ERK activation depends on a novel interaction between TLR4 and TLR2. TLR2 plays a dual role in the induction of intracellular signals that impair MTAL function, both through cooperation with TLR4 to mediate ERK signaling by LPS and through a TLR4-independent signaling pathway activated by Gram-positive bacterial ligands. Regulation of TLR2 expression and its interaction with TLR4 may provide new mechanisms for controlling and therapeutic targeting of TLR4-mediated LPS responses.