Bacterial lipoprotein induces resistance to Gram-negative sepsis in TLR4-deficient mice via enhanced bacterial clearance

TLRs are highly conserved pathogen recognition receptors. As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis. We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality. In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection. Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S. typhimurium (0.25 x 10(6) CFU/mouse). This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ). Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcgammaIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ). There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice. However, a significant augmented uptake, ingestion, and intracellular killing of S. typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance. These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.     

Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice

Endotoxin from Gram-negative bacteria bound to CD14 signals through Toll-like receptor (TLR) 4, while components of Gram-positive bacteria, fungi, and Mycobacterium tuberculosis (M.tb.) preferentially use TLR2 signaling. We asked whether TLR4 plays any role in host resistance to M.tb. infection in vivo. Therefore, we infected the TLR4 mutant C3H/HeJ mice and their controls, C3H/HeN mice, with M.tb. by aerosol. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10-100 times higher CFU organ levels than the wild-type mice and succumbed within 5-7 mo, whereas most of the wild-type mice controlled infection and survived the duration of the experiment. The lungs of TLR4 mutant mice showed chronic pneumonia with increased neutrophil infiltration, reduced macrophages recruitment, and abundant acid-fast bacilli. Furthermore, the pulmonary expression of TNF-alpha, IL-12p40, and monocyte chemoattractant protein 1 was significantly lower in C3H/HeJ mice when compared with the wild-type controls. C3H/HeJ-derived macrophages infected in vitro with M.tb. produced lower levels of TNF-alpha. Finally, the purified mycobacterial glycolipid, phosphatidylinositol mannosides, induced signaling in both a TLR2- and TLR4-dependent manner, thus suggesting that recognition of phosphatidylinositol mannosides in vivo may influence the development of protective immunity. In summary, macrophage recruitment and the proinflammatory response to M.tb. are impaired in TLR4 mutant mice, resulting in chronic infection with impaired elimination of mycobacteria. Therefore, TLR4 signaling is required to mount a protective response during chronic M.tb. infection.     

Fusobacterium nucleatum induces fetal death in mice via stimulation of TLR4-mediated placental inflammatory response

Intrauterine infection plays a pivotal role in preterm birth (PTB) and is characterized by inflammation. Currently, there is no effective therapy available to treat or prevent bacterial-induced PTB. Using Fusobacterium nucleatum, a Gram-negative anaerobe frequently associated with PTB, as a model organism, the mechanism of intrauterine infection was investigated. Previously, it was shown that F. nucleatum induced preterm and term stillbirth in mice. Fusobacterial-induced placental infection was characterized by localized bacterial colonization, inflammation, and necrosis. In this study, F. nucleatum was shown to activate both TLR2 and TLR4 in vitro. In vivo, the fetal death rate was significantly reduced in TLR4-deficient mice (C57BL/6 TLR4(-/-) and C3H/HeJ (TLR4(d/d))), but not in TLR2-deficient mice (C57BL/6 TLR2(-/-)), following F. nucleatum infection. The reduced fetal death in TLR4-deficient mice was accompanied by decreased placental necroinflammatory responses in both C57BL/6 TLR4(-/-) and C3H/HeJ. Decreased bacterial colonization in the placenta was observed in C3H/HeJ, but not in C57BL/6 TLR4(-/-). These results suggest that inflammation, rather than the bacteria per se, was the likely cause of fetal loss. TLR2 did not appear to be critically involved, as no difference in bacterial colonization, inflammation, or necrosis was observed between C57BL/6 and C57BL/6 TLR2(-/-) mice. A synthetic TLR4 antagonist, TLR4A, significantly reduced fusobacterial-induced fetal death and decidual necrosis without affecting the bacterial colonization in the placentas. TLR4A had no bactericidal activity nor did it affect the birth outcome in sham-infected mice. TLR4A could have promise as an anti-inflammatory agent for the treatment or prevention of bacterial-induced preterm birth.     

Role of Toll-like receptor 4 in endotoxin-induced acute renal failure

Toll-like receptor 4 (TLR4) is present on monocytes and other cell types, and mediates inflammatory events such as the release of TNF after exposure to LPS. C3H/HeJ mice are resistant to LPS-induced mortality, due to a naturally occurring mutation in TLR4. We therefore hypothesized that LPS-induced acute renal failure (ARF) requires systemic TNF release triggered by LPS acting on extrarenal TLR4. We injected C3H/HeJ mice and C3H/HeOuJ controls with 0.25 mg of LPS, and sacrificed them 6 h later for analysis of blood urea nitrogen (BUN) and kidney tissue (n = 8 per group). In contrast to C3H/HeOuJ controls, C3H/HeJ mice were completely resistant to LPS-induced ARF (6-h BUN of 32.3 +/- 1.1 vs 61.7 +/- 5.6 mg/dl). C3H/HeJ mice released no TNF into the circulation at 2 h (0.00 vs 1.24 +/- 0.16 ng/ml), had less renal neutrophil infiltration (6.4 +/- 1.0 vs 11.4 +/- 1.3 neutrophils per high power field), and less renal apoptosis, as assessed by DNA laddering. Transplant studies showed that C3H/HeJ recipients of wild-type kidneys (n = 9) were protected from LPS-induced ARF, while wild-type recipients of C3H/HeJ kidneys (n = 11) developed severe LPS-induced ARF (24-h BUN 44.0 +/- 4.1 vs 112.1 +/- 20.0 mg/dl). These experiments support our hypothesis that LPS acts on extrarenal TLR4, thereby leading to systemic TNF release and subsequent ARF. Renal neutrophil infiltration and renal cell apoptosis are potential mechanisms by which endotoxemia leads to functional ARF.     

Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury

Activation of macrophages via toll-like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non-pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFkappaB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2-/-) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild-type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin-1beta mRNA. In TLR2-/- mice, locomotor recovery also was impaired relative to SCI wild-type controls and novel patterns of myelin pathology existed within ventromedial white matter--an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post-injury sequelae and perhaps in regulating inflammation and gliosis after SCI.     

Systemic inflammation and remote organ injury following trauma require HMGB1

High-mobility group box 1 (HMGB1) is a 30-kDa DNA-binding protein that displays proinflammatory cytokine-like properties. HMGB1-dependent inflammatory processes have been demonstrated in models of sterile injury, including ischemia-reperfusion injury and hemorrhagic shock. Here, we tested the hypothesis that the systemic inflammatory response and associated remote organ injury that occur after peripheral tissue injury are highly dependent on HMGB1. Toll-like receptor 4 (TLR4) wild-type (WT) mice subjected to bilateral femur fracture after treatment with neutralizing antibodies to HMGB1 had lower serum IL-6 and IL-10 levels compared with mice treated with nonimmune control IgG. Similarly, compared with injured mice treated with control IgG, anti-HMGB1 antibody-treated mice had lower serum alanine aminotransferase levels and decreased hepatic and gut mucosal NF-kappaB DNA binding. TLR4 mutant (C3H/HeJ) mice subjected to bilateral femur fracture had less systemic inflammation and liver injury than WT controls. Residual trauma-induced systemic inflammation and hepatocellular injury were not ameliorated by treatment with a polyclonal anti-HMGB1 antibody, even though HMGB1 levels were transiently elevated just 1 h after injury in both WT and C3H/HeJ mice. Collectively, these data demonstrate a critical role for a TLR4-HMGB1 pathway in the initiation of systemic inflammation and end-organ injury following isolated peripheral tissue injury.     

Hepatic ischemia/reperfusion injury involves functional TLR4 signaling in nonparenchymal cells

Endogenous ligands from damaged cells, so-called damage-associated molecular pattern molecules, can activate innate immunity via TLR4 signaling. Hepatic warm ischemia and reperfusion (I/R) injury and inflammation is largely TLR4 dependent. We produced TLR4 chimeric mice to assess whether the TLR4-dependent injury required TLR4 expression on liver parenchymal or nonparenchymal cells. Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mouse bone marrow. Wild-type chimeric mice bearing TLR4 mutant hemopoietic cells and TLR4 mutant mice transplanted with their own bone marrow-derived cells were protected from hepatic I/R and exhibited decreased JNK and NF-kappaB activation compared with WT chimeric mice transplanted with their own bone marrow. In contrast, TLR4 mutant mice transplanted with TLR4 WT bone marrow were not protected from liver I/R and demonstrated pronounced increases in JNK and NF-kappaB activation when compared with autochthonous transplanted mutant mice. In addition, depletion of phagocytes taking up gadolinium chloride failed to provide any additional protection to TLR4 mutant mice, but substantially reduced damage in WT mice after hepatic I/R. Together, these results demonstrate that TLR4 engagement on actively phagocytic nonparenchymal cells such as Kupffer cells is required for warm I/R-induced injury and inflammation in the liver.     

Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a Toll-like receptor 4 mutation

Obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. We have provided in vitro evidence that saturated fatty acids, which are released from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for Toll-like receptor 4 (TLR4) to induce the inflammatory changes in macrophages. Here we show the attenuation of adipose tissue inflammation in C3H/HeJ mice carrying a functional mutation in the TLR4 gene relative to control C3H/HeN mice during a 16-week high-fat diet. We also find that adiponectin mRNA expression is significantly reduced by co-culture of hypertrophied 3T3-L1 adipocytes and C3H/HeN peritoneal macrophages, which is reversed, when co-cultured with C3H/HeJ peritoneal macrophages. This study provides in vivo evidence that TLR4 plays a role in obesity-related adipose tissue inflammation and thus helps to identify the therapeutic targets that may reduce obesity-induced inflammation and the metabolic syndrome.     

Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product

The human homologue of Drosophila Toll (hToll), also called Toll-like receptor 4 (TLR4), is a recently cloned receptor of the IL-1/Toll receptor family. Interestingly, the TLR4 gene has been localized to the same region to which the Lps locus (endotoxin unresponsive gene locus) is mapped. To examine the role of TLR4 in LPS responsiveness, we have generated mice lacking TLR4. Macrophages and B cells from TLR4-deficient mice did not respond to LPS. All these manifestations were quite similar to those of LPS-hyporesponsive C3H/HeJ mice. Furthermore, C3H/HeJ mice have, in the cytoplasmic portion of TLR4, a single point mutation of the amino acid that is highly conserved among the IL-1/Toll receptor family. Overexpression of wild-type TLR4 but not the mutant TLR4 from C3H/HeJ mice activated NF-kappaB. Taken together, the present study demonstrates that TLR4 is the gene product that regulates LPS response.     

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.     

Cutting edge: high-mobility group box 1 preconditioning protects against liver ischemia-reperfusion injury

High mobility group box 1 (HMGB1) is a NF released extracellularly as a late mediator of lethality in sepsis and as an early mediator of inflammation following injury. Here we demonstrate that in contrast to the proinflammatory role of HMGB1, preconditioning with HMGB1 results in protection following hepatic ischemia/reperfusion (I/R). Pretreatment of mice with HMGB1 significantly decreased liver damage after I/R. The protection observed in mice pretreated with HMGB1 was associated with a higher expression of IL-1R-associated kinase-M, a negative regulator of TLR4 signaling, compared with controls. We thus explored the possibility that HMGB1 preconditioning was mediated through TLR4 activation. HMGB1 preconditioning failed to provide protection in TLR4 mutant (C3H/HeJ) mice, but successfully reduced damage in TLR4 wild-type (C3H/HeOuj) mice. Our studies demonstrate that in contrast to the role of HMGB1 as an early mediator of inflammation and organ damage in hepatic I/R, HMGB1 preconditioning can be protective.     

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.     

Membrane TNF confers protection to acute mycobacterial infection

Background

Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).

Methods

C57BL/6, TNF KO and mem-TNF mice were infected with M. tuberculosis H37Rv (Mtb at 100 CFU by intranasal administration) and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.

Results

While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.

Conclusion

Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.     

BCG-induced enhancement of endotoxin sensitivity in C3H/HeJ mice. I. In vivo studies

C3H/HeJ mice exhibit a marked insensitivity to bacterial lipopolysaccharide (LPS) in vivo. Pretreatment of these mice with viable BCG organisms 11 days before LPS administration renders them sensitive to the lethal effects of a highly purified, phenol-extracted LPS. Other in vivo responses to LPS are increased in BCG-infected C3H/HeJ mice in parallel with enhanced lethality. These include 1) the elevation of serum interferon, 2) the production of the acute phase reactant, serum amyloid A (SAA), and 3) hypoglycemia. However, BCG infection has only a minimal effect on anti-LPS antibody production. BCG-infected C3H/HeJ mice approach the LPS sensitivity of normal C3H/HeN mice, but the enhanced LPS sensitivity is transient and decreases over a 2-month period. The ability of BCG to induce LPS sensitivity in C3H/HeJ mice demonstrates that LPS unresponsiveness is not due to an absolute defect in this strain, but rather, a partially reversible state of hyporesponsiveness. In addition, these findings, in conjunction with other observations, suggest that the enhancement of LPS sensitivity induced by BCG infection is mediated primarily through an effect on T cells and/or macrophages rather than B lymphocytes.     

Oral administration of poly-gamma-glutamate induces TLR4- and dendritic cell-dependent antitumor effect

Previously, we reported that the oral administration of high molecular mass poly-γ-glutamate (γ-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass γ-PGA induced secretion of tumor necrosis factor (TNF)-α from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2^−/−) mice, but not those of myeloid differentiation factor 88 knockout (MyD88^−/−) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-γ-inducible protein 10 (IP-10) in response to treatment with γ-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, γ-PGA induced productions of TNF-α and IP-10 could not be blocked by polymyxin B. Furthermore, γ-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88^−/− and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited γ-PGA-induced TNF-α secretion from the RAW264.7 cells. γ-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of γ-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa γ-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of γ-PGA is mediated by TLR4. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Defective tumoricidal capacity of macrophages from C3H/HeJ mice

Peritoneal macrophages from C3H/HeN mice treated i.p. with T cell mitogens or viable BCG organisms were cytotoxic to syngeneic tumor cells in vitro. Macrophages from endotoxin-unresponsive C3H/HeJ mice treated with BCG or T cell mitogens, however, were not tumoricidal. Furthermore, unlike cells from C3H/HeN mice, macrophages from C3H/HeJ mice could not be activated for tumor cytotoxicity after in vitro treatment with bacterial endotoxins or with lymphokine-rich supernatants. The subnormal induction of cytotoxic macrophages after in vitro or in vivo treatments in C3H/HeJ mice appears to be a highly selective defect. Macrophage responses (yield, phagocytosis, or peroxidase staining) in inflammatory exudates induced by BCG, T cell mitogens, or heterologous serum in C3H/HeJ or C3H/HeN mice were identical. C3H/HeJ macrophages also responded normally in vitor to chemotactic lymphokines. Thus, C3H/HeJ macrophages possess a profound and selective defect in tumoricidal capacity. This defect was not dependent upon exogenous endotoxins. Defective macrophage cytotoxic responses may reflect non-LPS related functions regulated by the LPS gene.     

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.     

Novel interactions of a microbial superantigen with TLR2 and TLR4 differentially regulate IL-17 and Th17-associated cytokines

Mycoplasma arthritidis, an inflammatory murine pathogen, secretes a potent superantigen, Mycoplasma arthritidis mitogen (MAM) that contributes to toxic shock, arthritis and skin necrosis. Previously we showed that MAM induced type 2 T-cell cytokines in mice that express functional TLR2 and TLR4, but type 1 cytokines in mice that lack TLR4 function. We show here that IL-17, pSTAT3 and retinoid-related orphan nuclear receptorγt are rapidly expressed in wild-type C3H/HeSnJ (TLR2+/4+) mice but are significantly delayed in mutant C3H/HeJ (TLR2+/4-) mice. This marked kinetic difference was associated with a high level of IL-6 in TLR2+/4+ mice versus high levels of IL-1β and TNFα in TLR2+/4- mice. Also antibodies to IL-6 and IL-23, suppressed IL-17 responses to MAM in TLR2+/4+ mice whereas anti-IL-1β, but not anti-TNFα, enhanced IL-17 in TLR2+/4- mice. Antibody blocking of TLR4 in TLR2+/4+ mice decreased IL-17 and IL-6 but not IL-23. In addition both IL-17 and IL-6 but not IL-23 were elevated in TLR2 KO mice versus wild-type TLR2+/4+ mice given MAM. We conclude that the MAM interaction with TLR2 versus TLR4 leads to distinct cytokine pathways mediated primarily by IL-1β or IL-6/IL-17 signalling respectively. Our findings suggest that the differential interaction of MAM with different TLRs might play an important role in disease outcomes by M. arthritidis.     

Lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS

The present study deals with whether lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) is related to LPS-susceptibility of either mother or fetus and how LPS or LPS-induced TNF causes IUFD. LPS-susceptible C3H/HeN or -hypo-susceptible C3H/HeJ pregnant mice and the mice mated reciprocally with these mice were used on days 14 to 16 of gestation for experiments. All of fetuses in pregnant C3H/HeN mice mated with either C3H/HeN males [HeN(HeN)] or C3H/HeJ males [HeN(HeJ)] were killed within 24 hr when injected intravenously (i.v.) with 50 or 100 microg of LPS. On the other hand, the majority of fetuses in C3H/HeJ females mated with either C3H/HeJ males [HeJ(HeJ)] or C3H/HeN males [HeJ(HeN)] survived when injected i.v. with even 400 microg of LPS. These findings indicate that LPS-induced IUFD depends on the maternal LPS-responsiveness. LPS injected into mothers could pass through placenta to fetuses, since an injection with 125I-labeled LPS or IgG into pregnant mice resulted in considerable levels of radioactivity in fetuses as well as placenta. Cultured peritoneal macrophages derived from F1 mice of HeJ(HeN) or HeN(HeJ) mice, produced nitric oxide (NO) and tumor necrosis factor (TNF) in response to LPS, although the levels of NO and TNF were lower in comparison with those of C3H/HeN macrophage cultures, suggesting a possibility that the fetus as well as F1 cells might be responsible to LPS. LPS-induced IUFD was not blocked by treatment with anti-TNF antibody which inhibited LPS-induced TNF production in pregnant females, although an injection of recombinant TNFalpha instead of LPS could induce IUFD, suggesting that the cause of IUFD cannot be attributed to mother-derived TNF alone. The roles of LPS passed through placenta and LPS-induced mediators on IUFD were discussed.     

Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma

Macrophage synthesis of nitrite and nitrate after activation by BCG infection or by treatment in vitro with both T cell-derived (lymphokines (LK) or recombinant murine interferon-gamma (IFN-gamma] and bacterial (lipopolysaccharide (LPS) and heat-killed bacillus Calmette-Guerin (hk BCG] agents was studied by using macrophages from C3H/He and C3H/HeJ mice. Spleen and peritoneal macrophages isolated from BCG-infected donors that were producing nitrate continued to synthesize nitrite and nitrate in culture. LPS treatment in vitro (25 or 50 micrograms/ml) additionally increased this nitrite/nitrate synthesis. Thioglycolate-elicited macrophages from non-infected C3H/HeJ mice treated with LK also produced nitrite/nitrate, and concurrent LPS (0.1 to 50 micrograms/ml) treatment resulted in enhanced synthesis. Recombinant IFN-gamma also stimulated nitrite/nitrate synthesis by C3H/He and CeH/HeJ macrophages as did LPS (C3H/He only) and hk BCG. When given concurrently with either LPS or hk BCG, IFN-gamma enhanced C3H/He and C3H/HeJ macrophage nitrite/nitrate synthesis over that produced by macrophages treated with either LPS or hk BCG alone. Macrophages activated in vitro exhibited a 4 to 12 hr lag time before engaging in nitrite/nitrate synthesis, which then proceeded for 36 to 42 hr at linear rates. Daily medium renewal did not alter the synthesis kinetics but increased the total amount of nitrite/nitrate produced. Nitrate and nitrite were stable under the conditions of culture and when added did not influence additional macrophage synthesis. Taken together, these results indicate that T cell lymphokines and IFN-gamma are powerful modulators of macrophage nitrite/nitrate synthesis during BCG infection and in vitro, and nitrite/nitrate synthesis appears to be common property of both primed and fully activated macrophage populations.