TLR4 KO Research Articles

Contribution of IPS-1 to polyI:C-induced cytokine production in conjunctival epithelial cells

We previously demonstrated that ocular surface epithelium expressed TLR3 and that its ligand, polyI:C, stimulation induced the secretion of inflammatory cytokines and type I IFN. It was recently reported that RIG-I and MDA5 also recognize viral dsRNA mimicking polyI:C. In this study, we investigated whether RIG-I and/or MDA5 contribute to polyI:C-inducible responses in conjunctival epithelium. The expression of RIG-I, MDA5, and TLR3 in human conjunctival epithelium was examined by RT-PCR and their up-regulation after polyI:C stimulation by quantitative RT-PCR and immunoblot analysis. Human conjunctival epithelial cells also expressed RIG-I, MDA5 and TLR3 mRNA and protein. The expression of RIG-I and MDA5, but not of TLR3, was markedly up-regulated upon polyI:C stimulation. We also examined the function of IPS-1 (an adaptor molecule common to RIG-I and/or MDA5) and TLR3 in conjunctival epithelium using IPS-1 KO and TLR3 KO mice. To analyze in vivo murine conjunctival epithelial cells, 10μl of a 100μg/ml polyI:C solution were delivered subconjunctivally and as eye drops, then conjunctival epithelial cells were subjected to gene expression analysis. We focused on 10 transcripts up-regulated in murine conjunctival epithelium upon polyI:C stimulation. Cxcl10, Mx1, Ifi44, Ifi203, Iigp2 and Rtp4 were dominantly regulated by IPS-1, Ccl5 by TLR3, and Rsad2, Mx2 and Cmpk2 were regulated by TLR3 and IPS-1. Our results showed that conjunctival epithelial cells express RIG-I and MDA5, and IPS-1, an adaptor molecule common to RIG-I and MDA5, contributes to polyI:C-inducible cytokine production in conjunctival epithelial cells.

The role of TLR2 in nerve injury‐induced neuropathic pain is essentially mediated through macrophages in peripheral inflammatory response

Activation of macrophages/microglia via toll-like receptors (TLRs) plays an important role in inflammation and host defense against pathogens. Pathogen-associated molecular patterns bind TLRs, thereby triggering NF-κB signaling and production of proinflammatory cytokines. Recent data suggest that nonpathogenic molecules resulting from trauma can also trigger inflammation via TLRs. We sought to determine whether peripheral nerve injury could induce the expression of TLR2 on the site of injury-damaged nerves and/or in the central nervous system and to investigate whether TLR2 is necessary for the development of nerve injury-induced neuropathic pain. We observed a significant increase in TLR2, IκB-α, and TNF-α mRNAs in damaged nerves. Increased inflammation-related molecules were found essentially on ED1(+) macrophages. Expression of both IκB-α and TNF-α in peripheral injured nerves was reduced in TLR2 deficient mice where the recruitment of ED1(+) cells is significantly impaired. Although after peripheral nerve injury, spinal microglia became highly activated showing an increase in Iba-1 immunoreactivity and an enlargement of their cell bodies, neither TLR2 mRNA nor IκB-α mRNA was detected in activated microglia. Nerve injury-evoked spinal microglial activation was not significantly altered in TLR2 KO mice. Paw withdrawal threshold and latency in response to mechanical and heat stimuli, respectively, decreased shortly after nerve lesion in wild type mice. In TLR2 KO mice, nerve injury-induced thermal hyperalgesia was completely abolished contrary to that seen in wild-type mice, whereas mechanical allodynia was partially reduced. We suggest that TLR2 is necessary for the development of neuropathic pain and its contribution is more important in thermal hypersensitivity than that of mechanical allodynia.

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.

PS204. Toll-like Receptors 2 and 4, but Not RAGE Deficiency Results in Greater Muscle Necrosis 2 Weeks After Hind-limb Ischemia: Implications for Muscle Recovery After Ischemic Injury

Innate immunity is the first line of defense against invading pathogens through receptors such as TLR2 and TLR4. These receptors also mediate responses to tissue injury through endogenous danger signals. We have shown that HMGB1, a danger signal, is released by hypoxic cells and promotes angiogenesis in vitro. Anti-HMGB1 antibody increases muscle necrosis in murine hind-limb ischemia but did not change endothelial density, supporting a role for HMGB1 and innate immunity in skeletal muscle recovery following ischemia. In this study, we examined the roles of the HMGB1 receptors TLR2, TLR4 and RAGE in mediating this protection. 10-12 wk old mice genetically deficient for TLR4 (C3H/HeJ, control HeOuJ; N = 4), TLR2 (N = 3), or RAGE (N = 4; control C57B/6, N = 4) underwent right femoral artery ligation. At 2wks, calf muscles were fixed, sectioned, and stained with H&E to quantify muscle necrosis (% area) and vWF for endothelial density. Statistical significance was determined by ANOVA. TLR4 mutant mice developed greater muscle necrosis following hind-limb ischemia than control HeOuJ mice (16.4% ± 4.6 vs 4.6 ± 2.1; p = 0.04). Muscle necrosis was also markedly increased in TLR2 KO (23.5 ± 17.3; P-0.04) while no necrosis was detected in the C57B/6 controls. RAGE KO mice also showed little necrosis. In contrast, the C57B/6 and RAGE KO mice exhibited significant muscle regeneration in the ischemic hind-limb. Endothelial density was similar between the knockout and control mice. The absence of TLR2 and TLR4 signaling is associated with increased skeletal muscle necrosis following ischemia and suggests that these receptors are necessary for muscle protection. In contrast, RAGE signaling is not involved. The protection offered by TLR2 and TLR4 did not involve endothelial proliferation but may involve regenerative processes. These data suggest that TLRs may be critical in tissue recovery from ischemia independent of angiogenesis.

74 Toll-Like Receptor (TLR) 2 in Kupffer Cells Plays a Crucial Role in the Development of Steatohepatitis in Mice

Background: Gut-derived bacterial products are candidates for a promoting factor to progress non-alcoholic steatohepatitis (NASH). Toll-like receptors are pattern recognition receptors that sense microbial products such as LPS. We have previously shown that TLR9, a receptor for bacterial DNA, plays an important role in the development of NASH. However, little information on TLR2, a receptor for Gram-positive bacterial products such as peptidoglycan, is available in that process. In addition, the role of TLR2 in Kupffer cells, hepatic macrophages, remain unknown. Materials and Methods: WT and TLR2 KO were fed choline-deficient (CDAA) or Choline-supplemented control (CSAA) diet for 22 weeks. Steatosis, inflammation and fibrosis were assessed by histological examination, quantitative real-time PCR and ELISA. To examine the role of Kupffer cells in the development of steatohepatitis, WT mice were injected clodronate to deplete Kupffer cells, followed by CDAA diet feeding for 2 weeks. To further explore whether Kupffer cells mediate TLR2-dependent inflammation and fibrosis, we generated TLR2-chimeric mice using a combination of Kupffer cell depletion, irradiation and bone-marrow transplantation, followed by CDAA diet feeding for 22 weeks. Results: After 22 weeks of CDAA diet feeding, WT mice showed typical steatohepatitis characterized by severe steatosis, inflammatory cell infiltration and hepatocyte ballooning as well as perisinusoidal fibrosis. In contrast, TLR2 KOmice exhibited less inflammatory cell infiltration and hepatocyte ballooning. NAFLD activity score in TLR2KO mice was significantly lower than that in WT mice. In addition, TLR2KO mice showed a significant reduction in fibrosis compared with WT mice. In Kupffer cells-depleted WT mice, diet-induced steatohepatitis and fibrosis were significantly suppressed compared with WT mice without Kupffer cell depletion. In chimeric mice, full reconstitution of Kupffer cells was achieved, but hepatic stellate cells were not replaced by bone marrow-derived cells. WT mice containing Tlr2deficient bone marrow showed suppressed steatohepatitis and fibrosis, whereas Tlr2-deficient mice containing wild-type bone marrow showed advanced steatohepatitis and fibrosis. Conclusion: TLR2 expression in Kupffer cells plays a crucial role in CDAA diet-induced steatohepatitis and fibrosis in mice.

Toll-like receptor 4 (TLR4) and interferon-gamma (IFN-r) are required for HMGB1-induced TNF release from macrophages. (34.18)

Abstract High mobility group box 1 (HMGB1) has been implicated as a cytokine mediator in the pathogenesis of inflammatory diseases (Yang et al. BBA, 2009). To study mechanisms of HMGB1 action, we produced mammalian HMGB1, devoid of bacterial products, and examined receptor signaling for HMGB1 mediated macrophage cytokine release. HMGB1 induced significant TNF release in macrophages from TLR2 KO, RAGE KO and wild type (WT) mice (n = 4-5 experiments). HMGB1 induced TNF release was completely abolished in TLR4 KO macrophages as compared to WT. Interestingly, we observed that HMGB1 induced TNF mRNA expression, but not TNF protein release, from TLR4 KO mouse macrophages. Pre-treatment of TLR4 KO macrophages with IFN-r restored HMGB1-induced TNF protein release. Thus, our results indicate that TLR4 is required for HMGB1-mediated cytokine release from macrophages, and that IFN-r can restore HMGB1-induced TNF release in TLR4 KO macrophages. (supported in part by grant from NIH, NIGMS, to KJT).

Hemorrhagic shock primes for IL-1β processing: role of upregulated TLR2 (136.1)

Abstract IL-1β contributes to post-trauma sepsis, and its release requires processing in inflammasomes. Hemorrhagic shock (HS) primes for IL-1β release in response to LPS via yet unclear mechanisms. We have reported that HS-activated neutrophils (PMN) augment LPS/TLR4 signaling upregulation of TLR2 in alveolar macrophages (AM), thereby sensitizing the AM to TLR2 ligands. Here, we addressed the role of upregulated TLR2 in the HS-primed IL-1β processing. WT, TLR4-mutant and TLR2 KO mice were subjected to HS, followed by intratracheal LPS (30 µg/kg B.W.) and PGN (30 µg/kg B.W.) at 2h and 4h after HS, respectively. Bronchoalveolar lavage fluids (BALF) were collected at 2 h after PGN for detection of IL-1β. In vitro, AM were co-cultured with PMN isolated from HS mice, and treated with LPS, PMN was then removed and PGN was added at 2h. IL-1β and caspase-1 maturation in the AM were assessed. In vivo treatments of LPS and PGN caused a marked increase in IL-1β in BALF in the WT/HS mice, but not in the WT/sham, TLR4-mutant/HS or TLR2 KO/HS mice. HS-activated PMN increased TLR2 expression in AM in response to LPS, and subsequently increased caspase-1 and IL-1β maturation in the AM as well as IL-1β release in response to PGN. Moreover increasing extracellular [K+] failed to prevent the TLR2 signal-induced IL-1β maturation. In HS, amplified TLR2 signaling acts as a second signal in stimulating caspase-1 and IL-1β maturation via mechanisms independent of K+ efflux.

TLR4 and RAGE Function Are Not Essential for Angiogenesis in a Model of Hind-Limb Ischemia

Introduction: Inflammation is an important feature of the angiogenic response. Innate immunity is the first line of defense against invading pathogens through pattern recognition receptors such as TLR4 and RAGE. It also mediates inflammation following tissue injury through endogenous ligands called danger signals that bind the same receptors. We have previously shown that HMGB-1, a highly conserved nuclear protein and danger signal, is released by ischemic skeletal muscle and hypoxic cells and promotes angiogenic activity in vitro. HMGB-1 blocking antibody dramatically inhibits endothelial tube formation and increases tissue necrosis in murine hind-limb ischemia, suggesting that innate immunity mediates angiogenesis. Receptors for HMGB-1 include TLR4 and RAGE. Our hypothesis is that signaling through these receptors may be necessary for ischemia-induced angiogenesis. Methods: C3H/HeJ (TLR4 mutant; N=6) and C3H/HeOuJ (wildtype; N=6) mice underwent femoral artery ligation on the right hind limb and sham dissection on the left. Laser doppler perfusion imaging (LDPI) was used to measure tissue perfusion and results are expressed as a ratio of right limb:left limb perfusion. Hind-limb ischemia was also created in RAGE knockout mice (N=4) and control C57/B6 mice (N=4). Muscle samples were collected at 2 wks, sectioned, and stained for von Willebrand's factor. Results: Femoral artery ligation resulted in significant ischemia in both HeJ and HeOuJ mice, resulting in limping and toe necrosis. By 2 wks, the ratio of perfusion in ischemic compared to nonischemic limbs was ∼2-fold greater in HeJ mice than in HeOuJ mice (0.89±0.30 vs. 0.48±0.23, p=0.023; Figure). In RAGE KO and control mice, femoral artery ligation resulted in initial limb dysfunction. By 2 wks, the function of ischemic legs returned essentially to normal in both RAGE KO and control mice. Vascular density in the ischemic muscle was similar in RAGE KO and control mice (13.1±0.5 vs 14.9±0.8 vessels/HPF). Interestingly, the severity of ischemic injury and degree of recovery varied significantly between the mouse strains with C3H demonstrating greater tissue injury with less recovery vs C57/B6 mice. Conclusions: Our results reveal greater recovery of perfusion following hind-limb ischemia in mice lacking functional TLR4. Our previous studies revealed HMGB-1 to be pro-angiogenic and contrary to our hypothesis, our current experiments suggest that angiogenesis following ischemia does not require intact TLR4 signaling. Similarly, the presence of functional RAGE did not increase vascular density after ischemic injury. Thus, other HMGB-1 receptors may be involved in mediating effects on angiogenesis. Alternatively, the absence of TLR4 or RAGE may upregulate other innate immune pathways that compensate for the absence of the receptor. Finally, differences in the response to hind-limb ischemia between C3H and C57/B6 mice may compromise the ability to compare the angiogenic responses between TLR4 and RAGE KO mice.

MYD88‐dependent and ‐independent activation of TREM‐1 via specific TLR ligands

Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in myeloid cell-activated inflammatory responses. Although TLR ligands such as LPS and lipoteichoic acid have been shown to upregulate TREM-1 expression in macrophage and neutrophils, the role of specific TLR in inducing the expression of TREM-1 remains unclear. In this study, we investigated whether the presence of TLR is necessary for the expression of TREM-1. We show that BM-derived macrophages from TLR4 and TLR2 KO mice failed to induce expression of TREM-1 message and protein in response to their specific ligands. Interestingly, the expression of TREM-1 in response to LPS is not altered in myeloid differentiation factor 88 (MyD88) KO macrophages, suggesting that downstream of TLR a MyD88-independent pathway induces the expression of TREM-1. Inhibiting toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) expression by siRNA decreased TREM-1 expression in response to LPS, suggesting that the expression of TREM-1 in response to LPS was mediated by the TRIF signaling pathway. On the other hand, the expression of TREM-1 in response to lipoteichoic acid is dependent on MyD88 expression. These data indicate that the expression of TREM-1 in response to TLR ligands occurs secondary to downstream signaling events and that the presence of TLR is necessary for the expression of TREM-1 in response to their specific ligands. However, the downstream signaling required for the expression of TREM-1 is dependent on the stimulus and the surface receptor through which the signaling is initiated.

Increased circulating heat shock protein 60 induced by menopause, stimulates apoptosis of osteoblast-lineage cells via up-regulation of toll-like receptors

Postmenopausal osteoporosis is a heterogeneous disorder characterized by accelerated bone loss after natural or surgical menopause and an increased risk of fractures. The bone loss in estrogen deficiency results from the increased bone resorption and impaired ability of osteoblastic bone formation. Previous studies have reported that the HSP60 stimulates osteoclast formation and bone resorption. Here we found that plasma HSP60 levels were significantly higher in postmenopausal (median 1152.4 ng/ml; range 724.7–2123.4 ng/ml) than in premenopausal (median 316.3 ng/ml; range 164.6–638.4 ng/ml) women. In primary human bone marrow stromal cells (hBMSC) and the HS-5 hBMSC cell line, HSP60 significantly reduced cell viability and increased caspase-dependent apoptosis. Consistent with these observations, HSP60 activated caspase-3 and -9, but not caspase-8 in HS-5 cells, and increased the release of mitochondrial cytochrome c into the cytosol. In addition, HSP60 activated p38 and NFκB, but not ERK or JNK; importantly, inhibitors of p38 (SB203580) and NFκB (PDTC) abolished HSP60-induced apoptosis. Furthermore, Western blotting showed that HSP60 up-regulated TLR-2 and TLR-4 expression, and pretreatment with blocking antibodies for TLR-2 and TLR-4 almost completely eliminated the effects of HSP60 on apoptosis, caspase-3 and -9 activation, and activation of NFκB and p38 MAPK. Most notably, ovariectomy-induced bone loss was attenuated in TLR-2 KO mice. In conclusion, up-regulation of TLR-2 by HSP60 may play a critical role in promoting bone loss in the estrogen-deficient state.

Abstract 350: Toll-like Receptor 2 Activation, Especially In Circulating Leukocytes, Mediates Myocardial Ischemia/Reperfusion Injury In Mice

OBJECTIVES. Myocardial ischemia/reperfusion (MI/R) injury is characterized by an inflammatory response through NF-κB, increase of infarct size and worsening of cardiac function. Toll-like receptors (TLRs) are part of innate immunity and initiate the same inflammatory reaction. Here we studied in vivo to what extent TLR2 is involved in myocardial damage and what the relative contribution is of TLR2 expression in parenchymal cells and leukocytes to myocardial damage during MI/R in mice. METHODS. C57Bl6J mice underwent 30 minutes ischemia - 24 hours of reperfusion. Four experimental groups were studied: TLR2 knock-out (TLR2 KO) mice (n=10), saline treated wild-type (WT) mice (n=10), generated chimeric WT mice with TLR2 KO bone marrow (BLOOD KO; n=7) and chimeric TLR2 KO mice with WT hematopoietic cells (ORGAN KO; n=7). Saline was administered via the tail vein 5 minutes prior to reperfusion. After 24 hours, the LCA was ligated again at the level marked by the suture left in place. Mice were terminated and infarct size (IS) was measured as a percentage of the area at risk (AAR) using 4% Evans’ blue dye injection in the aortic root and triphenyltetrazolium chloride (TTC) staining (fig. 1). Data are presented as Mean±SEM. RESULTS. The AAR as percentage of the left ventricle was similar between groups: TLR2 KO 41%, saline 41%, Blood KO 41%, Organ KO 42%. Saline treatment resulted in 34.5%±3.3 of infarction, whereas in TLR2 KO mice infarct size decreased to 23.0%±2.9 (p=0.029 vs. saline). Infarct size in BLOOD KO mice was 22.9%±2.7 (p=0.024 vs. saline), while ORGAN KO mice had 33.9%±3.2 (p=0.998 vs. saline) of infarction within the area at risk (fig. 2). CONCLUSION. TLR2 deficiency significantly reduces infarct size with ~33% compared to saline treatment in mice after 30 minutes of ischemia and 24 hours of reperfusion. We show for the first time that TLR2 expression in circulating leukocytes plays an important role in infarction after MI/R injury. Systemic inhibition of TLR2 may be a potential therapeutic target in the treatment of patients with acute myocardial infarction.

Lymphocytic Choriomeningitis Virus (LCMV) infection of CNS glial cells results in TLR2-MyD88/Mal-dependent inflammatory responses

In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild-type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-α. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.

Histoplasma capsulatum yeast phase-specific protein Yps3p induces Toll-like receptor 2 signaling

Histoplasma capsulatum is a common cause of fungal infection in certain geographic areas, and although most infections are asymptomatic, it is capable of causing histoplasmosis, a disseminated, life-threatening disease, especially in immunocompromised individuals. A deeper understanding of this host-pathogen interaction is needed to develop novel therapeutic strategies to counter lethal infection. Although several lines of evidence suggest that this fungus is neurotropic in HIV patients, little is known about the immunobiology of Histoplasma infection in the central nervous system [CNS]. The goal of the present study was to understand the innate neuroimmune mechanisms that recognize H. capsulatum during the initial stages of infection. Using a 293T stable cell line expressing murine Toll-like receptor 2 [TLR2], we show here that TLR2 recognizes H. capsulatum cell wall protein Yps3p and induces the activation of NF-κB. In further experiments, we tested the ability of Yps3p to induce signaling from TLR2 in primary microglial cells, the resident brain macrophages of the CNS. Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2. Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors. This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

Decreased implantation rate after cervicovaginal exposure to lipopolysaccharide, a potential pathway of decreased uterine receptivity by Toll-Like receptor activation

To determine whether cervicovaginal exposure to lipopolysacharride influences implantation efficiency in the mouse and, if so, to determine whether a potential mechanism includes activation of the innate immune system via ligation of a Toll-Like receptor, TLR-4. Comparison of number and size of embryos in both wild type (WT) and TLR-4 knockout mice (TLR4 KO) after cervico-vaginal exposure to lipopolysacharride (LPS), a specific ligand of TLR-4. Female wild-type (WT), n = 8, and TLR-4 knockout mice (TLR4 KO) n = 4, were treated daily with ultra-pure lipopolysacharride (LPS), 1 μg per day in PBS carrier, or an equal volume of PBS carrier for controls. Exposure to LPS was via atraumatic placement in the vagina. All animals were housed in a pathogen-free environment and treated for 3 to 4 days prior to pairing with males of the same respective background (WT or TLR4 KO). Treatment continued until six days post vaginal plug visualization. On days ten to twelve post-mating, the animals were sacrificed via CO2 asphyxiation and inspected for embryo number and size. None of the WT mice that were treated with LPS showed evidence of embryonic implantation. All other mice became pregnant after mating, including the TLR-4 KO mice that were treated with LPS, with similar numbers and sizes of viable implanted embryos. Cervicovaginal exposure to LPS is associated with altered implantation rates in the wild-type mouse. As suggested by this study, activation of TLR-4 by LPS may play an integral role in creating an intrauterine environment that is suboptimal for implantation. The influence of innate immune system activation via Toll-like receptors on embryonic implantation represents an attractive area for future investigation. Though the endometrium appears to be a likely candidate for TLR activation, further work is needed to confirm the location of activated TLR's, including those which may be expressed by the pre-implantation embryo, and to define the related molecular signals that produce an environment that is inhospitable to implantation.

Activation of toll-like receptor 3 protects against DSS-induced acute colitis

Mimetics of bacterial DNA, given orally or subcutaneously, protect mice from experimental colitis via a toll-like receptor (TLR)-9-dependent mechanism. The goal of the study was to define whether synthetic viral RNA, polyinosinic acid:cytidylic acid [poly(I:C)], which is also a potent immunomodulator, might also affect murine colitis and, if so, define whether such effects were mediated by TLR3, which is one of at least 4 known receptors for this viral RNA analog. Mice (C57BL6, IL-10KO, or TLR3 KO) were administered 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days. Two hours before treatment with DSS, mice were given phosphate-buffered saline (PBS) or poly(I:C) 20 mug subcutaneously (s.c.), or 100 mug intragastrically (i.g.). In wildtype mice s.c. administration of poly(I:C) dramatically protected against DSS-induced colitis as assessed by every parameter analyzed, which included body weight, rectal bleeding, colonic myeloperoxidase, histopathology, serum keratinocyte-derived chemokine, serum amyloid A, and lipocalin-2. In contrast, i.g. administration of poly(I:C) offered no protection in this colitis model nor did its administration activate the innate immune system as assessed by serologic parameters. Subcutaneous poly(I:C) protected against DSS-induced colitis equally well in C57BL6 and IL-10KO mice, indicating that this antiinflammatory cytokine is not required for such protection. Protection against colitis given by poly(I:C) treatment was ablated in TLR3 KO, indicating that the protective action of this viral RNA analog was mediated by this receptor. Activation of TLR3 on cells that are accessible by systemic, but not oral, administration of synthetic viral RNA results in protection against the acute inflammation that can ensue upon damage of the gut epithelium. Thus, this viral RNA analog, which is under clinical trials for other inflammatory disorders (e.g., lupus), may also have therapeutic value for inflammatory bowel disease.

Modulation of TLR2 induces cardioprotection through a Phosphoinositide 3‐Kinase Dependent Mechanism

We have reported that the immune modulator, glucan, rapidly induces cardioprotection. TLR2 is required for glucan activation of cellular signaling. However, the role of TLR2 in cardioprotection has not been investigated. Peptidoglycan (PDG) activates cellular signaling specifically through TLR2. In this study we examined the effect of modulation of TLR2 on myocardial I/R injury. TLR2 KO (n=8) and wild type mice (n=8) were treated with PDG (100 ƒÝg/25g) or glucan (1 mg/25g) one hr before the hearts were subjected to ischemia (1 hr)/reperfusion (4 hrs). Untreated mice (n=8) served as control. To investigate the role of PI3K/Akt in modulation of TLR2-induced cardioprotection, we administered PDG (100 ƒÝg/25g) or glucan (1 mg/25g) to kdAkt transgenic mice (n=8) one hr before I/R. Myocardial infarction was determined by TTC staining. Infarct size was significantly reduced in PDG (10.5 ¡Ó 3.03% vs 30.1 ¡Ó 7.59%, p<0.01) and glucan (11.6 ¡Ó 2.38% vs 36.1 ¡Ó 3.48%, p<0.01) treated mice vs untreated mice. Both PDG and glucan-induced cardioprotection were completely abolished in TLR2 KO mice. The cardioprotection induced by either direct (PDG) or indicate (glucan) modulation of TLR2 was also abolished in kdAkt mice. The results suggest that the mechanism of modulation of TLR2-induced cardioprotection is mediated through a PI3K/Akt dependent mechanism and that there is a link between TLR2 and PI3K/Akt signaling during I/R.

Toll‐Like Receptor and Heme Oxygenase‐1 Signaling in Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll-like receptor 4 (TLR4) signaling is specifically required in initiating antigen-independent IRI leading to liver inflammation, whereas local induction of anti-oxidant heme oxygenase-1 (HO-1) is cytoprotective. This study analyzes in vivo interactions between HO-1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90-min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2-deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzuki's scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF-alpha production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO-1 expression. In contrast, tin protoporphyrin-mediated HO-1 inhibition restored hepatic damage in otherwise IRI-resistant TLR4 mutant/KO mice. CoPP-induced HO-1 overexpression ameliorated hepatic damage in IRI-susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO-1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO-1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO-1 repressor.

Non-mannose-capped lipoarabinomannan induces lung inflammation via toll-like receptor 2.

Non-mannose-capped lipoarabinomannan (AraLAM) is part of the cell membrane of atypical mycobacteria. To determine the capacity of AraLAM to induce lung inflammation in vivo and to determine the signaling receptors involved herein, wild-type (WT) mice, lipopolysaccharide binding protein knockout mice, CD14-deficient (CD14 KO) mice, Toll-like receptor (TLR) 4 mutant mice, or TLR2 KO mice were intranasally inoculated with purified AraLAM. AraLAM induced high lung levels of tumor necrosis factor, interleukin-1beta, interleukin-6, and cytokine-induced neutrophil chemoattractant (KC) and an influx of neutrophils into the pulmonary compartment of WT mice. Lipopolysaccharide binding protein knockout, CD14 KO, and TLR4 mutant mice displayed similar inflammatory responses as WT mice, whereas in TLR2 KO mice, AraLAM-induced lung inflammation was strongly diminished. In addition, TLR2 KO mice, but not CD14 KO or TLR4 mutant mice, displayed a delayed clearance of pulmonary infection with the atypical AraLAM expressing Mycobacterium smegmatis. These data indicate that TLR2 is the signaling receptor for purified AraLAM in the lung in vivo and that this receptor contributes to an effective clearance of M. smegmatis from the pulmonary compartment.

エンドトキシン不応答性ノックアウトマウスの作製と解析

Toll in Drosophila is a receptor that is required for dorsoventral polarity during development, and also involved in host defence against fungal infection. Recently, mammalian homologues of Toll, designated as Toll-like receptors (TLRs) have been identified. The TLR family harbors an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic domain that is homologous to that of the IL-1R family. Analogous to the IL-1R, TLR recruits IRAK via adaptor MyD88, and then induces activation of TRAF6, NIK and finally NF-κB.In order to examine the role of TLR family, we have generated mice lacking TLR2, TLR4, and MyD88. TLR4 KO mice are hyporesponsive to LPS. On the other hand, TLR2 KO mice are hyporesponsive to peptidoglycan from gram-positive bacteria, but not to LPS. MyD88 KO mice are unresponsive to LPS. Taken together, these results demonstrate that responses to bacterial cell wall components are differentially mediated by TLR2 and TLR4, and that LPS signaling is mediated by TLR4 via MyD88.

Iliac crest periosteum responds to the level of physical activity in human adults

The aim of this study was to characterize the formation and progression of experimentally induced periapical lesions in TLR2 knockout (TLR2 KO) mice.Periapical lesions were induced in molars of 28 wild type (WT) and 27 TLR2 KO mice. After 7, 21, and 42 days, the animals were euthanized, and the mandibles were subjected to histotechnical processing. Hematoxylin-eosin–stained sections were examined under conventional light microscopy for the description of pulpal, apical, and periapical features and under fluorescence microscopy for the determination of the periapical lesion size. The subsequent sections were evaluated by tartrate resistant acid phosphatase histoenzymology (osteoclasts), Brown and Brenn staining (bacteria), and immunohistochemistry (RANK, RANKL, and OPG). Data were analyzed by the Mann-Whitney U and Kruskal-Wallis tests (α = 0.05).The WT group showed significant differences (P < .05) in the periapical lesion size and the osteoclast number between 7 and 42 days and between 21 and 42 days. In the TLR2 KO group, significant differences (P < .05) in the periapical lesion size and the osteoclast number were found between 7 days and the other periods. There was a significant difference (P < .05) between the 2 types of animal regarding the periapical lesion size, which was larger in the TLR2 KO animals. No significant differences (P > .05) were found between WT and TLR2 KO mice related to the pulpal, apical, and periapical features; bacteria localization; and immunohistochemical results (except for RANK expression).TLR2 KO animals developed larger periapical lesions with a greater number of osteoclasts, indicating the important role of this receptor in the host's immune and inflammatory response to root canal and periradicular infection.