Short report: Association of macrophage inflammatory response and cell death after in vitro Borrelia burgdorferi infection with arthritis resistance

Susceptibility to Borrelia burgdorferi infection and subsequent arthritis is genetically determined in mice and determined by innate immunity. Accordingly, macrophage responses to B. burgdorferi challenge may differ between mouse strains. Bone marrow-derived macrophages were infected ex vivo with clonal B. burgdorferi strain N40. Interleukin-12 and tumor necrosis factor-alpha (TNF-alpha) production were higher in macrophages from resistant C57Bl/6 mice than in macrophages from susceptible C3H/HeJ mice. However, TNF-alpha production was observed in lower concentrations in C3H/HeJ (toll-like receptor-4(-/-)) macrophages than in C3H/FeJ (TLR4(+/+)) macrophages, suggesting that TLR4 might contribute to the response to B. burgdorferi. A higher cytokine response to B. burgdorferi was associated with cell death in macrophages from resistant C57Bl/6 mice. Understanding variability in the response of macrophages to B. burgdorferi may contribute to understanding Lyme arthritis.     

Neutrophil recruitment and resistance to urinary tract infection.

This study examined the role of neutrophil leukocytes for the antibacterial defense at mucosal infection sites. Urinary tract infection (UTI) was established by injection into the bladder lumen of Escherichia coli 1177, a fully virulent clinical isolate. Infection of C3H/HeN (lpsn, lpsn) mice recruited neutrophils into the urinary tract, and bacteria were cleared from kidneys and bladders. The neutrophil response was absent in C3H/HeJ (lpsd, lpsd) mice, and bacteria persisted in the tissues. Peripheral neutrophil depletion of C3H/HeN mice was subsequently achieved by pretreatment with the granulocyte-specific antibody RB6-8C5. The E. coli-induced neutrophil recruitment was inhibited, as shown by immunohistochemistry and tissue myeloperoxidase quantitation. As a consequence, bacterial clearance from kidneys and bladders was drastically impaired. Antibody treatment of C3H/HeJ mice had only a marginal effect. The results show that neutrophils are essential for bacterial clearance from the urinary tract and that the neutrophil recruitment deficiency in C3H/HeJ mice explains their susceptibility to gram-negative UTI.     

In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4

Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.     

Increased expression of toll-like receptor 4 enhances endotoxin-induced hepatic failure in partially hepatectomized mice

BACKGROUND/AIMS: Liver failure associated with infections after hepatectomy remains a cause of mortality. It has recently been reported that toll-like receptor 4 (TLR4) is involved in recognizing lipopolysaccharides (LPS). The aim of this study was to investigate the role of TLR4 in endotoxin-induced liver injury after hepatectomy. METHODS: C3H/HeN and C3H/HeJ mice underwent 70% hepatectomy or sham surgery, and LPS was administered 48 h after surgery. Expression of TLR4 mRNA, nuclear factor-kappaB (NF-kappaB) activation, tumor necrosis factor-alpha (TNF-alpha) and serum ALT levels, histological findings, and myeloperoxidase content were examined. Survival after LPS administration was also determined. RESULTS: Hepatic expression of TLR4 was significantly increased 6-72 h after hepatectomy. In mice with endotoxemia after hepatectomy, hepatic NF-kappaB activation was greatly increased. Hepatic mRNA and serum levels of TNF-alpha, and ALT levels were significantly elevated compared with sham operated controls. Focal necrosis with neutrophil infiltration was apparent, which is consistent with increased myeloperoxidase contents in endotoxemia after hepatectomy in C3H/HeN mice. These were completely absent in C3H/HeJ mice. Survival of C3H/HeN mice with endotoxemia after hepatectomy was significantly lower than that of C3H/HeJ mice. CONCLUSIONS: Upregulated TLR4 expression and function after hepatectomy plays a pivotal role in endotoxin-induced liver injury after hepatectomy.     

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6

Aims/hypothesis

Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function.

Methods

Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed.

Results

Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1β secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression.

Conclusions/interpretation

We conclude that islet macrophages are major contributors to islet IL-1β secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1β- and IL-6-mediated decreased insulin gene expression.     

Murine retroviruses activate B cells via interaction with toll-like receptor 4

Although most retroviruses require activated cells as their targets for infection, it is not known how this is achieved in vivo. A candidate protein for the activation of B cells by either mouse mammary tumor virus (MMTV) or murine leukemia virus is the toll-like receptor 4 (TLR4), a component of the innate immune system. MMTV caused B cell activation in C3H/HeN mice but not in C3H/HeJ or BALB/c (C.C3H Tlr4(lps-d)) congenic mice, both of which have a mutant TLR4 gene. This activation was independent of viral gene expression, because it occurred after treatment of MMTV with ultraviolet light or 2,2'-dithiodipyridine and in azidothymidine-treated mice. Nuclear extracts prepared from the lymphocytes of MMTV-injected C3H/HeN but not C3H/HeJ mice showed increased nuclear factor kappaB activity. Additionally, the MMTV- and Moloney murine leukemia virus envelope proteins coimmunoprecipitated with TLR4 when expressed in 293T cells. The MMTV receptor failed to coimmunoprecipitate with TLR4, suggesting that MMTV/TLR4 interaction is independent of virus attachment and fusion. These results identify retroviral proteins that interact with a mammalian toll receptor and show that direct activation by such viruses may initiate in vivo infection pathways.     

Toll-like receptor 4 participates in gastric mucosal protection through Cox-2 and PGE2

AimTo elucidate the role of Toll-like receptors (TLRs) in gastric cytoprotection after ethanol injury.MethodsC57BL/6J, C3H/HeOuJ and C3H/HeJ mice were used. All mice were killed 4 h after ethanol administration. TLR4, cyclooxygenase-2 (Cox-2) and prostaglandin E₂ (PGE₂) expression were measured by immunohistochemistry, western blotting and enzyme-linked immunosorbent assay (ELISA) separately.ResultsThe expression of TLR4 increased in C57BL/6J mice stomach 4 h after ethanol injury. The cells expressing TLR4 included Cox-2 expressing cells and macrophages. The injury in C3H/HeJ mice was more severe than in C3H/HeOuJ mice 4 h after ethanol injury. The expression of Cox-2 and PGE₂ only increased in C3H/HeOuJ mice. The number of macrophages and the expression of macrophage-inflammatory protein-2 (MIP-2) also increased only in C3H/HeOuJ mice.ConclusionTLR4 signal is activated in mice stomach 4 h after ethanol injury. The protective effects of TLR4 signalling are mediated through the induction of Cox-2 expression and the production of PGE₂.     

Endogenous interleukin (IL)-1 alpha and IL-1 beta are crucial for host defense against disseminated candidiasis

BACKGROUND: Interleukin (IL)-1 alpha and IL-1 beta are protective proinflammatory cytokines involved in host defense against Candida albicans. It is, however, unknown whether they provide protection through similar mechanisms. We investigated the effect of endogenous IL-1 alpha and IL-1 beta on disseminated C. albicans infection. METHODS: Mice deficient in the genes encoding IL-1 alpha (IL-1 alpha-/-), IL-1 beta (IL-1 beta-/-), or both molecules (IL-1 alpha-/- beta-/-) were used. Survival and C. albicans outgrowth in the kidneys was assessed after intravenous injection of C. albicans. RESULTS: Both mortality and C. albicans outgrowth in the kidneys were significantly increased in IL-1 alpha-/- and IL-1 beta-/- mice, compared with those in control mice, with the IL-1 alpha-/- beta-/- mice being most susceptible to disseminated candidiasis. The host defense mechanisms triggered by IL-1 alpha and IL-1 beta differed from one another. IL-1 beta-/- mice showed decreased recruitment of granulocytes in response to an intraperitoneal C. albicans challenge, and generation of superoxide production was diminished in IL-1 beta-/- granulocytes. IL-1 alpha-/- mice had a reduced capacity to damage C. albicans pseudohyphae. Protective type 1 responses were deficient in both IL-1 alpha-/- and IL-1 beta-/- mice, as assessed by production of interferon-gamma by splenocytes in response to heat-killed C. albicans. CONCLUSION: Although IL-1 alpha and IL-1 beta have differential effects on the various arms of host defense, both cytokines are essential for mounting a protective host response against invasive C. albicans infection.     

TOLL样受体4对小鼠脑缺血再灌注损伤中的保护作用

目的探讨TOLL样受体4(TLR4)在脑缺血再灌注中的保护作用。方法C3H/HeJ小鼠16只采用线栓法制作TLR4先天缺损大脑中动脉缺血再灌注模型(C3H/HeJ组),以C3H/HeN小鼠16只作对照(C3H/HeN组),观察缺血6h及再灌注24h时,两组小鼠神经功能缺损评分、脑含水量、脑梗死体积及血清TNF-α水平。光镜及电镜观察两者缺血再灌注脑组织损伤情况。结果与C3H/HeN组比较,再灌注24h时,C3H/HeJ组小鼠的神经功能缺损评分明显减小(P<0.05);脑含水量明显降低,脑梗死体积显著缩小(P<0.01);血清中TNF-α浓度明显降低(P<0.05);脑缺血皮质区的神经细胞超微结构改变轻于C3H/HeN组小鼠。结论C3H/HeJ小鼠脑缺血再灌注损伤明显减轻,TLR4介导了脑缺血再灌注损伤。其机制可能与炎性细胞因子减少有关。     

TLR4 mediates LPS-induced HO-1 expression in mouse liver： Role of TNF-α and IL-lβ

AIM: Heme oxygenase (HO)-I catalyzes the conversion of heme to biliverdin, iron and carbon monoxide. HO-1 is induced by many stimuli including heme, Hb, heat stress,lipopolysaccharide (LP5) and cytokines. Previous studies demonstrated that LP5 induced HO-1 gene activation and HO-1 expression in liver. However, the mechanisms of LPS-induced HO-1 expression in liver remain unknown. The effect of toll-like receptor-4 (TLR4) on LPS-induced liver HO-1 expression and the role of TNF-α and IL-1β in this condition were determined.METHODS: HO-1 expression was determined by immunofluorescent staining and immunoblotting. Double immunofluorescent staining was performed to determine the cell type of HO-1 expression in liver.RESULTS: A low dose of LPS significantly increased HO-1 expression in the liver which was localized in Kupffer cells only. Furthermore, HO-1 expression was enhanced by three doses of LPS. HO-1 expression was significantly inhibited in the liver of TLR4 mutant mice. While the liver HO-1 expression in TNF KO mice was much lower than that in C57 mice following the same LPS treatment, IL-1β KO had a slight influence on liver HO-1 expression following LPS treatment.CONCLUSION: The preserfl: results confirm that macrophages are the major source of HO-1 in the liver induced by LPS.This study demonstrates that TLR4 plays a dominant role in mediating HO-1 expression following LPS. LPS-induced HO-1 expression is mainly mediated by endogenous TNF-α, but only partially by endogenous IL-1β.     

Staphylococcal enterotoxin B induces hepatic injury and lethal shock in endotoxin-resistant C3H/HeJ mice despite a deficient macrophage response

Bacterial toxins, including endotoxin/LPS as well as superantigens, are major causative agents of multi-organ failure associated with sepsis and liver disease. However, the precise mechanisms initiating cell activation by the toxins have not been clarified. We compared lethal shock and cytokine production in response to LPS with responses to the superantigen staphylococcal enterotoxin B (SEB) in both LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice treated with D-galactosamine (GalN). LPS was not lethal and did not induce production of TNF-alpha in C3H/HeJ mice. In contrast, SEB produced lethal shock associated with liver failure and induced cytokines such as TNF-alpha, IFN-gamma, and IL-2 in both C3H/HeN and C3H/HeJ mice. Peritoneal macrophages from C3H/HeJ mice did not produce TNF-alpha in vitro in response to SEB or LPS. However, no significant difference was observed in production of TNF-alpha in response to stimulation in vitro by SEB between C3H/HeN and C3H/HeJ splenic lymphocytes. We have demonstrated that SEB causes lethal toxicity associated with liver injury in LPS-hyporesponsive C3H/HeJ mice and that as the underlying mechanism, the normal T-cell function in these mice still maintained the sensitivity to SEB since the genetic defect of C3H/HeJ mice unresponsive to LPS and SEB is restricted in macrophages/monocytes and does not extend to T cells.     

Role of Toll-like receptor 4 in lung inflammation following exposure to swine barn air

The authors tested a hypothesis that lung inflammation and airway hyperresponsiveness (AHR) induced following barn air exposure are dependent on Toll-like receptor 4 (TLR4) by exposing C3HeB/FeJ (intact TLR4, wild type [WT]) and C3H/HeJ (defective TLR4, mutant) mice either to the barn air (8 hours/day for 1, 5, or 20 days) or ambient air. Both strains of mice, compared to their respective controls, showed increased AHR following 5 exposures but dampened AHR after 20 exposures to show lack of effect of TLR4 on AHR. However, swine barn air induced lung inflammation with recruitment of inflammatory cells and cytokine expression was observed in WT but not in mutant mice. These data show different roles of TLR4 in lung inflammation and AHR in mice exposed to swine barn air.     

Legionella pneumophila mediated activation of dendritic cells involves CD14 and TLR2

In this study, we analyzed the activation of bone-marrow derived dendritic cells (BMDCs) from mice lacking the cd14-gene with purified Legionella pneumophila lipopolysaccharide and with viable or formalin-killed L. pneumophila. We found that low concentrations of LPS and doses of L. pneumophila that are relevant to infection are dependent on CD14 to activate BMDCs. Higher concentrations of LPS are able to overcome the lack of CD14 indicating that other receptors areinvolved. We, therefore, included studies using BMDCs from mice lacking functional TLR2 and/or TLR4 molecules. We found that purified L. pneumophila LPS as well as L. pneumophila either viable or formalin-killed are able to activate BMDCs from TLR4-deficient C3H/HeJ mice but fail to activate BMDCs from TLR2-knockout mice. Our data show that not only purified LPS from L. pneumophila but also the microorganism itself stimulate BMDCs via TLR2 and that this stimulation is dependent on CD14 in this mouse model.     

Aged mice exhibit greater mortality concomitant to increased brain and plasma TNF-alpha levels following intracerebroventricular injection of lipopolysaccharide

BACKGROUND: Age-related defects in the development of peripheral inflammatory responses have been observed in rodents and humans. OBJECTIVE: We examined the effects of age on a centrally injected endotoxin-induced cytokine production and cellular activation in mice. METHODS: Male C57BL/6J (B6) mice, C3H/HeN mice, and C3H/HeJ mice received an intracerebroventricular injection of lipopolysaccharide (LPS) and were sacrificed at various times (2, 4, 8 h) thereafter. ELISA for IL-1beta, IL-6, IL-12, and TNF-alpha were conducted on forebrain tissue homogenates as well as plasma samples, and lectin staining to detect activated microglia was prepared for selected brain slices. RESULTS: Intracerebroventricular injection of LPS in B6 mice produced an age-associated increase in mortality which was paralleled with a significant increase in brain and plasma levels of TNF-alpha. AntiTNF-alpha- and IL-6-immunoreactive cells possessed macrophagelike morphologies and were observed along the LPS injection tract and scattered throughout the hilus of the dorsal hippocampus and cerebral cortices. This LPS-mediated response was found to be specific in that the LPS-hyporesponsive mouse strain (C3H/HeJ) failed to demonstrate significant brain or plasma levels of TNF-alpha after LPS administration compared to C3H/HeN mice. CONCLUSION: These results suggest that the age-related increases in TNF-alpha production and mortality following the intracerebroventricular administration of LPS may be due to an increased endotoxin hypersensitivity of brain microglia/macrophages within aged animals.     

Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529

Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood. The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells. Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case. Similarly, the intravenous administration of these AGPs induced resistance to L. monocytogenes infection as measured by survival or reduction of bacteria in the spleen. Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes. Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.     

Signaling mechanisms in the restoration of impaired immune function due to diet-induced obesity

Our previous data have linked obesity with immune dysfunction. It is known that physical exercise with dietary control has beneficial effects on immune function and the comorbidities of obesity. However, the mechanisms underlying the improvement of immune function in obesity after physical exercise with dietary control remain unknown. Here we show that moderate daily exercise with dietary control restores the impaired cytokine responses in diet-induced obese (DIO) mice and improves the resolution of Porphyromonas gingivalis-induced periodontitis. This restoration of immune responses is related to the reduction of circulating free fatty acids (FFAs) and TNF. Both FFAs and TNF induce an Akt inhibitor, carboxyl-terminal modulator protein (CTMP). The expression of CTMP is also observed increased in bone marrow-derived macrophages (BMMΦ) from DIO mice and restored after moderate daily exercise with dietary control. Toll-like receptor 2 (TLR2), which increases CTMP induction by FFAs, is inhibited in BMMΦ from DIO mice or after either FFA or TNF treatment, but unexpectedly is not restored by moderate daily exercise with dietary control. Furthermore, BMMΦ from DIO mice display reduced histone H3 (Lys-9) acetylation and NF-κB recruitment to TNF, IL-10, and TLR2 promoters after P. gingivalis infection. However, moderate daily exercise with dietary control restores these defects at promoters for TNF and IL-10, but not for TLR2. Thus, metabolizing FFAs and TNF by moderate daily exercise with dietary control improves innate immune responses to infection in DIO mice via restoration of CTMP and chromatin modification.     

Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4

Our laboratory develops protocols to prevent or reverse ongoing anti‐hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9^?/Y mice develop high titre anti‐hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9^?/Y mice have mild anti‐hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX‐specific B‐ and T‐cell responses in BALB/c and C3H/HeJ F9^?/Y mice trigger the difference in anti‐hFIX immune responses. BALB/c and C3H/HeJ F9^?/Y mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti‐hFIX ELISA, Bethesda assay for inhibitors and B‐cell ELISpot on bone marrow and spleen cells. T‐cell studies measured the T_H1 (IFN‐γ) and T_H2 (IL‐4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9^?/Y mice with restored toll‐like receptor 4 (TLR4) function. BALB/c F9^?/Y mice have a T_H2 skewed response and a reduction in anti‐hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9^?/Y mice have a mixed T_H1 and T_H2 response (mainly T_H2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti‐hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.