Ozone-induced lung injury and sterile inflammation. Role of toll-like receptor 4

Inhalation of toxic doses of ozone is associated with a sterile inflammatory response characterized by an accumulation of macrophages in the lower lung which are activated to release cytotoxic/proinflammatory mediators that contribute to tissue injury. Toll-like receptor 4 (TLR4) is a pattern recognition receptor present on macrophages that has been implicated in sterile inflammatory responses. In the present studies we used TLR4 mutant C3H/HeJ mice to analyze the role of TLR4 in ozone-induced lung injury, oxidative stress and inflammation. Acute exposure of control C3H/HeOuJ mice to ozone (0.8ppm for 3h) resulted in increases in bronchoalveolar lavage (BAL) lipocalin 24p3 and 4-hydroxynonenal modified protein, markers of oxidative stress and lipid peroxidation. This was correlated with increases in BAL protein, as well as numbers of alveolar macrophages. Levels of surfactant protein-D, a pulmonary collectin known to regulate macrophage inflammatory responses, also increased in BAL following ozone inhalation. Ozone inhalation was associated with classical macrophage activation, as measured by increased NF-κB binding activity and expression of TNFα mRNA. The observation that these responses to ozone were not evident in TLR4 mutant C3H/HeJ mice demonstrates that functional TLR4 contributes to ozone-induced sterile inflammation and macrophage activation.     

MHCII, Tlr4 and Nramp1 genes control host pulmonary resistance against the opportunistic bacterium Pasteurella pneumotropica

MHCII, Tlr4, and Nramp1 genes are each independently important in pulmonary immunity. To determine the effect of these genes on host resistance, mice carrying various combinations of functional alleles for these three genes were experimentally challenged with the opportunistic bacterium, Pasteurella pneumotropica. MHCII-/-, Tlr4d/d, and Nramp1s/s mice were significantly more susceptible to experimental infections by P. pneumotropica after intranasal challenge compared to mice carrying functional alleles at only one of those genes. P. pneumotropica were cultured from the lungs of challenged mice, and the severity of the pneumonia strongly correlated with the number of isolated bacteria. Mice with the genotype MHCII-/- Tlr4n/n genotype were less susceptible to pneumonia than MHCII+/+, Tlr4d/d mice. It is interesting that the Nramp1 gene contribution to host resistance was apparent only in the absence of functional MHCII or Tlr4 genes. These data suggest that MHCII, Tlr4, and Nramp1 genes are important to pulmonary bacterial resistance.     

Differential involvement of TLR2 and TLR4 in host survival during pulmonary infection with Chlamydia pneumoniae

The relevance of TLR2 and TLR4 for recognizing Chlamydia pneumoniae in vivo during pulmonary infection and to survive the infection was explored. We found that early immune responses triggered by C. pneumoniae partially depended on TLR2, but not on TLR4. The chemokines MIP-2 and MIP-1alpha were not induced, while IL-12p40 levels were higher in TLR2(-/-) mice compared to wild-type mice. Secretion of TNF, keratinocyte-derived chemokine and monocyte chemoattractant protein-1 was attenuated in TLR2(-/-) mice, while IFN-gamma was increased as in wild-type mice. The pulmonary cyto- and chemokine response of TLR2(-/-) x TLR4(d/d) was similar to TLR2(-/-) mice. TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice also attracted fewer polymorphonuclear neutrophils into the lung, while TLR4(d/d) mice recruited them. Attenuated recruitment of polymorphonuclear neutrophils correlated with reduced weight loss in TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice and a lower chlamydial burden 3 days post infection. At 9 days post infection, TLR2(-/-) and TLR2(-/-) x TLR4(d/d) mice produced cyto- and chemokines as efficiently as wild-type mice, indicating that the involvement of TLR in inflammation varies over time. All TLR2(-/-) x TLR4(d/d) mice succumbed to the infection, while about 50% of TLR2(-/-) mice died. Taken together, the function of TLR2 and TLR4 is required to survive pulmonary infection with C. pneumoniae.     

Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden

The roles of innate immune responses in protection from or pathogenesis of severe leptospirosis remain unclear. We examined the role of Toll-like receptors (TLRs) in mouse infection and macrophage responses to Leptospira. C3H/HeJ mice (TLR4 deficient) and C3H/HeJ-SCID mice, but not C3H/OuJ mice (TLR4 intact), died after intraperitoneal infection with Leptospira interrogans serovar Icterohaemorrhagiae. Death in both C3H/HeJ mouse strains was associated with jaundice and pulmonary hemorrhage, similar to the patient from whom the isolate was obtained. In chronic sublethal infection, TLR4-deficient mice harbored more leptospires in liver, lung, and kidney than control mice. Heat-killed Leptospira stimulated macrophages to secrete proinflammatory cytokines, tumor necrosis factor alpha, interleukin-6, and macrophage inflammatory protein 2 not inhibited by polymyxin B, suggesting that leptospiral lipopolysaccharide (LPS) did not drive these responses. Anti-TLR4 and anti-MD-2 but not anti-CD14 monoclonal antibodies inhibited cytokine production. Peritoneal macrophages from CD14-/- and TLR2-/- mice exhibited no defect in cytokine responses to Leptospira compared to controls. Macrophages from C3H/HeJ, TLR4-/-, and MyD88-/- mice secreted far-lower levels of cytokines than wild-type macrophages in response to Leptospira. TLR4 plays a crucial role in protection from acute lethal infection and control of leptospiral burden during sublethal chronic infection. Cytokine responses in macrophages correlated with leptospiral clearance. These TLR4-dependent but CD14/TLR2-independent responses are likely mediated by a leptospiral ligand(s) other than LPS.     

A role for complement C5 in organism containment and granulomatous response during murine tuberculosis

The molecular mechanisms underlying protective granuloma formation and control of bacterial growth during infection with Mycobacterium tuberculosis (MTB) are not yet completely understood. MTB-infected mice with natural deficiency in complement component C5 are unable to develop productive granulomatous responses, and are impaired in limiting organism growth within the lung. To address the molecular basis for this histologic dysfunction, congenic complement C5-sufficient (B10.D2-H2d H2-T18c Hcl/nSnJ) and complement C5-deficient strains (B10.D2-H2d H2-T18c Hco/oSnJ) congenic mice were infected with Mycobacterium tuberculosis, and cytokine and chemokine responses were examined. Twelve and 28 days after infection, lungs showed elevated messages for multiple inflammatory cytokines in both congenic strains. Interleukin (IL)-12(p40) mRNA was also induced during infection in C5-deficient mice, although levels were significantly decreased compared to C5-sufficient congenics. C5-deficient mice also demonstrated reduced KC, MIP-2, IP-10, and MCP-1 mRNA. The defect may directly involve C5-mediated effects on macrophage responses; C5-deficient bone marrow derived macrophages had significantly reduced secretion of KC, MIP-1 alpha and MIP-2 compared to C5-sufficient macrophages following in vitro infection. These findings indicate a role for C5 in mediation of chemotactic and activation events that are the basis for granulomatous responses during murine tuberculosis.     

gammadeltaT cell-mediated regulation of chemokine producing macrophages during Listeria monocytogenes infection-induced inflammation

Infection of gammadeltaT cell-deficient (TcRdelta-/-) mice with the intracellular bacterium Listeria monocytogenes (Lm) results in an exacerbated inflammatory response characterized by the accumulation of activated macrophages and necrotic liver lesions. Here we investigated whether changes in chemokine production by Lm-elicited macrophages contribute to this abnormal inflammatory response. In response to Lm infection, activated macrophages accumulate in the primary sites of infection in TcRdelta-/- mice and express high amounts of mRNA encoding the chemokines CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CXCL2 (MIP-2) and CXCL10 (IP-10). In the infected tissues of TcRdelta-/- the number of chemokine-synthesizing macrophages was higher than in wild-type (WT) mice, with the amount of MIP-1alpha and MIP-1beta secreted by individual macrophages in the spleen of TcRdelta-/- mice also being significantly higher than in WT mice. By contrast, protease activity and NO production in individual splenic macrophages of Lm-infected TcRdelta-/- and WT mice were comparable. Pathogen-elicited macrophages in TcRdelta-/- mice also expressed high levels of the CCL3 and CCL4 receptor, CCR5. In macrophage-gammadeltaT cell co-cultures, chemokine-producing macrophages were killed by cytotoxic Vgamma1+ T cells in a Fas-FasL-dependent manner consistent with the high levels of chemokine-producing macrophages seen in infected TcRdelta-/- mice being due to the absence of Vgamma1+ T cells. Together these findings highlight the importance of gammadeltaT cells in regulating macrophage anti-microbial responses.     

Cytokine responses to fungal pathogens in Kupffer Cells are Toll-like receptor 4 independent and mediated by tyrosine kinases

Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in cytokine production, using primary cultures of rat and murine Kupffer cells exposed to Aspergillus fumigatus and Candida albicans hyphae and conidia. All fungal components induced the release of tumour necrosis factor-alpha (TNF-alpha), but with delayed kinetics compared with lipopolysaccharide (LPS). Candida albicans was the most potent inducer of TNF-alpha protein and mRNA and the only inducer of interleukin-10 (IL-10) in rat Kupffer cells. All fungal components induced enhanced mRNA levels of macrophage inhibitory protein-2 (MIP-2) in the cells, similar to LPS. Inhibitors of Src tyrosine kinases added to cells prior to stimulation led to attenuation in the release of both TNF-alpha (60%, P < 0.05) and IL-10 (70%, P < 0.05) induced by C. albicans conidia but did not influence the LPS-mediated cytokine release. Murine Kupffer cells (C57BL/10J) also released TNF-alpha as well as the chemokines keratinocyte-derived chemokine (KC) and MIP-2 in response to fungal component. Surprisingly, Kupffer cells from TLR4-deficient C57BL/ScCr mice exhibited significantly enhanced production of KC and MIP-2 upon stimulation by fungal components compared with control littermates (P < 0.05). Our study demonstrates that Aspergillus and Candida components induce cytokine production in rat Kupffer cells and that the response to C. albicans conidia involves Src tyrosine kinases. The experiments with TLR4-deficient Kupffer cells suggest that the cytokine response in these cells to fungal component is not mediated by TLR4.     

Dysregulated Toll-like receptor expression and signaling in bone marrow-derived macrophages at the onset of diabetes in the non-obese diabetic mouse

The expression, responsiveness and regulation of mouse Toll-like receptors (TLRs) in bone marrow-derived macrophages (BM-?) were investigated prior to and following the development of diabetes. Expression of TLR3 and TLR5 was significantly higher in newly diabetic non-obese diabetic (NOD) mice when compared with pre-diabetic and control strains of mice. The TLR3 ligand poly(I)poly(C) triggered up-regulation of its own receptor in NOR and pre-diabetic NOD, but TLR3 was already highly expressed in diabetic NOD mice. Expression levels of TLR3 correlated with poly(I)poly(C)-triggered IFN activity. LPS triggered down-regulation of TLR4 in pre-diabetic NOD, NOR and BALB/c, while levels of TLR4 remained consistently elevated in type 1 diabetic NOD and type 2 diabetic NZL mice. Dysregulation of TLR4 expression in the diabetic state correlated with increased nuclear factor kappa B (NF-kappaB) activation in response to the TLR4 ligand LPS and higher expression of IL-12p40, tumor necrosis factor alpha (TNFalpha), IL-6 and inducible nitric oxide synthase but lowered expression of IL-10. Exposure of bone marrow precursor cells from NOD mice to a hyperglycemic environment during differentiation into macrophages resulted in elevated levels of TLR2 and TLR4 and the cytokine TNFalpha. The results indicate that macrophage precursors are influenced by systemic changes in diabetes favoring altered TLR expression and sensitivity that may influence susceptibility to macrophage-mediated diabetes complications and explain inappropriate responses to infection in diabetes.     

Isolation of Pasteurella ureae from reproductive tracts of congenic mice.

Infertility noted in two congenic strains of mice was associated with abortion, metritis, and stillbirths. Pasteurella pneumotropica was recovered from affected uterine tracts of ATL mice. Tetracycline treatment for 6 weeks was instituted in an attempt to minimize Pasteurella-associated infertility. Nine ATL mice and 12 ATH mice were examined 4 and 12 months after cessation of tetracycline therapy, respectively. Histopathologically, all animals appeared normal. P. pneumotropica was recovered from six of nine prepuces of both ATH and ATL mice; similarly, two of three vaginal culture of the ATL mice yielded this bacterium. Pasteurella ureae was isolated from three of eight reproductive tracts of the female ATH mice, Negative indole and ornithine decarboxylase reactions and positive acid production for mannitol differentiated P. ureae from P. pneumotropica. This report represents the first confirmed isolation of P. urea from animals. Biochemical profiles of atypical strains of P. pneumotropica isolated from animals are discussed and compared with those of P. ureae. The need for careful biochemical characterization of all Pasteurella isolated from rodents is emphasized.     

Toll-like receptor 4 (TLR4)-deficient murine macrophage cell line as an in vitro assay system to show TLR4-independent signaling of Bacteroides fragilis lipopolysaccharide

Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating signaling through toll-like receptor 4 (TLR4). We and others have hypothesized that LPS derived from different bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, we have generated using a nonviral transformation procedure a bone marrow-derived macrophage cell line called 10ScNCr/23 from mouse strain C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing the mouse strain to be nonresponsive to stimulation by LPS from Escherichia coli while responding normally to other bacterial substrates, such as lipoteichoic acid (LTA) from Staphylococcus aureus, which signal TLR4 independently. Stimulation with LTA induces five- and sixfold increases in 10ScNCr/23 cell line tumor necrosis factor alpha and macrophage inflammatory protein-2 (MIP-2) secretion, but no increases in either cytokine were found when cells were stimulated with E. coli LPS. Bacteroides fragilis-derived LPS, however, can effectively stimulate MIP-2 expression in the absence of functional TLR4 in the 10ScNCr/23 cell line. This gives rise to the notion that LPS from some bacterial species will utilize alternative receptors to stimulate the innate immune response.     

Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins

The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3alpha (MIP-3alpha), and the MyD88-independent molecules beta interferon (IFN-beta), nitric oxide, and IFN-gamma-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-alpha, IL-1beta, and MIP-3alpha, but significantly less IFN-beta, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-beta, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-alpha and MIP-3alpha in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-alpha release but did not influence nitric oxide release. IFN-beta polyclonal antibody and IFN-alpha/beta receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.     

The lung responds to zymosan in a unique manner independent of toll-like receptors, complement, and dectin-1

In vitro studies indicate that the inflammatory response to zymosan, a fungal wall preparation, is dependent on Toll-like receptor (TLR) 2, and that this response is enhanced by the dectin-1 receptor. Complement may also play an important role in this inflammatory response. However, the relevance of these molecules within the in vivo pulmonary environment remains unknown. To examine pulmonary in vivo inflammatory responses of the lung to zymosan, zymosan was administered by intratracheal aerosolization to C57BL/6, TLR2- TLR4-, MyD88-, and complement-deficient mice. Outcomes included bronchoalveolar fluid cell counts. We next examined effects of dectin-1 inhibition on response to zymosan in alveolar macrophages in vitro and in lungs of C57BL/6, TLR2-, and complement-deficient mice. Finally, the effect of alveolar macrophage depletion on in vivo pulmonary responses was assessed. Marked zymosan-induced neutrophil responses were unaltered in TLR2-deficient mice despite a TLR2-dependent response seen with synthetic TLR2 agonists. TLR4, MyD88, and complement activation were not required for the inflammatory response to zymosan. Although dectin-1 receptor inhibition blocked the inflammatory response of alveolar macrophages to zymosan in vitro, in vivo pulmonary leukocyte recruitment was not altered even in the absence of TLR2 or complement. Depletion of alveolar macrophages did not affect the response to zymosan. Neither complement, macrophages, nor TLR2, TLR4, MyD88, and/or dectin-1 receptors were involved in the pulmonary in vivo inflammatory response to zymosan.     

Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila

The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2(-/-)), TLR4(-/-), and wild-type (WT) littermate (C57BL/6 x 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2(-/-) macrophages was significantly lower than those of WT and TLR4(-/-) macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.     

Genetic control of natural resistance in mouse macrophages regulating intracellular Legionella pneumophila multiplication in vitro.

It is known that Legionella pneumophila proliferates in peritoneal macrophage cultures derived from A/J mice but not in macrophage cultures derived from many other strains, including C57BL/6 mice. To analyze the genetic control of this trait and the location of the Legionella resistance-susceptibility gene, we prepared segregating progeny of A/J and C57BL/6 mice and determined the levels of susceptibility of individual mice. Peritoneal macrophages were collected by injecting thioglycolate medium, and macrophage monolayers were infected in vitro with L. pneumophila Philadelphia-1. Counting of colonies on buffered charcoal yeast extract agar plates and Gimenez staining of macrophage monolayers were carried out daily. There was a 10-fold increase in bacterial burden 1 day after infection and a 100-fold increase after 2 days in A/J (susceptible) macrophages. The increase in bacterial burden was always less than 10-fold in macrophages from C57BL/6 (resistant) progenitors, A/J x C57BL/6 F1 hybrids, and C57BL/6 x F1 backcross progeny. The ratios of resistant individuals to susceptible individuals were 22:6 for F2 progeny and 20:22 for A/J x F1 backcross progeny. The fact that the organism did not proliferate in macrophages from B10.A mice demonstrated that major histocompatibility antigens did not regulate the macrophage resistance of C57BL/6-derived mice. The sex and coat color genes of mice were not linked to the resistance-susceptibility gene. We suggest that resistance and susceptibility are controlled by a single gene or closely linked genes which are autosomal and that the resistance allele is dominant. The results of a comparison of the strain distribution pattern of this trait with the distribution pattern of 185 allelic markers in A/J x C57BL/6 and C57BL/6 x A/J recombinant inbred strains suggest that this susceptibility-resistance gene is located in the proximal part of chromosome 15.     

The role of macrophage activation and of Bcg-encoded macrophage function(s) in the control of Mycobacterium avium infection in mice.

Following the intraperitoneal inoculation of 2.5 x 10(8) colony-forming units of Mycobacterium avium strain ATCC 25291, there was bacillary growth in the liver, spleen and peritoneal cavity of C57BL/6, C57BL/10, DBA/1 and BALB/c mice whereas DBA/2, C3H/He, CBA/Ca and CD-1 mice controlled the infection showing constant or slightly decreasing numbers of viable bacteria in the liver and spleen and effective clearance of the bacilli from the peritoneal cavities. The acquisition of non-specific resistance (NSR) to Listeria monocytogenes during the infection by M. avium was high in C57BL/6, BALB/c and C3H/He mice and negligible in DBA/2 and CD-1 mice. The magnitude of the acquisition of NSR was reduced in T cell-deficient mice and was directly proportional to the dose of the inoculum of M. avium. The production of hydrogen peroxide by phorbol myristate acetate-stimulated peritoneal macrophages of M. avium-infected mice was higher in C57BL/6 and BALB/c mice than in CD-1, DBA/2 and C3H/He animals. BALB/c. Bcgr (C.D2) mice, unlike their congenic strain BALB/c, restricted bacterial growth following the intravenous inoculation of 2.5 x 10(8) CFU of M. avium as efficiently as DBA/2 mice. C.D2 and BALB/c peritoneal macrophages from infected mice produced similar amounts of H2O2 but BALB/c mice developed higher levels of NSR to listeria than C.D2 mice. The production of nitrite by peritoneal macrophages from infected mice was found to be enhanced in DBA/2 and C3H/He but not in BALB/c, C57BL/6, DC-1 and C.D2 mice. Resident peritoneal macrophages from C.D2 mice were more bacteriostatic in vitro for M. avium than macrophages from BALB/c mice. The same relative differences between the two macrophage populations were observed when the cells were activated with lymphokines. The results show that the populations were observed when the cells were activated with lymphokines. The results show that the resistance to M. avium infection in mice is under the control of the Bcg gene and that susceptibility may be due to some defect in macrophage antibacterial function not completely overcome by the activation of this phagocyte in the susceptible strains of mice.     

Lung defenses against Pseudomonas aeruginosa in C5-deficient mice with different genetic backgrounds.

Lung defenses against Pseudomonas aeruginosa were investigated in C5-deficient strains of mice with different genetic backgrounds. We studied pulmonary clearance and cell responses after aerosol exposure to P. aeruginosa in C5-deficient B10.D2/oSnJ and DBA/2J mice and their closest C5-sufficient counterparts, B10.D2/nSnJ and DBA/1J mice. Different patterns of lung clearance and pulmonary cell responses were found for the two C5-deficient strains. C5-deficient B10.D2/oSnJ mice showed defective lung clearance of P. aeruginosa 4 h after challenge compared with C5-sufficient B10.D2/nSnJ animals. This finding was associated with a decreased number of polymorphonuclear leukocytes recruited into the airways during the same time. Interestingly, C5-deficient DBA/2J mice recruited higher numbers of polymorphonuclear leukocytes than did C5-sufficient DBA/1J mice by 4 h after aerosolization. Nevertheless, lung clearance of P. aeruginosa in DBA/2J mice was not as effective as in C5-sufficient DBA/1J mice, suggesting that other functions of C5 besides chemotaxism could be involved. Lung clearance of P. aeruginosa was also investigated in C5-deficient and -sufficient hybrids sharing the same genetic background (DBA/2J X B10.D2). The results suggested that murine lung clearance of P. aeruginosa is markedly affected by lack of C5 in a specific genetic background (B10.D2).     

Hirsutella sinensis mycelium regulates autophagy of alveolar macrophages via TLR4/NF-κB signaling pathway

Background: Hirsutella sinensis mycelium (HSM) has potent anti-pulmonary fibrotic activities and has been proposed as an effective treatment for idiopathic pulmonary fibrosis. Macrophages are the main innate immune cells in the lung tissue, playing key roles in pulmonary fibrosis repair and homeostasis. Excessive macrophage autophagy plays a vital role in pulmonary fibrosis. The protective effect of HSM on macrophages of bleomycin (BLM)-induced pulmonary fibrotic mice remain unclear.Methods: In this study, we collected lung tissue and bronchoalveolar lavage fluid (BALF) samples from pulmonary fibrotic mice. Meanwhile, alveolar macrophages were isolated and murine macrophage RAW264.7 cell line was cultured for further study of HSM autophagy.Results: First, we found that HSM decreased the number of autophagosomes, as well as the levels of LC3B and ATG5, and increased the protein level of P62 during the development of pulmonary fibrosis. Meanwhile, HSM reduced alveolar macrophages infiltration into the BALF and inhibited their accumulation in the fibrotic lung tissue. Flow cytometry analysis showed that HSM administration inhibited the autophagy marker LC3B expression in CD11b^loCD11c^hi alveolar macrophages in BLM-induced lung fibrosis without affecting CD11b^hiCD11c^lo interstitial macrophages. Transmission electron microscopy and JC-1 staining for mitochondrial membrane potential of alveolar macrophages also verified that the HSM significantly decreased autophagy in the alveolar macrophages of BLM-treated mice. In vitro, autophagosomes-lysosome fusion inhibitor chloroquine (CQ) was pre-incubated with RAW264.7 cells, and HSM reduced CQ-induced autophagosomes accumulation. TLR4 signaling inhibitor CLI095 reversed the above effects, suggesting HSM could reduce the cumulation of autophagosomes dependent on TLR4. Furthermore, lipopolysaccharide (LPS)-stimulated TLR4-related autophagy was significantly inhibited by HSM treatment. In addition, the protein expressions of TLR4 and phospho-NF-κB p65 were markedly inhibited in cells treated with HSM.Conclusions: These results indicated that HSM could inhibit the autophagy of alveolar macrophages through TLR4/NF-κB signaling pathway to achieve anti-fibrotic effect.