LPS-binding protein-deficient mice have an impaired defense against Gram-negative but not Gram-positive pneumonia

LPS-binding protein (LBP) can facilitate the transfer of cell wall components of both Gram-negative bacteria (LPS) and Gram-positive bacteria (lipoteichoic acid) to inflammatory cells. Although LBP is predominantly produced in the liver, recent studies have indicated that this protein is also synthesized locally in the lung by epithelial cells. To determine the role of LBP in the immune response to pneumonia, LBP gene-deficient (-/-) and normal wild-type (WT) mice were intra-nasally infected with either Streptococcus pneumoniae or Klebsiella pneumoniae, common Gram-positive and Gram-negative pathogens, respectively. Pneumococcal pneumonia was associated with a 7-fold rise in LBP concentrations in bronchoalveolar lavage fluid of WT mice; LBP-/- mice infected with S. pneumoniae showed a similar survival and a similar bacterial burden in their lungs 48 h post-infection. In Klebsiella pneumonia, however, LBP-/- mice demonstrated a diminished survival together with an enhanced bacterial outgrowth in their lungs. These data suggest that LBP is important for a protective immune response in Klebsiella pneumonia, but does not contribute to an effective host response in pneumococcal pneumonia.     

Impairment of host defence by exotoxin A in Pseudomonas aeruginosa pneumonia in mice

Exotoxin A (P-ExA) is considered to be a major virulence factor of Pseudomonas aeruginosa. Neutrophils, cytokines and nitric oxide (NO) have been implicated as important components of an effective host defence against bacterial respiratory tract infection. To study the role of P-ExA in the pathogenesis of P. aeruginosa pneumonia, C57Bl/6 mice were inoculated intranasally with wild-type PA103 or a mutant P. aeruginosa strain that did not produce P-ExA, PA103-29. P-ExA facilitated the outgrowth of P. aeruginosa in lungs, as reflected by an increasing number of cfu during pneumonia with strain PA103, whereas the number of cfu decreased during pulmonary infection with strain PA103-29. Influx of neutrophils was similar in broncho-alveolar lavage fluids (BALF) during pneumonia with strains PA103 and PA103-29. Lung levels of cytokines (tumor necrosis factor-alpha, interleukin-6) and chemokines (macrophage inflammatory protein-2, KC) were higher in mice inoculated with strain PA103, whereas BALF concentrations of NO were similar in mice treated with strains PA103 and PA103-29. These data suggest that P-ExA impairs host defence during pneumonia caused by P. aeruginosa by a mechanism that does not involve effects on neutrophil influx, cytokines, chemokines or NO formation.     

Surfactant proteins A and D enhance pulmonary clearance of Pseudomonas aeruginosa

Surfactant protein (SP)-A and SP-D, members of the collectin family, are involved in innate host defenses against various bacterial and viral pathogens. In this study, we asked whether SP-A and SP-D enhance clearance of a nonmucoid strain of Pseudomonas aeruginosa from the lungs. We infected mice deficient in SP-A (SP-A-/-), SP-D (SP-D-/-) and both pulmonary collectins (SP-AD-/-) by intratracheal administration of P. aeruginosa. Six hours after infection, bacterial counts were significantly higher in SP-A-/-, SP-D-/-, and SP-AD-/- compared with wild-type (WT) mice. Forty-eight hours after infection, bacterial counts were significantly higher in SP-A-/- mice compared with WT mice and in SP-AD-/- mice compared with WT, SP-A-/-, and SP-D-/- mice. Phagocytosis of the bacteria by alveolar macrophages was decreased in SP-A-/- and SP-D-/- mice. Levels of macrophage inflammatory peptide-2 and IL-6 were more elevated in the lungs of SP-D and SP-AD-/- mice compared with WT mice. There was more infiltration by neutrophils in the lungs of SP-D-/- compared with WT and SP-A-/- mice 48 h after infection. This study shows that SP-A and SP-D enhance pulmonary clearance of P. aeruginosa by stimulating phagocytosis by alveolar macrophages and by modulating the inflammatory response in the lungs. These findings also show that the functions of SP-A and SP-D are not completely redundant in vivo.     

Interleukin 18 participates in the early inflammatory response and bacterial clearance during pneumonia caused by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a common gram-negative respiratory pathogen. To determine the role of the proinflammatory cytokine interleukin 18 (IL-18) during NTHi pneumonia, normal wild-type (WT) and IL-18 knockout (KO) mice were intranasally infected with NTHi. IL-18 KO mice displayed a delayed clearance of NTHi from the respiratory tract, resulting in >20-fold higher bacterial loads in their lungs at 24 h after infection, preceded by a strongly attenuated pulmonary innate immune response as determined by cytokine and chemokine induction and histopathology. These data identify IL-18 as part of an adequate innate immune response during NTHi pneumonia.     

Role of interferon-gamma in inflammatory responses in murine respiratory infection with Legionella pneumophila

The role of interferon (IFN)-gamma in host inflammatory responses, including inflammatory cytokine production, in experimental pneumonia with Legionella pneumophila was examined in IFN-gamma knockout (IFN-gamma-/-) mice. IFN-gamma-/- mice and wild-type BALB/cA mice were inoculated intranasally with L. pneumophila strain KC. The survival rate of IFN-gamma-/- mice was significantly lower than that of control mice. Viable bacterial counts in lungs and blood showed a rapid and continuous increase in IFN-gamma-/- mice, in contrast to a gradual decrease in the lungs and an intermittent bacteraemia in control mice. Histopathological analysis of L. pneumophila-infected lung tissues demonstrated mild pneumonia in control mice, whereas severe pneumonia was shown in IFN-gamma-/- mice. During the late stages of infection, the number of total bronchoalveolar lavage (BAL) cells was significantly higher in IFN-gamma-/- than in control mice. The concentrations of tumour necrosis factor-alpha and interleukin-1beta in sera of IFN-gamma-/- mice were significantly lower in control mice during the early stages of infection, suggesting suppressed production of inflammatory cytokines in IFN-gamma-/- mice. In contrast, during the late stages of infection, the levels of these cytokines were significantly higher in sera of IFN-gamma-/- mice than in control mice, suggesting severe and systemic infection in IFN-gamma-/- mice. The findings suggest that retardation of host immune responses, including inflammatory cytokine production caused by deficiency of IFN-gamma, might allow the bacteria to grow and cause fulminant pneumonia.     

Influence of gender and interleukin-10 deficiency on the inflammatory response during lung infection with Pseudomonas aeruginosa in mice

Cystic fibrosis females have a worse prognosis compared to male patients. Furthermore, cystic fibrosis patients infected with Pseudomonas aeruginosa have been shown to have dysregulated cytokine profiles, as higher levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and lower levels of IL-10 are found in the bronchoalveolar lavage fluid compared to healthy controls. The present study was aimed at investigating the importance of gender and IL-10 in the susceptibility of C57BL/6 mice to pulmonary infection with Pseudomonas aeruginosa. We found that wildtype females were more susceptible than males to infection, as we observed greater weight loss, higher bacterial load, and inflammatory mediators in their lungs. IL-10 knockout mice, both females and males, had higher levels of TNF-alpha in the lungs compared to wildtype mice and maintained higher levels of polymorphonuclear cells and lower levels of macrophages for a longer period of time. Our results demonstrate that the number of bacteria recovered from the lungs of IL-10 knockout male mice was significantly higher than that observed in their wildtype male counterparts and we show that neutralization of IL-10 in infected female mice for a prolonged period of time leads to increased susceptibility to infection. Results reported in this study clearly demonstrate that females, both wildtype and IL-10 knockout mice are more susceptible to Pseudomonas aeruginosa infection than males, and that they mount a stronger inflammatory response in the lungs.     

Receptor-interacting protein 2 controls pulmonary host defense to Escherichia coli infection via the regulation of interleukin-17A

Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2(-/-) mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2(-/-) mice after infection. RIP2(-/-) mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-κB and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2(-/-) mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2(-/-) mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2(-/-) mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.     

Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice

To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.     

Phenotypic switching in Cryptococcus neoformans contributes to virulence by changing the immunological host response

Cryptococcus neoformans is an encapsulated opportunistic organism that can undergo phenotypic switching. In this process, the parent smooth colony (SM) switches to a more virulent mucoid colony (MC) variant. The host responses mounted against the SM and MC variants differ, and lower tissue interleukin 10 (IL-10) levels are consistently observed in lungs of MC-infected C57BL/6 and BALB/c mice. This suggested different roles of this cytokine in SM and MC infections. The objective of this study was to compare survival rates and characterize the host responses of SM- and MC-infected IL-10-depleted (IL-10(-/-)) mice, which exhibit a Th1-polarized immune response and are considered resistant hosts. As expected, SM-infected IL-10(-/-) mice survived longer than wild-type mice, whereas MC-infected IL-10(-/-) mice did not exhibit a survival benefit. Consistent with this observation, we demonstrated marked differences in the inflammatory responses of SM- and MC-infected IL-10(-/-) and wild-type mice. This included a more Th1-polarized inflammatory response with enhanced recruitment of macrophages and natural killer and CD8 cells in MC- than in SM-infected IL-10(-/-) and wild-type mice. In contrast, both SM-infected IL-10(-/-) and wild-type mice exhibited higher recruitment of CD4 cells, consistent with enhanced survival and differences in recruitment and Th1/Th2 polarization. Lung tissue levels of IL-21, IL-6, IL-4, transforming growth factor beta, IL-12, and gamma interferon were higher in MC-infected IL-10(-/-) and wild-type mice than in SM-infected mice, whereas tumor necrosis factor alpha levels were higher in SM-infected IL-10(-/-) mice. In conclusion, the MC variant elicits an excessive inflammatory response in a Th1-polarized host environment, and therefore, the outcome is negatively affected by the absence of IL-10.     

Contrasting roles of IL-12p40 and IL-12p35 in the development of hapten-induced colitis.

IL-12(p70), a heterodimer composed of two subunits (p35 and p40), is a key cytokine for Th1 mediated inflammatory responses. We dissected the role of IL-12 in the development of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis by studying mice deficient in IL-12p40, IL-12p35, or IL-12Rbeta1. TNBS-treated IL-12Rbeta1(-/-) and IL-12p35(-/-) mice developed only a mild disease associated with low level IL-18 expression in IL-12p35(-/-) mice. In contrast, IL-12p40(-/-) mice developed more severe colitis than wild-type mice associated with high level colonic IL-18 expression. Administration of IL-12p40 neutralizing mononuclear antibody dramatically increased pathology in IL-12p35(-/-) mice similar to disease scored in IL-12p40(-/-) mice. Numbers of IFN-gamma-producing cells infiltrating the lamina propria were comparably augmented in the different groups of IL-12-mutant and wild-type mice. These results demonstrate that IL-12p40, in contrast to IL-12p70, inhibits TNBS-induced colitis and IL-18 expression independent of IFN-gamma.     

Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1-type immunopathology mediated by local production of IFN-gamma, TNF-alpha, and NO. To analyze whether the proinflammatory cytokines IL-12 and IL-18 play a role in the induction of immunopathology, IL-12p35/p40(-/-) and IL-18(-/-) mice were orally infected with T. gondii. Wild-type mice developed massive necrosis in their small intestines and died 7-10 days post infection. Even though IL-12p35/40(-/-) mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL-18(-/-) mice died during the acute phase of infection. Compared to wild-type mice, IL-12p35/p40(-/-) but not IL-18(-/-) mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite-associated inflammatory foci in their livers. IFN-gamma production was similar in infected wild-type and IL-18(-/-) mice but significantly decreased in IL-12p35/p40(-/-) mice. Treatment of mice with anti-IL-12- or anti-IL-18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL-12 and IL-18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL-12 is dominant over IL-18 in the host defense against parasite replication. Therefore, neutralization of IL-18 (rather than TNF-alpha, IL-12, and IFN-gamma) may be a safe strategy for the treatment of Th1-associated diseases.     

A regulatory role for interleukin 4 in differential inflammatory responses in the lung following infection of mice primed with Th1- or Th2-inducing pertussis vaccines

Protection against infectious pathogens at mucosal surfaces is dependent on local antibody responses, production of inflammatory mediators, and recruitment of immune effector cells to the site of infection. Since Th1 and Th2 cells produce cytokines with pro- and anti-inflammatory activities, immunization with vaccines that induce these T-cell subtypes may regulate the subsequent inflammatory response to infection. We have demonstrated that immunization of mice with pertussis whole-cell or acellular vaccines (Pw or Pa) selectively induces Th1 and Th2 cells, respectively. In this study we have used a murine respiratory-infection model to demonstrate that priming with a Th1- or Th2-inducing pertussis vaccine can influence the local inflammatory response and immune effector cells in the lung following aerosol challenge with Bordetella pertussis. Analysis of bronchoalveolar lavage (BAL) fluid taken during the course of B. pertussis infection of na?ve mice or mice immunized with Pw revealed an early influx of neutrophils and local production of interleukin 1beta (IL-1beta) in the lungs. In contrast, neutrophil infiltration and IL-1beta production were not observed following challenge of mice immunized with the Th2-inducing Pa. Conversely, during infection local production of IL-6 and IL-1ra was significantly greater in mice immunized with Pa than in those immunized with Pw. Studies of knockout mice revealed neutrophil and lymphocyte infiltration in the lungs following B. pertussis infection of IL-4-defective (IL-4(-/-)) mice but not in wild-type mice immunized with Pa. Furthermore, the levels of IL-1beta, IL-6, and IL-1ra in Pa-immunized IL-4(-/-) mice were comparable to those in mice immunized with Pw. These results demonstrate distinct influences of Th1- and Th2-inducing vaccines on the protective inflammatory responses in the lungs following challenge with B. pertussis and implicate IL-4 as an important regulator of inflammatory-cell recruitment.     

Protection against pulmonary infection with Klebsiella pneumoniae in mice by interferon-gamma through activation of phagocytic cells and stimulation of production of other cytokines.

The study was designed to determine the role of interferon (IFN)-gamma in inflammatory responses against experimentally induced pneumonia caused by Klebsiella pneumoniae. The host immunological responses in IFN-gamma gene knockout (IFN-gamma(-/-)) mice and immunocompetent control mice were compared. K. pneumoniae strain T-113 was inoculated intranasally into anaesthetised mice to induce pneumonia. Infected control mice survived significantly longer than infected IFN-gamma(-/-) mice. Viable bacterial counts in lungs and blood abruptly increased in IFN-gamma(-/-) mice; in contrast, a gradual decrease in the number of bacteria was noted in control mice. During the early stages of infection, the concentrations of interleukin (IL)-1beta and IL-6 in broncho-alveolar lavage fluid and IL-1beta in serum of IFN-gamma(-/-) mice were significantly lower than in control mice. During the late stage of infection, serum IL-6 level in IFN-gamma(-/-) mice was significantly higher than in control mice. These results suggest that the defective immunological host response, including inflammatory cytokine production caused by deficiency of IFN-gamma, is one of the mechanisms that allow the progression of pulmonary infection to systemic septicaemia.     

TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection

To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.     

Endotoxin priming improves clearance of Pseudomonas aeruginosa in wild-type and interleukin-10 knockout mice

Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-gamma) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogen Pseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance of Pseudomonas aeruginosa was measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN-gamma and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN-gamma and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice cleared Pseudomonas aeruginosa from lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearing Pseudomonas aeruginosa after systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance of Pseudomonas aeruginosa and that this effect was augmented by neutralization of IL-10.     

Absence of interleukin-4 determines less severe pulmonary paracoccidioidomycosis associated with impaired Th2 response

Host resistance to paracoccidiodomycosis, the main deep mycosis in Latin America, is mainly due to cellular immunity and gamma interferon (IFN-gamma) production. To assess the role of interleukin-4 (IL-4), a Th2-inducing cytokine, pulmonary paracoccidioidomycosis was studied in IL-4-deficient (IL-4(-/-)) and wild-type (WT) C57BL/6 mice at the innate and acquired phases of immune response. Forty-eight hours after infection, equivalent numbers of viable Paracoccidioides brasiliensis yeast cells were recovered from the lungs of IL-4(-/-) and WT mice intratracheally infected with one million fungal cells. Alveolar macrophages from infected IL-4(-/-) mice controlled in vitro fungal growth more efficiently than macrophages from WT mice and secreted higher levels of nitric oxide. Compared with WT mice, IL-4(-/-) animals presented increased levels of pulmonary IFN-gamma and augmented polymorphonuclear leukocyte influx to the lungs. Decreased pulmonary fungal loads were characterized in deficient mice at week 2 postinfection, concomitant with diminished presence of IL-10. At week 8, lower numbers of yeasts were recovered from lungs and liver of IL-4(-/-) mice associated with increased production of IFN-gamma but impaired synthesis of IL-5 and IL-10. However, a clear shift to a Th1 pattern was not characterized, since IL-4(-/-) mice did not alter delayed-type hypersensitivity anergy or IL-2 levels. In addition, IL-4 deficiency resulted in significantly reduced levels of pulmonary IL-12, granulocyte-macrophage colony-stimulating factor, IL-3, monocyte chemotactic protein 1, and specific antibody isotypes. In IL-4(-/-) mice, well-organized granulomas restraining fungal cells replaced the more extensive lesions containing high numbers of fungi and inflammatory leukocytes developed by IL-4-sufficient mice. These results clearly showed that genetically determined deficiency of IL-4 can exert a protective role in pulmonary paracoccidioidomycosis.     

Pulmonary inflammation induced by Pseudomonas aeruginosa lipopolysaccharide, phospholipase C, and exotoxin A: role of interferon regulatory factor 1

Chronic pulmonary infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) patients. P. aeruginosa lipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta-6, gamma interferon (IFN-gamma), MIP-1 alpha MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-alpha and was a weak inducer of IL-1 beta, IL-6, macrophage inflammatory protein 1 alpha (MIP-1 alpha), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-gamma. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-alpha, IL-1 beta, and IFN-gamma than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1 beta and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1 alpha and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8(+) and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors from P. aeruginosa induce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.     

TLR9 activation is a key event for the maintenance of a mycobacterial antigen-elicited pulmonary granulomatous response

Type 1 (Th1) granulomas can be studied in mice sensitized with mycobacterium antigens followed by challenge of agarose beads covalently coupled to purified protein derivative. TLR9 is known to play a role in the regulation of Th1 responses; thus, we investigated the role of TLR9 in granuloma formation during challenge with mycobacterium antigens and demonstrated that mice deficient in TLR9 had increased granuloma formation, but a dramatically altered cytokine phenotype. Th1 cytokine levels of IFN-gamma and IL-12 in the lungs were decreased in TLR9(-/-) mice when compared to wild-type mice. In contrast, Th2 cytokine levels of IL-4, IL-5, and IL-13 were increased in TLR9(-/-) mice. The migration of CD4(+) T cells in the granuloma was impaired, while the number of F4/80(+) macrophages was increased in TLR9(-/-) mice. Macrophages in the lungs of the TLR9-deficient animals with developing granulomas expressed significantly lower levels of the classically activated macrophage marker, nitric oxide synthase, but higher levels of the alternatively activated macrophage markers such as 'found in inflammatory zone-1' antigen and Arginase-1. These results suggest that TLR9 plays an important role in maintaining the appropriate phenotype in a Th1 granulomatous response.     

MD-2-dependent pulmonary immune responses to inhaled lipooligosaccharides: effect of acylation state

Endotoxins represent one of the most potent classes of microbial immunoactive components that can cause pulmonary inflammation. The aim of this study was to compare the inflammatory potency of two types of Neisseria meningitidis endotoxins (lipooligosaccharides) in lungs: wild type (hexaacylated, LOS(wt)) and mutant type (pentaacylated, LOS(msbB)), and to determine the importance of MD-2 in endotoxin responses in lungs in vivo. Endotoxin-normoresponsive mice (BALB/c) were exposed to selected doses of penta- and hexaacylated lipooligosaccharides (LOS) by nasal aspiration. Cellular and cytokine/chemokine inflammatory responses in bronchoalveolar lavage were measured at 1-, 4-, 8-, 16-, 24-, and 48-hour time points. MD-2-null mice were exposed to one dose of hexaacylated LOS and inflammatory responses were measured after 4 and 24 hours. Inhalation of hexaacylated LOS resulted in strong inflammatory responses, while pentaacylated LOS was much less potent in inducing increases of neutrophils, TNF-alpha, macrophage inflammatory protein-1 alpha, IL-6, granulocyte colony-stimulating factor, and IL-1 beta concentration in bronchoalveolar lavage. Similar kinetics of inflammatory responses in lungs were found in both types of endotoxin exposures. Inhalation of hexaacylated LOS in MD-2-null mice resulted in significantly lower numbers of neutrophils in bronchoalveolar lavage than in normoresponsive mice. Markedly lower inflammatory potency of pentaacylated LOS was observed compared with hexaacylated LOS. Hyporesponsiveness in MD-2-null mice after nasal aspiration of wild-type LOS indicate its essential role in airway responsiveness to endotoxin.     

Limited role for interleukin-18 in the host protection response to pulmonary infection with Pseudomonas aeruginosa in mice

We report that clearance of Pseudomonas aeruginosa, accumulation of neutrophils, and synthesis of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the infected lung were not largely different in interleukin-18 (IL-18) knockout or transgenic mice compared with control mice. Our results suggest a limited role for IL-18 in the host defense against P. aeruginosa.