In this study, experiments were performed to determine the contribution of TLR9 to the generation of protective innate immunity against virulent bacterial pathogens of the lung. In initial studies, we found that the intratracheal administration of Klebsiella pneumoniae in wild-type (WT) BALB/c mice resulted in the rapid accumulation of dendritic cells (DC) expressing TLR9. As compared with WT mice, animals deficient in TLR9 (TLR9−/−) displayed significantly increased mortality that was associated with a >50-fold increase in lung CFU and a >400-fold increase in K. pneumoniae CFU in blood and spleen, respectively. Intrapulmonary bacterial challenge in TLR9−/− mice resulted in reduced lung DC accumulation and maturation as well as impaired activation of lung macrophages, NK cells, and αβ and γδ T cells. Mice deficient in TLR9 failed to generate an effective Th1 cytokine response following bacterial administration. The adoptive transfer of bone marrow-derived DC from syngeneic WT but not TLR9−/− mice administered intratracheally reconstituted antibacterial immunity in TLR9−/− mice. Collectively, our findings indicate that TLR9 is required for effective innate immune responses against Gram-negative bacterial pathogens and that approaches to maximize TLR9-mediated DC responses may serve as a means to augment antibacterial immunity in pneumonia.

Tumor associated macrophages (TAMs) constitute a major component of the immune cell infiltrate observed in the tumor microenvironment (TME). Factors present in the TME including TGF-β, allow tumors to circumvent host mediated immune responses to promote tumor progression. However, the molecular mechanism(s) involved are not clear. Toll-like receptors (TLRs) are important mediators of innate immune responses by immune cells, whose activation triggers the production of molecules required for anti-tumoral responses. Interleukin receptor associated kinase (IRAK)-M is an inactive serine/threonine kinase, predominantly expressed in macrophages and is a potent negative regulator of TLR signaling. Here we show that TAMs express significantly higher levels of IRAK-M compared to peritoneal macrophages (PEMs) in a syngeneic mouse model of lung cancer. Subcutaneous implantation of LLC cells in IRAK-M−/− mice resulted in a five-fold reduction in tumor growth, as compared to tumors in wild type animals. Furthermore, compared to WT TAMs, TAMs isolated from IRAK-M−/− mice displayed features of a classically activated (M1) rather than alternatively activated (M2) phenotype, as manifest by greater expression of IL-12, IFN-γ, and iNOS. Human lung cancer cells induced IRAK-M expression in human PBMCs when co-cultured together. Tumor cell-induced expression of IRAK-M was dependent on the activation of TGF-β pathway. Similarly, treatment of human PBMCs or mouse macrophage cell line, RAW 264.4, with TGF-β, induced IRAK-M expression. Interestingly, IRAK-M gene expression in 439 human lung adenocarcinoma tumors correlated with poor survival in patients with lung cancer. Together, our data demonstrates that TGF-β-dependent induction of IRAK-M expression is an important, clinically relevant mechanism by which tumors may circumvent anti-tumor responses of macrophages.

Influenza virus is a common cause of respiratory infection and morbidity, which is often due to deleterious host immune responses directed against the pathogen. We investigated the role of IL-1 receptor-associated kinase-M (IRAK-M), an inhibitor of MyD88-dependent TLR signaling, in modulating the innate inflammatory response during influenza pneumonia using a murine model. The intranasal administration of influenza resulted in the upregulation of IRAK-M mRNA and protein levels in the lungs within 2 d after infectious challenge. Pulmonary influenza infection in mice deficient in IRAK-M (IRAK-M−/−) resulted in substantially increased mortality compared with similarly treated wild-type animals. Increased mortality in IRAK-M−/− mice was associated with enhanced early influx of neutrophils, high permeability edema, apoptosis of lung epithelial cells, markedly increased expression of inflammatory cytokines/chemokines, and release of neutrophil-derived enzymes, including myeloperoxidase and neutrophil elastase. Early viral clearance was not different in mutant mice, whereas viral titers in lungs and blood were significantly higher in IRAK-M−/−mice compared with wild-type animals. Increased lethality observed in IRAK-M−/− mice after influenza challenge was abrogated by Ab-mediated blockade of CXCR2. Collectively, our findings indicate that IRAK-M is critical to preventing deleterious neutrophil-dependent lung injury during influenza infection of the respiratory tract.

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, CRAMP, to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp−/−) demonstrate impaired lung bacterial clearance, increased bacterial dissemination and reduced survival in response to i.t. K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 hrs in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.

Experiments were performed to determine the contribution of TLR9 to the generation of protective immunity against the intracellular respiratory bacterial pathogen Legionella pneumophila. In initial studies, we found that the intratracheal (i.t.) administration of L. pneumophila to mice deficient in TLR9 (TLR9

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9+/+) and TLR-deficient (TLR9−/−) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b+ dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4+ and CD8+ T cells, CD11b+ lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9−/− mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b+ DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9+/+ and TLR9−/− mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9+/+ mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Sepsis results in a profound state of immunosuppression, which is temporally associated with impaired leukocyte function. The mechanism of leukocyte reprogramming in sepsis is incompletely understood. In this study, we explored mechanisms contributing to dysregulated inflammatory cytokine expression by pulmonary macrophages during experimental sepsis. Pulmonary macrophages (PM) recovered from the lungs of mice undergoing cecal ligation and puncture (CLP) display transiently reduced expression of some, but not all innate genes in response to LPS. Impaired expression of TNF-α and iNOS was associated with reduced acetylation and methylation of specific histones (AcH4 and H3K4me3) and reduced binding of RNA polymerase II to the promoters of these genes. Transient impairment in LPS-induced cytokine responses in septic PM temporally correlated with induction of IRAK-M mRNA and protein, which occurred in a MyD88-dependent fashion. PM isolated from IRAK-M−/− mice were largely refractory to CLP-induced impairment in cytokine expression, chromatin remodeling, recruitment of RNA polymerase II, and induction of histone deacetylase-2 observed during sepsis. Our findings indicate that systemic sepsis induces epigenetic silencing of cytokine gene expression in lung macrophages, and IRAK-M appears to be a critical mediator of this response.

To determine whether TLR9 signaling contributes to the development of the adaptive immune response to cryptococcal infection, wild-type (TLR9؉/؉) and TLR9 knockout (TLR9؊/؊) BALB/c mice were infected intratracheally with 10 4 C. neoformans 52D. We evaluated 1) organ microbial burdens, 2) pulmonary leukocyte recruitment, 3) pulmonary and systemic cytokine induction , and 4) macrophage activation profiles. TLR9 deletion did not affect pulmonary growth during the innate phase, but profoundly impaired pulmonary clearance during the adaptive phase of the immune response (a 1000-fold difference at week 6). The impaired clearance in TLR9؊/؊ mice was associated with: 1) significantly reduced CD4