Personalized training pathways for translational science trainees: Building on a framework of knowledge, skills, and abilities across the translational science spectrum.

BACKGROUND: In order to conduct translational science, scientists must combine domain-specific expertise with knowledge on how to identify and cross translational hurdles, and insights on positioning discoveries for the next translational stage. Expert educators from the Clinical and Translational Science Awards (CTSA) Consortium identified 97 knowledge, skills, and abilities (KSAs) important to include in training programs for translational scientists. To assist educators and trainees to use these KSAs, a conceptual model called "Personalized Pathways" was developed that prioritizes KSAs based on trainee background, research area, or phenotype, and expertise on the research team. PURPOSE: To understand how CTSA educators prioritize specific KSAs when developing personalized training plans for different translational phenotypes and to identify areas of similarity and difference across phenotypes. METHODS: A web-based, cross-sectional survey of CTSA educators was done. For a selected phenotype, respondents recommended one of four levels of mastery for each of the 97 KSAs. Results were tabulated by frequency, weighted by importance, and divided into tertiles representing high, middle, and lower priority KSAs. Agreement across phenotypes was compared using Krippendorff's alpha. RESULTS: Ten KSAs were high training priority for Preclinical, Clinical, and Community-Engaged phenotypes. These address research methods, responsible conduct of research, team building, and communicating research results. Nine KSAs were in the next tertile for priority reflecting KSAs in biostatistics, bioinformatics, regulatory precepts, and translating implications of research findings. CONCLUSION: A smaller set of KSAs can be prioritized for training Preclinical-, Clinical-, and Community-Engaged researchers. Future work should explore this approach for other phenotypes.

Unique Lipid Signatures of Extracellular Vesicles from the Airways of Asthmatics.

Asthma is a chronic inflammatory disease process involving the conductive airways of the human lung. The dysregulated inflammatory response in this disease process may involve multiple cell-cell interactions mediated by signaling molecules, including lipid mediators. Extracellular vesicles (EVs) are lipid membrane particles that are now recognized as critical mediators of cell-cell communication. Here, we compared the lipid composition and presence of specific lipid mediators in airway EVs purified from the bronchoalveolar lavage (BAL) fluid of healthy controls and asthmatic subjects with and without second-hand smoke (SHS) exposure. Airway exosome concentrations were increased in asthmatics, and correlated with blood eosinophilia and serum IgE levels. Frequencies of HLA-DR+ and CD54+ exosomes were also significantly higher in asthmatics. Lipidomics analysis revealed that phosphatidylglycerol, ceramide-phosphates, and ceramides were significantly reduced in exosomes from asthmatics compared to the non-exposed control groups. Sphingomyelin 34:1 was more abundant in exosomes of SHS-exposed asthmatics compared to healthy controls. Our results suggest that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.

Distal airway microbiome is associated with immunoregulatory myeloid cell responses in lung transplant recipients.

BACKGROUND: Long-term survival of lung transplant recipients (LTRs) is limited by the occurrence of bronchiolitis obliterans syndrome (BOS). Recent evidence suggests a role for microbiome alterations in the occurrence of BOS, although the precise mechanisms are unclear. In this study we evaluated the relationship between the airway microbiome and distinct subsets of immunoregulatory myeloid-derived suppressor cells (MDSCs) in LTRs. METHODS: Bronchoalveolar lavage (BAL) and simultaneous oral wash and nasal swab samples were collected from adult LTRs. Microbial genomic DNA was isolated, 16S rRNA genes amplified using V4 primers, and polymerase chain reaction (PCR) products sequenced and analyzed. BAL MDSC subsets were enumerated using flow cytometry. RESULTS: The oral microbiome signature differs from that of the nasal, proximal and distal airway microbiomes, whereas the nasal microbiome is closer to the airway microbiome. Proximal and distal airway microbiome signatures of individual subjects are distinct. We identified phenotypic subsets of MDSCs in BAL, with a higher proportion of immunosuppressive MDSCs in the proximal airways, in contrast to a preponderance of pro-inflammatory MDSCs in distal airways. Relative abundance of distinct bacterial phyla in proximal and distal airways correlated with particular airway MDSCs. Expression of CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP), an endoplasmic (ER) stress sensor, was increased in immunosuppressive MDSCs when compared with pro-inflammatory MDSCs. CONCLUSIONS: The nasal microbiome closely resembles the microbiome of the proximal and distal airways in LTRs. The association of distinct microbial communities with airway MDSCs suggests a functional relationship between the local microbiome and MDSC phenotype, which may contribute to the pathogenesis of BOS.

Subsets of airway myeloid-derived regulatory cells distinguish mild asthma from chronic obstructive pulmonary disease.

BACKGROUND: Subsets of myeloid-derived regulatory cells (MDRCs), which are phenotypically similar to the myeloid-derived suppressor cells found in patients with cancer, have recently been appreciated as critical regulators of airway inflammation in mouse models of asthma. OBJECTIVE: We test the hypothesis that subsets of airway MDRCs contribute differentially to the inflammatory milieu in human asthma and chronic obstructive pulmonary disease (COPD). METHODS: We used bronchoalveolar lavage to identify and characterize human airway MDRCs from 10 healthy subjects, 9 patients with mild asthma, and 8 patients with COPD, none of whom were treated with inhaled or systemic corticosteroids. We defined subsets of airway MDRCs using flow cytometry, the molecular mediators they produce, and their abilities to regulate proliferation of polyclonally activated autologous T lymphocytes. RESULTS: We found substantial differences in the functional potential of MDRC subsets in healthy subjects, patients with asthma, and patients with COPD, with these differences regulated by the nitrosative and oxidative free radicals and cytokines they produced. Nitric oxide-producing MDRCs suppressed and superoxide-producing MDRCs enhanced proliferation of polyclonally activated autologous CD4 T cells. HLA-DR(+)CD11b(+)CD11c(+)CD163(-) superoxide-producing MDRCs, which stimulated proliferation of autologous T cells, comprised a high fraction of MDRCs in the airways of patients with mild asthma or COPD but not those of healthy control subjects. CD11b(+)CD14(+)CD16(-)HLA-DR(-) nitric oxide-producing MDRCs, which suppressed T-cell proliferation, were present in high numbers in airways of patients with mild asthma but not patients with COPD or healthy control subjects. CONCLUSION: Subsets of airway MDRCs conclusively discriminate patients with mild asthma, patients with COPD, and healthy subjects from each other. The distinctive activities of these MDRCs in patients with asthma or COPD might provide novel targets for new therapeutics for these common disorders. [Corrected]

IL-22-producing neutrophils contribute to antimicrobial defense and restitution of colonic epithelial integrity during colitis.

IL-22 plays an important role in mucosal epithelial cell homeostasis. Using a dextran sodium sulfate-induced mouse model of acute colitis, we observed an IL-23-dependent up-regulation of IL-22 in the middle and distal colon at the onset of epithelial cell damage. This heightened IL-22 correlated with an influx of innate immune cells, suggesting an important role in colonic epithelial protection. Freshly isolated colon-infiltrating neutrophils produced IL-22 contingent upon IL-23 signaling, and IL-22 production was augmented by TNF-α. Importantly, the depletion of neutrophils resulted in diminished IL-22 levels in the colon, and the transfer of IL-22-competent neutrophils to Il22a-deficient mice protected the colonic epithelium from dextran sodium sulfate-induced damage. In addition, IL-22-producing neutrophils targeted colonic epithelial cells to up-regulate the antimicrobial peptides, RegIIIß and S100A8. This study establishes a role for neutrophils in providing IL-22-dependent mucosal epithelial support that contributes to the resolution of colitis.

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism.

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.

Inflammasome-derived IL-1ß regulates the production of GM-CSF by CD4(+) T cells and γδ T cells.

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αß and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1ß upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αß and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1ß and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.

Vitronectin inhibits neutrophil apoptosis through activation of integrin-associated signaling pathways.

Vitronectin is present in large concentrations in serum and the extracellular matrix. Although vitronectin is known to modulate neutrophil adhesion and chemotaxis, and to contribute to neutrophil-associated proinflammatory processes, a role in apoptosis has not been demonstrated. In the present studies, we found that neutrophils demonstrated more rapid progression to spontaneous or TNF-related apoptosis-inducing ligand-induced apoptosis when incubated under vitronectin-free conditions than when vitronectin was present. The ability of native vitronectin to delay neutrophil apoptosis was not recapitulated by the vitronectin somatomedin B domain. In contrast, inclusion of the cyclo[Arg-Gly-Asp-D-Phe-Val] peptide in cultures containing vitronectin resulted in enhanced neutrophil apoptosis, showing that the vitronectin RGD motif (Arg-Gly-Asp motif) was responsible for the antiapoptotic effects of vitronectin. Addition of antibodies to ß(1), ß(3), or ß(5), but not to ß(2) or ß(4) integrins, reversed the ability of vitronectin to diminish neutrophil apoptosis. The ability of vitronectin to enhance neutrophil viability was dependent on activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 kinases, but not on the p38 kinase. Increased numbers of apoptotic neutrophils were present in the lungs of LPS-treated transgenic vitronectin-deficient mice, as compared with control mice. These results demonstrate a novel antiapoptotic function for vitronectin.

Regulatory role of antigen-induced interleukin-10, produced by CD4(+) T cells, in airway neutrophilia in a murine model for asthma.

It has been suggested that interleukin (IL)-10 exerts immunosuppressive effects on allergic inflammation, including asthma, mainly through inhibition of Th2 cell-mediated eosinophilic airway inflammation. In a model of experimental asthma utilizing multiple intratracheal antigen challenges in sensitized mice, IL-10 production as well as eosinophilia and neutrophilia in the lung were induced by the multiple challenges. In this study, we set out to reveal the cellular source of endogenously produced IL-10, and the roles of IL-10 in airway leukocyte inflammation using an anti-IL-10 receptor monoclonal antibody. Balb/c mice were sensitized i.p. with ovalbumin+Al(OH)(3), and then challenged by intratracheal administration of ovalbumin 4 times. Flow cytometric analyses revealed that the cellular source of IL-10 was CD4(+) T cells lacking the transcription factor, forkhead box P3. Treatment with anti-IL-10 receptor monoclonal antibody prior to the 4th challenge significantly augmented airway neutrophilia as well as the production of IL-1ß, and CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2, but not airway eosinophilia, Th2 cytokine (IL-4 and IL-5) production, or a late-phase increase in specific airway resistance. Approximately 40% of IL-10 receptor(+) cells expressed the macrophage marker F4/80, whereas only 3-4% of the IL-10 receptor(+) cells were granulocyte differentiation antigen (Gr)-1(high) cells (neutrophils). In conclusion, multiple airway antigen challenges induced the proliferation of IL-10-expressing CD4(+) T cells in regulating airway neutrophilia. Systemic blockade of IL-10 function coincided with increases in IL-1ß and CXC chemokines. Thus, IL-1ß and CXC chemokines may be targets for development of novel pharmacotherapy for neutrophilic asthma.

AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils.

Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46 ± 7.8 or 85 ± 26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21 ± 1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.

Neutrophils produce interleukin 17A (IL-17A) in a dectin-1- and IL-23-dependent manner during invasive fungal infection.

We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogen Aspergillus fumigatus. Here we have shown that culturing lung cells from A. fumigatus-challenged mice ex vivo demonstrated Dectin-1-dependent IL-17A production. In this system, neutralization of IL-23 but not IL-6, IL-1ß, or IL-18 resulted in attenuated IL-17A production. Il23 mRNA expression was found to be lower in lung cells from A. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response to A. fumigatus in vitro. Addition of recombinant IL-23 augmented IL-17A production by wild-type (WT) and Dectin-1-deficient lung cells, although the addition of IL-6 or IL-1ß did not augment the effect of IL-23. Intracellular cytokine staining of lung cells revealed lower levels of CD11b(+) IL-17A(+) and Ly-6G(+) IL-17A(+) cells in A. fumigatus-challenged Dectin-1-deficient mice. Ly-6G(+) neutrophils purified from the lungs of A. fumigatus-challenged Dectin-1-deficient mice displayed lower Il17a mRNA expression but surprisingly had intact Rorc and Rora mRNA expression. We further demonstrated that Ly-6G(+) neutrophils required the presence of myeloid cells for IL-17A production. Finally, upon in vitro stimulation with A. fumigatus, thioglycolate-elicited peritoneal neutrophils were positive for intracellular IL-17A expression and produced IL-17A in a Dectin-1- and IL-23-dependent manner. In summary, Dectin-1-dependent IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b(+) Ly-6G(+) neutrophils in an IL-23-dependent manner.

Inhibition of neutrophil apoptosis by PAI-1.

Increased circulating and tissue levels of plasminogen activator inhibitor 1 (PAI-1) are often present in severe inflammatory states associated with neutrophil activation and accumulation and correlate with poor clinical outcome from many of these conditions. The mechanisms by which PAI-1 contributes to inflammation have not been fully delineated. In the present experiments, we found that addition of PAI-1 to neutrophil cultures diminished the rate of spontaneous and TNF-related apoptosis-inducing ligand-induced apoptotic cell death. The effects of PAI-1 on cell viability were associated with activation of antiapoptotic signaling pathways, including upregulation of PKB/Akt, Mcl-1, and Bcl-x(L). Although urokinase-plasminogen activator receptor, lipoprotein receptor-related protein, and vitronectin are primary ligands for PAI-1, these molecules were not involved in mediating its antiapoptotic properties. In contrast, blocking pertussis toxin-sensitive G protein-coupled receptors and selective inhibition of phosphatidylinositide 3-kinase reversed the ability of PAI-1 to extend neutrophil viability. The antiapoptotic effects of PAI-1 were also evident under in vivo conditions during LPS-induced acute lung injury, where enhanced apoptosis was present among neutrophils accumulating in the lungs of PAI-1(-/-) compared with PAI-1(+/+) mice. These results demonstrate a novel antiapoptotic role for PAI-1 that may contribute to its participation in neutrophil-associated inflammatory responses.

Important role of neutrophils in the late asthmatic response in mice.

AIMS: Neutrophils have been found increasingly in the lungs of patients with severe asthma; however, it is unclear whether the neutrophils contribute to the induction of the airway obstruction. We determined using a murine model whether neutrophils are involved in the late asthmatic response (LAR), and analyzed mechanisms underlying the antigen-induced airway neutrophilia. MAIN METHODS: BALB/c mice sensitized by ovalbumin (OVA)+Al(OH)(3) were challenged 4 times by intratracheal administration of OVA. Airway mechanics were measured as specific airway resistance. KEY FINDINGS: Induction of the LAR after the 4th challenge coincided with airway neutrophilia. In contrast, eosinophil infiltration was established prior to the 4th challenge. A treatment with an anti-Gr-1 monoclonal antibody (mAb) before the 4th challenge selectively suppressed increases in the neutrophil number and myeloperoxidase (MPO) level in bronchoalveolar lavage fluid (BALF), and attenuated the magnitude of LAR by 60-70%. Selective suppression of eosinophilia by anti-IL-5 mAb had little effect on the LAR. The increases in neutrophil number and MPO level were partially inhibited by an anti-CD4 mAb treatment. The CD4(+) cell depletion also significantly inhibited increases in neutrophil chemoattractants, IL-17A, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in BALF. However, blockade of FcγRII/III failed to suppress the neutrophilia. SIGNIFICANCE: These data suggest that neutrophils are key inducers of the LAR, and that the antigen-induced neutrophilia is partially dependent on activated CD4(+) cells that are involved in the production of IL-17A, KC and MIP-2.

Elevated levels of NO are localized to distal airways in asthma.

The contribution of nitric oxide (NO) to the pathophysiology of asthma remains incompletely defined despite its established pro- and anti-inflammatory effects. Induction of the inducible nitric oxide synthase (iNOS), arginase, and superoxide pathways is correlated with increased airway hyperresponsiveness in asthmatic subjects. To determine the contributions of these pathways in proximal and distal airways, we compared bronchial wash (BW) to traditional bronchoalveolar lavage (BAL) for measurements of reactive nitrogen/oxygen species, arginase activation, and cytokine/chemokine levels in asthmatic and normal subjects. Levels of NO were preferentially elevated in the BAL, demonstrating higher level NOS activation in the distal airway compartment of asthmatic subjects. In contrast, DHE(+) cells, which have the potential to generate reactive oxygen species, were increased in both proximal and distal airway compartments of asthmatics compared to controls. Different patterns of cytokines and chemokines were observed, with a predominance of epithelial cell-associated mediators in the BW compared to macrophage/monocyte-derived mediators in the BAL of asthmatic subjects. Our study demonstrates differential production of reactive species and soluble mediators within the distal airways compared to the proximal airways in asthma. These results indicate that cellular mechanisms are activated in the distal airways of asthmatics and must be considered in the development of therapeutic strategies for this chronic inflammatory disorder.

Inhibition of the catalytic function of activation-induced cytidine deaminase promotes apoptosis of germinal center B cells in BXD2 mice.

OBJECTIVE: To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. METHODS: We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice. RESULTS: Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice. CONCLUSION: Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

Follicular dendritic cell makes environmental sense.

Follicular dendritic cells (FDCs) are key organizers of B cell follicles and germinal centers. In this issue of Immunity, Suzuki et al. (2010) and Garin et al. (2010) identify the roles of Toll-like receptors in the responses of FDCs, providing a unique link between innate and adaptive immunity.

Overview of the immune response.

The immune system has evolved to protect the host from a universe of pathogenic microbes that are themselves constantly evolving. The immune system also helps the host eliminate toxic or allergenic substances that enter through mucosal surfaces. Central to the immune system's ability to mobilize a response to an invading pathogen, toxin, or allergen is its ability to distinguish self from nonself. The host uses both innate and adaptive mechanisms to detect and eliminate pathogenic microbes, and both of these mechanisms include self-nonself discrimination. This overview identifies key mechanisms used by the immune system to respond to invading microbes and other exogenous threats and identifies settings in which disturbed immune function exacerbates tissue injury.

Critical role of macrophages and their activation via MyD88-NFκB signaling in lung innate immunity to Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp), a common cause of pneumonia, is associated with asthma; however, the mechanisms underlying this association remain unclear. We investigated the cellular immune response to Mp in mice. Intranasal inoculation with Mp elicited infiltration of the lungs with neutrophils, monocytes and macrophages. Systemic depletion of macrophages, but not neutrophils, resulted in impaired clearance of Mp from the lungs. Accumulation and activation of macrophages were decreased in the lungs of MyD88(-/-) mice and clearance of Mp was impaired, indicating that MyD88 is a key signaling protein in the anti-Mp response. MyD88-dependent signaling was also required for the Mp-induced activation of NFκB, which was essential for macrophages to eliminate the microbe in vitro. Thus, MyD88-NFκB signaling in macrophages is essential for clearance of Mp from the lungs.