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A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03346-0.
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Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.
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Inflamassomos , Nucleosídeo Difosfato Quinase D , Animais , Inflamassomos/genética , Inflamassomos/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Mitocôndrias/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleosídeo Difosfato Quinase D/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009395.].
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The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.
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Infecções por Burkholderia/imunologia , Burkholderia cenocepacia/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Macrófagos/imunologia , Animais , Citosol/imunologia , Citosol/microbiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The liver connects the intestinal portal vasculature with the general circulation, using a diverse array of immune cells to protect from pathogens that translocate from the gut1. In liver lobules, blood flows from portal triads that are situated in periportal lobular regions to the central vein via a polarized sinusoidal network. Despite this asymmetry, resident immune cells in the liver are considered to be broadly dispersed across the lobule. This differs from lymphoid organs, in which immune cells adopt spatially biased positions to promote effective host defence2,3. Here we used quantitative multiplex imaging, genetic perturbations, transcriptomics, infection-based assays and mathematical modelling to reassess the relationship between the localization of immune cells in the liver and host protection. We found that myeloid and lymphoid resident immune cells concentrate around periportal regions. This asymmetric localization was not developmentally controlled, but resulted from sustained MYD88-dependent signalling induced by commensal bacteria in liver sinusoidal endothelial cells, which in turn regulated the composition of the pericellular matrix involved in the formation of chemokine gradients. In vivo experiments and modelling showed that this immune spatial polarization was more efficient than a uniform distribution in protecting against systemic bacterial dissemination. Together, these data reveal that liver sinusoidal endothelial cells sense the microbiome, actively orchestrating the localization of immune cells, to optimize host defence.
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Microbioma Gastrointestinal/imunologia , Fígado/imunologia , Fígado/microbiologia , Simbiose/imunologia , Animais , Bactérias/imunologia , Bactérias/isolamento & purificação , Separação Celular , Quimiocina CXCL9/imunologia , Células Endoteliais/citologia , Células Endoteliais/imunologia , Feminino , Humanos , Células de Kupffer/citologia , Células de Kupffer/imunologia , Células de Kupffer/metabolismo , Fígado/irrigação sanguínea , Fígado/citologia , Linfócitos/imunologia , Masculino , Camundongos , Modelos Imunológicos , Imagem Molecular , Células Mieloides/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Simbiose/genética , TranscriptomaRESUMO
Despite existing evidence for tuning of innate immunity to different classes of bacteria, the molecular mechanisms used by macrophages to tailor inflammatory responses to specific pathogens remain incompletely defined. By stimulating mouse macrophages with a titration matrix of TLR ligand pairs, we identified distinct stimulus requirements for activating and inhibitory events that evoked diverse cytokine production dynamics. These regulatory events were linked to patterns of inflammatory responses that distinguished between Gram-positive and Gram-negative bacteria, both in vitro and after in vivo lung infection. Stimulation beyond a TLR4 threshold and Gram-negative bacteria-induced responses were characterized by a rapid type I IFN-dependent decline in inflammatory cytokine production, independent of IL-10, whereas inflammatory responses to Gram-positive species were more sustained due to the absence of this IFN-dependent regulation. Thus, disparate triggering of a cytokine negative feedback loop promotes tuning of macrophage responses in a bacteria class-specific manner and provides context-dependent regulation of inflammation dynamics.
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Retroalimentação Fisiológica , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Positivas/imunologia , Interferon Tipo I/metabolismo , Macrófagos/imunologia , Pneumonia Bacteriana/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
The nuclear factor-κB (NF-κB) signaling pathway is one of the best understood immune-related pathways thanks to almost four decades of intense research. NF-κB signaling is activated by numerous discrete stimuli and is a master regulator of the inflammatory response to pathogens and cancerous cells, as well as a key regulator of autoimmune diseases. In this regard, the role of NF-κB signaling in immunity is not unlike that of the macrophage. The dynamics by which NF-κB proteins shuttle between the cytoplasm and the nucleus to initiate transcription have been studied rigorously in fibroblasts and other non-hematopoietic cells, but many questions remain as to how current models of NF-κB signaling and dynamics can be translated to innate immune cells such as macrophages. In this review, we will present recent research on the dynamics of NF-κB signaling and focus especially on how these dynamics vary in different cell types, while discussing why these characteristics may be important. We will end by looking ahead to how new techniques and technologies should allow us to analyze these signaling processes with greater clarity, bringing us closer to a more complete understanding of inflammatory transcription factor dynamics and how different cellular contexts might allow for appropriate control of innate immune responses.
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Macrófagos/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Animais , Humanos , Imunidade Inata/imunologia , Inflamação/imunologiaRESUMO
Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1-/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.
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Cromatina/metabolismo , Fator de Transcrição Ikaros/metabolismo , Inflamação/imunologia , Macrófagos/fisiologia , Animais , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica/imunologia , Humanos , Fator de Transcrição Ikaros/genética , Inflamação/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Ligação Proteica , Células RAW 264.7RESUMO
Streptococcus pneumoniae is a leading cause of invasive bacterial infections, with nasal colonization an important first step in disease. While cigarette smoking is a strong risk factor for invasive pneumococcal disease, the underlying mechanisms remain unknown. This is partly due to a lack of clinically relevant animal models investigating nasal pneumococcal colonization in the context of cigarette smoke exposure. We present a model of nasal pneumococcal colonization in cigarette smoke-exposed mice and document, for the first time, that cigarette smoke predisposes to invasive pneumococcal infection and mortality in an animal model. Cigarette smoke increased the risk of bacteremia and meningitis without prior lung infection. Mechanistically, deficiency in interleukin 1α (IL-1α) or platelet-activating factor receptor (PAFR), an important host receptor thought to bind and facilitate pneumococcal invasiveness, did not rescue cigarette smoke-exposed mice from invasive pneumococcal disease. Importantly, we observed cigarette smoke to attenuate nasal inflammatory mediator expression, particularly that of neutrophil-recruiting chemokines, normally elicited by pneumococcal colonization. Smoking cessation during nasal pneumococcal colonization rescued nasal neutrophil recruitment and prevented invasive disease in mice. We propose that cigarette smoke predisposes to invasive pneumococcal disease by suppressing inflammatory processes of the upper respiratory tract. Given that smoking prevalence remains high worldwide, these findings are relevant to the continued efforts to reduce the invasive pneumococcal disease burden.
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Portador Sadio/imunologia , Mucosa Nasal/microbiologia , Infecções Pneumocócicas/imunologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Streptococcus pneumoniae/crescimento & desenvolvimento , Animais , Bacteriemia/microbiologia , Bacteriemia/prevenção & controle , Portador Sadio/prevenção & controle , Modelos Animais de Doenças , Resistência à Doença , Meningite Pneumocócica/microbiologia , Meningite Pneumocócica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Nasal/imunologia , Neutrófilos/imunologia , Infecções Pneumocócicas/prevenção & controle , Streptococcus pneumoniae/imunologiaRESUMO
Macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor (cA-SR) that recognizes and phagocytoses a wide variety of pathogens. Most cA-SRs that contain a C-terminal scavenger receptor cysteine-rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signaling is less clear. The discovery of a naturally occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signaling and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant-negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-like receptor 2 (TLR2)-mediated pro-inflammatory signaling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, whereas MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signaling and MARCO-mediated cellular adhesion.
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Processamento Alternativo/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Sequência de Aminoácidos , Animais , Adesão Celular , Forma Celular , Clonagem Molecular , Endocitose , Células HEK293 , Humanos , Ligantes , Receptores de Lipopolissacarídeos/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/metabolismo , Streptococcus pneumoniae/fisiologia , Relação Estrutura-Atividade , Receptor 2 Toll-Like/metabolismoRESUMO
RATIONALE: Nontypeable Haemophilus influenzae (NTHi) causes acute exacerbation of chronic obstructive pulmonary disease (AECOPD). IL-17A is central for neutrophilic inflammation and has been linked to COPD pathogenesis. OBJECTIVES: We investigated whether IL-17A is elevated in NTHi-associated AECOPD and required for NTHi-exacerbated pulmonary neutrophilia induced by cigarette smoke. METHODS: Experimental studies with cigarette smoke and NTHi infection were pursued in gene-targeted mice and using antibody intervention. IL-17A was measured in sputum collected from patients with COPD at baseline, during, and after AECOPD. MEASUREMENTS AND MAIN RESULTS: Exacerbated airway neutrophilia in cigarette smoke-exposed mice infected with NTHi was associated with an induction of IL-17A. In agreement, elevated IL-17A was observed in sputum collected during NTHi-associated AECOPD, compared with samples collected before or after the event. NTHi-exacerbated neutrophilia and induction of neutrophil chemoattractants over the background of cigarette smoke, as observed in wild-type mice, was absent in Il17a(-/-) mice and in mice treated with a neutralizing anti-IL-17A antibody. Further studies revealed that IL-1 receptor (R)1 signaling was required for IL-17A-dependent neutrophilia. Moreover, deficiency or therapeutic neutralization of IL-17A did not increase bacterial burden or delay bacterial clearance. CONCLUSIONS: IL-17A is induced during NTHi-associated AECOPD. Functionally, IL-1R1-dependent IL-17A is required for NTHi-exacerbated pulmonary neutrophilia induced by cigarette smoke. Targeting IL-17A in AECOPD may thus be beneficial to reduce neutrophil recruitment to the airways.
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Infecções por Haemophilus/metabolismo , Haemophilus influenzae , Interleucina-17/metabolismo , Neutrófilos/fisiologia , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/metabolismo , Idoso , Animais , Modelos Animais de Doenças , Feminino , Infecções por Haemophilus/complicações , Humanos , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Fumar/efeitos adversosRESUMO
Tumor necrosis factor (TNF), a potent inflammatory cytokine, and mitochondrial DNA (mtDNA), a product of inflammation-induced tissue damage, increase with age ("inflammaging") and many chronic diseases. Peripheral blood neutrophils, a critical component of innate immunity, have also been shown to be altered with age, and are exceptionally sensitive to external stimuli. Herein, we describe that the phenotype of neutrophils from the advanced-age, frail elderly (ELD) is determined by levels of circulating TNF and mtDNA. Neutrophils from ELD donors are morphologically immature, and have higher levels of intracellular reactive oxygen species (ROS) and expression of the activation markers CD11b and HLA-DR. The frequency of CD11b(++) neutrophils correlated with plasma TNF, and recombinant TNF elevated neutrophil CD11b ex vivo and in vivo. Furthermore, neutrophils from aged TNF-deficient mice expressed CD11b similar to young counterparts. The frequency of HLA-DR(+) neutrophils, on the other hand, positively correlated with circulating mtDNA, which increased neutrophil HLA-DR expression in a dose-dependent manner ex vivo. Cell-surface TLR-9 expression, however, was unaltered on neutrophils from ELD donors. In summary, we provide novel evidence that products of age-related inflammation modulate neutrophil phenotype in vivo. Given this, anti-inflammatory therapies may prove beneficial in improving neutrophil functionality in the elderly.
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DNA Mitocondrial/sangue , Idoso Fragilizado , Inflamação/imunologia , Neutrófilos/imunologia , Fator de Necrose Tumoral alfa/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno CD11b/biossíntese , Doença Crônica , Feminino , Antígenos HLA-DR/biossíntese , Humanos , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Ativação de Neutrófilo/imunologia , Neutrófilos/fisiologia , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Receptor Toll-Like 9/biossíntese , Fator de Necrose Tumoral alfa/genética , Adulto JovemRESUMO
Pneumonia caused by Streptococcus pneumoniae is a major cause of death and an economic burden worldwide. S. pneumoniae is an intermittent colonizer of the human upper respiratory tract, and the ability to control asymptomatic colonization determines the likelihood of developing invasive disease. Recognition of S. pneumoniae by resident macrophages via Toll-like receptor 2 (TLR-2) and the macrophage receptor with collagenous structure (MARCO) and the presence of interleukin-17 (IL-17)-secreting CD4(+) T cells are required for macrophage recruitment and bacterial clearance. Despite the fact that the primary cellular effectors needed for bacterial clearance have been identified, much of the underlying regulatory mechanisms are unknown. Herein, we demonstrate that the small, noncoding RNA microRNA-155 (mir-155) is critical for the effective clearance of S. pneumoniae. Our studies show that mir-155-deficient mice maintain the ability to prevent acute invasive pneumococcal infection but have significantly higher bacterial burdens following colonization, independently of macrophage recognition by TLR-2, MARCO expression, or bactericidal capacity. The observed defects in bacterial clearance parallel reduced IL-17A and gamma interferon CD4(+) T-cell responses in vivo, lower IL-17A mRNA levels in the nasopharynx, and a reduced capacity to induce Th17 cell polarization. Given that knockout mice are also limited in the capacity to generate high-titer S. pneumoniae-specific antibodies, we conclude that mir-155 is a critical mediator of the cellular effectors needed to clear primary and secondary S. pneumoniae colonizations.
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MicroRNAs/metabolismo , Nasofaringe/microbiologia , Streptococcus pneumoniae/fisiologia , Animais , Portador Sadio , Regulação da Expressão Gênica/imunologia , Macrófagos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Nasofaringe/imunologia , FagocitoseRESUMO
Vaccination remains the most effective prophylactic intervention for infectious disease in the healthcare professional's toolkit. However, the efficacy and effectiveness of vaccines decrease with age. This becomes most apparent after an individual reaches 65-70 years old, and results from complex changes in the immune system that occur during aging. As such, new vaccine formulations and strategies that can accommodate age-related changes in immunity are required to protect this expanding population. Here, we summarize the consequences of immunosenescence on vaccination and how novel vaccination strategies can be designed to accommodate the aging immune system. We conclude that current vaccination protocols are not sufficient to protect our aging population and, in some cases, are an inefficient use of healthcare resources. However, researchers and clinicians are developing novel vaccination strategies that include modifying who and when we vaccinate and capitalize on existing vaccines, in addition to formulating new vaccines specifically tailored to the elderly in order to remedy this deficiency.
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As we age, the composition of our peripheral leukocytes changes dramatically. Many of these alterations contribute to the general immune dysfunction that burdens the elderly, which in turn, contributes to increased susceptibility to disease. MDSCs represent a heterogeneous population of immunosuppressive leukocytes that are elevated in the peripheral blood of cancer patients. Given the relation between cancer incidence and age, this study examined the frequency of peripheral blood CD33(+)HLA-DR(-) MDSCs across three cohorts: healthy adults (19-59 years old), community-dwelling seniors (61-76 years old), and frail elderly (67-99 years old). This analysis is the first to demonstrate that MDSCs and specifically the CD11b(+)CD15(+) MDSC subset are increased with age. Proinflammatory cytokines that are required for the differentiation of MDSCs (e.g., TNF-α, IL-6, and IL-1ß) were similarly found to be increased in the serum of the frail elderly. Furthermore, the proportion of MDSCs and the CD11b(+)CD15(+) subset were found to be elevated significantly in elderly donors with a history of cancer. This age-related elevation in the frequency of MDSCs may contribute to the increased cancer incidence that occurs with age. Further investigation into the functional consequences of elevated MDSCs will provide valuable insight into the progression of age-related pathologies.
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Envelhecimento/patologia , Antígenos HLA-DR/genética , Células Mieloides/patologia , Neoplasias/patologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/sangue , Envelhecimento/imunologia , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Diferenciação Celular , Feminino , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Expressão Gênica , Antígenos HLA-DR/imunologia , Humanos , Imunofenotipagem , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Antígenos CD15/genética , Antígenos CD15/imunologia , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/sangue , Neoplasias/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Streptococcus pneumoniae is a common human pathogen that accounts for >1 million deaths every year. Colonization of the nasopharynx by S. pneumoniae precedes pulmonary and other invasive diseases and, therefore, is a promising target for intervention. Because the receptors scavenger receptor A (SRA), macrophage receptor with collagenous structure (MARCO), and mannose receptor (MR) have been identified as nonopsonic receptors for S. pneumoniae in the lung, we used scavenger receptor knockout mice to study the roles of these receptors in the clearance of S. pneumoniae from the nasopharynx. MARCO(-/-), but not SRA(-/-) or MR(-/-), mice had significantly impaired clearance of S. pneumoniae from the nasopharynx. In addition to impairment in bacterial clearance, MARCO(-/-) mice had abrogated cytokine production and cellular recruitment to the nasopharynx following colonization. Furthermore, macrophages from MARCO(-/-) mice were deficient in cytokine and chemokine production, including type I IFNs, in response to S. pneumoniae. MARCO was required for maximal TLR2- and nucleotide-binding oligomerization domain-containing (Nod)2-dependent NF-κB activation and signaling that ultimately resulted in clearance. Thus, MARCO is an important component of anti-S. pneumoniae responses in the murine nasopharynx during colonization.