Proteomic analysis of lung responses to SARS-CoV-2 infection in aged non-human primates: clinical and research relevance.

With devastating health and socioeconomic impact worldwide, much work is left to understand the Coronavirus Disease 2019 (COVID-19), with emphasis in the severely affected elderly population. Here, we present a proteomics study of lung tissue obtained from aged vs. young rhesus macaques (Macaca mulatta) and olive baboons (Papio Anubis) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using age as a variable, we identified common proteomic profiles in the lungs of aged infected non-human primates (NHPs), including key regulators of immune function, as well as cell and tissue remodeling, and discuss the potential clinical relevance of such parameters. Further, we identified key differences in proteomic profiles between both NHP species, and compared those to what is known about SARS-CoV-2 in humans. Finally, we explored the translatability of these animal models in the context of aging and the human presentation of the COVID-19.

HIV infection impairs the host response to Mycobacterium tuberculosis infection by altering surfactant protein D function in the human lung alveolar mucosa.

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections.

Extracellular Delivery of Functional Mitochondria Rescues the Dysfunction of CD4+ T Cells in Aging.

Mitochondrial dysfunction alters cellular metabolism, increases tissue oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, it is investigated whether the transfer of functional mitochondria into CD4+ T cells that are isolated from old mice (aged CD4+ T cells), can abrogate aging-associated mitochondrial dysfunction, and improve the aged CD4+ T cell functionality. The results show that the delivery of exogenous mitochondria to aged non-activated CD4+ T cells led to significant mitochondrial proteome alterations highlighted by improved aerobic metabolism and decreased cellular mitoROS. Additionally, mito-transferred aged CD4+ T cells showed improvements in activation-induced TCR-signaling kinetics displaying markers of activation (CD25), increased IL-2 production, enhanced proliferation ex vivo. Importantly, immune deficient mouse models (RAG-KO) showed that adoptive transfer of mito-transferred naive aged CD4+ T cells, protected recipient mice from influenza A and Mycobacterium tuberculosis infections. These findings support mitochondria as targets of therapeutic intervention in aging.

Animal Models of COVID-19: Transgenic Mouse Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged in December 2019 in Wuhan, China, and rapidly spread throughout the world, threatening global public health. An animal model is a valuable and a crucial tool that allows understanding of nature in the pathogenesis of SARS-CoV-2 and its associated COVID-19 disease. Here we introduce detailed protocols of SARS-CoV-2 infection and COVID-19 disease using C57BL/6 (B6) transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) from the human cytokeratin 18 promoter (K18 hACE2). To mimic natural SARS-CoV-2 infection, K18 hACE2 transgenic mice are infected intranasally under anesthesia. Upon infection, viral pathogenesis is determined by monitoring changes in body weight (morbidity) and monitoring survival (mortality), cytokine/chemokine responses, gross-lung pathology, histopathology, and viral replication in tissues. The presence of the virus and viral replication is evaluated by immunohistochemistry (IHC) and viral titrations, respectively, from the upper (nasal turbinate) and the lower (lungs) respiratory tracts, and nervous system (brain). Also, the immune response to SARS-CoV-2 infection is measured by cytokine/chemokine enzyme-linked immunosorbent assay (ELISA) from lung, spleen and brain homogenates to characterize the cytokine storm that hallmarks as one of the major causes of death caused by SARS-CoV-2 infection. This small rodent animal model based on the use of K18 hACE2 transgenic mice represents an excellent option to understand the pathogenicity of natural SARS-CoV-2 strains and its recently described Variants of Concern (VoC), and will be applicable to the identification and characterization of prophylactic (vaccine) and therapeutic (antiviral and/or neutralizing monoclonal antibodies) strategies for the prevention or treatment of SARS-CoV-2 infection or its associated COVID-19 disease.

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

Correction to: Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T-cell immunity.

Following publication of the original article [1], the author reported the wrong version of Figs. 5 and 7 have been published. The correct version of the figures can be found below.

Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity.

BACKGROUND: Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. METHODS: We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. RESULTS: Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. CONCLUSION: Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer.

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

Multivalent Forms of the Notch Ligand DLL-1 Enhance Antitumor T-cell Immunity in Lung Cancer and Improve Efficacy of EGFR-Targeted Therapy.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.

Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages.

Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human.

A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy.

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery.

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.

Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients.

Cystic fibrosis (CF) is accompanied with heightened inflammation worsened by drug resistant Burkholderia cenocepacia. Human CF macrophage responses to B. cenocepacia are poorly characterized and variable in the literature. Therefore, we examined human macrophage responses to the epidemic B. cenocepacia J2315 strain in order to identify novel anti-inflammatory targets. Peripheral blood monocyte derived macrophages were obtained from 23 CF and 27 non-CF donors. Macrophages were infected with B. cenocepacia J2315 and analyzed for cytokines, cytotoxicity, and microscopy. CF macrophages demonstrated significant increases in IL-1ß, IL-10, MCP-1, and IFN-γ production in comparison to non-CF controls. CF patients on prednisone exhibited globally diminished cytokines compared to controls and other CF patients. CF macrophages also displayed increased bacterial burden and cell death. In conclusion, CF macrophages demonstrate exaggerated IL-1ß, IL-10, MCP-1, and IFN-γ production and cell death during B. cenocepacia infection. Treatment with corticosteroids acutely suppressed cytokine responses.

Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization.

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1ß, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1ß processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1ß release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1ß release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1ß response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1ß processing and release.

Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepacia in a model of cystic fibrosis.

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1ß. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Asc-dependent and independent mechanisms contribute to restriction of legionella pneumophila infection in murine macrophages.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase(-1-/-) and Nlrc4(-/-) allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc(-/-) macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.

Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes.

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.

Burkholderia cenocepacia O polysaccharide chain contributes to caspase-1-dependent IL-1beta production in macrophages.

Burkholderia cenocepacia infections in CF patients involve heightened inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory IL-1ß secretion is important in airway inflammation and tissue damage. However, little is known about this pathway in macrophages upon B. cenocepacia infection. We report here that murine macrophages infected with B. cenocepacia K56-2 produce proinflammatory cytokine IL-1ß in a TLR4 and caspase-1-mediated manner. We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to IL-1ß production and pyroptotic cell death. Furthermore, we showed that the malfunction of the CFTR channel augmented IL-1ß production upon B. cenocepacia infection of murine macrophages. Taken together, we identified eukaryotic and bacterial factors that contribute to inflammation during B. cenocepacia infection, which may aid in the design of novel approaches to control pulmonary inflammation.

Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4(-/-) (Ipaf(-/-)), and caspase-1(-/-) derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7(-/-) macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.