Biology subjects

Chavez, R. A.

Publications and source records attributed to Chavez, R. A..

3 recordsLinked to original sources

bioRxiv2019

New mutant mouse models clarify the role of NAIPs, phosphorylation, NLRP3, and tumors in NLRC4 inflammasome activation

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates Caspase-1 and initiates potent downstream immune responses. Structural, biochemical, and genetic data all demonstrate that the NAIP proteins act as receptors for specific bacterial ligands, while NLRC4 is a downstream adaptor protein that multimerizes with NAIPs to form a macromolecular structure called an inflammasome. However, several aspects of NLRC4 biology remain unresolved. For example, in addition to its clear function in responding to bacteria, NLRC4 has also been proposed to initiate anti-tumor responses, though the underlying mechanism is unknown. NLRC4 has also been shown to be phosphorylated on serine 533, and this modification was suggested to be important for NLRC4 function. In the absence of S533 phosphorylation, it was further proposed that another inflammasome component, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line as well as mice encoding phosphomimetic S533D and non-phosphorylatable S533A NLRC4 proteins. Using these genetic models in vivo and in vitro, we fail to observe a role for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results simplify and clarify our understanding of the mechanism of NAIP/NLRC4 activation and its biological functions.

immunology↗

bioRxiv2019

IRG1 and iNOS act redundantly with other interferon gamma induced factors to restrict intracellular replication of Legionella pneumophila.

Interferon gamma (IFN{gamma}) restricts the intracellular replication of many pathogens, but how IFN{gamma} confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN{gamma}, but consistent with prior results, no individual genetic deficiency we tested compromised IFN{gamma}-mediated control. Intriguingly, however, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN{gamma}-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN{gamma}. Instead, we found that 2DG rescues bacterial replication predominantly by inhibiting the induction of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirm that loss of both iNOS and IRG1, but not individual deficiency in each gene, partially reduces IFN{gamma}-mediated restriction of L. pneumophila. Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we find that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3 and NOX2) results in a total loss of L. pneumophila restriction by IFN{gamma} in primary bone marrow macrophages. There are few, if any, other examples in which the complete set of cell-intrinsic factors required for IFN{gamma}-mediated restriction of an intracellular bacterial pathogen have been genetically identified. Our results highlight the combinatorial strategy used by hosts to block the exploitation of macrophages by pathogens.\n\nImportanceLegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN{gamma}) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has determined the exact molecular factors induced by IFN{gamma} that are required for its activity. We generated macrophages lacking different combinations of IFN{gamma}-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN{gamma}-mediated restriction of L. pneumophila. We successfully identified six genes that comprise the totality of the IFN{gamma}-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN{gamma} and highlight how redundancy downstream of IFN{gamma} is key to prevent exploitation of the macrophage niche by pathogens.

immunology↗

bioRxiv2019

Inflammasome-mediated antagonism of type I interferon enhances Rickettsia pathogenesis

Inflammasomes and interferons constitute two critical arms of innate immunity. Most facultative bacterial pathogens that inhabit the host cell cytosol avoid activating inflammasomes and are often resistant to killing by type I interferon (IFN-I). We report that the human pathogen Rickettsia parkeri, an obligate intracellular pathogen that resides in the cytosol, is sensitive to IFN-I. The mechanism of IFN-I-dependent restriction requires the transcription factor IRF5, which upregulates anti-rickettsial factors including guanylate-binding proteins and iNOS. However, R. parkeri curtails cGAS-dependent IFN-I production by causing caspase-11-dependent pyroptosis. In vivo, inflammasome activation antagonizes IFN-I production, enhancing R. parkeri abundance in the spleen. Mice lacking either IFN-I or IFN-{gamma} signaling are resistant to infection, but mice lacking both rapidly succumb, revealing that both interferons are required to control R. parkeri. This study illuminates how an obligate cytosolic pathogen exploits the intrinsic trade-off between cell death and cytokine production to escape killing by innate immunity.\n\nHighlightsO_LIRickettsia killed by GBPs activates caspase-11 and GSDMD, promoting pyroptosis\nC_LIO_LIRickettsia exploits pyroptosis to avoid cGAS-dependent type I interferon\nC_LIO_LIIRF5, GBPs, and iNOS contribute to controlling R. parkeri infection\nC_LIO_LIIfnar-/-Ifngr-/- mice succumb to infection, uncovering a mouse model to study R. parkeri\nC_LI

immunology↗