Project description:Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures comprised of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent Salmonella enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using a STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin converting enzyme (ACE) defines a granuloma macrophage population that is non-permissive for intracellular bacteria and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.
Project description:Monocyte-macrophage lineage cells, crucial components of the innate immune system, can uniquely form bone-resorbing osteoclasts upon exposure to the cytokine receptor activator of NF-κB ligand (RANKL) in the bone microenvironment. Recent studies have also begun to uncover extensive extraskeletal roles of RANKL. However, how monocyte-macrophage lineage cells respond to RANKL outside of the bone, and the impact that this signaling pathway exerts on the host immune response, is not fully understood. In this study, we sought to define how RANKL exposure shapes the macrophage inflammatory response to pathogens by using the model intracellular bacterium Salmonella enterica serovar Typhimurium (STm), which co-opts macrophages to cause life-threatening infections. We found that exposing both mouse and human macrophages to sub-osteoclastogenic levels of RANKL increased intracellular STm burdens and decreased pro-inflammatory cytokine production. RNA sequencing revealed downregulation of pattern recognition receptor (PRR) signaling pathways in RANKL-treated macrophages during the early stages of infection. Therefore, we hypothesized that RANKL impairs PRR-dependent signaling pathways that are important for pro-inflammatory cytokine production. We discovered that RANKL-treated macrophages exhibit reduced NF-κB and IRF3 activation, specifically in response to Toll-like receptor (TLR) 2 and TLR4 stimulation. We determined that prior RANKL exposure decreases abundance of the TLR2 and TLR4 adaptor proteins TRAM and TIRAP. Together, these data suggest that RANKL exposure negatively impacts the macrophage TLR-mediated inflammatory response to bacteria.
Project description:SrfJ is an effector of the type III secretion systems of the Gram-negative intracellular pathogen Salmonella enterica serovar Typhimurium. To study the effects of this effector on global gene expression in host cells, we have infected murine RAW264.7 macrophages with two strains of Salmonella enterica serovar Typhimurium. The comparison between cells infected with the wild-type strain and cells infected with a srfJ mutant revealed a number of genes that are differentially expressed when SrfJ is present.
Project description:Investigation of whole genome gene expression level changes in a Salmonella enterica serovar Typhimurium UK1 delta-iacP mutant, compared to the wild-type strain. IacP is resoponsible for the secretion of virulence effector proteins via the type III secretion system, thereby contributing the virulence of S. Typhimurium. The mutants analyzed in this study are further described in Kim et al. 2011. Role of Salmonella Pathogenicity Island 1 Protein IacP in Salmonella enterica Serovar Typhimurium Pathogenesis. Infection and Immunity 79(4):1440-1450 (PMID 21263021). A chip study using total RNA recovered from two separate wild-type cultures of Salmonella enterica serovar Typhimurium UK1 and two separate cultures of a mutant strain, Salmonella enterica serovar Typhimurium UK1 delta-iacP. Each chip measures the expression level of 4,302 genes from Salmonella enterica serovar Typhimurium.
Project description:Investigation of whole genome gene expression level changes in a Salmonella enterica serovar Typhimurium 14028 delta GidA mutant The mutant described in this study is further analyzed in Shippy, D. C., N. M. Eakley, P. N. Bochsler, and A. A. Fadl. 2011. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene. Microb Pathog. A single chip study using three separate cultures of wild-type Salmonella enterica serovar Typhimurium 14028 and three separate cultures of a single mutant, delta GidA Salmonella enterica serovar Typhimurium 14028.
Project description:Investigation of whole genome gene expression level changes in a Salmonella enterica serovar Typhimurium 14028 delta GidA mutant The mutant described in this study is further analyzed in Shippy, D. C., N. M. Eakley, P. N. Bochsler, and A. A. Fadl. 2011. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene. Microb Pathog.
Project description:Transcriptomic analysis in a Salmonella enterica Serovar Typhimurium SL 1344 that constitutively expresses stdE and stdF compared with a strain carrying an stdEF deletion A four chip study using total RNA recovered from two separate cultures of Salmonella enterica Serovar Typhimurium SL 1344 constitutively expressing stdE and stdF and two separate cultures of Salmonella enterica Serovar Typhimurium SL 1344 lacking stdE and stdF.
Project description:Wild type and Irg1 knockout mouse bone marrow derived macrophages (BMDMs) were processed with a redox proteomics workflow and analyzed by LC-MS/MS with TMT based quantification.