Contribution of Toll-Like Receptor 4 to Infection-Induced Hemophagocytosis

McDonald, Erin Michelle

Graduate Thesis Or Dissertation

Contribution of Toll-Like Receptor 4 to Infection-Induced Hemophagocytosis Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/pn89d666g

Abstract

Hemophagocytes are a unique macrophage subset that accumulate during infection with many different microbes and are identified by their engulfment of erythrocytes and leukocytes. Hemophagocytes express anti-inflammatory markers and persist into chronic stages of infection; however, how hemophagocytes recognize blood cells for uptake and the signaling pathway(s) required to initiate hemophagocytosis during Salmonella infection are unknown. I identified both resident and inflammatory monocytes as the main splenic hemophagocyte populations in Salmonella enterica serovar Typhimurium-infected mice. Furthermore, I established that structurally conserved microbial products stimulate macrophages to hemophagocytose through Toll-like receptor (TLR) recognition and subsequent NF-κB activation. Previous work in the laboratory showed that hemophagocytic macrophages harbor live S. Typhimurium during chronic infection and that both S. Typhimurium and Yersinia pseudotuberculosis stimulated hemophagocytosis in vitro. Since these pathogens survive within macrophages, I hypothesized that prolonged exposure to microbes is key to stimulating hemophagocytosis. To support this hypothesis, I showed that macrophages become hemophagocytic upon prolonged exposure to heat-killed S. Typhimurium, Y. pseudotuberculosis, Bacillus subtilis, or Mycobacterium marinum. Furthermore, conserved microbial products were sufficient to stimulate macrophages to hemophagocytose. S. Typhimurium LPS (a TLR4 agonist) induced hemophagocytosis in resting and IFN-γ-pretreated macrophages, whereas lipoteichoic acid (a TLR2 agonist) and synthetic unmethylated deoxycytidine-deoxyguanosine dinucleotides, which mimic bacterial DNA (a TLR9 agonist), induced hemophagocytosis only in IFN-γ-pretreated macrophages. I identified a requirement for TLR4 in the induction of hemophagocytosis: chemical inhibition of TLR4 or genetic ablation of TLR4 prevented both Salmonella- and LPS-stimulated hemophagocytosis. Thus, LPS is the major constituent of S. Typhimurium that stimulates hemophagocytosis during infection. To demonstrate that signaling pathways downstream of TLR4 were required, I blocked activation of the transcription factor NF-κB and inhibited hemophagocytosis. In addition, I investigated whether phosphatidylserine (PS) recognition was required for erythrocyte uptake; treatment with antibodies to block PS-receptor interactions decreased the fraction of LPS- and Salmonella-induced hemophagocytes in IFN-γ-pretreated macrophages; there was no effect on resting macrophages. These findings show that prolonged TLR signaling and activation of NF-κB reprograms a subset of primary macrophages to hemophagocytose and that macrophage recognition of PS on erythrocytes is not the dominant mechanism driving hemophagocytosis

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