Integrin Αdβ2 (Cd11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection
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ORIGINAL RESEARCH published: 24 May 2018 doi: 10.3389/fimmu.2018.01128 Integrin αDβ2 (CD11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection Danielle de Oliveira Nascimento 1, Adriana Vieira-de-Abreu 1,2, Angélica F. Arcanjo 1, Patricia Torres Bozza 1, Guy A. Zimmerman 2* and Hugo Caire Castro-Faria-Neto 1* 1Laboratório de Immunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil, 2Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, United States Edited by: β2 integrins are critical in host defense responses to invading pathogens and inflam- Celio Geraldo Freire-de-Lima, Universidade Federal do Rio de mation. Previously, we reported that genetic deficiency of integrin αDβ2 in mice altered Janeiro, Brazil outcomes in experimental systemic infections including accelerated mortality in animals Reviewed by: infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of Juliana Dutra Barbosa Da Rocha, αDβ2 results in impaired accumulation of leukocytes in response to peritoneal infection University of Toronto, Canada Eden Ramalho Ferreira, by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination Federal University of São Paulo, Brazil by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process Laura Noelia Cariddi, α β National University of Río Cuarto, triggered by Salmonella. Salmonella-infected animals deficient in D 2 had increased Argentina levels of peritoneal cytokines in addition to other markers of pyroptosis, which may *Correspondence: contribute to inflammatory injury and increased mortality in the context of impaired Guy A. Zimmerman bacterial killing. These observations indicate important contributions of leukocyte integrins [email protected]; Hugo Caire Castro-Faria-Neto to the host response in experimental Salmonella infection and reveal previous activities of [email protected] αDβ2 in bacterial infection. Specialty section: Keywords: Salmonella enterica serovar Typhimurium pathogenesis, non-typhoidal Salmonella, myeloid leukocytes, This article was submitted to macrophage, integrin, pyroptosis Microbial Immunology, a section of the journal Frontiers in Immunology INTRODUCTION Received: 07 March 2018 Accepted: 04 May 2018 Trafficking of leukocytes in tissues and organs, and cell–cell interactions between immune effector Published: 24 May 2018 cells, are essential for defense against pathogens, innate and acquired immunity, and physiologic and Citation: pathologic inflammation (1, 2). Integrins are critical in these processes. Integrins are transmembrane Nascimento DdO, Vieira-de-Abreu A, heterodimers that are broadly expressed in varying patterns on metazoan cells (3, 4). In addition to Arcanjo AF, Bozza PT, Zimmerman cellular trafficking, integrins mediate adhesion-dependent cellular localization, signaling, intercel- GA and Castro-Faria-Neto HC (2018) lular communication, interactions with matrix, and regulation of apoptosis (3, 5). A subfamily of Integrin α β (CD11d/CD18) D 2 integrins called the β2 or CD11/CD18 integrins, also termed leukocyte integrins or leukointegrins, Modulates Leukocyte Accumulation, consists of four members: αLβ2 (6), αMβ2 (CD11b/CD18, MAC-1, CR3), αXβ2 (CD11c/CD18), Pathogen Clearance, and Pyroptosis and α β (CD11d/CD18) (3, 7). As in all integrins, the α subunits are not expressed on the in Experimental Salmonella D 2 Typhimurium Infection. cell surface without non-covalent heterodimerization with the β subunit, and vice versa (3–5). Front. Immunol. 9:1128. Leukocyte integrin heterodimers are expressed in varying patterns on circulating myeloid leukocytes doi: 10.3389/fimmu.2018.01128 and lymphocytes and on tissue leukocytes, including macrophages and dendritic cells, and have Frontiers in Immunology | www.frontiersin.org 1 May 2018 | Volume 9 | Article 1128 Nascimento et al. αD β2 Modulates Responses to Salmonella important activities in infection, inflammation, and immunity Cayman-Chemical. Fluorochrome inhibitor of caspase (FLICA) (8–10). Critical roles of leukocyte integrins in defense against was purchased from Immunochemistry Technologies (Blooming- bacterial infection, innate immune responses, wound surveillance, ton, MN, USA). Necrostatin-1 (Nec-1) was obtained from Sigma- and tissue repair are clearly demonstrated by heritable human Aldrich (Taufkirchen, Germany). Nec-1 is an inhibitor of the leukocyte adhesion deficiency syndromes in which the expression receptor interacting protein 1 kinase and acts as an inhibitor of of β2 integrins is absent or dramatically reduced, or inside-out necroptosis (19). signaling of integrin heterodimers is impaired (10). Experimental models in which β2 integrins are genetically deleted or blocked with specific antibodies confirm essential physiologic contribu- Mice α (α–/–) tions of leukocyte integrins in host defense and inflammation (5, C57Bl/6 WT mice and mice with targeted deletion of D D to yield deficiency of αDβ2 weighing 20–25 g of both sexes were 7–10). Additional integrins of the β1 and other integrin subclasses obtained from the Oswaldo Cruz Foundation breeding unit. We are also found on individual leukocyte types depending on their –/– have previously characterized these αD mice in detail. The ani- lineage and activation state (5, 7, 9, 10). ◦ mals were kept at constant temperature (25 C) with free access The most recently discovered member of the leukocyte, or β2, to food and water in a room with a 12-h light/dark cycle. The integrin subfamily is αDβ2 (CD11d/CD18), which is expressed on circulating leukocytes and tissue macrophages in humans and protocols employed in this work were approved by the Oswaldo on a subpopulation of blood leukocytes and distinct subsets of Cruz Foundation Animal Welfare Committee under the license macrophages in uninfected mice (6, 10–14). Experiments with number 0011-00. primary leukocytes from humans or mice or with transfected cell lines indicate that αDβ2 recognizes ICAM-3, ICAM-1, VCAM- Bacterial Strains 1, and various matrix proteins (6, 11, 13–15). In vivo studies Salmonella enterica serovar Typhimurium (16) ISC 5302/2004 –/– of wild type (WT) and αD demonstrate that αDβ2 expression was a generous gift from the Department of Microbiology of the can be dynamically modulated in response to infection and that Oswaldo Cruz Foundation. The bacteria were cultured in Luria- it has complex activities in immune regulation and systemic Bertani broth (Guria Broth Miller; Sigma-Aldrich) for 16–18 h ◦ inflammatory responses (12, 13). Nevertheless, little is known at 37 C to obtain stationary growth phase cultures and were ◦ about the functions of αDβ2 in infectious and inflammatory then centrifuged (1,000 × g) for 10 min at 4 C. The pellets were syndromes (10). resuspended in PBS to an OD of 0.1 at 660 nm, corresponding to We previously reported that αDβ2 expression is altered on 8 –/– 10 CFU/mL. WT and αD mice were infected by intraperitoneal splenic and hepatic macrophages in response to the rodent malar- (i.p.) injection of 200 μL of the bacteria suspension (105 CFU ial pathogen, Plasmodium berghei Anka, and that targeted dele- Salmonella/mouse). Control WT mice were sham injected (saline tion of αD conferred a survival advantage in this model of alone) in parallel. severe malaria (13). In parallel, we examined mice infected with As YVAD (N-Ac–Tyr–Val–Ala–Asp–CMK) is a selective and Salmonella enterica serovar Typhimurium (16) to determine if irreversible inhibitor of interleukin-1 beta converting enzyme genetic deletion of αDβ2 has stereotyped or, conversely, differen- –/– (Caspase-1), the effect of caspase-1 action on the absence of αD tial effects in lethal systemic infections. S. enterica is an intracellu- during Salmonella Typhimurium infection was analyzed in vivo. lar facultative anaerobe that is one of the leading causes of enteric For this, in some experiments, 15 min after S. Typhimurium diseases in the United States. Salmonella can cause diseases such –/– infection, WT and αD mice were injected i.p. with YVAD as typhoid, gastroenteritis, bacteremia, and chronic asymptomatic (200 μg/animal) or Nec-1 (1.65 mg Nec-1/kg body weight). Ani- carriage (17). S. enterica serovar Typhimurium, the causative agent mals injected with sterile saline or DMSO served as controls. of gastroenteritis, has developed different strategies to evade host defense mechanisms (18). In contrast to increased survival at early time points in the experimental malaria model, mortality Leukocytes Counts –/– was accelerated in αD infected with S. Typhimurium (13). Here, Total leukocyte counts were performed in Neubauer chambers we further explore functions of αDβ2 in animals challenged with by means of an optical microscope after diluting the peritoneal S. Typhimurium. wash in Turk’s solution (2% acetic acid). Differential leukocyte analysis was performed under an oil immersion objective on cytocentrifuged cells stained with May-Grunwald–Giemsa dye. MATERIALS AND METHODS Reagents Cytokine Measurements Lipopolysaccharide (LPS) from Escherichia coli (serotype 0127:b8) Levels of IL-6, TNF-α, MIP-1α, and IL-1β in peritoneal fluid were and from S. enterica serotype Typhimurium were purchased evaluated by enzyme-linked immunosorbent assay measurements from Sigma-Aldrich. Live/Dead BacLight bacterial viability kit using specific monoclonal antibodies, according to manufacturer and latex beads were