Proliferation of Resident Macrophages Is Dispensable for Protection during Giardia duodenalis Infections Marc Y. Fink, Jenny Maloney, Aleksander Keselman, Erqiu Li, Samantha Menegas, Christopher Downloaded from Staniorski and Steven M. Singer ImmunoHorizons 2019, 3 (8) 412-421 doi: https://doi.org/10.4049/immunohorizons.1900041 http://www.immunohorizons.org/content/3/8/412 http://www.immunohorizons.org/ This information is current as of September 29, 2021. Supplementary http://www.immunohorizons.org/content/suppl/2019/08/27/3.8.412.DCSupp Material lemental References This article cites 70 articles, 19 of which you can access for free at: http://www.immunohorizons.org/content/3/8/412.full#ref-list-1 by guest on September 29, 2021 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE Infectious Disease Proliferation of Resident Macrophages Is Dispensable for Protection during Giardia duodenalis Infections Marc Y. Fink, Jenny Maloney,1 Aleksander Keselman,2 Erqiu Li, Samantha Menegas, Christopher Staniorski, and Steven M. Singer Department of Biology, Georgetown University, Washington, DC 20057 Downloaded from ABSTRACT Infection with the intestinal parasite Giardia duodenalis is one of the most common causes of diarrheal disease in the world. Previous http://www.immunohorizons.org/ work has demonstrated that the cells and mechanisms of the adaptive immune system are critical for clearance of this parasite. However, the innate system has not been as well studied in the context of Giardia infection. We have previously demonstrated that Giardia infection leads to the accumulation of a population of CD11b+, F4/80+, ARG1+, and NOS2+ macrophages in the small intestinal lamina propria. In this report, we sought to identify the accumulation mechanism of duodenal macrophages during Giardia infection and to determine if these cells were essential to the induction of protective Giardia immunity. We show that F4/80+, CD11b+, CD11cint, CX3CR1+, MHC class II+, Ly6C2, ARG1+, and NOS2+ macrophages accumulate in the small intestine during infections in mice. Consistent with this resident macrophage phenotype, macrophage accumulation does not require CCR2, and the macrophages incorporate EdU, indicating in situ proliferation rather than the recruitment of monocytes. Depletion of macrophages using anti-CSF1R did not impact parasite clearance nor development of regulatory T cell or Th17 cellular responses, suggesting that these macrophages are dispensable for protective Giardia immunity. ImmunoHorizons, 2019, 3: 412–421. by guest on September 29, 2021 INTRODUCTION associated with childhood stunting yet not necessarily correlated to symptomatic diarrhea; thus, infections with Giardia contribute Giardia duodenalis (syn. G. intestinalis, G. lamblia)isoneofthe to childhood malnutrition (1). Paradoxically, Giardia is also asso- most common intestinal parasites in the world. Giardia is spread ciated with a reduced risk of moderate-to-severe diarrhea (2–5). through fecal-oral transmission with infections initiated through Acute infections with this noninvasive protozoan can manifest the ingestion of cyst-contaminated drinking water or food. As such, with symptoms typical of intestinal distress or be subclinical. disease burdens are highest in regions where access to clean water In addition, long-term effects such as postinfectious irritable is limited. Recently, the Malnutriotion and Enteric Dysfunction bowel syndrome and chronic fatigue syndrome have been reported (MAL-ED) study found that subclinical Giardia infections were (6–10). Adding to this intrigue, symptomatic patients may not Received for publication May 30, 2019. Accepted for publication August 6, 2019. Address correspondence and reprint requests to: Dr. Steven M. Singer, Department of Biology, Georgetown University, Reiss Science Building, Room 406, 37th and O Streets NW, Washington, DC 20057. E-mail address: [email protected] ORCIDs: 0000-0002-6405-883X (J.M.); 0000-0001-5719-7535 (S.M.S.). 1Current address: Environmental Microbial and Food Safety Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD. 2Current address: Beckman Coulter Life Sciences, Indianapolis, IN. This work was supported by National Institutes of Health Grants AI-094492 and AI-109591 (to S.M.S.), an American Association of Immunologists Careers in Immunology Fellowship (to J.M.), and a Crohn’s and Colitis Foundation of America Summer Student Fellowship (to C.S.). Georgetown Lombardi Shared Resources are partially supported by Grants CA-51008 and NCATS 8 UL1 TR000101 from the National Cancer Institute. The Georgetown University Barrier Animal Facility was partially supported by Grant RR-025828 from the National Center for Research Resources. Abbreviations used in this article: ARG1, arginase 1; CM, complete medium; EdU, 5-ethynyl-29-deoxyuridine; MHCII, MHC class II; NOS2, NO synthase 2; qRT-PCR, quantitative real-time PCR; WT, wild-type. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2019 The Authors 412 https://doi.org/10.4049/immunohorizons.1900041 ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons RESIDENT MACROPHAGES DURING GIARDIA INFECTIONS 413 exhibit mucosal inflammation despite having diarrhea, diffuse that induction of critical Th17 cellular responses were not impacted, shortening of the intestinal microvilli, absorptive deficiencies, and suggesting that macrophages are dispensable for protection. epithelial barrier disruption (11–14). The causes for this variation in clinical manifestations and outcomes are still poorly understood, but the host immune response has been reported to play a role in MATERIALS AND METHODS the symptoms associated with Giardia infection. Previous work from our laboratory suggests that T cells are critical for clearance Parasites culture, infections, and counts of Giardia infections (15, 16) and also contribute to immunopa- G. lamblia (strain GS/M/H7) was obtained from American Type thology (17). Recent work has also shown that IL-17 is the major Culture Collection, Manassas, VA (catalog no. 50581). Parasites were T cell cytokine necessary for protective immunity against Giardia cultured in standard TYI-S-33 medium supplemented with bovine (18–22). However, it is unclear which innate cells are responsible bile, L-cysteine, ascorbic acid, and an antibiotic-antimycotic solution for the initial recognition of the Giardia parasite and the activation (Sigma-Aldrich, St. Louis, MO). In preparation for infection, Giardia of T cells. trophozoites were thoroughly washed in PBS for several cycles and Myeloid cells, such as macrophages and dendritic cells, func- then resuspended in PBS. Mice were infected via gavage with 1 3 106 tion as initiators of immunity through the use of pattern recogni- Giardia trophozoites in 0.1 ml of PBS. Following euthanasia, parasites Downloaded from tion receptors and also activators of T cell proliferation and were counted from intestinal segments extracted from infected differentiation. Dendritic cells and their importance to protective animals by icing duodenum segments (3–5 cm immediately distal to Giardiaimmunity has been described, as these cells are a significant the ligament of Treitz) for 30 min in PBS. Intestinal sections were source of IL-6 during Giardia infections, and infected IL-6– then minced, and trophozoites were counted on a hemocytometer. deficient mice have a defect in parasite clearance (23–25). In http://www.immunohorizons.org/ contrast, the role of macrophages has not been as well studied, Mice despite their ability to recognize pathogens and contribute to acti- C57BL/6J, CCR22/2 (B6.129S4-Ccr2tm1Ifc/J) and CX3CR1-GFP vation of downstream immune responses. Moreover, little work has (B6.129P2(Cg)-Cx3cr1tm1Litt/J) mice were obtained from The been done to understand the role of macrophages in vivo during Jackson Laboratory (Bar Harbor, ME) and kept under specific Giardia infections. pathogen-free conditions at Georgetown University.CX3CR1-GFP Our laboratory has previously identified an increase in a popula- were bred at Georgetown University, and all littermates were tion of intestinal macrophages following G. duodenalis infection used for comparative infection experiments. All experiments that expresses both NO synthase 2 (NOS2) and arginase 1 (ARG1) were performed with the approval of the Georgetown University (26), markers that are used to distinguish between canonical Animal Care and Use Committee. macrophage subsets: inflammatory M1 (NOS2+) and regulatory by guest on September 29, 2021 M2 (ARG1+) macrophages. Similarly, following G. muris infections Isolation of intestinal lamina propria cells in IL-10–deficient mice, there is an expansion of colonic CD11b+, Intestinal segments from individual mice were digested to liberate CD11c2 macrophages that resulted in the inflammation of the cells residing within the duodenal lamina propria of the small colon, suggesting that macrophages have a role in downregulating intestine (32). Briefly, segments were excised, Peyer patches inflammatory signals in wild-type (WT) mice (27). In murine