Sex impacts Th1 cells, Tregs, and DCs in both intestinal and systemic immunity in a mouse strain and location-dependent manner

Elderman et al. Biology of Sex Differences (2016) 7:21 DOI 10.1186/s13293-016-0075-9 RESEARCH Open Access Sex impacts Th1 cells, Tregs, and DCs in both intestinal and systemic immunity in a mouse strain and location-dependent manner Marlies Elderman1,2, Adriaan van Beek1,3, Eelke Brandsma1,2, Bart de Haan2, Huub Savelkoul3, Paul de Vos1,2 and Marijke Faas2,4* Abstract Background: Males and females have a different predisposition for the development of intestinal disorders, like inflammatory bowel disease (IBD). We hypothesized that sex specific differences in intestinal immune responses may underlie this bias. To test this hypothesis, we studied sex differences in immune cell populations in the Peyer’s patches (PP). For comparison with systemic immunity, we studied spleen cells. Methods: Two mouse strains with different susceptibility for developing colitis (BALB/c and C57Bl/6) were used. Using flow cytometry, we measured the percentage of T cells, Th1, Th17, and Treg cells in the PP and spleen. In addition, we measured the percentages of NK cells, macrophages, myeloid, and lymphoid dendritic cells (DCs) and their expression of CD80 and CD103. Moreover, we measured percentages of monocyte subsets in the peripheral circulation. Results were tested using two-way ANOVA, p < 0.05. Results: Males had a lower percentage of T cells in both PP and spleen (PP BALB/c 22.1 %, B6 13.6 %; spleen BALB/c 4.7 %, B6 19.9 %) but a higher percentage of Th1 cell in both tissues (PP BALB/c 350 %, B6 109.5 %; spleen BALB/c 48.7 %, B6 41.9 %) than females. They also had a higher percentage of Tregs in the spleen than females (BALB/c 20.5 %, B6 4.5 %). Furthermore, males had a higher percentage of CD80+ DCs in both the PP and spleen (lymphoid DCs in PP BALB/c 104.7 %, B6 29.6 %; spleen BALB/c 72.2 %, B6 44.2 %; myeloid DCs in PP BALB/c 80.5 %, B6 93.3 %; spleen BALB/c 88.5 %, B6 50.8 %) and a higher percentage of lymphoid CD103+ DCs in the spleen than females (BALB/c 41.5 %, B6 28.3 %). The percentage of NK cells was decreased in the spleen (BALB/c 12.5 %, B6 25.1 %) but increased in the PP (BALB/c 75.7 %, B6 78.6 %) of males as compared with females. Strain differences were also found in the PP; BALB/c mice had a higher percentage of T cells (males 58.1 %, females 75.5 %), a higher Th/Tc ratio (males 81.0 %, females 134.2 %), less FoxP3+CD25− T cells (males 14.6 %, females 30.0 %), more DCs (males 14.8 %, females 15.7 %) and macrophages (males 67.9 %, females 141.2 %), and more NK cells (males 160 %, females164.3 %) than BALB/c mice. Conclusions: In this study, we show sex differences in intestinal and peripheral immune populations. These differences may underlie sex differences in intestinal disorders like IBD, and this information may be an important knowledge for the treatment of intestinal-related diseases. Keywords: Sex differences, Peyer’s patches, Intestinal immune cells, T cells, Dendritic cells, Macrophages, Natural killer cells, C57Bl/6, BALB/c * Correspondence: 2 Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Centre Groningen, Groningen, The Netherlands 4 Department of Obstetrics and Gynaecology, University of Groningen and University Medical Centre Groningen, Groningen, The Netherlands Full list of author information is available at the end of the article © 2016 Elderman et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Elderman et al. Biology of Sex Differences (2016) 7:21 Background Females have a higher risk of developing autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus than males [1]. Sex differences in peripheral immune responses are thought to underlie this bias in autoimmune diseases [2]. Females generally react stronger and more vigorously with their adaptive immune response, whereas males have increased innate immune responses [3, 4]. Also, susceptibility to develop inflammatory bowel disease (IBD), such as Crohn’s disease, may be sex dependent; in European countries, females have a higher incidence of the disease, whereas in Asian Countries, men have a higher incidence [5, 6]. As IBD is characterized by chronic inflammation of the intestine [7], we hypothesized that sex differences in IBD may be due to sex differences in the intestinal immune response. The intestinal immune system, also referred to as gutassociated lymphoid tissue (GALT), is in close contact with intestinal microbes and dietary antigens, making it distinct from the peripheral immune system [8]. The main challenge of the GALT is to distinguish harmless from harmful substances and to respond appropriately. The GALT consists of immune cells scattered through the lamina propria and organized lymph structures like the Peyer’s patches (PP) and mesenteric lymph nodes (MLN) [9]. Dendritic cells (DCs) in the PP expressing integrin sub-unit CD103 are able to differentiate T helper (Th) cells into FoxP3+ T regulatory cells (Tregs) [10]. Tregs can produce Il-10 and are important in controlling other T helper responses, preventing inflammation. Both CD103+ DCs and Tregs play an important role in intestinal homeostasis and tolerance and in the prevention of IBD [11, 12]. An intestinal immune response is induced by DCs and macrophages upon encountering an antigen. Depending on the type of antigen, DCs and macrophages induce the differentiation of T cells into effector T cells, such as T helper 1 (Th1), T helper 2 (Th2), and T helper 17 (Th17) cells, as well as the abovementioned Treg cells [13]. Other immune cells that play a role in intestinal homeostasis are natural killer (NK) cells. NKp46+ NK cells co-expressing transcription factor ROR t in the intestine can produce interleukin 22 (IL-22) [14], which is involved in regulating mucosal barrier homeostasis and antimicrobial host defense [15]. Whether these intestinal immune cell subsets are sex dependent is still to be determined. The aim of this study was therefore to test the hypothesis that sex affects intestinal immune cell populations. We focused on Th cell subsets, DCs, macrophages, and NK cells. In humans, susceptibility to develop IBD is related to genetic variation [16]. Therefore, sex differences in intestinal immune cells were researched in two mouse strains with different genetic backgrounds. The colitis- Page 2 of 15 susceptible C57Bl/6 mice and the more resistant BALB/c mice were used [17, 18]. We used the PP as intestinal target (...truncated)