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B cell activation in the cecal patches during the development of an experimental colitis model
Biochemical and Biophysical Research Communications 496 (2018) 367e373
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
B cell activation in the cecal patches during the development of an experimental colitis model Taro Watabe a, 1, Takashi Nagaishi b, 1, Naoya Tsugawa a, Yudai Kojima a, Nisha Jose a, Akinori Hosoya a, Michio Onizawa b, Yasuhiro Nemoto a, Shigeru Oshima a, Tetsuya Nakamura b, Hajime Karasuyama c, Takahiro Adachi d, Mamoru Watanabe a, * a
Department of Gastroenterology, Graduate School of Medical Science, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Department of Advanced Therapeutics for GI Diseases, Graduate School of Medical Science, TMDU, Tokyo, Japan Department of Immune Regulation, Graduate School of Medical Science, TMDU, Tokyo, Japan d Department of Immunology, Medical Research Institute, TMDU, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 January 2018 Accepted 9 January 2018 Available online 10 January 2018
Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca2þ signaling in CP B cells during the early phase of colitis development. These findings suggested that the CP B cells may be involved in the pathogenesis of colitis including inflammatory bowel diseases in humans. © 2018 Elsevier Inc. All rights reserved.
Keywords: Colitis Oxazolone Intravital imaging Cecal patches B cells
1. Introduction Previous studies reported that appendectomy attenuates experimental colitis models such as spontaneous colitis in T cell receptor a chain-mutant mice (TCRa/-) [1] and dextran sodium sulfate (DSS)-induced colitis [2]. From these findings, it has been suggested that appendiceal lymphoid tissues seem to serve some
Abbreviations: BCR, B cell receptor; Ca2þ, calcium ion; CD, Crohn's disease; CFP, cyan fluorescent protein; CP, cecal patches; DSS, dextran sodium sulfate; ELISA, enzyme-linked immunosorbent assay; EtOH, ethanol; FAE, follicular-associated epithelia; FRET, fluorescence resonance energy transfer; GALT, gut-associated lymphoid tissues; H&E, hematoxylin and eosin; IBD, inflammatory bowel diseases; IFN-g, interferon-g; IL, interleukin; LPL, lamina propria lymphocytes; mAb, monoclonal antibody; mMT, mice deficient for the membrane exon of the m heavy chain gene; NKT, natural killer T; n.s., not significant; Ox, oxazolone; TCRa, T cell receptor a chain; Th, helper T cells; UC, ulcerative colitis; YC, yellow cameleon; YFP, yellow fluorescent protein. * Corresponding author. Department of Gastroenterology and Hepatology, Graduate School of Medical Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. E-mail address: [email protected] (M. Watanabe). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2018.01.053 0006-291X/© 2018 Elsevier Inc. All rights reserved.
significant immunological functions. Recently, it was reported that appendiceal lymphoid follicle, called cecal patches (CP) in mice, is a major site for generation of IgA-secreting cells that migrate to the large intestine [3], but the role of this in colitis has not been elucidated. There are several animal models developed to explore the mechanisms of colitis pathogenesis [4], and oxazolone-induced colitis is an often used model. Sensitization to oxazolone by intrarectal administration to mice can induce colonic injury accompanied by the upregulation of T-helper (Th) 2 cytokines [5]. The histopathological features of oxazolone colitis is similar to that of inflammatory bowel diseases (IBD), especially ulcerative colitis (UC) in humans [6], and this model has been proven useful in assessing causal relationships between specific treatments and colitis [7]. The initiation process of oxazolone colitis is thought to be dependent on the T cell response since colitis does not develop in severe combined immune-deficient mice [8]. On the other hand, one of the natural killer T (NKT) cell subsets has been reported to be involved in the pathogenesis of human UC [9], and involvement of invariant NKT cells in oxazolone colitis in mice is also supported by previous observations [10,11]. As described above, oxazolone colitis
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shares pathological similarities to human UC, so we employed this model to assess the importance of CP in colitis. 2. Materials and methods 2.1. Mice
(1 cm in length) followed by exteriorization of the appendix. The appendix was divided between two ligatures (absorbable surgical suture, Ethicon) that were placed proximal to the border of cecum and appendix. In the sham-operated group, mice were anesthetized and underwent a midline incision but without excision of the appendix. In both groups, the abdominal wall was closed in two layers, using a running suture technique.
Wild-type C57BL/6 mice (WT) were obtained from CLEA Japan (Tokyo, Japan). Mice lacking mature B cells (mMT) were kindly provided by Dr. Klaus Rajewsky (Max Delbrück Center for Molecular Medicine, Germany), and the yellow cameleon 3.60 (YC3.60) mice were from Dr. Atsushi Miyawaki (RIKEN Center for Advanced Photonics, Japan). YC3.60 mice were intercrossed with CD19-Cre transgenic mice (CD19-Cre/YC3.60) to induce specific transgene expressions in B cells. All mice were maintained in the animal facility of Tokyo Medical and Dental University (TMDU) under specific pathogen free conditions in accordance with guidelines of the Institutional Animal Care and Use Committee of TMDU. All experimental procedures on animals were approved by the Institutional Animal Care and Use Committee of TMDU, and all experiments were carried out in accordance with approved guidelines.
We analyzed Ca2þ signaling as previously described [13]. CP of mice anesthetized by inhalation of isoflurane were imaged as following: CP were surgically exteriorized, immobilized on a microscope stage, and maintained at 37 C. All image acquisition and analysis were performed using an A1® laser scanning confocal system with a 20 objective and software NIS-Elements® (Nikon). The yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) signal ratio was obtained by excitation at 458 nm.
2.2. Induction of colitis
The results were expressed as the means ± SEM. Statistical analysis were performed with unpaired Student's t-test. Differences were considered to be statistically significant when p value < .05.
Colitis was induced in 8e12 week-old mice by pre-sensitizing with 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone, Sigma-Aldrich) at a concentration of 3% in 100% ethanol (EtOH) by skin painting 1 week before intrarectal administration at a concentration of 1% in 50% EtOH on day 0, as previously described [11]. On day 5, or on day 1 in some experiments, mice were euthanized and analyzed. 2.3. Determination of cytokine production Colonic lamina propria lymphocytes (LPL) were isolated from freshly obtained colonic specimens as described previously [12]. The isolated LPL (5 105 cells/well) on day 5 were stimulated with 5 mg/ml plate-bound antiemouse CD3ε mAb (145-2C11, BD Biosciences) and 2 mg/ml soluble antiemouse CD28 mAb (37.51, BD Biosciences). Culture supernatants were harvested 48 h after the incubation. Concentrations of IFN-g and IL-4 were measured with OptEIA® ELISA sets (BD Biosciences), and IL-17 was measured with Quantikine® ELISA kit (R&D systems), according to the manufacturers' instruction. 2.4. Histological assessment of colitis Colonic tissues were removed on day 5, embedded in OCT compound (Sakura Finetech) and frozen at 80 C. Eight mm cryosections were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (H&E). The degree of inflammation in the colon was graded semi-quantitatively by a modified method from previously described criteria [11]. Briefly, five criteria (hypervascularization, presence of mononuclear cells, epithelial hyperplasia, epithelial injury, and presence of granulocytes) were each scored from 0 to 3. The cumulative degree of each was calculated as a total histological score ranging from 0 (no colitis) to 15 (maximal colitis activity). Such evaluation was performed in a blinded fashion. 2.5. Appendectomy Surgical procedures were performed as described previously [2]. Eight week-old mice were anesthetized by inhalation of isoflurane. Under sterile conditions, mice underwent a midline laparotomy
2.6. Intravital imaging
2.7. Statistical analysis
3. Results 3.1. The inflammation accompanied with ulceration is induced by oxazolone treatment in cecal mucosa as well as colonic tissues in mice To first assess whether ileocecal immune response is involved in the pathogenesis of our colitis model, WT pre-sensitized with oxazolone were given rectal administration of oxazolone, and then observed for 5 days (Fig. 1a). We observed diarrhea and significant wasting of oxazolone-treated mice from day 1 compared to vehicle (EtOH)-treated control. However, the latter subsequently recovered and even surpassed the original body weight on day 3e5 (Fig. 1a), which was apparently caused by systemic edema. Mice were then euthanized for histologic analysis on day 5. The oxazolone-treated mice showed significant shortening of colon length compared to that of vehicle-treated control groups (Fig. 1b and Supplementary Fig. 1). However, we observed profound infiltration of lymphocytes and granulocytes in the colonic tissues of oxazolone-treated mice (Fig. 1c), even though there were occasional ulcerations. And interestingly, we were also able to observe remarkable ulcerations particularly at the follicular-associated epithelia (FAE) on CP with the follicular expansion in the oxazolone-treated mice (Fig. 1c). Taken together, histopathological assessment revealed that oxazolone-treated mice had significant colitis (Fig. 1d) accompanied by the cecal inflammation. These observations were associated with significantly increased pro-inflammatory cytokine production, such as IFN-g, IL-4 and IL-17, from the colonic LPL (Fig. 1e). These results are consistent with previous observations [5,6,10,11,14]. 3.2. Immune responses in cecal patches are involved in the pathogenesis of oxazolone-induced colitis Given the remarkable ulceration at the FAE overlying CP in oxazolone-treated mice, we hypothesized that the immune response in CP may be associated with the pathogenesis of colitis. We therefore focused on the function of CP in the setting of colitis. WT underwent either appendectomy or sham-operation two weeks prior were subjected to oxazolone colitis by the same
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operation (Fig. 2a). Regarding the length of colons, oxazolonetreated mice were significantly shorten compared to EtOHtreated mice in the sham-operated group. However, oxazolonetreated mice had less colon shortening in the appendectomyoperated group, and the difference between these groups were not significantly different (Fig. 2b and Supplementary Fig. 2). Consistent with this, histopathological assessment showed significant attenuation of lymphocyte and granulocyte infiltrations in the colonic lamina propria of oxazolone-treated mice in the appendectomy-operated group compared to that of mice in the sham-operated group (Fig. 2c and d). These observations were associated with significantly abrogated productions of IFN-g, IL-4 and IL-17, from colonic LPL isolated from the oxazolone-treated mice with appendectomy compared to the oxazolone-treated mice with sham-operation (Fig. 2e). 3.3. The functions of B cells are involved in the pathogenesis of oxazolone-induced colitis
Fig. 1. Oxazolone treatment results in inflammation and ulceration in both cecum and colon. (a) Wasting, as defined by percentage of initial body weight, in WT treated with either vehicle control (EtOH) or oxazolone (Ox). Data are expressed as means ± SEM. Each group contained eight mice. *p < .05. (b) Colon length of mice treated with either vehicle (EtOH) or oxazolone (Ox). Data are expressed as means ± SEM. n ¼ 8. *p < .05. (c) H&E-stained microscopic features of colons and cecal patches (CP) from mice treated with either vehicle (EtOH) or oxazolone (Ox). Representative features from each group are shown. (d) Quantitative histopathological assessments of colitis severity from mice treated with either vehicle (EtOH) or oxazolone (Ox). Data are expressed as means ± SEM. n ¼ 8. *p < .05. (e) Productions of pro-inflammatory cytokines such as IFN-g, IL-4 and IL-17 from colonic LPL in mice treated with either vehicle (EtOH) or oxazolone (Ox). Data are indicated as means ± SEM. n ¼ 8. *p < .05.
procedures as above. A series of pilot experiments showed that surgical procedures alone did not affect the gastrointestinal tract functionally or morphologically before the oxazolone treatment (data not shown). There was no significant difference in any clinical, histological, or biochemical parameters between sham- and appendectomy-operated mice without colitis induction. We also compared oxazolone-treated non-operated mice to oxazolonetreated sham-operated mice, and there was no significant difference in these as well (data not shown). Therefore, we considered that oxazolone-treated sham-operated mice were suitable controls for oxazolone-treated appendectomy-operated mice. We observed significant wasting 1 day after oxazolone treatment in both groups that underwent appendectomy and sham-
Given the results of the attenuated colitis in oxazolone-treated mice with appendectomy, we next hypothesized that certain population in CP may initiate the inflammation in this model. In general, most of secondary lymphoid follicles, including ones in the gut-associated lymphoid tissues (GALT), mainly consist of T cells and B cells. Previous studies have already established the importance of T cells in the induction of acquired immune responses even in the context of oxazolone colitis. We want to focus on the function of B cells in this model. Therefore, same model was induced in mMT. Oxazolone-treated mMT were showing less body weight during recovery from day 2 to day 5 compared to that of oxazolone-treated WT (Fig. 3a). However, the shortening of colon by oxazolone treatment in mMT was rather attenuated compared to that of WT (Fig. 3b and Supplementary Fig. 3). Associated with the less shortening of colon, histopathological assessment revealed significantly attenuated colitis severity as well as the ulceration at CP in oxazolone-treated mMT compared to that of oxazolone-treated WT (Fig. 3c and d). Moreover, such attenuation of colitis was associated with significantly suppressed productions of IL-4 and IL-17 but not IFN-g, from colonic LPL in oxazolone-treated mMT compared to that of oxazolone-treated WT (Fig. 3e). These studies imply that B cells in GALT, such as CP, may be responsible for the induction of colonic inflammation in the setting of colitis. 3.4. Frequent Ca2þ flux in cecal patch B cells is induced at early phase of colitis development To assess the immunological response in CP dynamically, we worked on intravital imaging using B cell-specific YC3.60 transgenic mice [13]. YC3.60 is a Ca2þ sensor adopting the fluorescence resonance energy transfer (FRET) technology consisting of CFP and YFP linked by the sequence of calmodulin and calmodulin-binding peptide M13. When Ca2þ binds to calmodulin, it results in structural change and an increase in the efficiency of the energy transfer [15]. CFP is excited at 458 nm under low Ca2þ concentration. However, when Ca2þ concentration becomes higher in the cells, not only CFP but also YFP can be excited. Thus, the increased emission ratio of YFP to CFP (YFP/CFP) signal excited at 458 nm reflects the intracellular Ca2þ flux. CD19-Cre/YC3.60 were treated with either oxazolone or vehicle, and intravital Ca2þ signaling in CP B cells was monitored 1 day after intrarectal administration. The Ca2þ flux, defined as YFP/CFP ratio >1.2, in CP B cells was frequently observed in oxazolone-treated mice, although that was hardly observed in EtOH-treated mice (Fig. 4a and b and Supplementary Fig. 4 and 5). The time-lapse observation revealed that Ca2þ flux in single cells were activated
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Fig. 2. Oxazolone colitis is attenuated in mice that underwent appendectomy. (a) Wasting, as defined by percentage of initial body weight, in mice underwent the following procedures: sham operation followed by vehicle control treatment (sham EtOH), sham followed by oxazolone treatment (sham Ox), appendectomy followed by vehicle treatment (appe EtOH), or appendectomy followed by oxazolone treatment (appe Ox). Data are expressed as mean values ± SEM. Each group contained six mice. *p < .05. (b) Colon length of mice in each group. Data are expressed as mean values ± SEM. n ¼ 6. n.s., not significant. *p < .05. (c) H&E-stained microscopic features of colons from oxazolone-treated mice that had either sham operation (sham Ox) or appendectomy (appe Ox). Representative features from each group are shown. (d) Histopathological assessments of colitis severity from each group. Data are expressed as means ± SEM. n ¼ 6. *p < .05. (e) Productions of pro-inflammatory cytokines such as IFN-g, IL-4 and IL-17 by colonic LPL isolated from each group. Data are indicated as means ± SEM. n ¼ 6. *p < .05.
either intermittently or continuously in oxazolone-treated mice (Fig. 4a, c and Supplementary Fig. 5). We had also attempted to observe Ca2þ signaling in CP B cells 3 or 5 days after the administration, but interestingly, the frequency of activating cells decreased significantly as time progressed (data not shown). These results suggest that B cells in CP contribute to the onset of colitis. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.01.053. 4. Discussion In this study, we hypothesized that CP serves an important role during colitis. In fact, our studies with appendectomy or deficiency of mature B cells suggested that the functions of CP or B cells in GALT may be involved in the pathogenesis of oxazolone colitis.
Moreover, the activated Ca2þ signaling in CP B cells observed by intravital imaging suggested that CP B cells contribute to the onset of colitis. In experimental models of colitis, such as spontaneous colitis in TCRa/- and DSS-induced colitis, severity is also attenuated by appendectomy. In humans, a large-scale epidemiological study had previously shown an inverse relation between UC and appendectomy [16,17], especially if performed before the age of 20 [18]. This inverse relation was limited to patients who underwent appendectomy due to appendicitis or lymphadenitis. These studies implied that some latent abnormal immunological response in appendix may induce chronic inflammation in colon, but the detailed mechanism has not been elucidated. There are many lymphoid follicles in human appendix, and the equivalent tissues in murine GALT are called CP. A recent report showed that CP is a
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Fig. 3. Oxazolone colitis is attenuated by the deficiency of mature B cells. (a) Wasting defined by percentage of initial body weight in WT and mMT induced colitis. Mice were treated with either vehicle control (EtOH) or oxazolone (Ox). Data are expressed as mean values ± SEM. Each group contained three mice. *p < .05. (b) Colon length of WT with vehicle control (WT EtOH), mMT with vehicle (mMT EtOH), WT with oxazolone (WT Ox) and mMT with oxazolone (mMT Ox). Data are expressed as mean values ± SEM. n ¼ 3. n.s., not significant. *p < .05. (c) Microscopic features of colons and CP from both WT and mMT treated with oxazolone. Representative features from each group are shown. (d) Histopathological evaluations of colitis severity from each group. Data are expressed as means ± SEM. n ¼ 3. *p < .05. (e) Productions of cytokines such as IFN-g, IL-4 and IL-17 by colonic LPL isolated from each group. Data are indicated as means ± SEM. n ¼ 3. n.s., not significant. *p < .05.
major site where IgA-secreting cells are generated to migrate to the large intestine, and therefore, it serves to regulate intestinal microbiota [3]. However, the function of CP in colitis pathogenesis is still unsolved. In the previous study of spontaneous colitis in TCRa/- by others, the increase of B cells in CP were observed and the activation of B cells was suggested in this model [1]. Thus, we focused on the function of CP B cells. To analyze this, we employed oxazolone colitis, which is characterized by the colonic injury associated with the upregulation of Th2 cytokines, characteristics similar to that of human UC [6,7].
In our studies, the production of IL-4 from colonic LPL was upregulated in oxazolone-treated mice, which is consistent with prior report [5]. In addition, the production of IFN-g from colonic LPL was also up-regulated in these mice, which is also consistent with previous observation [6]. Moreover, we found that IL-17 production was also up-regulated, which is consistent with the analysis with semi-quantitative polymerase chain reaction reported by others [14]. These results indicated that Th1, Th2 and Th17 responses are all involved in the pathogenesis of oxazolone colitis model. Another interesting observation in our studies is the finding
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Fig. 4. Frequent Ca2þ flux in cecal patch B cells is induced by oxazolone-treatment. (a) Intravital images of Ca2þ signaling in CP B cells of CD19-Cre/YC3.60 mice. Representative observations under the confocal microscopy one day after the treatment with either vehicle (Ctrl) or oxazolone (Ox) are shown. Representatives of intermittently and continuously activated cells are indicated by yellow and red arrowhead, respectively. The highest signal of YFP is modified into red color by computer processing methods in these images. (b) Distribution of time-integrated intracellular Ca2þ concentrations in CP B cells of CD19Cre/YC3.60 one day after the treatment with either vehicle or oxazolone. n ¼ 50, frame ¼ 20. Percentages of cells expressing YFP/CFP ratio >1.2 are also indicated. (c) Kinetics for YFP/CFP ratio at excitation of 458 nm in representative CP B cells one day after the treatment with either vehicle or oxazolone are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
of ulceration at CP accompanied by the follicular expansion, which suggests an involvement of CP in these acquired immune responses associated with colitis. After appendectomy, the severity of oxazolone colitis was attenuated, and the productions of IFN-g, IL-4 and IL-17 from colonic LPL were significantly downregulated. Colitis was also attenuated when mature B cells were absent, and this was associated with decreased productions of IL-4 and IL-17 from colonic LPL. IFN-g production in mMT was also slightly downregulated, but it
was not statistically significant compared to that of WT. These results indicate the involvement of CP B cells in the pathogenesis of oxazolone colitis, especially in the aspects of Th2 and Th17 responses. Regarding the different observations of cytokine profile between the two different conditions (appendectomy and mature B cell deficiency in Figs. 2 and 3), it is suggested that not only B cells but also other cell types, such as dendritic cells and macrophages, in CP were removed during appendectomy, and consequently Th1 response becomes downregulated. In addition, we have observed
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relatively reduced body weight in oxazolone-treated mMT on day 3e5 compared to oxazolone-treated WT in Fig. 3a, and this was reproducible in several rounds of experiments even though the difference was not statistically significant. The potential interpretation regarding such discrepancy would be that the increase of body weight during day 3e5 in oxazolone-treated mice may partially be caused by systemic edema due to increased production of Th2 and/or Th17 cytokines as seen in Fig. 1a. Therefore, oxazolone-treated mMT, whose colonic LPL showed significantly suppressed production of IL-4 and IL-17 but not IFN-g, had relatively more body weight loss with less systemic edema. We employed intravital imaging to assess the contribution of B cells, which enabled dynamic observation of cellular activities in the setting of colitis. It has been well-known that the Ca2þ signaling mediated by B cell receptors plays an important role in B cell activation and differentiation [15]. We were able to observe frequent Ca2þ signaling in CP B cells of colitis-induced CD19-Cre/ YC3.60 one day after oxazolone treatment. A subset of B cells in secondary lymphoid follicles is generally suggested to be supported by follicular helper T cells, which are activated by antigen presenting cells, in order to differentiate into plasma cells to secrete immunoglobulins. Therefore, our observation of B cell activation in CP would suggest B cell differentiation into plasma cells. On the other hand, there were some B cells that had already been activated at the early phase of the colitis development, and such activation was immediately attenuating during the recovery. Therefore, the results from our observation with intravital imaging and amelioration of colitis by appendectomy and mature B cell deficiency would suggest that CP B cells may be involved in the onset of the colitis pathogenesis. There have been an increase number of patients diagnosed with IBD, such as UC and Crohn's disease (CD). Although many efforts have revealed that IBD can be caused by various factors, such as environmental, genetic, immunologic, and intestinal microbiota [19,20], there are still many questions that need to be elucidated, including the involvement of appendix. Our results imply that B cells in human appendiceal lymphoid follicles may be involved in the pathogenesis of IBD. Further analysis of Ca2þ signaling in B cells may shed light on the novel functions of CP in the immune response in the gut. Grant This study was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (MW: 22117508), Challenging Exploratory Research (T. Nagaishi: 15K15288), Scientific Research-S (MW: 26221307), and Scientific Research-B (T. Nagaishi: 16H05286) from the Japanese Ministry of Education, Culture, Sports, Science and Technology; Research Center Network Program for the Realization of Regenerative Medicine (MW) from the Japan Science and Technology Agency and Japan Agency for Medical Research and Development (AMED); Practical Research Project for Rare/Intractable Diseases (MW: 16eK0109047h0003) from AMED; Memorial Fund of Nihon Univ. Medical Alumni Association (T. Nagaishi); Collaboration Research Program of TMDU Medical Research Institute (T. Nagaishi, and TA); Naoki Tsuchida Memorial Resarch Grant (T. Nagaishi, and TA); Otsuka Toshimi Scholarship Foundation (NJ). Acknowledgments We are grateful to Drs. K. Rajewsky and A. Miyawaki for providing the mMT and YC3.60 mice, respectively, A. Tokai for the technical support, and Dr. K. Yoshikawa for confocal microscopy use.
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Disclosures The authors have no financial conflict of interest. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.01.053. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.01.053. References [1] A. Mizoguchi, E. Mizoguchi, C. Chiba, A.K. Bhan, Role of appendix in the development of inflammatory bowel disease in TCR-alpha mutant mice, J. Exp. Med. 184 (2) (1996) 707e715. Epub 1996/08/01. [2] C.F. Krieglstein, W.H. Cerwinka, F.S. Laroux, M.B. Grisham, G. Schurmann, M. Bruwer, et al., Role of appendix and spleen in experimental colitis, J. Surg. Res. 101 (2) (2001) 166e175. [3] K. Masahata, E. Umemoto, H. Kayama, M. Kotani, S. Nakamura, T. Kurakawa, et al., Generation of colonic IgA-secreting cells in the caecal patch, Nat. Commun. 5 (2014) 3704. [4] S. Wirtz, C. Neufert, B. Weigmann, M.F. Neurath, Chemically induced mouse models of intestinal inflammation, Nat. Protoc. 2 (3) (2007) 541e546. [5] M. Boirivant, I.J. Fuss, A. Chu, W. Strober, Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4, J. Exp. Med. 188 (10) (1998) 1929e1939. Epub 1998/11/17. [6] H. Iijima, M.F. Neurath, T. Nagaishi, J.N. Glickman, E.E. Nieuwenhuis, A. Nakajima, et al., Specific regulation of T helper cell 1-mediated murine colitis by CEACAM1, J. Exp. Med. 199 (4) (2004) 471e482. [7] V. Popp, K. Gerlach, S. Mott, A. Turowska, H. Garn, R. Atreya, et al., Rectal delivery of a DNAzyme that specifically blocks the transcription factor GATA3 and reduces colitis in mice, Gastroenterology 152 (1) (2017) 176e192 e5. [8] M. Taube, L. Svensson, H. Carlsten, T lymphocytes are not the target for estradiol-mediated suppression of DTH in reconstituted female severe combined immunodeficient (SCID) mice, Clin. Exp. Immunol. 114 (2) (1998) 147e153. Epub 1998/11/20. [9] I.J. Fuss, F. Heller, M. Boirivant, F. Leon, M. Yoshida, S. Fichtner-Feigl, et al., Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis, J. Clin. Invest. 113 (10) (2004) 1490e1497. Epub 2004/05/18. [10] S. Brozovic, T. Nagaishi, M. Yoshida, S. Betz, A. Salas, D. Chen, et al., CD1d function is regulated by microsomal triglyceride transfer protein, Nat. Med. 10 (5) (2004) 535e539. [11] F. Heller, I.J. Fuss, E.E. Nieuwenhuis, R.S. Blumberg, W. Strober, Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13producing NK-T cells, Immunity 17 (5) (2002) 629e638. [12] M.F. Neurath, B. Weigmann, S. Finotto, J. Glickman, E. Nieuwenhuis, H. Iijima, et al., The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease, J. Exp. Med. 195 (9) (2002) 1129e1143. [13] S. Yoshikawa, T. Usami, J. Kikuta, M. Ishii, T. Sasano, K. Sugiyama, et al., Intravital imaging of Ca(2þ) signals in lymphocytes of Ca(2þ) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset, Sci. Rep. 6 (2016) 18738. [14] V. Galitovskiy, J. Qian, A.I. Chernyavsky, S. Marchenko, V. Gindi, R.A. Edwards, et al., Cytokine-induced alterations of alpha7 nicotinic receptor in colonic CD4 T cells mediate dichotomous response to nicotine in murine models of Th1/ Th17- versus Th2-mediated colitis, J. Immunol. 187 (5) (2011) 2677e2687. Epub 2011/07/26. [15] T. Adachi, T. Tsubata, FRET-based Ca2þ measurement in B lymphocyte by flow cytometry and confocal microscopy, Biochem. Biophys. Res. Commun. 367 (2) (2008) 377e382. [16] A.B. Lowenfels, Appendectomy and ulcerative colitis, Gastroenterology 107 (5) (1994) 1570. Epub 1994/11/01. [17] M.G. Russel, E. Dorant, R.J. Brummer, M.A. van de Kruijs, J.W. Muris, J.M. Bergers, et al., Appendectomy and the risk of developing ulcerative colitis or Crohn's disease: results of a large case-control study. South Limburg Inflammatory Bowel Disease Study Group, Gastroenterology 113 (2) (1997) 377e382. [18] R.E. Andersson, G. Olaison, C. Tysk, A. Ekbom, Appendectomy and protection against ulcerative colitis, N. Engl. J. Med. 344 (11) (2001) 808e814. Epub 2001/03/15. [19] C. Fiocchi, Inflammatory bowel disease: etiology and pathogenesis, Gastroenterology 115 (1) (1998) 182e205. [20] Y.Z. Zhang, Y.Y. Li, Inflammatory bowel disease: pathogenesis, World J. Gastroenterol. 20 (1) (2014) 91e99.
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B cell activation in the cecal patches during the development of an experimental colitis model Taro Watabe a, 1, Takashi Nagaishi b, 1, Naoya Tsugawa a, Yudai Kojima a, Nisha Jose a, Akinori Hosoya a, Michio Onizawa b, Yasuhiro Nemoto a, Shigeru Oshima a, Tetsuya Nakamura b, Hajime Karasuyama c, Takahiro Adachi d, Mamoru Watanabe a, * a
Department of Gastroenterology, Graduate School of Medical Science, Tokyo Medical and Dental University (TMDU), Tokyo, Japan Department of Advanced Therapeutics for GI Diseases, Graduate School of Medical Science, TMDU, Tokyo, Japan Department of Immune Regulation, Graduate School of Medical Science, TMDU, Tokyo, Japan d Department of Immunology, Medical Research Institute, TMDU, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 January 2018 Accepted 9 January 2018 Available online 10 January 2018
Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca2þ signaling in CP B cells during the early phase of colitis development. These findings suggested that the CP B cells may be involved in the pathogenesis of colitis including inflammatory bowel diseases in humans. © 2018 Elsevier Inc. All rights reserved.
Keywords: Colitis Oxazolone Intravital imaging Cecal patches B cells
1. Introduction Previous studies reported that appendectomy attenuates experimental colitis models such as spontaneous colitis in T cell receptor a chain-mutant mice (TCRa/-) [1] and dextran sodium sulfate (DSS)-induced colitis [2]. From these findings, it has been suggested that appendiceal lymphoid tissues seem to serve some
Abbreviations: BCR, B cell receptor; Ca2þ, calcium ion; CD, Crohn's disease; CFP, cyan fluorescent protein; CP, cecal patches; DSS, dextran sodium sulfate; ELISA, enzyme-linked immunosorbent assay; EtOH, ethanol; FAE, follicular-associated epithelia; FRET, fluorescence resonance energy transfer; GALT, gut-associated lymphoid tissues; H&E, hematoxylin and eosin; IBD, inflammatory bowel diseases; IFN-g, interferon-g; IL, interleukin; LPL, lamina propria lymphocytes; mAb, monoclonal antibody; mMT, mice deficient for the membrane exon of the m heavy chain gene; NKT, natural killer T; n.s., not significant; Ox, oxazolone; TCRa, T cell receptor a chain; Th, helper T cells; UC, ulcerative colitis; YC, yellow cameleon; YFP, yellow fluorescent protein. * Corresponding author. Department of Gastroenterology and Hepatology, Graduate School of Medical Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. E-mail address: [email protected] (M. Watanabe). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2018.01.053 0006-291X/© 2018 Elsevier Inc. All rights reserved.
significant immunological functions. Recently, it was reported that appendiceal lymphoid follicle, called cecal patches (CP) in mice, is a major site for generation of IgA-secreting cells that migrate to the large intestine [3], but the role of this in colitis has not been elucidated. There are several animal models developed to explore the mechanisms of colitis pathogenesis [4], and oxazolone-induced colitis is an often used model. Sensitization to oxazolone by intrarectal administration to mice can induce colonic injury accompanied by the upregulation of T-helper (Th) 2 cytokines [5]. The histopathological features of oxazolone colitis is similar to that of inflammatory bowel diseases (IBD), especially ulcerative colitis (UC) in humans [6], and this model has been proven useful in assessing causal relationships between specific treatments and colitis [7]. The initiation process of oxazolone colitis is thought to be dependent on the T cell response since colitis does not develop in severe combined immune-deficient mice [8]. On the other hand, one of the natural killer T (NKT) cell subsets has been reported to be involved in the pathogenesis of human UC [9], and involvement of invariant NKT cells in oxazolone colitis in mice is also supported by previous observations [10,11]. As described above, oxazolone colitis
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shares pathological similarities to human UC, so we employed this model to assess the importance of CP in colitis. 2. Materials and methods 2.1. Mice
(1 cm in length) followed by exteriorization of the appendix. The appendix was divided between two ligatures (absorbable surgical suture, Ethicon) that were placed proximal to the border of cecum and appendix. In the sham-operated group, mice were anesthetized and underwent a midline incision but without excision of the appendix. In both groups, the abdominal wall was closed in two layers, using a running suture technique.
Wild-type C57BL/6 mice (WT) were obtained from CLEA Japan (Tokyo, Japan). Mice lacking mature B cells (mMT) were kindly provided by Dr. Klaus Rajewsky (Max Delbrück Center for Molecular Medicine, Germany), and the yellow cameleon 3.60 (YC3.60) mice were from Dr. Atsushi Miyawaki (RIKEN Center for Advanced Photonics, Japan). YC3.60 mice were intercrossed with CD19-Cre transgenic mice (CD19-Cre/YC3.60) to induce specific transgene expressions in B cells. All mice were maintained in the animal facility of Tokyo Medical and Dental University (TMDU) under specific pathogen free conditions in accordance with guidelines of the Institutional Animal Care and Use Committee of TMDU. All experimental procedures on animals were approved by the Institutional Animal Care and Use Committee of TMDU, and all experiments were carried out in accordance with approved guidelines.
We analyzed Ca2þ signaling as previously described [13]. CP of mice anesthetized by inhalation of isoflurane were imaged as following: CP were surgically exteriorized, immobilized on a microscope stage, and maintained at 37 C. All image acquisition and analysis were performed using an A1® laser scanning confocal system with a 20 objective and software NIS-Elements® (Nikon). The yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) signal ratio was obtained by excitation at 458 nm.
2.2. Induction of colitis
The results were expressed as the means ± SEM. Statistical analysis were performed with unpaired Student's t-test. Differences were considered to be statistically significant when p value < .05.
Colitis was induced in 8e12 week-old mice by pre-sensitizing with 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone, Sigma-Aldrich) at a concentration of 3% in 100% ethanol (EtOH) by skin painting 1 week before intrarectal administration at a concentration of 1% in 50% EtOH on day 0, as previously described [11]. On day 5, or on day 1 in some experiments, mice were euthanized and analyzed. 2.3. Determination of cytokine production Colonic lamina propria lymphocytes (LPL) were isolated from freshly obtained colonic specimens as described previously [12]. The isolated LPL (5 105 cells/well) on day 5 were stimulated with 5 mg/ml plate-bound antiemouse CD3ε mAb (145-2C11, BD Biosciences) and 2 mg/ml soluble antiemouse CD28 mAb (37.51, BD Biosciences). Culture supernatants were harvested 48 h after the incubation. Concentrations of IFN-g and IL-4 were measured with OptEIA® ELISA sets (BD Biosciences), and IL-17 was measured with Quantikine® ELISA kit (R&D systems), according to the manufacturers' instruction. 2.4. Histological assessment of colitis Colonic tissues were removed on day 5, embedded in OCT compound (Sakura Finetech) and frozen at 80 C. Eight mm cryosections were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin (H&E). The degree of inflammation in the colon was graded semi-quantitatively by a modified method from previously described criteria [11]. Briefly, five criteria (hypervascularization, presence of mononuclear cells, epithelial hyperplasia, epithelial injury, and presence of granulocytes) were each scored from 0 to 3. The cumulative degree of each was calculated as a total histological score ranging from 0 (no colitis) to 15 (maximal colitis activity). Such evaluation was performed in a blinded fashion. 2.5. Appendectomy Surgical procedures were performed as described previously [2]. Eight week-old mice were anesthetized by inhalation of isoflurane. Under sterile conditions, mice underwent a midline laparotomy
2.6. Intravital imaging
2.7. Statistical analysis
3. Results 3.1. The inflammation accompanied with ulceration is induced by oxazolone treatment in cecal mucosa as well as colonic tissues in mice To first assess whether ileocecal immune response is involved in the pathogenesis of our colitis model, WT pre-sensitized with oxazolone were given rectal administration of oxazolone, and then observed for 5 days (Fig. 1a). We observed diarrhea and significant wasting of oxazolone-treated mice from day 1 compared to vehicle (EtOH)-treated control. However, the latter subsequently recovered and even surpassed the original body weight on day 3e5 (Fig. 1a), which was apparently caused by systemic edema. Mice were then euthanized for histologic analysis on day 5. The oxazolone-treated mice showed significant shortening of colon length compared to that of vehicle-treated control groups (Fig. 1b and Supplementary Fig. 1). However, we observed profound infiltration of lymphocytes and granulocytes in the colonic tissues of oxazolone-treated mice (Fig. 1c), even though there were occasional ulcerations. And interestingly, we were also able to observe remarkable ulcerations particularly at the follicular-associated epithelia (FAE) on CP with the follicular expansion in the oxazolone-treated mice (Fig. 1c). Taken together, histopathological assessment revealed that oxazolone-treated mice had significant colitis (Fig. 1d) accompanied by the cecal inflammation. These observations were associated with significantly increased pro-inflammatory cytokine production, such as IFN-g, IL-4 and IL-17, from the colonic LPL (Fig. 1e). These results are consistent with previous observations [5,6,10,11,14]. 3.2. Immune responses in cecal patches are involved in the pathogenesis of oxazolone-induced colitis Given the remarkable ulceration at the FAE overlying CP in oxazolone-treated mice, we hypothesized that the immune response in CP may be associated with the pathogenesis of colitis. We therefore focused on the function of CP in the setting of colitis. WT underwent either appendectomy or sham-operation two weeks prior were subjected to oxazolone colitis by the same
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operation (Fig. 2a). Regarding the length of colons, oxazolonetreated mice were significantly shorten compared to EtOHtreated mice in the sham-operated group. However, oxazolonetreated mice had less colon shortening in the appendectomyoperated group, and the difference between these groups were not significantly different (Fig. 2b and Supplementary Fig. 2). Consistent with this, histopathological assessment showed significant attenuation of lymphocyte and granulocyte infiltrations in the colonic lamina propria of oxazolone-treated mice in the appendectomy-operated group compared to that of mice in the sham-operated group (Fig. 2c and d). These observations were associated with significantly abrogated productions of IFN-g, IL-4 and IL-17, from colonic LPL isolated from the oxazolone-treated mice with appendectomy compared to the oxazolone-treated mice with sham-operation (Fig. 2e). 3.3. The functions of B cells are involved in the pathogenesis of oxazolone-induced colitis
Fig. 1. Oxazolone treatment results in inflammation and ulceration in both cecum and colon. (a) Wasting, as defined by percentage of initial body weight, in WT treated with either vehicle control (EtOH) or oxazolone (Ox). Data are expressed as means ± SEM. Each group contained eight mice. *p < .05. (b) Colon length of mice treated with either vehicle (EtOH) or oxazolone (Ox). Data are expressed as means ± SEM. n ¼ 8. *p < .05. (c) H&E-stained microscopic features of colons and cecal patches (CP) from mice treated with either vehicle (EtOH) or oxazolone (Ox). Representative features from each group are shown. (d) Quantitative histopathological assessments of colitis severity from mice treated with either vehicle (EtOH) or oxazolone (Ox). Data are expressed as means ± SEM. n ¼ 8. *p < .05. (e) Productions of pro-inflammatory cytokines such as IFN-g, IL-4 and IL-17 from colonic LPL in mice treated with either vehicle (EtOH) or oxazolone (Ox). Data are indicated as means ± SEM. n ¼ 8. *p < .05.
procedures as above. A series of pilot experiments showed that surgical procedures alone did not affect the gastrointestinal tract functionally or morphologically before the oxazolone treatment (data not shown). There was no significant difference in any clinical, histological, or biochemical parameters between sham- and appendectomy-operated mice without colitis induction. We also compared oxazolone-treated non-operated mice to oxazolonetreated sham-operated mice, and there was no significant difference in these as well (data not shown). Therefore, we considered that oxazolone-treated sham-operated mice were suitable controls for oxazolone-treated appendectomy-operated mice. We observed significant wasting 1 day after oxazolone treatment in both groups that underwent appendectomy and sham-
Given the results of the attenuated colitis in oxazolone-treated mice with appendectomy, we next hypothesized that certain population in CP may initiate the inflammation in this model. In general, most of secondary lymphoid follicles, including ones in the gut-associated lymphoid tissues (GALT), mainly consist of T cells and B cells. Previous studies have already established the importance of T cells in the induction of acquired immune responses even in the context of oxazolone colitis. We want to focus on the function of B cells in this model. Therefore, same model was induced in mMT. Oxazolone-treated mMT were showing less body weight during recovery from day 2 to day 5 compared to that of oxazolone-treated WT (Fig. 3a). However, the shortening of colon by oxazolone treatment in mMT was rather attenuated compared to that of WT (Fig. 3b and Supplementary Fig. 3). Associated with the less shortening of colon, histopathological assessment revealed significantly attenuated colitis severity as well as the ulceration at CP in oxazolone-treated mMT compared to that of oxazolone-treated WT (Fig. 3c and d). Moreover, such attenuation of colitis was associated with significantly suppressed productions of IL-4 and IL-17 but not IFN-g, from colonic LPL in oxazolone-treated mMT compared to that of oxazolone-treated WT (Fig. 3e). These studies imply that B cells in GALT, such as CP, may be responsible for the induction of colonic inflammation in the setting of colitis. 3.4. Frequent Ca2þ flux in cecal patch B cells is induced at early phase of colitis development To assess the immunological response in CP dynamically, we worked on intravital imaging using B cell-specific YC3.60 transgenic mice [13]. YC3.60 is a Ca2þ sensor adopting the fluorescence resonance energy transfer (FRET) technology consisting of CFP and YFP linked by the sequence of calmodulin and calmodulin-binding peptide M13. When Ca2þ binds to calmodulin, it results in structural change and an increase in the efficiency of the energy transfer [15]. CFP is excited at 458 nm under low Ca2þ concentration. However, when Ca2þ concentration becomes higher in the cells, not only CFP but also YFP can be excited. Thus, the increased emission ratio of YFP to CFP (YFP/CFP) signal excited at 458 nm reflects the intracellular Ca2þ flux. CD19-Cre/YC3.60 were treated with either oxazolone or vehicle, and intravital Ca2þ signaling in CP B cells was monitored 1 day after intrarectal administration. The Ca2þ flux, defined as YFP/CFP ratio >1.2, in CP B cells was frequently observed in oxazolone-treated mice, although that was hardly observed in EtOH-treated mice (Fig. 4a and b and Supplementary Fig. 4 and 5). The time-lapse observation revealed that Ca2þ flux in single cells were activated
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Fig. 2. Oxazolone colitis is attenuated in mice that underwent appendectomy. (a) Wasting, as defined by percentage of initial body weight, in mice underwent the following procedures: sham operation followed by vehicle control treatment (sham EtOH), sham followed by oxazolone treatment (sham Ox), appendectomy followed by vehicle treatment (appe EtOH), or appendectomy followed by oxazolone treatment (appe Ox). Data are expressed as mean values ± SEM. Each group contained six mice. *p < .05. (b) Colon length of mice in each group. Data are expressed as mean values ± SEM. n ¼ 6. n.s., not significant. *p < .05. (c) H&E-stained microscopic features of colons from oxazolone-treated mice that had either sham operation (sham Ox) or appendectomy (appe Ox). Representative features from each group are shown. (d) Histopathological assessments of colitis severity from each group. Data are expressed as means ± SEM. n ¼ 6. *p < .05. (e) Productions of pro-inflammatory cytokines such as IFN-g, IL-4 and IL-17 by colonic LPL isolated from each group. Data are indicated as means ± SEM. n ¼ 6. *p < .05.
either intermittently or continuously in oxazolone-treated mice (Fig. 4a, c and Supplementary Fig. 5). We had also attempted to observe Ca2þ signaling in CP B cells 3 or 5 days after the administration, but interestingly, the frequency of activating cells decreased significantly as time progressed (data not shown). These results suggest that B cells in CP contribute to the onset of colitis. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.01.053. 4. Discussion In this study, we hypothesized that CP serves an important role during colitis. In fact, our studies with appendectomy or deficiency of mature B cells suggested that the functions of CP or B cells in GALT may be involved in the pathogenesis of oxazolone colitis.
Moreover, the activated Ca2þ signaling in CP B cells observed by intravital imaging suggested that CP B cells contribute to the onset of colitis. In experimental models of colitis, such as spontaneous colitis in TCRa/- and DSS-induced colitis, severity is also attenuated by appendectomy. In humans, a large-scale epidemiological study had previously shown an inverse relation between UC and appendectomy [16,17], especially if performed before the age of 20 [18]. This inverse relation was limited to patients who underwent appendectomy due to appendicitis or lymphadenitis. These studies implied that some latent abnormal immunological response in appendix may induce chronic inflammation in colon, but the detailed mechanism has not been elucidated. There are many lymphoid follicles in human appendix, and the equivalent tissues in murine GALT are called CP. A recent report showed that CP is a
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Fig. 3. Oxazolone colitis is attenuated by the deficiency of mature B cells. (a) Wasting defined by percentage of initial body weight in WT and mMT induced colitis. Mice were treated with either vehicle control (EtOH) or oxazolone (Ox). Data are expressed as mean values ± SEM. Each group contained three mice. *p < .05. (b) Colon length of WT with vehicle control (WT EtOH), mMT with vehicle (mMT EtOH), WT with oxazolone (WT Ox) and mMT with oxazolone (mMT Ox). Data are expressed as mean values ± SEM. n ¼ 3. n.s., not significant. *p < .05. (c) Microscopic features of colons and CP from both WT and mMT treated with oxazolone. Representative features from each group are shown. (d) Histopathological evaluations of colitis severity from each group. Data are expressed as means ± SEM. n ¼ 3. *p < .05. (e) Productions of cytokines such as IFN-g, IL-4 and IL-17 by colonic LPL isolated from each group. Data are indicated as means ± SEM. n ¼ 3. n.s., not significant. *p < .05.
major site where IgA-secreting cells are generated to migrate to the large intestine, and therefore, it serves to regulate intestinal microbiota [3]. However, the function of CP in colitis pathogenesis is still unsolved. In the previous study of spontaneous colitis in TCRa/- by others, the increase of B cells in CP were observed and the activation of B cells was suggested in this model [1]. Thus, we focused on the function of CP B cells. To analyze this, we employed oxazolone colitis, which is characterized by the colonic injury associated with the upregulation of Th2 cytokines, characteristics similar to that of human UC [6,7].
In our studies, the production of IL-4 from colonic LPL was upregulated in oxazolone-treated mice, which is consistent with prior report [5]. In addition, the production of IFN-g from colonic LPL was also up-regulated in these mice, which is also consistent with previous observation [6]. Moreover, we found that IL-17 production was also up-regulated, which is consistent with the analysis with semi-quantitative polymerase chain reaction reported by others [14]. These results indicated that Th1, Th2 and Th17 responses are all involved in the pathogenesis of oxazolone colitis model. Another interesting observation in our studies is the finding
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Fig. 4. Frequent Ca2þ flux in cecal patch B cells is induced by oxazolone-treatment. (a) Intravital images of Ca2þ signaling in CP B cells of CD19-Cre/YC3.60 mice. Representative observations under the confocal microscopy one day after the treatment with either vehicle (Ctrl) or oxazolone (Ox) are shown. Representatives of intermittently and continuously activated cells are indicated by yellow and red arrowhead, respectively. The highest signal of YFP is modified into red color by computer processing methods in these images. (b) Distribution of time-integrated intracellular Ca2þ concentrations in CP B cells of CD19Cre/YC3.60 one day after the treatment with either vehicle or oxazolone. n ¼ 50, frame ¼ 20. Percentages of cells expressing YFP/CFP ratio >1.2 are also indicated. (c) Kinetics for YFP/CFP ratio at excitation of 458 nm in representative CP B cells one day after the treatment with either vehicle or oxazolone are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
of ulceration at CP accompanied by the follicular expansion, which suggests an involvement of CP in these acquired immune responses associated with colitis. After appendectomy, the severity of oxazolone colitis was attenuated, and the productions of IFN-g, IL-4 and IL-17 from colonic LPL were significantly downregulated. Colitis was also attenuated when mature B cells were absent, and this was associated with decreased productions of IL-4 and IL-17 from colonic LPL. IFN-g production in mMT was also slightly downregulated, but it
was not statistically significant compared to that of WT. These results indicate the involvement of CP B cells in the pathogenesis of oxazolone colitis, especially in the aspects of Th2 and Th17 responses. Regarding the different observations of cytokine profile between the two different conditions (appendectomy and mature B cell deficiency in Figs. 2 and 3), it is suggested that not only B cells but also other cell types, such as dendritic cells and macrophages, in CP were removed during appendectomy, and consequently Th1 response becomes downregulated. In addition, we have observed
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relatively reduced body weight in oxazolone-treated mMT on day 3e5 compared to oxazolone-treated WT in Fig. 3a, and this was reproducible in several rounds of experiments even though the difference was not statistically significant. The potential interpretation regarding such discrepancy would be that the increase of body weight during day 3e5 in oxazolone-treated mice may partially be caused by systemic edema due to increased production of Th2 and/or Th17 cytokines as seen in Fig. 1a. Therefore, oxazolone-treated mMT, whose colonic LPL showed significantly suppressed production of IL-4 and IL-17 but not IFN-g, had relatively more body weight loss with less systemic edema. We employed intravital imaging to assess the contribution of B cells, which enabled dynamic observation of cellular activities in the setting of colitis. It has been well-known that the Ca2þ signaling mediated by B cell receptors plays an important role in B cell activation and differentiation [15]. We were able to observe frequent Ca2þ signaling in CP B cells of colitis-induced CD19-Cre/ YC3.60 one day after oxazolone treatment. A subset of B cells in secondary lymphoid follicles is generally suggested to be supported by follicular helper T cells, which are activated by antigen presenting cells, in order to differentiate into plasma cells to secrete immunoglobulins. Therefore, our observation of B cell activation in CP would suggest B cell differentiation into plasma cells. On the other hand, there were some B cells that had already been activated at the early phase of the colitis development, and such activation was immediately attenuating during the recovery. Therefore, the results from our observation with intravital imaging and amelioration of colitis by appendectomy and mature B cell deficiency would suggest that CP B cells may be involved in the onset of the colitis pathogenesis. There have been an increase number of patients diagnosed with IBD, such as UC and Crohn's disease (CD). Although many efforts have revealed that IBD can be caused by various factors, such as environmental, genetic, immunologic, and intestinal microbiota [19,20], there are still many questions that need to be elucidated, including the involvement of appendix. Our results imply that B cells in human appendiceal lymphoid follicles may be involved in the pathogenesis of IBD. Further analysis of Ca2þ signaling in B cells may shed light on the novel functions of CP in the immune response in the gut. Grant This study was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (MW: 22117508), Challenging Exploratory Research (T. Nagaishi: 15K15288), Scientific Research-S (MW: 26221307), and Scientific Research-B (T. Nagaishi: 16H05286) from the Japanese Ministry of Education, Culture, Sports, Science and Technology; Research Center Network Program for the Realization of Regenerative Medicine (MW) from the Japan Science and Technology Agency and Japan Agency for Medical Research and Development (AMED); Practical Research Project for Rare/Intractable Diseases (MW: 16eK0109047h0003) from AMED; Memorial Fund of Nihon Univ. Medical Alumni Association (T. Nagaishi); Collaboration Research Program of TMDU Medical Research Institute (T. Nagaishi, and TA); Naoki Tsuchida Memorial Resarch Grant (T. Nagaishi, and TA); Otsuka Toshimi Scholarship Foundation (NJ). Acknowledgments We are grateful to Drs. K. Rajewsky and A. Miyawaki for providing the mMT and YC3.60 mice, respectively, A. Tokai for the technical support, and Dr. K. Yoshikawa for confocal microscopy use.
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Disclosures The authors have no financial conflict of interest. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.01.053. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.01.053. References [1] A. Mizoguchi, E. Mizoguchi, C. Chiba, A.K. Bhan, Role of appendix in the development of inflammatory bowel disease in TCR-alpha mutant mice, J. Exp. Med. 184 (2) (1996) 707e715. Epub 1996/08/01. [2] C.F. Krieglstein, W.H. Cerwinka, F.S. Laroux, M.B. Grisham, G. Schurmann, M. Bruwer, et al., Role of appendix and spleen in experimental colitis, J. Surg. Res. 101 (2) (2001) 166e175. [3] K. Masahata, E. Umemoto, H. Kayama, M. Kotani, S. Nakamura, T. Kurakawa, et al., Generation of colonic IgA-secreting cells in the caecal patch, Nat. Commun. 5 (2014) 3704. [4] S. Wirtz, C. Neufert, B. Weigmann, M.F. Neurath, Chemically induced mouse models of intestinal inflammation, Nat. Protoc. 2 (3) (2007) 541e546. [5] M. Boirivant, I.J. Fuss, A. Chu, W. Strober, Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4, J. Exp. Med. 188 (10) (1998) 1929e1939. Epub 1998/11/17. [6] H. Iijima, M.F. Neurath, T. Nagaishi, J.N. Glickman, E.E. Nieuwenhuis, A. Nakajima, et al., Specific regulation of T helper cell 1-mediated murine colitis by CEACAM1, J. Exp. Med. 199 (4) (2004) 471e482. [7] V. Popp, K. Gerlach, S. Mott, A. Turowska, H. Garn, R. Atreya, et al., Rectal delivery of a DNAzyme that specifically blocks the transcription factor GATA3 and reduces colitis in mice, Gastroenterology 152 (1) (2017) 176e192 e5. [8] M. Taube, L. Svensson, H. Carlsten, T lymphocytes are not the target for estradiol-mediated suppression of DTH in reconstituted female severe combined immunodeficient (SCID) mice, Clin. Exp. Immunol. 114 (2) (1998) 147e153. Epub 1998/11/20. [9] I.J. Fuss, F. Heller, M. Boirivant, F. Leon, M. Yoshida, S. Fichtner-Feigl, et al., Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis, J. Clin. Invest. 113 (10) (2004) 1490e1497. Epub 2004/05/18. [10] S. Brozovic, T. Nagaishi, M. Yoshida, S. Betz, A. Salas, D. Chen, et al., CD1d function is regulated by microsomal triglyceride transfer protein, Nat. Med. 10 (5) (2004) 535e539. [11] F. Heller, I.J. Fuss, E.E. Nieuwenhuis, R.S. Blumberg, W. Strober, Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13producing NK-T cells, Immunity 17 (5) (2002) 629e638. [12] M.F. Neurath, B. Weigmann, S. Finotto, J. Glickman, E. Nieuwenhuis, H. Iijima, et al., The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease, J. Exp. Med. 195 (9) (2002) 1129e1143. [13] S. Yoshikawa, T. Usami, J. Kikuta, M. Ishii, T. Sasano, K. Sugiyama, et al., Intravital imaging of Ca(2þ) signals in lymphocytes of Ca(2þ) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset, Sci. Rep. 6 (2016) 18738. [14] V. Galitovskiy, J. Qian, A.I. Chernyavsky, S. Marchenko, V. Gindi, R.A. Edwards, et al., Cytokine-induced alterations of alpha7 nicotinic receptor in colonic CD4 T cells mediate dichotomous response to nicotine in murine models of Th1/ Th17- versus Th2-mediated colitis, J. Immunol. 187 (5) (2011) 2677e2687. Epub 2011/07/26. [15] T. Adachi, T. Tsubata, FRET-based Ca2þ measurement in B lymphocyte by flow cytometry and confocal microscopy, Biochem. Biophys. Res. Commun. 367 (2) (2008) 377e382. [16] A.B. Lowenfels, Appendectomy and ulcerative colitis, Gastroenterology 107 (5) (1994) 1570. Epub 1994/11/01. [17] M.G. Russel, E. Dorant, R.J. Brummer, M.A. van de Kruijs, J.W. Muris, J.M. Bergers, et al., Appendectomy and the risk of developing ulcerative colitis or Crohn's disease: results of a large case-control study. South Limburg Inflammatory Bowel Disease Study Group, Gastroenterology 113 (2) (1997) 377e382. [18] R.E. Andersson, G. Olaison, C. Tysk, A. Ekbom, Appendectomy and protection against ulcerative colitis, N. Engl. J. Med. 344 (11) (2001) 808e814. Epub 2001/03/15. [19] C. Fiocchi, Inflammatory bowel disease: etiology and pathogenesis, Gastroenterology 115 (1) (1998) 182e205. [20] Y.Z. Zhang, Y.Y. Li, Inflammatory bowel disease: pathogenesis, World J. Gastroenterol. 20 (1) (2014) 91e99.