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Inhibition of ASM activity ameliorates DSS-induced colitis in mice
Prostaglandins and Other Lipid Mediators 140 (2019) 26–30
Contents lists available at ScienceDirect
Prostaglandins and Other Lipid Mediators journal homepage: www.elsevier.com/locate/prostaglandins
Original Research Article
Inhibition of ASM activity ameliorates DSS-induced colitis in mice a
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Yan Xiong , Xiao-Dan Zhu , Ping Wan , Yu-Ping Ren , Chong Wang , Run-Wei Yan , Yuan Guo , ⁎ Ai-Ping Baia, a
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China Transplant Center, Department of Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266555, China Department of Rheumatology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China d Department of Pharmacy, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Acid sphingomyelinase Macrophage Colitis Cytokine Inhibition
Acid sphingomyelinase (ASM) is a membrane lipid hydrolase, acting to generate ceramide and regulate cell functions and inflammatory responses.The roles of ASM in mediating T cell functions are postulated whereas its function in regulation of macrophages remains uncertain. The study was performed to explore ASM activity in control of macrophage functions. RAW 264.7 cells were pretreated with desipramine, an ASM inhibitor, prior to LPS challenge in vitro. LPS initiated ASM activity in RAW 264.7 cells. Conversely, inhibition of ASM activity by desipramine diminished LPS induced ASM activities and TNF production of RAW 264.7 cells. The DSS colitis in mice was induced, and desipramine was administered to the mice two days post induction of colitis. Murine colitis was characterized by elevation of ASM activities in colon tissues. Desipramine administration overrode ASM activities in colon, and ameliorated DSS-induced colitis evidenced with the reduced disease activities and the decreased cytokine levels. Together, our data show a crucial role of ASM activity in regulation of macrophage functions and responses, and suggest that ASM represents a novel therapeutic approach for the management of immune diseases.
1. Introduction Innate immune cells including macrophages are the main components of human immune system, playing the pivotal roles in maintaining immune homeostasis. In the presence of bacteria and virus infections, macrophages become activated and serve to clear those pathogens. However, when improperly activated, macrophages initiate aberrant immune responses and contribute to autoimmune diseases including inflammatory bowel disease (IBD) [1]. IBD is a serious intestinal immune disorder with poor prognosis. During the progression of IBD, macrophages become excessively activated, and release substantive proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1β, resulting in the sustained inflammation and tissue damage in the colon [1,2]. Inhibition of macrophage functions has been proposed as one of the targets for the treatment of human immune diseases including IBD [3,4]. Acid sphingomyelinase (ASM) is a membrane lipid hydrolase. Through hydrolysis of sphingomyelin to generate signal messenger, i.e., ceramide, ASM exhibits the pivotal roles in mediating cell signals [5,6]. Others and our group have noted that ASM determines immune cell functions inclusive of natural killer (NK) cells and T helper (Th) cells ⁎
[7–9]. Upon stimulation of cellular receptors by their ligands, ASM bioactivity is initiated, and subsequently mediates T cell activation [10,11]. Conversely, inhibition of ASM bioactivity dampens pathogenic Th responses [7]. ASM activity is postulated to mediate T cell activation and functions, whereas its roles in regulation of macrophage functions remain uncertain. Here we reported that upon inflammatory stimuli, ASM regulated macrophage activation and mediated the progression of dextran sulfate sodium (DSS)-induced colitis in mice, an innate immune cells-driven inflammation in colon [12]. 2. Methods 2.1. Cell culture Murine macrophage cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin G (100U/mL), and streptomycin (100 μg/mL) at 37 °C. For in vitro study, about 1 × 106/mL RAW264.7 cells were seeded in 12 or 96-well plates. The cells were treated with vehicle, or titrated desipramine (Sigma-Aldrich) for 30 min, prior to lipopolysaccharide
Corresponding author. E-mail address: [email protected] (A.-P. Bai).
https://doi.org/10.1016/j.prostaglandins.2018.12.002 Received 20 July 2018; Received in revised form 6 December 2018; Accepted 10 December 2018 Available online 11 December 2018 1098-8823/ © 2018 Elsevier Inc. All rights reserved.
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on a scoring system modified from a previous study [18], and a combined score of inflammatory cell infiltration and tissue damage was determined as follows: score 0, normal colonic mucosa and occasional inflammatory cells in the lamina propria; 1, loss of one-third of the crypts; 2, loss of two-thirds of the crypts; 3, the lamina propria is covered with a single layer of epithelium and mild inflammatory cell infiltration is present; and 4, erosions and transmural extension of infiltrate.
(LPS, from Escherichia coli 0111:B4; Sigma-Aldrich) challenge. 2.2. Enzyme-linked immunosorbent assays (ELISA) Cytokine concentrations in the supernatants or colon tissue homogenates were determined by ELISA, regarding to the manufacturer’s instructions (R&D Systems, Inc) [13–15]. Briefly, polyclonal rat antimouse cytokine antibodies were used as capturing antibodies and biotinylated polyclonal rat anti-mouse cytokine antibodies for detection, and the standard curve of cytokines was set up meanwhile. Color changes were determined at 450 nm.
2.6. Immunohistochemistry Colon tissues of mice were taken and fixed immediately in 10% buffered formalin, embedded in paraffin, and cut into 4 μm sections. After blockade of inner peroxidase, sections were incubated sequentially with the first antibody solution including rabbit anti-NF-κB p65 (phospho S536, from Abcam) antibody [19]. After three washes in PBS (pH 7.4), the sections were then incubated in secondary goat anti-rabbit immunoglobulin (Ig)G conjugated with peroxidase labeled polymer, prior to colorization using diaminobenzidine reaction and counterstained with haematoxylin. Negative controls were established using rabbit IgG instead of the first antibodies. The cells stained with anti-NFκB P65 antibody in both cytoplasm andthe nucleus were counted as NFκB positive cells, whereas those without nucleus stain were regarded as NF-κB negative cells [14,18]. The sections were evaluated using light microscopy, and 100 cells in lamina propria per high power field were calculated for statistical analysis.
2.3. ASM activity measurement ASM activity in cellular extracts or colon tissue extracts was determined using Amplex Red sphingomyelinase assay kit (Invitrogen) according to the manufacturer’s protocol. Briefly, cellular extracts or colon tissue extracts were diluted in 50 mM sodium acetate (pH 5.0), and mixed with 5 mM sphingomyelin solution. After adequate incubation, the samples above were mixed sequentially with the Amplex Red reagent/HRP/cholineoxidase/alkaline phosphatase working solution. The fluorescence intensity in each well was determined in a fluorescence reader with the excitation at 530 nm and the emission at 590 nm respectively. 2.4. Animals
2.7. Statistical analysis
Female C57/BL6 mice were provided by the Experimental Animal Center of Nanchang University, and fed underspecific pathogen-free conditions. 7–8 week-old mice were randomly divided into three groups (10 mice per group) in the study, weighing approximately 22 g. All protocols for the projects using the mice were reviewed and approved by the Institutional Animal Care Committee of Nanchang University.
All data in the text and figures are expressed as mean ± the standard error of the mean. Comparisons of more than two groups were made with a one-way analysis of variance using Tukey’s post hoc test. When appropriate, comparison with two groups was made using Student’s t-test for unpaired data. Differences were considered statistically significant if P < 0.05.
2.5. Induction of dextran sulphate sodium (DSS) colitis
3. Results
Acute DSS colitis was induced in C57/BL6 mice according to the previously published method with minor modification [16,17]. The mice were fed 4% (w/v) DSS (molecular mass, 36–50 kDa; MP Biomedicals) dissolved in the drinking water on day one. Fresh DSS solution was provided every other day. Control mice drank only distilled water. Desipramine (20 mg/kg body weight) was administered intraperitoneally (i.p.) to the mice drinking DSS (DSS + Des group) since day three and repeated daily until the mice were killed on day eight. Disease symptoms of colitis were assessed daily by measurement of body weight, evaluation of stool consistency and detection of bloody stools. Disease severity was scored using a clinical disease activity index (DAI) ranging from 0 to 4, calculated as described previously [14,18] using the following parameters: stool consistency, presence or absence of fecal blood and weight loss. The mice were sacrificed on day eight, and the middle section of colon was fixed in 10% formaldehyde-saline. Hematoxylin and eosin stain(HE)-stained sections were graded based
3.1. Inhibition of ASM activity dampens cytokine release by LPS-induced macrophages To induce ASM bioactivities by inflammatory stimulations, we exposed RAW264.7, a murine macrophage cell line, to LPS, and noted that LPS immediately initiated ASM activities in a dose dependent manner (Fig. 1A). Meanwhile, minimal changes of ASM expression in mRNA levels in murine macrophages post LPS challenge were noticed (data not shown). To inhibit ASM activity, we pretreated the cells with desipramine 30 min prior to LPS challenge. As shown in Fig. 1B, administration of desipramine dampened LPS induced ASM activities in the cells in a dose dependent manner. As TNF is one of the key cytokines mediating inflammatory responses [20], we next determined TNF production by macrophage post
Fig. 1. Inhibition of ASM activity dampens LPS induced macrophage activation. (A) RAW 264.7 cells were treated with LPS for 60 min, and ASM activity was determined thereby. (B-C) RAW 264.7 cells were pretreated with titrated desipramine (Des) for 30 min prior to LPS challenge. ASM activity (B) and TNF levels (C) were determined 60 min and 24 h post LPS stimulation, respectively. N = 4–5 per group, *, P < 0.05; **, P < 0.01. 27
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Fig. 2. Blockade of ASM activity ameliorates murine DSS colitis. Colitis was induced in mice by administration of 4% DSS dissolved in drinking water. Desipramine (DSS + Des group) or vehicle (DSS group) were administered intraperitoneally 2 days after induction of colitis, and repeated daily until the mice were sacrificed onday 8. Control mice drank only distilled water. (A) Body weight changes of the mice in each group (n = 10 per group). (B) DAI was scored on day 7 using the following parameters: stool consistency, presence or absence of fecal blood and weight loss (n = 10 per group). (C) ASM activities in colon tissue extracts were determined respectively after euthanasia of the mice at day 8 (n = 6 per group). (D) HE staining was performed and histological scores were graded (n = 10 per group). Scale bars: 250 μm. *, P < 0.05; **, P < 0.01.
NF-κB is one of the pivotal transcriptional factors regulating immune responses [22]. Once activated by stimulations, NF-κB signals regulate inflammatory molecules expression and dominate inflammation progression including colitis [14]. Next, we studied whether ASM inhibition treatment could impact on NF-κB activation. As shown in Fig. 2C, DSS colitis was characterized by drastic increase of phosphorNF-κB p65 expression in lamina propria immune cells. In contrast, administration of desipramine decreased phosphor-NFκBp65 positive cell numbers in lamina propria, indicating significant blockade of NFκB activation in immune cells by inhibitionof ASM activity and signaling.
LPS stimulation. LPS dramatically induced TNF release by RAW264.7 cells, whereas desipramine inhibited LPS-induced TNF production in a dose dependent manner (Fig. 1C). The data indicate that blockade of ASM activity inhibits inflammatory stimulation-induced macrophage activation. 3.2. Inhibition of ASM activity ameliorates DSS-induced colitis To study the roles of ASM in mediating the progression of inflammation in colon, we induced murine DSS colitis by administering 4% DSS in drinking water and treated the mice with desipramine since day 3. DSS-induced colitis was characterized by body weight loss (starting on day 4), bloody diarrhea (on day 6), and the sustained increase of ASM activity in colon tissues (Fig. 2A-C). Conversely, administration of desipramine decreased ASM activity in colon tissues, and ameliorated colitis symptoms, including quick regain of body weight and lower disease activity indexes (Fig. 2A-C). Histological examination showed that DSS colitis was characterized by tissue damages in colon, including loss of crypts, reduction of goblet cells, focal ulcerations, extensive destruction of mucosal layer, and sufficient infiltration of inflammatory cells (Fig. 2D). However, desipramine-treated mice showed minimal loss of crypts, reduction of goblet cell loss, and less infiltration of inflammatory cells in the mucosa, as shown in Fig. 2D. The data indicates that inhibition of ASM attenuates DSS-induced inflammation in colon.
4. Discussion As a plasma membrane, ASM has been noted to mediate cell signaling upon extracellular stimulations, and play the pivotal roles in regulating cell growth, proliferation, apoptosis, and functions [5,6]. For instance, upon stimulations of cellular receptors, ASM activities are initiated, followed by subsequent activation of T cell signals [7,10,11]. Conversely, inhibition of ASM bioactivity dampens T cell signals and pathogenic T cell responses [7,9]. Indeed, ASM exhibits the key roles in maintaining immune homeostasis. Patients with Niemann–Pick disease, who have mutations in the ASM gene, display neurological symptoms and/or visceral organ abnormalities [23]. Meanwhile, the patients with Niemann–Pick disease are susceptible to pathogen infections [10], indicating the association between ASM deficiency and aberrant immune responses. The roles of ASM in regulation of T cell functions are postulated [7,10,11]. However, whether ASM dominates innate immune cell activation inclusive of macrophages remains to be further elucidated. Most of studies have reported that ASM triggers macrophage bioactivities, i.e., undergoing apoptosis by certain stimulations [24]. In this study, we have extended the current findings and noted that ASM bioactivities are association with macrophage activation. Inflammatory stimuli, e.g., LPS, initiated ASM activities in macrophages, resulting in subsequent cell activation and proinflammatory cytokine production. Intriguingly, inhibition of ASM bioactivities diminished LPS-induced cytokine release by macrophages.
3.3. Inhibition of ASM dampens immune responses of DSS colitis in mice TNF and IL-1β are the key cytokines mainly released by the activated macrophages, and mediate immune responses during inflammation [21]. Hence, we determined TNF and IL-1β productions in colon tissues by ELISA. The mice with DSS colitis exhibited high levels of cytokines in colonic homogenates, whereas treatment of desipramine decreased those cytokine productions in colon tissues as shown in Fig. 3A and B. The data imply that inhibition of ASM activity by desipramine significantly alleviates DSS-induced inflammatory responses in colon. 28
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Fig. 3. Inhibition of ASM dampens immune responses of DSS colitis in mice. (A-B) TNF (A) and IL-1β (B) levels in colonic homogenates of each group of mice were determined by ELISA (n = 8 per group). (C) Immunohistochemical staining of phosphor-NF-κB p65 was performed in sections from colonic tissues of the mice. Phosphor-NF-κB p65 positive cell numbers in per 100 cells in lamina propria of the mice were counted and summarized, as shown in the lower panel (n = 6 per group). Scale bars: 50 μm.*, P < 0.05; **, P < 0.01.
multiple immune diseases in mice including sepsis and inflammations in lung, joint, and liver [31–33]. The data suggest ASM activities and signals as the target for the treatment of human inflammatory diseases. Currently, diverse functional inhibitors of acid sphingomyelinase (FIASMA) have been reported [34]. Tricyclic anti depressants and analogs such as desipramine, imipramine, and amitriptyline, have been used as ASM inhibitors [35], to prevent human disease or/and animal models inclusive of cystic fibrosis [32,36]. Meanwhile, we noted the limited efficacy of those ASM inhibitors. For instance, ASM inhibitors including imipramine and desipramine can only partially block ASM bioactivities and signals, as shown in the both in vitro and in vivo systems reported, in comparison with control groups which were set up as the normalization controls in this study. In another word, those ASM inhibitors did not thoroughly inhibit ASM bioactivities to the normal or physical levels. At this end, to better take advantage of ASM inhibition in immune responses and inflammatory diseases, development of more efficient inhibitors of ASM is desired. In summary, inhibition of ASM activities may provide potential alternatives to current therapeutic approaches to control macrophage responses, and help to design new strategies for treatment of human immune diseases.
In the study, ASM bioactivities have been further associated with immune diseases, i.e., DSS colitis, a murine IBD model. IBD is a serious intestinal immune disorder. The etiology of IBD remains unclear so far, but it is proposed that dysregulated immune responses contribute to the progression of IBD [25]. In addition, excessive activation of innate immune cells including macrophages determines disease prognosis [25,26]. During the development of IBD, when exposed to abundant intestinal bacterial antigens, macrophages in lamina propria become activated, and thereby release substantive proinflammatory cytokines such as TNF and IL-1β [26]. Innate immune cells including macrophages, in combination with proinflammatory cytokines, play critical roles in mediating the uncontrolled inflammatory responses, which eventually result in intestinal tissue damage as seen in IBD [27]. Here we induced DSS-driven colitis model in mice, an established model with macrophage and other innate immune cell dominated inflammation [12], to study ASM activities in regulation of immune responses. The mice with DSS colitis displayed severe disease symptoms, exhibited augment of ASM activities and increase of cytokine production in colon tissues. Nevertheless, inhibition of ASM activities reduced immune responses in colon, and attenuated DSS-induced colitis, in consistent with other studies [28], indicating the potential roles of ASM signaling in the progression of immune responses and disease development. Clinical studies have reported that increase of ASM levels is associated with mortality in intensive care unit patients after systemic inflammation [29], suggesting induction of ASM bioactivities, at least partly in immune cells, during the progression of human inflammations. We have previously noted that during T cell activation, increase of ASM activities may contribute to T cell responses as seen in human IBD [9]. The parallel studies further confirmed ASM activation in other immune diseases inclusive of sickle cell disease [30]. In addition, besides our current colitis model, ASM inhibition has been reported to attenuate
Conflict of interest The authors declare no conflict of interest. Author contributions AB designed the study, YX, TY, XZ, PW, YR, CW, RY, YG, and AB conducted the experiments and collected the data, YX, TY, XZ, YG and AB performed data analysis, AB, YX, XZ, and YG wrote and finalized the 29
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manuscript. All authors approved the final version for publication.
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Acknowledgements This work was supported by National Natural Science Foundation of China, No. 81770555, 81470828, 81270472(AB); Principle Investigator Program of Jiangxi Province, No. 20153BCB22010 (AB); and Natural Science Foundation of Jiangxi Province, No. 20142BAB205048 (AB). References [1] S.T. Gren, O. Grip, Role of monocytes and intestinal macrophages in crohn’s disease and ulcerative colitis, Inflamm. Bowel Dis. 22 (8) (2016) 1992–1998. [2] M.F. Neurath, Cytokines in inflammatory bowel disease, nature reviews, Immunology 14 (5) (2014) 329–342. [3] J.M. Davies, M.T. Abreu, The innate immune system and inflammatory bowel disease, Scand. J. Gastroenterol. 50 (1) (2015) 24–33. [4] A. Bai, Z. Peng, Biological therapies of inflammatory bowel disease, Immunotherapy 2 (5) (2010) 727–742. [5] E.H. Schuchman, Acid sphingomyelinase, cell membranes and human disease: lessons from Niemann-Pick disease, FEBS Lett. 584 (9) (2010) 1895–1900. [6] A. Bai, S. Robson, Beyond ecto-nucleotidase: CD39 defines human Th17 cells with CD161, Purinergic Signall. 11 (3) (2015) 317–319. [7] A. Bai, E. Kokkotou, Y. Zheng, S.C. Robson, Role of acid sphingomyelinase bioactivity in human CD4(+) T-cell activation and immune responses, Cell Death Dis. 6 (2015) e1828. [8] C. Hollmann, S. Werner, E. Avota, D. Reuter, L. Japtok, B. Kleuser, E. Gulbins, K.A. Becker, J. Schneider-Schaulies, N. Beyersdorf, Inhibition of acid sphingomyelinase allows for selective targeting of CD4+ conventional versus Foxp3+ regulatory T cells, J. Immunol. 197 (8) (2016) 3130–3141. [9] A. Bai, A. Moss, E. Kokkotou, A. Usheva, X. Sun, A. Cheifetz, Y. Zheng, M.S. Longhi, W. Gao, Y. Wu, S.C. Robson, CD39 and CD161 modulate Th17 responses in crohn’s disease, J. Immunol. 193 (7) (2014) 3366–3377. [10] A. Bai, Y. Guo, Acid sphingomyelinase mediates human CD4(+) T-cell signaling: potential roles in T-cell responses and diseases, Cell Death Dis. 8 (7) (2017) e2963. [11] L.M. Boucher, K. Wiegmann, A. Futterer, K. Pfeffer, T. Machleidt, S. Schutze, T.W. Mak, M. Kronke, CD28 signals through acidic sphingomyelinase, J. Exp. Med. 181 (6) (1995) 2059–2068. [12] H. Tlaskalova-Hogenova, L. Tuckova, R. Stepankova, T. Hudcovic, L. PalovaJelinkova, H. Kozakova, P. Rossmann, D. Sanchez, J. Cinova, T. Hrncir, M. Kverka, L. Frolova, H. Uhlig, F. Powrie, P. Bland, Involvement of innate immunity in the development of inflammatory and autoimmune diseases, Ann. N.Y. Acad. Sci. 1051 (2005) 787–798. [13] Y. Guo, Y. Zhang, K. Hong, F. Luo, Q. Gu, N. Lu, A. Bai, AMPK inhibition blocks ROS-NFkappaB signaling and attenuates endotoxemia-induced liver injury, PloS One 9 (1) (2014) e86881. [14] K. Hong, Y. Zhang, Y. Guo, J. Xie, J. Wang, X. He, N. Lu, A. Bai, All-trans retinoic acid attenuates experimental colitis through inhibition of NF-kappaB signaling, Immunol. Lett. 162 (1 Pt A) (2014) 34–40. [15] P. Wan, X.D. Zhu, X.P. Liu, T. Yu, R.W. Yan, Y. Guo, A.P. Bai, Inhibition of neddylation ameliorates DSS-induced colitis, Cell. Mol. Immunol. 15 (6) (2018) 649–650. [16] A. Bai, N. Lu, Y. Guo, J. Chen, Z. Liu, Modulation of inflammatory response via alpha2-adrenoceptor blockade in acute murine colitis, Clin. Exp. Immunol. 156 (2) (2009) 353–362. [17] P. Wan, X. Liu, Y. Xiong, Y. Ren, J. Chen, N. Lu, Y. Guo, A. Bai, Extracellular ATP mediates inflammatory responses in colitis via P2 x 7 receptor signaling, Sci. Rep. 6 (2016) 19108.
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Contents lists available at ScienceDirect
Prostaglandins and Other Lipid Mediators journal homepage: www.elsevier.com/locate/prostaglandins
Original Research Article
Inhibition of ASM activity ameliorates DSS-induced colitis in mice a
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a
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a
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T d
Yan Xiong , Xiao-Dan Zhu , Ping Wan , Yu-Ping Ren , Chong Wang , Run-Wei Yan , Yuan Guo , ⁎ Ai-Ping Baia, a
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China Transplant Center, Department of Surgery, The Affiliated Hospital of Qingdao University, Qingdao, 266555, China Department of Rheumatology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China d Department of Pharmacy, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Acid sphingomyelinase Macrophage Colitis Cytokine Inhibition
Acid sphingomyelinase (ASM) is a membrane lipid hydrolase, acting to generate ceramide and regulate cell functions and inflammatory responses.The roles of ASM in mediating T cell functions are postulated whereas its function in regulation of macrophages remains uncertain. The study was performed to explore ASM activity in control of macrophage functions. RAW 264.7 cells were pretreated with desipramine, an ASM inhibitor, prior to LPS challenge in vitro. LPS initiated ASM activity in RAW 264.7 cells. Conversely, inhibition of ASM activity by desipramine diminished LPS induced ASM activities and TNF production of RAW 264.7 cells. The DSS colitis in mice was induced, and desipramine was administered to the mice two days post induction of colitis. Murine colitis was characterized by elevation of ASM activities in colon tissues. Desipramine administration overrode ASM activities in colon, and ameliorated DSS-induced colitis evidenced with the reduced disease activities and the decreased cytokine levels. Together, our data show a crucial role of ASM activity in regulation of macrophage functions and responses, and suggest that ASM represents a novel therapeutic approach for the management of immune diseases.
1. Introduction Innate immune cells including macrophages are the main components of human immune system, playing the pivotal roles in maintaining immune homeostasis. In the presence of bacteria and virus infections, macrophages become activated and serve to clear those pathogens. However, when improperly activated, macrophages initiate aberrant immune responses and contribute to autoimmune diseases including inflammatory bowel disease (IBD) [1]. IBD is a serious intestinal immune disorder with poor prognosis. During the progression of IBD, macrophages become excessively activated, and release substantive proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1β, resulting in the sustained inflammation and tissue damage in the colon [1,2]. Inhibition of macrophage functions has been proposed as one of the targets for the treatment of human immune diseases including IBD [3,4]. Acid sphingomyelinase (ASM) is a membrane lipid hydrolase. Through hydrolysis of sphingomyelin to generate signal messenger, i.e., ceramide, ASM exhibits the pivotal roles in mediating cell signals [5,6]. Others and our group have noted that ASM determines immune cell functions inclusive of natural killer (NK) cells and T helper (Th) cells ⁎
[7–9]. Upon stimulation of cellular receptors by their ligands, ASM bioactivity is initiated, and subsequently mediates T cell activation [10,11]. Conversely, inhibition of ASM bioactivity dampens pathogenic Th responses [7]. ASM activity is postulated to mediate T cell activation and functions, whereas its roles in regulation of macrophage functions remain uncertain. Here we reported that upon inflammatory stimuli, ASM regulated macrophage activation and mediated the progression of dextran sulfate sodium (DSS)-induced colitis in mice, an innate immune cells-driven inflammation in colon [12]. 2. Methods 2.1. Cell culture Murine macrophage cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin G (100U/mL), and streptomycin (100 μg/mL) at 37 °C. For in vitro study, about 1 × 106/mL RAW264.7 cells were seeded in 12 or 96-well plates. The cells were treated with vehicle, or titrated desipramine (Sigma-Aldrich) for 30 min, prior to lipopolysaccharide
Corresponding author. E-mail address: [email protected] (A.-P. Bai).
https://doi.org/10.1016/j.prostaglandins.2018.12.002 Received 20 July 2018; Received in revised form 6 December 2018; Accepted 10 December 2018 Available online 11 December 2018 1098-8823/ © 2018 Elsevier Inc. All rights reserved.
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on a scoring system modified from a previous study [18], and a combined score of inflammatory cell infiltration and tissue damage was determined as follows: score 0, normal colonic mucosa and occasional inflammatory cells in the lamina propria; 1, loss of one-third of the crypts; 2, loss of two-thirds of the crypts; 3, the lamina propria is covered with a single layer of epithelium and mild inflammatory cell infiltration is present; and 4, erosions and transmural extension of infiltrate.
(LPS, from Escherichia coli 0111:B4; Sigma-Aldrich) challenge. 2.2. Enzyme-linked immunosorbent assays (ELISA) Cytokine concentrations in the supernatants or colon tissue homogenates were determined by ELISA, regarding to the manufacturer’s instructions (R&D Systems, Inc) [13–15]. Briefly, polyclonal rat antimouse cytokine antibodies were used as capturing antibodies and biotinylated polyclonal rat anti-mouse cytokine antibodies for detection, and the standard curve of cytokines was set up meanwhile. Color changes were determined at 450 nm.
2.6. Immunohistochemistry Colon tissues of mice were taken and fixed immediately in 10% buffered formalin, embedded in paraffin, and cut into 4 μm sections. After blockade of inner peroxidase, sections were incubated sequentially with the first antibody solution including rabbit anti-NF-κB p65 (phospho S536, from Abcam) antibody [19]. After three washes in PBS (pH 7.4), the sections were then incubated in secondary goat anti-rabbit immunoglobulin (Ig)G conjugated with peroxidase labeled polymer, prior to colorization using diaminobenzidine reaction and counterstained with haematoxylin. Negative controls were established using rabbit IgG instead of the first antibodies. The cells stained with anti-NFκB P65 antibody in both cytoplasm andthe nucleus were counted as NFκB positive cells, whereas those without nucleus stain were regarded as NF-κB negative cells [14,18]. The sections were evaluated using light microscopy, and 100 cells in lamina propria per high power field were calculated for statistical analysis.
2.3. ASM activity measurement ASM activity in cellular extracts or colon tissue extracts was determined using Amplex Red sphingomyelinase assay kit (Invitrogen) according to the manufacturer’s protocol. Briefly, cellular extracts or colon tissue extracts were diluted in 50 mM sodium acetate (pH 5.0), and mixed with 5 mM sphingomyelin solution. After adequate incubation, the samples above were mixed sequentially with the Amplex Red reagent/HRP/cholineoxidase/alkaline phosphatase working solution. The fluorescence intensity in each well was determined in a fluorescence reader with the excitation at 530 nm and the emission at 590 nm respectively. 2.4. Animals
2.7. Statistical analysis
Female C57/BL6 mice were provided by the Experimental Animal Center of Nanchang University, and fed underspecific pathogen-free conditions. 7–8 week-old mice were randomly divided into three groups (10 mice per group) in the study, weighing approximately 22 g. All protocols for the projects using the mice were reviewed and approved by the Institutional Animal Care Committee of Nanchang University.
All data in the text and figures are expressed as mean ± the standard error of the mean. Comparisons of more than two groups were made with a one-way analysis of variance using Tukey’s post hoc test. When appropriate, comparison with two groups was made using Student’s t-test for unpaired data. Differences were considered statistically significant if P < 0.05.
2.5. Induction of dextran sulphate sodium (DSS) colitis
3. Results
Acute DSS colitis was induced in C57/BL6 mice according to the previously published method with minor modification [16,17]. The mice were fed 4% (w/v) DSS (molecular mass, 36–50 kDa; MP Biomedicals) dissolved in the drinking water on day one. Fresh DSS solution was provided every other day. Control mice drank only distilled water. Desipramine (20 mg/kg body weight) was administered intraperitoneally (i.p.) to the mice drinking DSS (DSS + Des group) since day three and repeated daily until the mice were killed on day eight. Disease symptoms of colitis were assessed daily by measurement of body weight, evaluation of stool consistency and detection of bloody stools. Disease severity was scored using a clinical disease activity index (DAI) ranging from 0 to 4, calculated as described previously [14,18] using the following parameters: stool consistency, presence or absence of fecal blood and weight loss. The mice were sacrificed on day eight, and the middle section of colon was fixed in 10% formaldehyde-saline. Hematoxylin and eosin stain(HE)-stained sections were graded based
3.1. Inhibition of ASM activity dampens cytokine release by LPS-induced macrophages To induce ASM bioactivities by inflammatory stimulations, we exposed RAW264.7, a murine macrophage cell line, to LPS, and noted that LPS immediately initiated ASM activities in a dose dependent manner (Fig. 1A). Meanwhile, minimal changes of ASM expression in mRNA levels in murine macrophages post LPS challenge were noticed (data not shown). To inhibit ASM activity, we pretreated the cells with desipramine 30 min prior to LPS challenge. As shown in Fig. 1B, administration of desipramine dampened LPS induced ASM activities in the cells in a dose dependent manner. As TNF is one of the key cytokines mediating inflammatory responses [20], we next determined TNF production by macrophage post
Fig. 1. Inhibition of ASM activity dampens LPS induced macrophage activation. (A) RAW 264.7 cells were treated with LPS for 60 min, and ASM activity was determined thereby. (B-C) RAW 264.7 cells were pretreated with titrated desipramine (Des) for 30 min prior to LPS challenge. ASM activity (B) and TNF levels (C) were determined 60 min and 24 h post LPS stimulation, respectively. N = 4–5 per group, *, P < 0.05; **, P < 0.01. 27
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Fig. 2. Blockade of ASM activity ameliorates murine DSS colitis. Colitis was induced in mice by administration of 4% DSS dissolved in drinking water. Desipramine (DSS + Des group) or vehicle (DSS group) were administered intraperitoneally 2 days after induction of colitis, and repeated daily until the mice were sacrificed onday 8. Control mice drank only distilled water. (A) Body weight changes of the mice in each group (n = 10 per group). (B) DAI was scored on day 7 using the following parameters: stool consistency, presence or absence of fecal blood and weight loss (n = 10 per group). (C) ASM activities in colon tissue extracts were determined respectively after euthanasia of the mice at day 8 (n = 6 per group). (D) HE staining was performed and histological scores were graded (n = 10 per group). Scale bars: 250 μm. *, P < 0.05; **, P < 0.01.
NF-κB is one of the pivotal transcriptional factors regulating immune responses [22]. Once activated by stimulations, NF-κB signals regulate inflammatory molecules expression and dominate inflammation progression including colitis [14]. Next, we studied whether ASM inhibition treatment could impact on NF-κB activation. As shown in Fig. 2C, DSS colitis was characterized by drastic increase of phosphorNF-κB p65 expression in lamina propria immune cells. In contrast, administration of desipramine decreased phosphor-NFκBp65 positive cell numbers in lamina propria, indicating significant blockade of NFκB activation in immune cells by inhibitionof ASM activity and signaling.
LPS stimulation. LPS dramatically induced TNF release by RAW264.7 cells, whereas desipramine inhibited LPS-induced TNF production in a dose dependent manner (Fig. 1C). The data indicate that blockade of ASM activity inhibits inflammatory stimulation-induced macrophage activation. 3.2. Inhibition of ASM activity ameliorates DSS-induced colitis To study the roles of ASM in mediating the progression of inflammation in colon, we induced murine DSS colitis by administering 4% DSS in drinking water and treated the mice with desipramine since day 3. DSS-induced colitis was characterized by body weight loss (starting on day 4), bloody diarrhea (on day 6), and the sustained increase of ASM activity in colon tissues (Fig. 2A-C). Conversely, administration of desipramine decreased ASM activity in colon tissues, and ameliorated colitis symptoms, including quick regain of body weight and lower disease activity indexes (Fig. 2A-C). Histological examination showed that DSS colitis was characterized by tissue damages in colon, including loss of crypts, reduction of goblet cells, focal ulcerations, extensive destruction of mucosal layer, and sufficient infiltration of inflammatory cells (Fig. 2D). However, desipramine-treated mice showed minimal loss of crypts, reduction of goblet cell loss, and less infiltration of inflammatory cells in the mucosa, as shown in Fig. 2D. The data indicates that inhibition of ASM attenuates DSS-induced inflammation in colon.
4. Discussion As a plasma membrane, ASM has been noted to mediate cell signaling upon extracellular stimulations, and play the pivotal roles in regulating cell growth, proliferation, apoptosis, and functions [5,6]. For instance, upon stimulations of cellular receptors, ASM activities are initiated, followed by subsequent activation of T cell signals [7,10,11]. Conversely, inhibition of ASM bioactivity dampens T cell signals and pathogenic T cell responses [7,9]. Indeed, ASM exhibits the key roles in maintaining immune homeostasis. Patients with Niemann–Pick disease, who have mutations in the ASM gene, display neurological symptoms and/or visceral organ abnormalities [23]. Meanwhile, the patients with Niemann–Pick disease are susceptible to pathogen infections [10], indicating the association between ASM deficiency and aberrant immune responses. The roles of ASM in regulation of T cell functions are postulated [7,10,11]. However, whether ASM dominates innate immune cell activation inclusive of macrophages remains to be further elucidated. Most of studies have reported that ASM triggers macrophage bioactivities, i.e., undergoing apoptosis by certain stimulations [24]. In this study, we have extended the current findings and noted that ASM bioactivities are association with macrophage activation. Inflammatory stimuli, e.g., LPS, initiated ASM activities in macrophages, resulting in subsequent cell activation and proinflammatory cytokine production. Intriguingly, inhibition of ASM bioactivities diminished LPS-induced cytokine release by macrophages.
3.3. Inhibition of ASM dampens immune responses of DSS colitis in mice TNF and IL-1β are the key cytokines mainly released by the activated macrophages, and mediate immune responses during inflammation [21]. Hence, we determined TNF and IL-1β productions in colon tissues by ELISA. The mice with DSS colitis exhibited high levels of cytokines in colonic homogenates, whereas treatment of desipramine decreased those cytokine productions in colon tissues as shown in Fig. 3A and B. The data imply that inhibition of ASM activity by desipramine significantly alleviates DSS-induced inflammatory responses in colon. 28
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Fig. 3. Inhibition of ASM dampens immune responses of DSS colitis in mice. (A-B) TNF (A) and IL-1β (B) levels in colonic homogenates of each group of mice were determined by ELISA (n = 8 per group). (C) Immunohistochemical staining of phosphor-NF-κB p65 was performed in sections from colonic tissues of the mice. Phosphor-NF-κB p65 positive cell numbers in per 100 cells in lamina propria of the mice were counted and summarized, as shown in the lower panel (n = 6 per group). Scale bars: 50 μm.*, P < 0.05; **, P < 0.01.
multiple immune diseases in mice including sepsis and inflammations in lung, joint, and liver [31–33]. The data suggest ASM activities and signals as the target for the treatment of human inflammatory diseases. Currently, diverse functional inhibitors of acid sphingomyelinase (FIASMA) have been reported [34]. Tricyclic anti depressants and analogs such as desipramine, imipramine, and amitriptyline, have been used as ASM inhibitors [35], to prevent human disease or/and animal models inclusive of cystic fibrosis [32,36]. Meanwhile, we noted the limited efficacy of those ASM inhibitors. For instance, ASM inhibitors including imipramine and desipramine can only partially block ASM bioactivities and signals, as shown in the both in vitro and in vivo systems reported, in comparison with control groups which were set up as the normalization controls in this study. In another word, those ASM inhibitors did not thoroughly inhibit ASM bioactivities to the normal or physical levels. At this end, to better take advantage of ASM inhibition in immune responses and inflammatory diseases, development of more efficient inhibitors of ASM is desired. In summary, inhibition of ASM activities may provide potential alternatives to current therapeutic approaches to control macrophage responses, and help to design new strategies for treatment of human immune diseases.
In the study, ASM bioactivities have been further associated with immune diseases, i.e., DSS colitis, a murine IBD model. IBD is a serious intestinal immune disorder. The etiology of IBD remains unclear so far, but it is proposed that dysregulated immune responses contribute to the progression of IBD [25]. In addition, excessive activation of innate immune cells including macrophages determines disease prognosis [25,26]. During the development of IBD, when exposed to abundant intestinal bacterial antigens, macrophages in lamina propria become activated, and thereby release substantive proinflammatory cytokines such as TNF and IL-1β [26]. Innate immune cells including macrophages, in combination with proinflammatory cytokines, play critical roles in mediating the uncontrolled inflammatory responses, which eventually result in intestinal tissue damage as seen in IBD [27]. Here we induced DSS-driven colitis model in mice, an established model with macrophage and other innate immune cell dominated inflammation [12], to study ASM activities in regulation of immune responses. The mice with DSS colitis displayed severe disease symptoms, exhibited augment of ASM activities and increase of cytokine production in colon tissues. Nevertheless, inhibition of ASM activities reduced immune responses in colon, and attenuated DSS-induced colitis, in consistent with other studies [28], indicating the potential roles of ASM signaling in the progression of immune responses and disease development. Clinical studies have reported that increase of ASM levels is associated with mortality in intensive care unit patients after systemic inflammation [29], suggesting induction of ASM bioactivities, at least partly in immune cells, during the progression of human inflammations. We have previously noted that during T cell activation, increase of ASM activities may contribute to T cell responses as seen in human IBD [9]. The parallel studies further confirmed ASM activation in other immune diseases inclusive of sickle cell disease [30]. In addition, besides our current colitis model, ASM inhibition has been reported to attenuate
Conflict of interest The authors declare no conflict of interest. Author contributions AB designed the study, YX, TY, XZ, PW, YR, CW, RY, YG, and AB conducted the experiments and collected the data, YX, TY, XZ, YG and AB performed data analysis, AB, YX, XZ, and YG wrote and finalized the 29
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manuscript. All authors approved the final version for publication.
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