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The ANXA1 released from intestinal epithelial cells alleviate DSS-induced colitis by improving NKG2A expression of Natural Killer cells
Biochemical and Biophysical Research Communications 478 (2016) 213e220
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The ANXA1 released from intestinal epithelial cells alleviate DSS-induced colitis by improving NKG2A expression of Natural Killer cells Z. Zou, D. Zuo, J. Yang, H. Fan* Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, 430022, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2016 Accepted 16 July 2016 Available online 18 July 2016
Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintenance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent antiinflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis. © 2016 Elsevier Inc. All rights reserved.
Keywords: ANXA1 Intestinal epithelial cells Colitis Natural killer cells NKG2A
1. Introduction In inflammatory conditions of the gastrointestinal tract, IBD is still a leading cause of morbidity and mortality all over the world [1,2]. It has been widely recognized that the development of IBD is followed by imbalance of intestinal immune homeostasis, which depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and IECs [2e4]. The gut epithelium composed by IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier
Abbreviations: IBD, inflammatory bowel disease; IECs, intestinal epithelial cells; NK, natural killer; UC, ulcerative colitis; CD, Crohn's disease; Treg, T regulatory; TGF-b, transforming growth factor-b; TSLP, thymic stromal lymphopoietin; TH1, T helper 1; FPRs, peptide receptors; EVs, extracellular vesicles; IFN-g, interferon g; TNF-a, tumor necrosis factor a; FITC, isothiocyanate; LPMCs, lamina propria mononuclear cells; MLN, mesenteric lymph nodes; HE, haematoxylin and eosin. * Corresponding author. E-mail address: [email protected] (H. Fan). http://dx.doi.org/10.1016/j.bbrc.2016.07.066 0006-291X/© 2016 Elsevier Inc. All rights reserved.
and the expression of antimicrobial peptides to maintain intestinal immune homeostasis [2,5]. Recently, more and more evidences have shown that some molecular events happened to IECs directly or indirectly affect the progression of IBD. For example, mice with IEC-specific knockout of FADD, an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis [3]. Obviously, IECs not only limit bacterial translocation across the mucosal barrier, but also maintain intestinal homeostasis by regulating innate and adaptive immune responses. Illustrating this point, intestinal alkaline phosphatase produced by IECs mediate lipopolysaccharide detoxification [6,7]. In addition, epithelial regeneration following intestinal injury is critical for barrier maintenance and organ function [8], such process relies on the immune microenvironment induced by pathogenic infection or other damage model. IECs may also influence intestinal homeostasis through the secretion of conditioning cytokines (IL-8, IL-12, IL-25 and IL-23) that affect adaptive responses primed by IECs [9e11].
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Fig. 1. DSS treatment increases the expression of ANXA1 and NK cells recruitment in colonic mucosal tissues. (A) Real-time PCR analysis of ANXA1 gene of mucosal tissues. (B C) Western blotting analysis of ANXA1 protein of mucosal tissues. (D) Real-time PCR analysis of ANXA1 gene of IECs. (E F) Western blotting analysis of ANXA1 protein of IECs. (G) Western blotting analysis of ANXA1 and CD63 protein of EVs from IECs. (H) Western blotting analysis of FPRL-1 of NK cells from spleen and mesenteric lymph nodes. (I K) LPMCs
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ANXA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, owning ability of regulating diverse cellular functions and exhibiting profound inhibitory actions on leukocyte transmigration and activation [12e14]. By binding to formyl peptide receptors (FPRs) expressed on responsive cells, such as phagocytes and epithelial cells [15,16], ANXA1 facilitates resolution of inflammation. ANXA1 can also promotes intestinal epithelial migration through activation of FPR1-, Rac1-, and NOX1dependent redox signaling, triggering the oxidative inactivation of regulatory phosphatases and subsequent modification of focal adhesion proteins conducive to regulating cell migration [17]. Another study demonstrated that epithelial cells released ANXA1 as a component of extracellular vesicles (EVs) to promote intestinal mucosal wound repair [15]. NK cells exert their diverse functions through direct cell-cell contact and secretion of cytokines such as interferon g (IFN-g) and tumor necrosis factor a (TNF-a) [18,19], which can induce the injury of IECs and the infiltration of inflammatory cells, accelerating the development of IBD [20]. Additionally, NK cells can suppress Th1-mediated colitis by controlling the responses of effector T cells to gut bacteria [21]. Moreover, some study have proved that NK cells are rapidly recruited to the inflamed colon and have a protective effect on DSS-induced colitis by regulating neutrophil function via the NKG2A receptor [19]. It remains obscure about the interaction between IECs and NK cells during progression of IBD, and whether ANXA1 released from IECs affect the function of NK cells and the ripple effect on colitis. Our results showed that IECs secreted excessive ANXA1 during DSS-induced colitis, and the ANXA1 was wrapped in EVs to influence the expression of NKG2A receptor in NK cells, alleviating the pathology of colitis. 2. Materials and methods Animals and in vivo studies. 8-weeks C57BL/6 mice were kept in a pathogen free conditions. Animal experiments were approved by the guidelines of the Chinese Council on Animal Care, and the procedures were performed according to the Tongji Medical college Committee (Wuhan, China). 3% DSS (36e50 kDa; MP Biomedicals) was dissolved in water and administered to mice for 7 days as described [22]. Mice received 50 mg anti-asialo GM1 (anti-AGM1) Ab (Wako Chemicals, Neuss, Germany) or anti-NKG2A Ab (Bioss Inc, Woburn, MA, USA) by intraperitoneal injections 3 days before DSS administration and repeated every 2 days to maintain depletion/ receptor blocking [19]. The isolation of IECs and LPMCs. IECs and lamina propria mononuclear cells (LPMCs) were isolated essentially as described previously [23]. The isolation NK cells. Spleens and mesenteric lymph nodes from mouse treated with DSS for 7 days were harvested for preparing cell suspension, from which the collected cells were used to isolate NK cells by magnetic cell sorting (MACS) with mouse NK cell isolation kit (Miltenyi Biotec, Bisley, UK). Isolation of neutrophils. Single-cell suspensions were prepared as described above. Neutrophils were isolated using PEeantiLy6G Ab (eBioscience), magnetic microbeads, and MiniMACS columns (Miltenyi Biotec) according to the manufacturers' protocol. EVs purification. EVs were isolated and purified according to a previously reported protocol [24]. The isolated IECs from normal and DSS treated mouse were suspended with DMED medium and
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maintained for 6 h at 37 C, 5% CO2. Afterwards, culture medium was subjected to differential centrifugation to remove cells, dead cells, and cell debris at a rate of 20,000 g for 20 min (microparticles isolation) to 100,000 g (for exosome isolation) for 60 min. Western blotting. Western blot analyses were performed as described previously [12]. Anti-annexin 1 (1:500, Santa Cruz, USA), b-actin (1:1000, CST, USA), Anti-CD63 (1:1000, Abcam, USA) were used as primary antibodies. Real-Time PCR. Total RNA was extracted using RNAprep pure Micro Kit (Tiangen Biotech CO., LTD.). The cDNAs were synthesized using a reverse transcription kit (Quant One Step RT-PCR kit, Tiangen, China). Real-time PCR was performed using SYBR green master mix (FastFire qPCR PreMix, Tiangen, China) on an AB iCycler system (AB, United States) according to the manufacturer's instructions. Primer sequences used are as follows: Ifn-g, 50 TGAGCTCATTGAATGCTTGG-30 and 50 - ACAGCAAGGCGAAAAAGGAT-3’; Tnf-a, 50 -AGGGTCTGGGCCATAGAACT-30 and 50 - CCACCACGCTCTTCTGTCTAC-3’; NKp46, 50 -GGCTGCTGTTCTCAACACCT-30 and 50 -GGCTCACAGAGGGACATAC-3’; Klrg1, 50 -CCTCAAGCCGATCCAGTAAA-30 and 50 -CTGTGCAGACAAAGGCTCAC-3’; NKG2D, 50 GTGGTTGCTGGGATTTGAAC-30 and 50 -TGCCTCTTAAGAATGCACCC3’; NKG2A, 50 -CGAAGGATTCCAGTCCATGA-30 and 50 -GGTGTC CTGCATTTCCAAAA-3’; Anxa1 50 -ATTTTCAAGAAAACGGGCCT-30 and 50 -AGAGTCTCTCTTCAGTCCCCG-3’. Flow Cytometry. Single cells suspensions were prepared and stained with fluorescein isothiocyanate (FITC)-anti-NK1.1 (1 mg/ 106 cells) or PE/Cy5®-Anti-Ly6g antibody (0.2 mg/106 cells). The stained cells were analyzed using a flow cytometer (FACS calibur; Becton Dickinson, Franklin Lakes, NJ), and data were analyzed by WinMDI2.9 software. Tissue Staining and Histological Analysis. Colon tissues were fixed in 4% paraformaldehyde, and they were embedded in paraffin and sliced into 6 mm sections and followed by dewaxing and rehydration. These sections were stained with haematoxylin and eosin (HE) according to standard histological procedures and histological scoring was performed as described previously [25]. Statistics. Data are presented as means ± SEM of several experiments. Statistical comparisons were performed by either Student's 2-tailed t-test or ANOVA with Tukey multiple comparison post-test, as appropriate. A P value of less than 0.05 was considered significant. 3. Results 3.1. The expression of ANXA1 increases in colonic mucosal tissues and IECs due to DSS treatment To investigate the change of ANXA1 expression in colonic mucosal tissues and IECs, mRNA and protein were extracted from colonic mucosal tissues and IECs of DSS treated mice and control mice for Quantitative real-time PCR and Western blotting analysis. As shown in Fig. 1A and C, the mRNA level of ANXA1 were increased in colonic mucosal tissues and IECs of DSS treated mice. Similarly, as shown in Fig. 1B and D the protein of ANXA1 were increased in colonic mucosal tissues and IECs following DSS treatment. In addition, we also collected the EVs secreted from IECs and detected the CD63 (a EVs marker of IECS) and ANXA1 protein of EVs using Western blotting method, the results showed that more ANXA1 protein were found in EVs from IECs of DSS treated mice (Fig. 1G). All of these results demonstrate that ANXA1 expression increases in
were prepared for flow cytomery analysis, the percentage of NK1.1 þ cells were shown. (J L) MLN cells were prepared for flow cytomery analysis, the percentage of NK1.1 þ cells were shown. (M) NK cell isolated from MLN of DSS treated mice were stained with CFSE and injected into the tail vein of mice treated with DSS for 5 days, two days later the colon tissues were harvested to produce frozen section, which were observed under fluorescence microscope to access the CFSE þ NK cells recruitment in colonic mucosal tissues. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
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Fig. 2. Depletion of NK cells or blocking of ANXA1 exacerbate DSS-induced colitis. Mice received 50 mg anti-AGM1 Ab with or without anti-ANXA1 Ab 3 days before DSS administration, and repeated every 2 days to maintain NK cells depletion or ANXA1 blocking. (A) After mice were treated DSS for 7 days, colon tissues were prepared for HE staining. (B) Overall histology score. (C) The percentage of weight change. (D) Colon length change. (E) LPMCs were isolated to detect the percentage of Ly6G þ cells (neutrophils) by flow cytomery analysis. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
colonic mucosal tissues, especially in IECs during colitis induced by DSS. 3.2. DSS treatment increases NK cells recruitment in colonic mucosal tissues To investigate whether NK cells are recruited to colonic mucosal tissues, LPMCs were isolated from normal and DSS treated mice prepared for flow cytomery analysis. As shown in Fig. 1 I and K, the percentage of NK1.1 þ cells increased in DSS treated mice compared with normal mice. Equally, mesenteric lymph nodes (MLN) cells from normal and DSS treated mouse were prepared for flow cytomery analysis, results showed that the percentage of NK1.1 þ cells were also increased in DSS treated mice (Fig. 1 J and L). Additionally, we isolated NK cells from spleen and MLN of DSS
treated mice, and isolated NK cells were stained with CFSE and injected into the tail vein of mice treated with DSS for 5 days, two days later colon tissues were harvested and prepared for frozen section, which were observed under fluorescence microscope to access the CFSE þ NK cells recruitment in colonic mucosal tissues. These results showed CFSE þ NK cells were recruited to colonic mucosal tissues during colitis (Fig. 1M). ANXA1 have been shown to function by signaling through formyl peptide receptors (FPR) [12], we also found NK cells can express FPR-1(Fig. 1H). 3.3. Depletion of NK cells or blocking of ANXA1 exacerbates DSSinduced colitis To investigate the function of recruited NK cells and the role of increased ANXA1 secreted from IECs in the progression of colitis
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Fig. 3. Transferation of NK cells stimulated by ANXA1 alleviates DSS-induced colitis, and ANXA1 accelerates the NKG2A expression of NK cells. A part of isolated NK cells were treated with ANXA1 or EVs, another part of isolated NK cells were used for co-culture with IECs present or absent anti-ANXA1 antibody in transwell. All of pretreated and untreated NK cells were stained with CFSE and transferred into mice respectively. (A) Experimental design for transferation of NK cells. (B) Colon tissues were prepared for HE staining. (C) Overall histology score. (D) The percentage of weight change. (E) Colon length change. (F) Real-time PCR analysis of IFN-g, TNF-a, NKp46, KLRG1, NKG2D and NKG2A genes of NK cells. (G) Western blotting analysis of NKG2A protein of NK cells. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
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Fig. 4. Blocking of NKG2A exacerbates DSS-induced colitis by affecting recruitment and activity of neutrophils. Mice received 50 mg anti- NKG2A Ab or IgG Ab after DSS administration for 3 days, and repeated every 2 days to maintain NKG2A blocking. (A) Colon tissues were prepared for HE staining. (B) The graph shows overall histology score. (C) The percentage of weight change. (D) Colon length change. (E) LPMCs were prepared for flow cytomery analysis, the percentage of Ly6G þ cells were shown. (F) Real-time PCR analysis of CD69, ROS, IL-6, IL-17A genes of isolated neutrophils. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
induced by DSS. As reported previously, anti-AGM1 Ab and antiANXA1 Ab could deplete NK cells and block ANXA1 activity. Mice received 50 mg anti-AGM1 Ab alone or in the present of anti-ANXA1 Ab 3 days before DSS administration by intraperitoneal injections,
and such procedures were repeated every 2 days to maintain NK cells depletion or ANXA1 blocking until the mice were sacrificed. After mice were treated DSS for 7 days, colon tissues were harvested and prepared for HE staining. As shown in Fig. 2A,B,C and D,
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DSS-induced colitis were exacerbated when NK cells were depleted or/and ANXA1 were blocked. Additionally, depletion of NK cells or/ and blockade of ANXA1 accelerate the recruitment of neutrophil in colonic mucosa during the development of colitis induced by DSS (Fig. 2E). These results demonstrated the protective role of NK cells and increased ANXA1 in colitis induced by DSS. 3.4. Transferation of NK cells stimulated by ANXA1 alleviates DSSinduced colitis To investigate the role of interaction between NK cells and ANXA1, a part of isolated NK cells were treated with ANXA1 or EVs, another part of isolated NK cells were used to co-culture with IECs present or absent anti-ANXA1 ab in transwell. All of pretreated and untreated NK cells were stained with CFSE and transferred into mice by tail vein injection respectively. Experimental design for transferation of NK cells was shown in Fig. 3A. Colon tissues were harvested and prepared for HE staining to access histology score. Results demonstrated that transferation of NK cells stimulated by ANXA1 or EVS alleviates DSS-induced colitis (Fig. 3B C D and E), moreover, transferation of NK cells co-cultured with IECs also play a similar role, which be reversed by anti-ANXA1 ab (Fig. 3B and C). All of these results suggest that the interaction between NK cells and IECs play a negative regulative role on DSS-induced colitis, such cross-talk might relay on ANXA1 released from IECs. 3.5. ANXA1 accelerates the NKG2A expression of NK cells To investigate the effect of ANXA1 on NK cells function, isolated NK cells were treated as methods described. Quantitative real-time PCR analysis was performed to detect the mRNA level of IFN-g, TNFa, NKp46, KLRG1, NKG2D and NKG2A genes of NK cells from different groups. As shown in Fig. 3D, the mRNA level of IFN-g, TNFa, NKp46, KLRG1, NKG2D remained unchanged, NKG2A level was increased due to ANXA1 or EVS stimulation. Meanwhile, NK cells co-cultured with IECs also expressed more NKG2A, which could be reversed by anti- ANXA1 ab. The protein level of NKG2A was consistent with the mRNA level (Fig. 3E). 3.6. Blocking of NKG2A exacerbates DSS-induced colitis by affecting recruitment and activity of neutrophils To investigate the effect of increased NKG2A on progression of colitis induced by DSS. Mice received 50 mg anti-NKG2A Ab or IgG Ab after DSS administration for 3 days by peritoneal injection, and such procedures were repeated every 2 days to maintain NKG2A blocking until the mouse were sacrificed (On the seventh day of DSS treatment). Colon tissues were harvested and prepared for HE staining. As shown in Fig. 4 A B C and D, treatment with anti-NKG2A Ab exacerbated DSS-induced colitis. Besides, we isolated LPMCs from two groups of mice and prepared for flow cytomery analysis, the percentage of Ly6G þ cells was increased (Fig. 4 E). Finally, we isolated neutrophils from LPMCs of the two groups mice to detect the mRNA level change of CD69, ROS, IL-6, IL-17A using Quantitative real-time PCR, results showed that mRNA level of CD69, ROS, IL-6, IL-17A (genes of neutrophils activation) were increased due to blocking of NKG2A (Fig. 4.F). All of these indicate that blocking of NKG2A exacerbates DSS-induced colitis by affecting recruitment and activity of neutrophils.
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ultimately fatal acute colitis in DSS-induced colitis model [19]. Our data reveals a new protective mechanism of IBD, in which ANXA1 released from IECs suppress the expression of NKG2A receptor in NK cells that is important for alleviating DSS-induced colitis. The anti-inflammatory effect of ANXA1 has been reported in models of endotoxemia, peritonitis, arthritis, as well as cerebral and myocardial ischemia [14,26]. Recent studies also demonstrated that ANXA1 has an important role in inhabiting IBD through the inhibition of leukocyte function and promotion of gastrointestinal mucosa wound healing ascribed to binding to ALX/FPRL-1 [27,28]. Interestingly, secretion of ANXA1 has been identified in inflamed intestinal mucosal tissues [29,30]. As expected, increased ANXA1 in IECs also have been found after induction of DSS in our study. In addition, we proved that more NK cells were recruited into intestinal mucosa after DSS treatment and the expression of FPR-1 was also detected in isolated NK cells, which give us a hint that the increased ANXA1 released from IECs may interact with FPR-1 to affect the function of NK cells. To test this hypothesis, the isolated NK Cells were stimulated with ANXA1, and the inflammation related genes (IFN-g, TNF-a, NKp46, KLRG1, NKG2D and NKG2A) were detected. As shown in Fig. 3, the NKG2A expression was increased after treatment with ANXA1. The isolated IECs from normal mice and DSS induced colitis mice were co-cultured with isolated NK cells, then the same genes in NK cells were detected and similar results are obtained. Similar to previous reports, we demonstrate that epithelial cells release ANXA1 as a component of EVs. To investigate if the EVs carrying ANXA1 protein affect the function of NK cells, we collected the EVs of IECs from normal mice and DSS induced colitis mice to stimulate NK cells respectively. Similarly, the NKG2A expression was increased after treatment with EVs from DSS induced colitis mice, and the effect of EVs was reversed by anti-ANXA1. All of these results demonstrate that ANXA1 released from IECs play an improving effect in regulating the NKG2A expression of NK cells. To explore the effect of the interaction between IECs and NK cells through EVs containing ANXA1 protein on the IBD, some isolated NK cells were stimulated with ANXA1 or EVs gathered from DSS induced colitis mice. Other NK cells were co-cultured with IECs from DSS induced colitis mice, then these NK cells were transferred to mice treated with DSS for five days respectively, three days later colons were harvested to assess the pathologic degree, as well as detecting the number and function of neutrophils. As shown in Figs. 3 and 4, the pathology of colonitis was alleviated owning to the transfer of these NK cells, and blocking of NKG2A exacerbates DSS-induced colitis by increasing recruitment and activity of neutrophils. These results indicate that the inhibiting effect of NK cells to colitis may be related to migration and activity of neutrophil. In conclusion, our findings identify a previously unknown immunoregulatory mechanism for NK cells during DSS-induced colitis, in which ANXA1 released from IECs affect the expression of NKG2A to mediate the recruitment and function of neutrophil. It cannot be ignored that the interaction among various kinds of cells in colon is the very essence of colitis, so more experiments need to be designed to investigate the mechanism. Anyway, our study may provide a new promising therapeutic strategy for intestinal diseases. Acknowledgments We thank the Natural Science Foundation of China for providing support (no: 81273906).
4. Discussion Appendix A. Supplementary data The gut epithelium made up of IECs has an important role on the maintenance of intestinal immune homeostasis [1]. NK cells can downregulate neutrophil function, preventing severe and
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.07.066.
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The ANXA1 released from intestinal epithelial cells alleviate DSS-induced colitis by improving NKG2A expression of Natural Killer cells Z. Zou, D. Zuo, J. Yang, H. Fan* Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, 430022, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2016 Accepted 16 July 2016 Available online 18 July 2016
Inflammatory bowel disease (IBD) arises when intestinal immune homeostasis is broken, the maintenance of such homeostasis is principally controlled by cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier and the expression of antimicrobial peptides to maintain intestinal immune homeostasis. During Colitis the IECs can express increased ANXA1, which is important for regeneration of intestinal mucosa and function as a potent antiinflammatory protein. Natural Killer (NK) cells can also suppress the progression of colitis. It is uncertain about the effect of the cross-talk between injured IECs and recruited NK cells during colitis. In this study, the expression of ANXA1 in IECS from DSS treated mice was increased, and more NK cells were recruited to intestinal mucosa. In addition, the expression of NKG2A was upregulated when co-cultured with NK cells. The results further proved that overexpression of NKG2A in NK cells was important for inhibiting the recruitment and activity of neutrophils to alleviate DSS-induced colitis. Here, we provide a new anti-inflammation mechanism about ANXA1 secreted from injured IECs, where ANXA1 can stimulate the expression of NKG2A in NK cells that affect the recruitment and activity of neutrophils necessary for pathology of colitis. © 2016 Elsevier Inc. All rights reserved.
Keywords: ANXA1 Intestinal epithelial cells Colitis Natural killer cells NKG2A
1. Introduction In inflammatory conditions of the gastrointestinal tract, IBD is still a leading cause of morbidity and mortality all over the world [1,2]. It has been widely recognized that the development of IBD is followed by imbalance of intestinal immune homeostasis, which depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and IECs [2e4]. The gut epithelium composed by IECs can prevent the contact between luminal bacteria with immune cells through the formation of a physical barrier
Abbreviations: IBD, inflammatory bowel disease; IECs, intestinal epithelial cells; NK, natural killer; UC, ulcerative colitis; CD, Crohn's disease; Treg, T regulatory; TGF-b, transforming growth factor-b; TSLP, thymic stromal lymphopoietin; TH1, T helper 1; FPRs, peptide receptors; EVs, extracellular vesicles; IFN-g, interferon g; TNF-a, tumor necrosis factor a; FITC, isothiocyanate; LPMCs, lamina propria mononuclear cells; MLN, mesenteric lymph nodes; HE, haematoxylin and eosin. * Corresponding author. E-mail address: [email protected] (H. Fan). http://dx.doi.org/10.1016/j.bbrc.2016.07.066 0006-291X/© 2016 Elsevier Inc. All rights reserved.
and the expression of antimicrobial peptides to maintain intestinal immune homeostasis [2,5]. Recently, more and more evidences have shown that some molecular events happened to IECs directly or indirectly affect the progression of IBD. For example, mice with IEC-specific knockout of FADD, an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis [3]. Obviously, IECs not only limit bacterial translocation across the mucosal barrier, but also maintain intestinal homeostasis by regulating innate and adaptive immune responses. Illustrating this point, intestinal alkaline phosphatase produced by IECs mediate lipopolysaccharide detoxification [6,7]. In addition, epithelial regeneration following intestinal injury is critical for barrier maintenance and organ function [8], such process relies on the immune microenvironment induced by pathogenic infection or other damage model. IECs may also influence intestinal homeostasis through the secretion of conditioning cytokines (IL-8, IL-12, IL-25 and IL-23) that affect adaptive responses primed by IECs [9e11].
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Fig. 1. DSS treatment increases the expression of ANXA1 and NK cells recruitment in colonic mucosal tissues. (A) Real-time PCR analysis of ANXA1 gene of mucosal tissues. (B C) Western blotting analysis of ANXA1 protein of mucosal tissues. (D) Real-time PCR analysis of ANXA1 gene of IECs. (E F) Western blotting analysis of ANXA1 protein of IECs. (G) Western blotting analysis of ANXA1 and CD63 protein of EVs from IECs. (H) Western blotting analysis of FPRL-1 of NK cells from spleen and mesenteric lymph nodes. (I K) LPMCs
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ANXA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, owning ability of regulating diverse cellular functions and exhibiting profound inhibitory actions on leukocyte transmigration and activation [12e14]. By binding to formyl peptide receptors (FPRs) expressed on responsive cells, such as phagocytes and epithelial cells [15,16], ANXA1 facilitates resolution of inflammation. ANXA1 can also promotes intestinal epithelial migration through activation of FPR1-, Rac1-, and NOX1dependent redox signaling, triggering the oxidative inactivation of regulatory phosphatases and subsequent modification of focal adhesion proteins conducive to regulating cell migration [17]. Another study demonstrated that epithelial cells released ANXA1 as a component of extracellular vesicles (EVs) to promote intestinal mucosal wound repair [15]. NK cells exert their diverse functions through direct cell-cell contact and secretion of cytokines such as interferon g (IFN-g) and tumor necrosis factor a (TNF-a) [18,19], which can induce the injury of IECs and the infiltration of inflammatory cells, accelerating the development of IBD [20]. Additionally, NK cells can suppress Th1-mediated colitis by controlling the responses of effector T cells to gut bacteria [21]. Moreover, some study have proved that NK cells are rapidly recruited to the inflamed colon and have a protective effect on DSS-induced colitis by regulating neutrophil function via the NKG2A receptor [19]. It remains obscure about the interaction between IECs and NK cells during progression of IBD, and whether ANXA1 released from IECs affect the function of NK cells and the ripple effect on colitis. Our results showed that IECs secreted excessive ANXA1 during DSS-induced colitis, and the ANXA1 was wrapped in EVs to influence the expression of NKG2A receptor in NK cells, alleviating the pathology of colitis. 2. Materials and methods Animals and in vivo studies. 8-weeks C57BL/6 mice were kept in a pathogen free conditions. Animal experiments were approved by the guidelines of the Chinese Council on Animal Care, and the procedures were performed according to the Tongji Medical college Committee (Wuhan, China). 3% DSS (36e50 kDa; MP Biomedicals) was dissolved in water and administered to mice for 7 days as described [22]. Mice received 50 mg anti-asialo GM1 (anti-AGM1) Ab (Wako Chemicals, Neuss, Germany) or anti-NKG2A Ab (Bioss Inc, Woburn, MA, USA) by intraperitoneal injections 3 days before DSS administration and repeated every 2 days to maintain depletion/ receptor blocking [19]. The isolation of IECs and LPMCs. IECs and lamina propria mononuclear cells (LPMCs) were isolated essentially as described previously [23]. The isolation NK cells. Spleens and mesenteric lymph nodes from mouse treated with DSS for 7 days were harvested for preparing cell suspension, from which the collected cells were used to isolate NK cells by magnetic cell sorting (MACS) with mouse NK cell isolation kit (Miltenyi Biotec, Bisley, UK). Isolation of neutrophils. Single-cell suspensions were prepared as described above. Neutrophils were isolated using PEeantiLy6G Ab (eBioscience), magnetic microbeads, and MiniMACS columns (Miltenyi Biotec) according to the manufacturers' protocol. EVs purification. EVs were isolated and purified according to a previously reported protocol [24]. The isolated IECs from normal and DSS treated mouse were suspended with DMED medium and
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maintained for 6 h at 37 C, 5% CO2. Afterwards, culture medium was subjected to differential centrifugation to remove cells, dead cells, and cell debris at a rate of 20,000 g for 20 min (microparticles isolation) to 100,000 g (for exosome isolation) for 60 min. Western blotting. Western blot analyses were performed as described previously [12]. Anti-annexin 1 (1:500, Santa Cruz, USA), b-actin (1:1000, CST, USA), Anti-CD63 (1:1000, Abcam, USA) were used as primary antibodies. Real-Time PCR. Total RNA was extracted using RNAprep pure Micro Kit (Tiangen Biotech CO., LTD.). The cDNAs were synthesized using a reverse transcription kit (Quant One Step RT-PCR kit, Tiangen, China). Real-time PCR was performed using SYBR green master mix (FastFire qPCR PreMix, Tiangen, China) on an AB iCycler system (AB, United States) according to the manufacturer's instructions. Primer sequences used are as follows: Ifn-g, 50 TGAGCTCATTGAATGCTTGG-30 and 50 - ACAGCAAGGCGAAAAAGGAT-3’; Tnf-a, 50 -AGGGTCTGGGCCATAGAACT-30 and 50 - CCACCACGCTCTTCTGTCTAC-3’; NKp46, 50 -GGCTGCTGTTCTCAACACCT-30 and 50 -GGCTCACAGAGGGACATAC-3’; Klrg1, 50 -CCTCAAGCCGATCCAGTAAA-30 and 50 -CTGTGCAGACAAAGGCTCAC-3’; NKG2D, 50 GTGGTTGCTGGGATTTGAAC-30 and 50 -TGCCTCTTAAGAATGCACCC3’; NKG2A, 50 -CGAAGGATTCCAGTCCATGA-30 and 50 -GGTGTC CTGCATTTCCAAAA-3’; Anxa1 50 -ATTTTCAAGAAAACGGGCCT-30 and 50 -AGAGTCTCTCTTCAGTCCCCG-3’. Flow Cytometry. Single cells suspensions were prepared and stained with fluorescein isothiocyanate (FITC)-anti-NK1.1 (1 mg/ 106 cells) or PE/Cy5®-Anti-Ly6g antibody (0.2 mg/106 cells). The stained cells were analyzed using a flow cytometer (FACS calibur; Becton Dickinson, Franklin Lakes, NJ), and data were analyzed by WinMDI2.9 software. Tissue Staining and Histological Analysis. Colon tissues were fixed in 4% paraformaldehyde, and they were embedded in paraffin and sliced into 6 mm sections and followed by dewaxing and rehydration. These sections were stained with haematoxylin and eosin (HE) according to standard histological procedures and histological scoring was performed as described previously [25]. Statistics. Data are presented as means ± SEM of several experiments. Statistical comparisons were performed by either Student's 2-tailed t-test or ANOVA with Tukey multiple comparison post-test, as appropriate. A P value of less than 0.05 was considered significant. 3. Results 3.1. The expression of ANXA1 increases in colonic mucosal tissues and IECs due to DSS treatment To investigate the change of ANXA1 expression in colonic mucosal tissues and IECs, mRNA and protein were extracted from colonic mucosal tissues and IECs of DSS treated mice and control mice for Quantitative real-time PCR and Western blotting analysis. As shown in Fig. 1A and C, the mRNA level of ANXA1 were increased in colonic mucosal tissues and IECs of DSS treated mice. Similarly, as shown in Fig. 1B and D the protein of ANXA1 were increased in colonic mucosal tissues and IECs following DSS treatment. In addition, we also collected the EVs secreted from IECs and detected the CD63 (a EVs marker of IECS) and ANXA1 protein of EVs using Western blotting method, the results showed that more ANXA1 protein were found in EVs from IECs of DSS treated mice (Fig. 1G). All of these results demonstrate that ANXA1 expression increases in
were prepared for flow cytomery analysis, the percentage of NK1.1 þ cells were shown. (J L) MLN cells were prepared for flow cytomery analysis, the percentage of NK1.1 þ cells were shown. (M) NK cell isolated from MLN of DSS treated mice were stained with CFSE and injected into the tail vein of mice treated with DSS for 5 days, two days later the colon tissues were harvested to produce frozen section, which were observed under fluorescence microscope to access the CFSE þ NK cells recruitment in colonic mucosal tissues. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
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Fig. 2. Depletion of NK cells or blocking of ANXA1 exacerbate DSS-induced colitis. Mice received 50 mg anti-AGM1 Ab with or without anti-ANXA1 Ab 3 days before DSS administration, and repeated every 2 days to maintain NK cells depletion or ANXA1 blocking. (A) After mice were treated DSS for 7 days, colon tissues were prepared for HE staining. (B) Overall histology score. (C) The percentage of weight change. (D) Colon length change. (E) LPMCs were isolated to detect the percentage of Ly6G þ cells (neutrophils) by flow cytomery analysis. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
colonic mucosal tissues, especially in IECs during colitis induced by DSS. 3.2. DSS treatment increases NK cells recruitment in colonic mucosal tissues To investigate whether NK cells are recruited to colonic mucosal tissues, LPMCs were isolated from normal and DSS treated mice prepared for flow cytomery analysis. As shown in Fig. 1 I and K, the percentage of NK1.1 þ cells increased in DSS treated mice compared with normal mice. Equally, mesenteric lymph nodes (MLN) cells from normal and DSS treated mouse were prepared for flow cytomery analysis, results showed that the percentage of NK1.1 þ cells were also increased in DSS treated mice (Fig. 1 J and L). Additionally, we isolated NK cells from spleen and MLN of DSS
treated mice, and isolated NK cells were stained with CFSE and injected into the tail vein of mice treated with DSS for 5 days, two days later colon tissues were harvested and prepared for frozen section, which were observed under fluorescence microscope to access the CFSE þ NK cells recruitment in colonic mucosal tissues. These results showed CFSE þ NK cells were recruited to colonic mucosal tissues during colitis (Fig. 1M). ANXA1 have been shown to function by signaling through formyl peptide receptors (FPR) [12], we also found NK cells can express FPR-1(Fig. 1H). 3.3. Depletion of NK cells or blocking of ANXA1 exacerbates DSSinduced colitis To investigate the function of recruited NK cells and the role of increased ANXA1 secreted from IECs in the progression of colitis
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Fig. 3. Transferation of NK cells stimulated by ANXA1 alleviates DSS-induced colitis, and ANXA1 accelerates the NKG2A expression of NK cells. A part of isolated NK cells were treated with ANXA1 or EVs, another part of isolated NK cells were used for co-culture with IECs present or absent anti-ANXA1 antibody in transwell. All of pretreated and untreated NK cells were stained with CFSE and transferred into mice respectively. (A) Experimental design for transferation of NK cells. (B) Colon tissues were prepared for HE staining. (C) Overall histology score. (D) The percentage of weight change. (E) Colon length change. (F) Real-time PCR analysis of IFN-g, TNF-a, NKp46, KLRG1, NKG2D and NKG2A genes of NK cells. (G) Western blotting analysis of NKG2A protein of NK cells. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
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Fig. 4. Blocking of NKG2A exacerbates DSS-induced colitis by affecting recruitment and activity of neutrophils. Mice received 50 mg anti- NKG2A Ab or IgG Ab after DSS administration for 3 days, and repeated every 2 days to maintain NKG2A blocking. (A) Colon tissues were prepared for HE staining. (B) The graph shows overall histology score. (C) The percentage of weight change. (D) Colon length change. (E) LPMCs were prepared for flow cytomery analysis, the percentage of Ly6G þ cells were shown. (F) Real-time PCR analysis of CD69, ROS, IL-6, IL-17A genes of isolated neutrophils. Data represent mean ± SEM; n ¼ 5 (*P < 0.05).
induced by DSS. As reported previously, anti-AGM1 Ab and antiANXA1 Ab could deplete NK cells and block ANXA1 activity. Mice received 50 mg anti-AGM1 Ab alone or in the present of anti-ANXA1 Ab 3 days before DSS administration by intraperitoneal injections,
and such procedures were repeated every 2 days to maintain NK cells depletion or ANXA1 blocking until the mice were sacrificed. After mice were treated DSS for 7 days, colon tissues were harvested and prepared for HE staining. As shown in Fig. 2A,B,C and D,
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DSS-induced colitis were exacerbated when NK cells were depleted or/and ANXA1 were blocked. Additionally, depletion of NK cells or/ and blockade of ANXA1 accelerate the recruitment of neutrophil in colonic mucosa during the development of colitis induced by DSS (Fig. 2E). These results demonstrated the protective role of NK cells and increased ANXA1 in colitis induced by DSS. 3.4. Transferation of NK cells stimulated by ANXA1 alleviates DSSinduced colitis To investigate the role of interaction between NK cells and ANXA1, a part of isolated NK cells were treated with ANXA1 or EVs, another part of isolated NK cells were used to co-culture with IECs present or absent anti-ANXA1 ab in transwell. All of pretreated and untreated NK cells were stained with CFSE and transferred into mice by tail vein injection respectively. Experimental design for transferation of NK cells was shown in Fig. 3A. Colon tissues were harvested and prepared for HE staining to access histology score. Results demonstrated that transferation of NK cells stimulated by ANXA1 or EVS alleviates DSS-induced colitis (Fig. 3B C D and E), moreover, transferation of NK cells co-cultured with IECs also play a similar role, which be reversed by anti-ANXA1 ab (Fig. 3B and C). All of these results suggest that the interaction between NK cells and IECs play a negative regulative role on DSS-induced colitis, such cross-talk might relay on ANXA1 released from IECs. 3.5. ANXA1 accelerates the NKG2A expression of NK cells To investigate the effect of ANXA1 on NK cells function, isolated NK cells were treated as methods described. Quantitative real-time PCR analysis was performed to detect the mRNA level of IFN-g, TNFa, NKp46, KLRG1, NKG2D and NKG2A genes of NK cells from different groups. As shown in Fig. 3D, the mRNA level of IFN-g, TNFa, NKp46, KLRG1, NKG2D remained unchanged, NKG2A level was increased due to ANXA1 or EVS stimulation. Meanwhile, NK cells co-cultured with IECs also expressed more NKG2A, which could be reversed by anti- ANXA1 ab. The protein level of NKG2A was consistent with the mRNA level (Fig. 3E). 3.6. Blocking of NKG2A exacerbates DSS-induced colitis by affecting recruitment and activity of neutrophils To investigate the effect of increased NKG2A on progression of colitis induced by DSS. Mice received 50 mg anti-NKG2A Ab or IgG Ab after DSS administration for 3 days by peritoneal injection, and such procedures were repeated every 2 days to maintain NKG2A blocking until the mouse were sacrificed (On the seventh day of DSS treatment). Colon tissues were harvested and prepared for HE staining. As shown in Fig. 4 A B C and D, treatment with anti-NKG2A Ab exacerbated DSS-induced colitis. Besides, we isolated LPMCs from two groups of mice and prepared for flow cytomery analysis, the percentage of Ly6G þ cells was increased (Fig. 4 E). Finally, we isolated neutrophils from LPMCs of the two groups mice to detect the mRNA level change of CD69, ROS, IL-6, IL-17A using Quantitative real-time PCR, results showed that mRNA level of CD69, ROS, IL-6, IL-17A (genes of neutrophils activation) were increased due to blocking of NKG2A (Fig. 4.F). All of these indicate that blocking of NKG2A exacerbates DSS-induced colitis by affecting recruitment and activity of neutrophils.
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ultimately fatal acute colitis in DSS-induced colitis model [19]. Our data reveals a new protective mechanism of IBD, in which ANXA1 released from IECs suppress the expression of NKG2A receptor in NK cells that is important for alleviating DSS-induced colitis. The anti-inflammatory effect of ANXA1 has been reported in models of endotoxemia, peritonitis, arthritis, as well as cerebral and myocardial ischemia [14,26]. Recent studies also demonstrated that ANXA1 has an important role in inhabiting IBD through the inhibition of leukocyte function and promotion of gastrointestinal mucosa wound healing ascribed to binding to ALX/FPRL-1 [27,28]. Interestingly, secretion of ANXA1 has been identified in inflamed intestinal mucosal tissues [29,30]. As expected, increased ANXA1 in IECs also have been found after induction of DSS in our study. In addition, we proved that more NK cells were recruited into intestinal mucosa after DSS treatment and the expression of FPR-1 was also detected in isolated NK cells, which give us a hint that the increased ANXA1 released from IECs may interact with FPR-1 to affect the function of NK cells. To test this hypothesis, the isolated NK Cells were stimulated with ANXA1, and the inflammation related genes (IFN-g, TNF-a, NKp46, KLRG1, NKG2D and NKG2A) were detected. As shown in Fig. 3, the NKG2A expression was increased after treatment with ANXA1. The isolated IECs from normal mice and DSS induced colitis mice were co-cultured with isolated NK cells, then the same genes in NK cells were detected and similar results are obtained. Similar to previous reports, we demonstrate that epithelial cells release ANXA1 as a component of EVs. To investigate if the EVs carrying ANXA1 protein affect the function of NK cells, we collected the EVs of IECs from normal mice and DSS induced colitis mice to stimulate NK cells respectively. Similarly, the NKG2A expression was increased after treatment with EVs from DSS induced colitis mice, and the effect of EVs was reversed by anti-ANXA1. All of these results demonstrate that ANXA1 released from IECs play an improving effect in regulating the NKG2A expression of NK cells. To explore the effect of the interaction between IECs and NK cells through EVs containing ANXA1 protein on the IBD, some isolated NK cells were stimulated with ANXA1 or EVs gathered from DSS induced colitis mice. Other NK cells were co-cultured with IECs from DSS induced colitis mice, then these NK cells were transferred to mice treated with DSS for five days respectively, three days later colons were harvested to assess the pathologic degree, as well as detecting the number and function of neutrophils. As shown in Figs. 3 and 4, the pathology of colonitis was alleviated owning to the transfer of these NK cells, and blocking of NKG2A exacerbates DSS-induced colitis by increasing recruitment and activity of neutrophils. These results indicate that the inhibiting effect of NK cells to colitis may be related to migration and activity of neutrophil. In conclusion, our findings identify a previously unknown immunoregulatory mechanism for NK cells during DSS-induced colitis, in which ANXA1 released from IECs affect the expression of NKG2A to mediate the recruitment and function of neutrophil. It cannot be ignored that the interaction among various kinds of cells in colon is the very essence of colitis, so more experiments need to be designed to investigate the mechanism. Anyway, our study may provide a new promising therapeutic strategy for intestinal diseases. Acknowledgments We thank the Natural Science Foundation of China for providing support (no: 81273906).
4. Discussion Appendix A. Supplementary data The gut epithelium made up of IECs has an important role on the maintenance of intestinal immune homeostasis [1]. NK cells can downregulate neutrophil function, preventing severe and
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.07.066.
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