Immunological Investigations, Early Online: 1–9, 2015 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2014.999937
High Frequency of Mononuclear Myeloid-Derived Suppressor Cells is Associated with Exacerbation of Inflammatory Bowel Disease Immunol Invest Downloaded from informahealthcare.com by University of Connecticut on 03/31/15 For personal use only.
Qinhua Xi,1* Yueqin Li,1 Juan Dai,1 and Weichang Chen1,2 1
Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China, and 2 Department of Clinical Immunology of Jiangsu Province, The First Affiliated Hospital of Soochow University, Suzhou, China Exacerbation and relapse of inflammatory bowel disease (IBD) is associated with reduced antibacterial immunity and increased immune regulatory activity, but the source of increased immune regulation during episodes of disease activity is unclear. Myeloid-derived suppressor cells (MDSCs) are a cell type with a well-recognized role in limiting immune reactions. MDSC function in IBD and its relation to disease activity, however, remains unexplored. Here we show that patients with either ulcerative colitis (UC) or Crohn’s disease (CD) have high peripheral blood levels of mononuclear MDSCs. Especially exacerbation of disease is associated with higher levels of mononuclear MDSC counts compared with those in remission of disease. Interestingly, chronic experimental colitis in mice coincides with increased MDCS mobilization. Thus, our results suggest that mononuclear MDCS are endogenous antagonists of immune system functionality in mucosal inflammation and the depression of antibacterial immunity associated with exacerbation of disease might involve increased activity of the MDSC compartment. Keywords Exacerbation, IBD, MDSC, remission
INTRODUCTION Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), represents a chronic relapsing inflammatory reaction of the intestine (Kaser et al., 2010; Xavier & Podolsky, 2007). Importantly, it is known that IBD patients are impaired with respect to immune responses and fail to control bacterial growth in the intestine (Braat et al., 2006). The mechanisms involved in limiting that preclude successful immune responses in the intestine remain, however, largely unclear. An attractive candidate to mediate tolerogenic regulation in IBD is the MDSC compartment. MDSCs exhibit a heterogeneous phenotype and are implicated in limiting infection and tumor immunity (Fujimura et al., 2010; Gabrilovich & Nagaraj, 2009). Mouse MDSCs are defined as CD11b+Gr-1+ cells (Youn et al., 2008). Although there are currently no uniform standards for *Authors Q. Xi and Y. Li equally contributed to the article. Correspondence: Weichang Chen, Department of Gastroenterology, and Department of Clinical Immunology of Jiangsu Province, The First Affiliated Hospital of Soochow University, Suzhou 215006, China. E-mail: [email protected]
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the definition of human MDSCs, HLA-DR-CD33+CD11b+ and CD14+HLADR-/low cells are the most widely recognized markers for identifying human MDSCs and constitute polymorphonuclear and mononuclear MDSCs respectively (Chalmin et al., 2010; Filipazzi et al., 2007; Hoechst et al., 2008; Lin et al., 2011; Poschke et al., 2010; Vuk- Pavlovic´ et al., 2010; Youn et al., 2008). MDSCs can exert their regulatory function through inhibition of effector T cell activity, enhancing regulatory T cell proliferation and by interacting with natural killer cells. The inhibitory effect of MDSCs on immune responses is attributed to production of arginase-1 (ARG-1) and release of nitric oxide as a consequence of inducible nitric oxide synthase (iNOS) expression. In addition, recent studies have documented that production of PGE2 through COX-2, and expression of B7-H1, CD80 and CD40 support the suppressive actions of MDSCs (Fujimura et al., 2012; Fujita et al., 2011; Mundy-Bosse et al., 2011; Obermajer et al., 2011; Pan et al., 2010). The importance of MDSCs in immune control raises questions as to their potential functionality during IBD and especially whether they are potentially involved in limiting anti-bacterial immunity during IBD exacerbation. Hitherto, most of the studies involving MDSCs have been limited to tumor immunity, and hence their role in other diseases remains poorly understood. Haile et al. (2008), however, showed that MDSCs attenuated experimental IBD in a specific murine IBD model that dependens on the introduction of antigen-specific CD8+ T cells. In addition these authors also provided evidence that MDSCs are involved in human IBD. Nevertheless it is fair to say that we have little insight into the size of the MDSC compartment during chronic inflammation and its relation to UC and CD specifically. Furthermore, it is unknown whether these cells are mobilized during a disease flare. Here we investigate the clinical significance of MDSC frequency in patients during both active disease and remission and we also attempt to relate results to MDSC numbers in experimental colitis. The results suggest that MDSCs are part of an endogenous response to increased mucosal inflammation during IBD flares and thus may explain the apparently paradoxical immune regulatory activity observed at disease exacerbation.
MATERIALS AND METHODS Patients A total of 69 IBD patients were included in this study and recruited from the First People’s Hospital of Soochow University, Second People’s Hospital of Changshu City, Suzhou City Hospital, Second People’s Hospital of Kunshan City, and First People’s Hospital of Taicang City. Diagnostic criteria used for all the cases were those set out in the guidelines as stipulated in ‘‘The Specification Consensus for Right Diagnosis and Treatment of Inflammatory Bowel Disease in China’’ by the Chinese Medical Association in 2007 (Ren et al., 2007). CD was classified as active disease or remission according to the CD activity Index (CDAI). The remission and active groups of UC were classified according to the Sourtherland disease activity index. Among the 33 CD cases (male, 27;
Mononuclear MDSC Associated with Exacerbation of IBD
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female, 6; mean age, 27 years), the numbers of patients in active and remission phases were 16 and 17, respectively. Of the 36 UC patients (male, 20; female, 16; mean age, 39 years), there were 21 and 15 of active and remission cases respectively. Thirty-six healthy volunteers (male, 22; female, 14; mean age, 35 years) without a family history of IBD or related diseases were recruited from the First People’s Hospital of Soochow University in the same period and used as normal controls. This study was approved by the ethical committee of the First People’s Hospital of Soochow University. Reagents PE-conjugated anti-human HLA-DR antibody (Clone: L243; Lot: 307606), FITC-conjugated anti-human CD14 antibody (Clone: HCD14; Lot: 325604), PE/Cy7-conjugated anti-mouse CD11b antibody (Clone: M1/70; Lot: 101216), FITC-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (RB6-8C5; Lot: 108406), and 10X RBC Lysis Buffer (Lot: B143823) were all purchased from Biolegend (San Diego, CA, USA). Animal Experimentation Ten SPF BALB/c male mice (age, 6-8 weeks), weighing 20 ± 1 g, were numbered, randomly allocated to control (A) and UC model (B) groups, and reared under SPF conditions. A 5% DSS (dextran sulphate sodium; molecular weight, 40 000) solution was prepared (50 g/L). Induction of experimental colitis using DSS was performed using previously described procedures (Dieleman et al., 1998). The animals were monitored daily for behavioral status, aspect of fur quality, stool, activity, diet, survival, etc. All the mice were killed after 22 days following induction of colitis and peripheral blood, spleen and bone marrow samples we collected for further analysis. Animal care and use was approved by the Institutional Animal Care and Use Committee of Soochow University. Humane care of the mice was thoroughly considered. Flow Cytometry Human MDSC were analyzed by flow cytometry (Beckman flow cytometer FC500, Pasadena, CA, USA) according to established protocols (Halle et al., 2008). For analysis of the mouse samples, heparinized peripheral blood was collected by retro-orbital bleeding. Spleens were harvested and filtered through a filter membrane to obtain single cells. Bone marrow cells were collected from one femur marrow cavity. Blood, bone marrow and spleen cells were subsequently incubated with anti-mouse Ly-6G/Ly-6C (Gr-1) antibody and anti-mouse CD11b antibody for 30 min at 4 C. After erythrocyte lysis, the levels of Gr-1+CD11b+cells were gated and analyzed by flow cytometry (FC 500, Beckman Coulter, Miami, FL, USA). Statistical Analysis All data are presented as means ± SEM and analyzed using Graphpad prism statistical software (version 5.01). The statistical significance of mean difference between the groups was determined using the t-test, whereas analysis of variance (ANOVA) was conducted to compare means among three or more groups. p 5 0.05 was considered statistically significant.
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RESULTS IBD is Characterized by Increased Frequency of Mononuclear MDSCs in the Peripheral Blood To gain insight into the size of the MDSC compartment in IBD, we quantified the proportions of polymorphonuclear and mononuclear MDSCs in the peripheral blood of UC and CD patients and compared the results to healthy controls. As shown in Figure 1, the proportion of mononuclear MDSCs in both CD (43.72 ± 4.012%) and UC (47.2 ± 4.38%) patients were significantly higher (p50.01) than those in healthy controls (HC) (16.75 ± 0.9%). However, between the UC and CD group, no statistically significant difference (p40.05; Figure 1A) with respect to mononuclear MDSC levels were detected. Interestingly, with respect to polymorphonuclear MDSCs no significant differences were found between the HC (0.2431 ± 0.0316%) group and CD (0.2238 ± 0.01748%) or UC (0.2273 ± 0.0231%) groups (p40.05), (Figure 1B).
Figure 1. Only circulating mononuclear but not polymorphonuclear related MDSCs, are augmented in the IBD patients. (A) Higher levels of circulating mononuclear MDSCs in patients with IBD. (Left) Representative flow cytometric diagram of monocytic MDSCs in healthy control (HC) and patients with UC and CD. IgG represnts IgG isotype control; (Right) Statistical analysis of the difference in monocytic MDSC levels among HC, UC, and CD groups using one-way ANOVA. ***p50.0001; ns, not significant. (B) Analysis of the levels of polymorphonuclear MDSCs in patients with IBD. (Left) Representative flow cytometric diagram of monocytic MDSCs in HC and patients with UC and CD; (Right) Statistical analysis of the difference in monocytic MDSC levels among HC, UC, and CD groups using one-way ANOVA. ns represents that the differences are not significant.
Mononuclear MDSC Associated with Exacerbation of IBD
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Also, no significant difference was found between CD and UC groups (p40.05) (Figure 2) with respect to polymorphonuclear MDSCs. Thus both CD and UC are associated with a specific increase in peripheral blood mononuclear MDSCs. Significant Increase of Mononuclear MDSCs in Active IBD Compared to Quiescent Disease We were interested in how mononuclear MDSC levels relate to IBD disease activity. In CD patients with active disease, the level of MDSCs was significantly higher compared to that for patients in remission (60.34 ± 4.191% vs 28.07 ± 3.932%; p50.01; Figures 2 and 3). Additionally, in UC patients with active disease, MDSCs levels were significantly increased as compared to patients with quiescent UC (66.21 ± 3.546% vs 20.83 ± 2.051%, respectively; p50.01; Figure 2). Levels of MDSCs of patients in remission remain, however, significantly higher when compared with a cohort of healthy controls (p50.01; data not shown). Abnormally Elevated Level MDSCs in Experimental Chronic Colitis To further confirm the findings in human IBD, we decided to test the levels of MDSC in the DSS-dependent chronic colitis mouse model (Dieleman et al., 1998). The bone marrow MDSC level in vehicle-challenged mice was significantly lower compared to those exposed to chronic DSS (58.20 ± 1.57% vs 74.86 ± 4.065%; p50.01; Figure 3B, left). Also in the spleen of the DSSchallenged animals, MDSC levels were significantly higher compared to vehicle control (2.81 ± 0.132% in the control group vs 7.85 + 0.708% in animals with chronic colitis; p50.001) or that in vehicle treated control mice (Figure 3B, middle). Finally, the proportion of MDSCs in the peripheral blood of DSS challenged animals was significantly higher than that of vehiclechallenged mice (5.42 ± 0.836% in control animals vs 12.46 ± 1.88% in DSSchallenged animals; p50.01; Figure 3B, right).
Figure 2. Analysis of the level of peripheral blood mononuclear MDSCs in UC versus CD patients at different disease stages of disease. Statistical analysis was performed using an unpaired t-test.
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Figure 3. Mobilization of MDSCs in DSS-mediated experimental chronic colitis. (A) A representative result of the flow cytometric analysis for MDSCs in the bone marrow (indicated as BM), spleen (indicated as SP), and peripheral blood (indicated as Blood) of vehicle-challenged and DSS-challenged mice respectively. (B) Depiction of the level of MDSCs in the bone marrow, spleen, and peripheral blood of vehicle-challenged and DSS-challenged mice respectively using unpaired t-test.
DISCUSSION IBD is a group of non-specific mucosal inflammatory diseases with unknown etiology. However, it is well recognized that the disease coincides with increased immune regulatory activity and concomitantly reduced bacterial clearance. The resulting bacterial overgrowth is thought to be involved in the pathogenesis of IBD (Braat et al., 2006). Sources of immune regulation in IBD remain, however, largely enigmatic but probably involve endogenous immune regulatory loops activated by the chronic character of the mucosal inflammation. The current study provides an important clue that the mononuclear MDSC compartment constitutes an element in such a loop. The excessive bacterial overgrowth resulting from MDSC mobilization may in turn provoke the production of the large amount of cytokines and inflammatory mediators, and eventually cause the tissue damage, pathological changes and clinical manifestations associated with IBD (Dharmani & Chadee, 2008; Wendelsdorf et al., 2010). MDSCs constitute a diverse population of cells derived from bone marrow progenitor cells that are at varying stages of differentiation, from early myeloid to a more granulocytic or monocytic phenotype. In murine tumor models, MDSCs have been isolated from peripheral blood, spleen, lymph nodes, and tumor sites and are known to have the ability to block both innate
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Mononuclear MDSC Associated with Exacerbation of IBD
and adaptive immunity (Diaz-Montero et al., 2014). Studies on MDSC functionality have almost entirely been focused on their role in tumor immune escape. However, the level and clinical significance of MDSCs in other immune-related diseases has rarely been examined (Gabrilovich & Nagaraj, 2009). In 2008, Hoechst et al. (2008) found that a group of CD14+HLA-DR-/low MDSCs were increased in the peripheral blood of patients with liver cancer, and that these cells displayed typical biological hallmarks of MDSCs. Subsequently, Haile et al. (2008) showed that the level of MDSCs is elevated in patients with IBD, and that these cells attenuated the progression of colitis in a CD8+ T cell-induced experimental model of IBD, suggesting that MDSCs are also of important clinical significance in this disease. However, the clinical significance of MDSCs in different subtypes and different pathological processes of IBD had not yet been reported. Here we show that the levels of mononuclear MDSCs in UC and CD patients were significantly higher than those in a control cohort, corroborating the earlier findings of Haile et al. (2008). Importantly, we now demonstrate that increased MDSC levels are a general characteristic of IBD. They are observed in both well-phenotyped UC and CD patients, the two manifestations of this IBD not displaying marked differences in this respect. Furthermore for both UC and CD, the levels of MDSCs in patients substantially increase upon exacerbation of disease. Thus we provide evidence that MDSC mobilization is an endogenous response to mucosal disease and probably constitutes an innate reaction of the body to chronic inflammation. In support of this notion, experimental induction of chronic colitis mobilized large numbers of Gr1+CD11b+ cells in mice. However, the mechanisms by which chronically inflamed mucosa signals to the bone marrow to induce increased MDSC levels warrant further investigation. One possibility is that the up-regulation of intestinal Toll-like receptor 4 (TLR4) and TLR-2, which is a characteristic of our DSS model but is also observed in human IBD, is involved. TLRs signals through Myd88 (myeloid differentiation primary response gene 88) to provoke expression of a large number of inflammatory mediators (Dauphinee & Karsan, 2006; Sekine et al., 2006). TLR-triggered activation of Myd88-dependent signaling appears crucial for the proliferation and activation of MDSCs (Delano et al., 2007), but further experimentation is necessary to substantiate this notion. In conclusion, this study documents that an increased level of mononuclear MDSCs is a general characteristic of IBD, that these levels are further elevated upon exacerbation of disease, and that this effect may constitute an endogenous response to chronic mucosal inflammation. As to whether the mobilization of MDSCs directly affects the progression of IBD (e.g., through reducing antibacterial immunity) needs further exploration.
DECLARATION OF INTEREST This work was supported by the National Natural Science Foundation of China (NO 81272737). The authors have declared no financial interest in the present work. The authors alone are responsible for the content and writing of the paper.
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REFERENCES Braat H, Peppelenbosch MP, Hommes DW. (2006). Immunology of Crohn’s disease. Ann N Y Acad Sci, 107, 135–54. Chalmin F, Ladoire S, Mignot G, et al. (2010). Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest, 120, 457–71. Dauphinee SM, Karsan A. (2006). Lipopolysaccharide signaling in endothelial cells. 2006. Lab Invest, 86, 9–22. Delano MJ, Scumpia PO, Weinstein JS. (2007). MyD88-dependent expansion of an immature GR-1 + CD11b population induces T cell suppression and Th2 polarization in sepsis. J Exp Med, 204, 1463–74. Dharmani P, Chadee K. (2008). Biologic therapies against inflammatory bowel disease: a dysregulated immune system and the cross talk with gastrointestinal mucosa hold the key. Curr Mol Pharmacol, 1, 195–212. Diaz-Montero CM, Finke J, Montero AJ. (2014). Myeloid-derived suppressor cells in cancer: therapeutic, predictive, and prognostic implications. Semin Oncol, 41, 174–84. Dieleman LA, Palmen MJ, Akol H, et al. (1998). Chronic experimental colitis induced by dextran sulphate sodium(DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol, 114, 385–91. Filipazzi P, Valenti R, Huber V, et al. (2007). Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol, 25, 2546–53. Fujimura T, Mahnke K, Enk AH. (2010). Myeloid derived suppressor cells and their role in tolerance induction in cancer. J Dermatol Sci, 59, 1–6. Fujimura T, Ring S, Umansky V, et al. (2012). Regulatory T cells stimulate B7-H1 expression in myeloid-derived suppressor cells in ret melanomas. Invest Dermatol, 132, 1239–46. Fujita M, Kohanbash G, Fellows-Mayle W, et al. (2011). COX-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells. Cancer Res, 71, 2664–74. Gabrilovich DI, Nagaraj S. (2009). Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol, 9, 162–74. Haile LA, von Wasielewski R, Gamrekelashvili J, et al. (2008). Myeloid-derived suppressor cells in inflammatory bowel disease: A new immunoregulatory pathway. Gastroenterology, 135, 871–81. Hoechst B, Ormandy LA, Ballmaier M, et al. (2008). A new population of myeloidderived suppressor cells in hepatocellular carcinoma patients induces CD4 + CD25 + Foxp3 + T cells. Gastroenterology, 135, 234–43. Kaser A, Zeissig S, Blumberg RS. (2010). Inflammatory bowel disease. Annu Rev Immunol, 28, 573–621. Lin Y, Gustafson MP, Bulur PA, et al. (2011). Immunosuppressive CD14+HLA-DR(low)/monocytes in B-cell non-Hodgkin lymphoma. 2011. Blood, 117, 872–81. Mundy-Bosse BL, Lesinski GB, Jaime-Ramirez AC, et al. (2011). Myeloid-derived suppressor cell inhibition of the IFN response in tumor-bearing mice. Cancer Res, 71, 5101–10. Obermajer N, Muthuswamy R, Odunsi K, et al. (2011). PGE(2)-induced CXCL12 production and CXCR4 expression controls the accumulation of human MDSCs in ovarian cancer environment. Cancer Res, 71, 7463–70. Pan PY, Ma G, Weber KJ, et al. (2010). Immune stimulatory receptor CD40 is required for T-cell suppression and T regulatory cell activation mediated by myeloid-derived suppressor cells in cancer. Cancer Res, 70, 99–108. Poschke I, Mougiakakos D, Hansson J, et al. (2010). Immature immunosuppressive CD14+HLA-DR-/low cells in melanoma patients are Stat3hi and overexpress CD80, CD83, and DC-sign. Cancer Res, 70, 4335–45.
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Ren WH, Lai M, Chen Y, et al. (2007). Validation of the mainland Chinese version of the inflammatory bowel disease questionnaire (IBDQ) for ulcerative colitis and Crohn’s disease. Inflam Bowel Dis, 13, 903–10. Sekine Y, Yumioka T, Yamamoto T, et al. (2006). Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages. J Immunol, 176, 380–9. Vuk-Pavlovic´ S, Bulur PA, Lin Y, et al. (2010). Immunosuppressive CD14+HLA-DRlow/monocytes in prostate cancer. Prostate, 70, 443–55. Wendelsdorf K, Bassaganva-Riera J, Hontecillas R, Eubank S. (2010). Modle of colonic inflammation: immune modulatory mechanism in inflammatory bowel disease. 2010. J Theor Boil, 264, 1225–39. Xavier RJ, Podolsky DK. (2007). Unravelling the pathogenesis of inflammatory bowel disease. Nature, 448, 427–34. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. (2008). Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol, 181, 5791–802.
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High Frequency of Mononuclear Myeloid-Derived Suppressor Cells is Associated with Exacerbation of Inflammatory Bowel Disease Immunol Invest Downloaded from informahealthcare.com by University of Connecticut on 03/31/15 For personal use only.
Qinhua Xi,1* Yueqin Li,1 Juan Dai,1 and Weichang Chen1,2 1
Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China, and 2 Department of Clinical Immunology of Jiangsu Province, The First Affiliated Hospital of Soochow University, Suzhou, China Exacerbation and relapse of inflammatory bowel disease (IBD) is associated with reduced antibacterial immunity and increased immune regulatory activity, but the source of increased immune regulation during episodes of disease activity is unclear. Myeloid-derived suppressor cells (MDSCs) are a cell type with a well-recognized role in limiting immune reactions. MDSC function in IBD and its relation to disease activity, however, remains unexplored. Here we show that patients with either ulcerative colitis (UC) or Crohn’s disease (CD) have high peripheral blood levels of mononuclear MDSCs. Especially exacerbation of disease is associated with higher levels of mononuclear MDSC counts compared with those in remission of disease. Interestingly, chronic experimental colitis in mice coincides with increased MDCS mobilization. Thus, our results suggest that mononuclear MDCS are endogenous antagonists of immune system functionality in mucosal inflammation and the depression of antibacterial immunity associated with exacerbation of disease might involve increased activity of the MDSC compartment. Keywords Exacerbation, IBD, MDSC, remission
INTRODUCTION Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), represents a chronic relapsing inflammatory reaction of the intestine (Kaser et al., 2010; Xavier & Podolsky, 2007). Importantly, it is known that IBD patients are impaired with respect to immune responses and fail to control bacterial growth in the intestine (Braat et al., 2006). The mechanisms involved in limiting that preclude successful immune responses in the intestine remain, however, largely unclear. An attractive candidate to mediate tolerogenic regulation in IBD is the MDSC compartment. MDSCs exhibit a heterogeneous phenotype and are implicated in limiting infection and tumor immunity (Fujimura et al., 2010; Gabrilovich & Nagaraj, 2009). Mouse MDSCs are defined as CD11b+Gr-1+ cells (Youn et al., 2008). Although there are currently no uniform standards for *Authors Q. Xi and Y. Li equally contributed to the article. Correspondence: Weichang Chen, Department of Gastroenterology, and Department of Clinical Immunology of Jiangsu Province, The First Affiliated Hospital of Soochow University, Suzhou 215006, China. E-mail: [email protected]
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the definition of human MDSCs, HLA-DR-CD33+CD11b+ and CD14+HLADR-/low cells are the most widely recognized markers for identifying human MDSCs and constitute polymorphonuclear and mononuclear MDSCs respectively (Chalmin et al., 2010; Filipazzi et al., 2007; Hoechst et al., 2008; Lin et al., 2011; Poschke et al., 2010; Vuk- Pavlovic´ et al., 2010; Youn et al., 2008). MDSCs can exert their regulatory function through inhibition of effector T cell activity, enhancing regulatory T cell proliferation and by interacting with natural killer cells. The inhibitory effect of MDSCs on immune responses is attributed to production of arginase-1 (ARG-1) and release of nitric oxide as a consequence of inducible nitric oxide synthase (iNOS) expression. In addition, recent studies have documented that production of PGE2 through COX-2, and expression of B7-H1, CD80 and CD40 support the suppressive actions of MDSCs (Fujimura et al., 2012; Fujita et al., 2011; Mundy-Bosse et al., 2011; Obermajer et al., 2011; Pan et al., 2010). The importance of MDSCs in immune control raises questions as to their potential functionality during IBD and especially whether they are potentially involved in limiting anti-bacterial immunity during IBD exacerbation. Hitherto, most of the studies involving MDSCs have been limited to tumor immunity, and hence their role in other diseases remains poorly understood. Haile et al. (2008), however, showed that MDSCs attenuated experimental IBD in a specific murine IBD model that dependens on the introduction of antigen-specific CD8+ T cells. In addition these authors also provided evidence that MDSCs are involved in human IBD. Nevertheless it is fair to say that we have little insight into the size of the MDSC compartment during chronic inflammation and its relation to UC and CD specifically. Furthermore, it is unknown whether these cells are mobilized during a disease flare. Here we investigate the clinical significance of MDSC frequency in patients during both active disease and remission and we also attempt to relate results to MDSC numbers in experimental colitis. The results suggest that MDSCs are part of an endogenous response to increased mucosal inflammation during IBD flares and thus may explain the apparently paradoxical immune regulatory activity observed at disease exacerbation.
MATERIALS AND METHODS Patients A total of 69 IBD patients were included in this study and recruited from the First People’s Hospital of Soochow University, Second People’s Hospital of Changshu City, Suzhou City Hospital, Second People’s Hospital of Kunshan City, and First People’s Hospital of Taicang City. Diagnostic criteria used for all the cases were those set out in the guidelines as stipulated in ‘‘The Specification Consensus for Right Diagnosis and Treatment of Inflammatory Bowel Disease in China’’ by the Chinese Medical Association in 2007 (Ren et al., 2007). CD was classified as active disease or remission according to the CD activity Index (CDAI). The remission and active groups of UC were classified according to the Sourtherland disease activity index. Among the 33 CD cases (male, 27;
Mononuclear MDSC Associated with Exacerbation of IBD
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female, 6; mean age, 27 years), the numbers of patients in active and remission phases were 16 and 17, respectively. Of the 36 UC patients (male, 20; female, 16; mean age, 39 years), there were 21 and 15 of active and remission cases respectively. Thirty-six healthy volunteers (male, 22; female, 14; mean age, 35 years) without a family history of IBD or related diseases were recruited from the First People’s Hospital of Soochow University in the same period and used as normal controls. This study was approved by the ethical committee of the First People’s Hospital of Soochow University. Reagents PE-conjugated anti-human HLA-DR antibody (Clone: L243; Lot: 307606), FITC-conjugated anti-human CD14 antibody (Clone: HCD14; Lot: 325604), PE/Cy7-conjugated anti-mouse CD11b antibody (Clone: M1/70; Lot: 101216), FITC-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (RB6-8C5; Lot: 108406), and 10X RBC Lysis Buffer (Lot: B143823) were all purchased from Biolegend (San Diego, CA, USA). Animal Experimentation Ten SPF BALB/c male mice (age, 6-8 weeks), weighing 20 ± 1 g, were numbered, randomly allocated to control (A) and UC model (B) groups, and reared under SPF conditions. A 5% DSS (dextran sulphate sodium; molecular weight, 40 000) solution was prepared (50 g/L). Induction of experimental colitis using DSS was performed using previously described procedures (Dieleman et al., 1998). The animals were monitored daily for behavioral status, aspect of fur quality, stool, activity, diet, survival, etc. All the mice were killed after 22 days following induction of colitis and peripheral blood, spleen and bone marrow samples we collected for further analysis. Animal care and use was approved by the Institutional Animal Care and Use Committee of Soochow University. Humane care of the mice was thoroughly considered. Flow Cytometry Human MDSC were analyzed by flow cytometry (Beckman flow cytometer FC500, Pasadena, CA, USA) according to established protocols (Halle et al., 2008). For analysis of the mouse samples, heparinized peripheral blood was collected by retro-orbital bleeding. Spleens were harvested and filtered through a filter membrane to obtain single cells. Bone marrow cells were collected from one femur marrow cavity. Blood, bone marrow and spleen cells were subsequently incubated with anti-mouse Ly-6G/Ly-6C (Gr-1) antibody and anti-mouse CD11b antibody for 30 min at 4 C. After erythrocyte lysis, the levels of Gr-1+CD11b+cells were gated and analyzed by flow cytometry (FC 500, Beckman Coulter, Miami, FL, USA). Statistical Analysis All data are presented as means ± SEM and analyzed using Graphpad prism statistical software (version 5.01). The statistical significance of mean difference between the groups was determined using the t-test, whereas analysis of variance (ANOVA) was conducted to compare means among three or more groups. p 5 0.05 was considered statistically significant.
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RESULTS IBD is Characterized by Increased Frequency of Mononuclear MDSCs in the Peripheral Blood To gain insight into the size of the MDSC compartment in IBD, we quantified the proportions of polymorphonuclear and mononuclear MDSCs in the peripheral blood of UC and CD patients and compared the results to healthy controls. As shown in Figure 1, the proportion of mononuclear MDSCs in both CD (43.72 ± 4.012%) and UC (47.2 ± 4.38%) patients were significantly higher (p50.01) than those in healthy controls (HC) (16.75 ± 0.9%). However, between the UC and CD group, no statistically significant difference (p40.05; Figure 1A) with respect to mononuclear MDSC levels were detected. Interestingly, with respect to polymorphonuclear MDSCs no significant differences were found between the HC (0.2431 ± 0.0316%) group and CD (0.2238 ± 0.01748%) or UC (0.2273 ± 0.0231%) groups (p40.05), (Figure 1B).
Figure 1. Only circulating mononuclear but not polymorphonuclear related MDSCs, are augmented in the IBD patients. (A) Higher levels of circulating mononuclear MDSCs in patients with IBD. (Left) Representative flow cytometric diagram of monocytic MDSCs in healthy control (HC) and patients with UC and CD. IgG represnts IgG isotype control; (Right) Statistical analysis of the difference in monocytic MDSC levels among HC, UC, and CD groups using one-way ANOVA. ***p50.0001; ns, not significant. (B) Analysis of the levels of polymorphonuclear MDSCs in patients with IBD. (Left) Representative flow cytometric diagram of monocytic MDSCs in HC and patients with UC and CD; (Right) Statistical analysis of the difference in monocytic MDSC levels among HC, UC, and CD groups using one-way ANOVA. ns represents that the differences are not significant.
Mononuclear MDSC Associated with Exacerbation of IBD
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Also, no significant difference was found between CD and UC groups (p40.05) (Figure 2) with respect to polymorphonuclear MDSCs. Thus both CD and UC are associated with a specific increase in peripheral blood mononuclear MDSCs. Significant Increase of Mononuclear MDSCs in Active IBD Compared to Quiescent Disease We were interested in how mononuclear MDSC levels relate to IBD disease activity. In CD patients with active disease, the level of MDSCs was significantly higher compared to that for patients in remission (60.34 ± 4.191% vs 28.07 ± 3.932%; p50.01; Figures 2 and 3). Additionally, in UC patients with active disease, MDSCs levels were significantly increased as compared to patients with quiescent UC (66.21 ± 3.546% vs 20.83 ± 2.051%, respectively; p50.01; Figure 2). Levels of MDSCs of patients in remission remain, however, significantly higher when compared with a cohort of healthy controls (p50.01; data not shown). Abnormally Elevated Level MDSCs in Experimental Chronic Colitis To further confirm the findings in human IBD, we decided to test the levels of MDSC in the DSS-dependent chronic colitis mouse model (Dieleman et al., 1998). The bone marrow MDSC level in vehicle-challenged mice was significantly lower compared to those exposed to chronic DSS (58.20 ± 1.57% vs 74.86 ± 4.065%; p50.01; Figure 3B, left). Also in the spleen of the DSSchallenged animals, MDSC levels were significantly higher compared to vehicle control (2.81 ± 0.132% in the control group vs 7.85 + 0.708% in animals with chronic colitis; p50.001) or that in vehicle treated control mice (Figure 3B, middle). Finally, the proportion of MDSCs in the peripheral blood of DSS challenged animals was significantly higher than that of vehiclechallenged mice (5.42 ± 0.836% in control animals vs 12.46 ± 1.88% in DSSchallenged animals; p50.01; Figure 3B, right).
Figure 2. Analysis of the level of peripheral blood mononuclear MDSCs in UC versus CD patients at different disease stages of disease. Statistical analysis was performed using an unpaired t-test.
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Figure 3. Mobilization of MDSCs in DSS-mediated experimental chronic colitis. (A) A representative result of the flow cytometric analysis for MDSCs in the bone marrow (indicated as BM), spleen (indicated as SP), and peripheral blood (indicated as Blood) of vehicle-challenged and DSS-challenged mice respectively. (B) Depiction of the level of MDSCs in the bone marrow, spleen, and peripheral blood of vehicle-challenged and DSS-challenged mice respectively using unpaired t-test.
DISCUSSION IBD is a group of non-specific mucosal inflammatory diseases with unknown etiology. However, it is well recognized that the disease coincides with increased immune regulatory activity and concomitantly reduced bacterial clearance. The resulting bacterial overgrowth is thought to be involved in the pathogenesis of IBD (Braat et al., 2006). Sources of immune regulation in IBD remain, however, largely enigmatic but probably involve endogenous immune regulatory loops activated by the chronic character of the mucosal inflammation. The current study provides an important clue that the mononuclear MDSC compartment constitutes an element in such a loop. The excessive bacterial overgrowth resulting from MDSC mobilization may in turn provoke the production of the large amount of cytokines and inflammatory mediators, and eventually cause the tissue damage, pathological changes and clinical manifestations associated with IBD (Dharmani & Chadee, 2008; Wendelsdorf et al., 2010). MDSCs constitute a diverse population of cells derived from bone marrow progenitor cells that are at varying stages of differentiation, from early myeloid to a more granulocytic or monocytic phenotype. In murine tumor models, MDSCs have been isolated from peripheral blood, spleen, lymph nodes, and tumor sites and are known to have the ability to block both innate
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Mononuclear MDSC Associated with Exacerbation of IBD
and adaptive immunity (Diaz-Montero et al., 2014). Studies on MDSC functionality have almost entirely been focused on their role in tumor immune escape. However, the level and clinical significance of MDSCs in other immune-related diseases has rarely been examined (Gabrilovich & Nagaraj, 2009). In 2008, Hoechst et al. (2008) found that a group of CD14+HLA-DR-/low MDSCs were increased in the peripheral blood of patients with liver cancer, and that these cells displayed typical biological hallmarks of MDSCs. Subsequently, Haile et al. (2008) showed that the level of MDSCs is elevated in patients with IBD, and that these cells attenuated the progression of colitis in a CD8+ T cell-induced experimental model of IBD, suggesting that MDSCs are also of important clinical significance in this disease. However, the clinical significance of MDSCs in different subtypes and different pathological processes of IBD had not yet been reported. Here we show that the levels of mononuclear MDSCs in UC and CD patients were significantly higher than those in a control cohort, corroborating the earlier findings of Haile et al. (2008). Importantly, we now demonstrate that increased MDSC levels are a general characteristic of IBD. They are observed in both well-phenotyped UC and CD patients, the two manifestations of this IBD not displaying marked differences in this respect. Furthermore for both UC and CD, the levels of MDSCs in patients substantially increase upon exacerbation of disease. Thus we provide evidence that MDSC mobilization is an endogenous response to mucosal disease and probably constitutes an innate reaction of the body to chronic inflammation. In support of this notion, experimental induction of chronic colitis mobilized large numbers of Gr1+CD11b+ cells in mice. However, the mechanisms by which chronically inflamed mucosa signals to the bone marrow to induce increased MDSC levels warrant further investigation. One possibility is that the up-regulation of intestinal Toll-like receptor 4 (TLR4) and TLR-2, which is a characteristic of our DSS model but is also observed in human IBD, is involved. TLRs signals through Myd88 (myeloid differentiation primary response gene 88) to provoke expression of a large number of inflammatory mediators (Dauphinee & Karsan, 2006; Sekine et al., 2006). TLR-triggered activation of Myd88-dependent signaling appears crucial for the proliferation and activation of MDSCs (Delano et al., 2007), but further experimentation is necessary to substantiate this notion. In conclusion, this study documents that an increased level of mononuclear MDSCs is a general characteristic of IBD, that these levels are further elevated upon exacerbation of disease, and that this effect may constitute an endogenous response to chronic mucosal inflammation. As to whether the mobilization of MDSCs directly affects the progression of IBD (e.g., through reducing antibacterial immunity) needs further exploration.
DECLARATION OF INTEREST This work was supported by the National Natural Science Foundation of China (NO 81272737). The authors have declared no financial interest in the present work. The authors alone are responsible for the content and writing of the paper.
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