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Original Article Increased circulating interleukin 10-secreting B cells in patients with dilated cardiomyopathy

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Original Article

Increased circulating interleukin 10-secreting

B cells in patients with dilated cardiomyopathy

Yujie Guo*, Zhihong Cen*, Bin Wei*, Weifeng Wu, Qiuxi Zhou

Department of Cardiology, First Affiliated Hospital of Guangxi Medical University, Guangxi Cardiovascular Institute,

Nanning 530021, China. *Equal contributors.

Received May 22, 2015; Accepted June 28, 2015; Epub July 1, 2015; Published July 15, 2015

Abstract: Objectives: Regulatory B cells (Bregs) have recently been implicated in the pathogenesis of autoimmune diseases. However, their role in the pathogenesis of dilated cardiomyopathy (DCM) remains uncertain. We exam-ined the levels of circulating interleukin 10 (IL-10)-secreting Bregs in patients with DCM. Methods: The frequency of CD19+IL-10+-, CD19+CD5+-, CD19+CD5+CD1d+-, and CD19+CD5+CD1d+IL-10+-B cells in peripheral blood from

DCM patients, heart failure (HF) control patients, and healthy controls (HCs) were analyzed by flow cytometry. Results: DCM and HF control patients showed significantly elevated heart rate, increased left ventricular end-diastolic and

left ventricular end-systolic diameters, higher levels of serum brain natriuretic peptide and reduced left ventricular

ejection fraction, compared to subjects in the HC group. DCM patients showed significantly elevated levels of cir -culating CD19+IL-10+-, CD19+CD5+-, CD19+CD5+CD1d+-, and CD19+CD5+CD1d+IL-10+-B cells than HF control and HC patients (all P < 0.05). Conclusions: DCM patients exhibited elevated peripheral blood IL-10-secreting B cell levels, suggesting that IL-10-secreting B cells may play an important role in the pathogenesis of DCM.

Keywords: IL-10-secreting B cells, regulatory B cells, dilated cardiomyopathy

Introduction

Dilated cardiomyopathy (DCM) is a condition of the heart characterized by systolic dysfunction and ventricular dilation that may progress into terminal heart failure and, eventually, heart transplantation [1, 2]. Myocyte injury and myo-cardial fibrosis, occurring as a result of overac-tive immune responses after viral heart infec-tion, are critical factors in the progression of DCM [3]. Nevertheless, the underlying mecha-nisms responsible for the development of DCM are not completely understood.

Regulatory B cells (Bregs) are a small subset of B cells that have important roles as suppres-sors of inflammation and other autoimmune responses [4, 5]. Various phenotypic variants of Bregs have been identified that express dif-ferent cell markers, with emerging evidence indicating that a subset of Bregs secrete inter-leukin-10 (IL-10), a cytokine that plays a key role in the suppression of autoimmune respons-es [6-8]. Bregs were first defined as a subset of B cells expressing the CD5 and CD1d cell

mark-ers [9]. Subsequent studies showed that CD5+ CD1d+ B cells were associated with the sup-pression of inflammation and autoimmune dis-ease through IL-10 secretion [10]. We previous-ly showed that the level of IL-10-producing B cells was increased in a mice model of CVB3-induced acute viral myocarditis (AVMC) [11]. Regardless of the close relationship between AVMC and DCM, little is known about the func-tion of Bregs in DCM.

In our present study we analyzed the percent-age of circulating CD19+IL-10+-, CD19+CD5+-, CD19+CD5+CD1d+-, and CD19+CD5+CD1d+IL-10+-B cells in patients with DCM. Our results may provide new insights into the role of Breg cells in DCM.

Materials and methods

Patients and controls

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IL-10-secreting B cells in dilated cardiomyopathy

stages of heart failure (DCM group); 30 patients presenting heart failure due to causes other than DCM, such as ischemic heart disease and valvular heart disease (HF control group); and 30 gender-, age-, and ethnicity-matched healthy individuals (HC group).

DCM was diagnosed in patients in accordance with World Health Organization criteria and guidelines [12]. HF controls were included in the study in an attempt to diminish the poten-tial confounding effects caused by the etiologi-cal heterogeneity of heart failure. A diagnosis of DCM was discarded in HC individuals by a nor-mal echocardiogram. Potential subjects pre-senting inflammatory or autoimmune disease, diabetes mellitus, or other underlying acute or chronic disease, were receiving anti-inflamma-tory or immunosuppressive medications, or had recently been treated with antibiotics were excluded from the cohort.

The Guangxi Medical University Ethics Com- mittee (Guangxi, China) approved the experi-mental protocol. Each subject provided written informed consent to participate in the study. Clinical characteristics of the study subjects are summarized in Table 1.

Laboratory tests

Venous blood was drawn from all study partici-pants on the morning following enrollment into

th FITC-anti-CD19 (BD Biosciences), PerCP-Cy5.5-anti-CD5 (eBioscience, San Diego, CA, USA), and APC-anti-CD1d (eBioscience). After treatment with a fixation solution and permea-bilization solution (BD Biosciences), intracellu-lar IL-10 cytokine was stained by adding PE-anti-IL-10 (BD Biosciences). Stained cells were ana-lyzed on a FACSCalibur flow cytometer (BD Bioscience). Data was acquired and analyzed using FlowJo 7.6 (Tree Star Inc., San Carlos, CA, USA).

Statistical analyses

Data were expressed as mean ± standard de- viation (SD). One-way analysis of variance (AN- OVA) was performed to compare mean values from different samples. Differences between sample means for which P < 0.05 were deemed to be statistically significant. Statistical analy-ses were performed using SPSS 17.0 software (IBM, USA).

Results

Clinical characteristics of study participants

DCM and HF control patients presented signifi-cantly elevated heart rate, increased left ven-tricular diastolic and left venven-tricular end-systolic diameters (LVEDD and LVESD, respec-tively), higher levels of serum BNP, and reduced left ventricular ejection fraction (LVEF), com-Table 1.Clinical characteristics of DCM patients and control

individu-als

Characteristics HC N = 30 HF N = 30 DCM N = 30 Age (years) 41.9 ± 6.4 43.6 ± 7.2 40.2 ± 6.1

Male/female (n) 19/11 20/10 19/11

Heart rate (beats/min) 70.9 ± 9.5** 83.6 ± 7.7 85.6 ± 8.6 Functional class (NYHA)

I 30 0 0

II 0 3 4

III 0 19 20

IV 0 8 6

LVEF (%) 63.1 ± 6.0** 37.4 ± 10.5 36.2 ± 10.1 LVEDD (mm) 45.3 ± 7.1** 59.8 ± 13.1 61.6 ± 11.7 LVESD (mm) 34.5 ± 7.3** 52.7 ± 11.8 53.1 ± 10.5 BNP (pg/ml) 54.7 ± 18.2** 2362.9 ± 2234.3 2686.5 ± 2164.7

DCM, dilated cardiomyopathy group; HF, heart failure control group; HC, healthy control group; NYHA, New York Heart Association; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; BNP, brain natriuretic peptide. Class I: no cardiac symptoms even with activity; Class II:

car-diac symptoms lead to some difficulties with usual activity; Class III: carcar-diac symptoms

make patient unable to perform usual physical activity; Class IV: cardiac symptoms pres-ent even at rest. Data expressed as mean ± SD; **: P < 0.01.

the study. The levels of serum brain natriuretic peptide (BNP) were deter- mined.

Flow cytometry analysis

Peripheral blood (500 µl) from individuals in each group was drawn in 50 U/ ml heparin and incubated in the presence of 25 ng/ ml phorbol myristate ace-tate (PMA; Sigma-Aldrich, St. Louis, MO, USA), 1 µg/ ml ionomycin (Sigma-Ald- rich), and 1 µl/106 cells

[image:2.612.90.394.96.267.2]
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pared to subjects in the HC group (Table 1; Figures 1 and 2).

Frequencies of circulating Breg subtypes in DCM patients and controls

We sorted and determined the frequency of CD19+IL-10+-, CD19+CD5+-, CD19+CD5+ CD1d+-, and CD19+CD5+CD1d+IL-10+-B cells in peripheral blood from DC, HF control, and HC individuals by flow cytometry analysis (Figure 3). The frequency of CD19+IL-10+ B cells was higher in peripheral blood from DCM patients (5.19 ± 2.64%) than in blood from HF controls (1.31 ± 0.63%) and HC individuals (2.25 ± 0.87%) (Figure 4A). Similarly, the frequency of CD19+CD5+ B cells was significantly higher in DCM patients than in HF control and HC indi-viduals (7.40 ± 4.06%, 2.16 ± 1.23%, and 2.30 ± 0.94%, respectively; P < 0.01; Figure 4B). Compared to HF control patients and HC indi-viduals, DCM patients showed a higher per-centage of CD19+CD5+CD1d+ B cells (1.15 ± 0.52%, 1.47 ± 0.63%, and 4.02 ± 2.41%, respectively; P<0.05) and CD19+CD5+CD1d+

[image:3.612.85.521.67.633.2]

Figure 1. Echocardiographic variables of DCM patients and controls. Echocardio-graphic variables were measured in DCM and HF control patients and in HC individ-uals. A. Left ventricular ejection fraction (LVEF). B. Left ventricular end-diastolic diameter (LVEDD). C. Left ventricular end-systolic diameter (LVESD). Data are pre-sented as mean values for each group, with error bars indicating the SD; ** indi-cates P < 0.01.

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[image:4.792.93.701.74.496.2]

IL-10-secreting B cells in dilated cardiomyopathy

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IL-10+ B cells (1.91 ± 1.13%, 1.57 ± 0.59%, and 4.89 ± 1.66%, respectively; P < 0.01) in peripheral blood (Figure 4C and 4D). Con- sistently, DCM patients showed significantly higher frequencies of each Breg subtype than HF patients and HC individuals. The differen- ces in Breg frequencies between HF control patients and HC individuals were not significant (P > 0.05).

Discussion

IL-10-secreting B cells play an important role in maintaining immune homeostasis [9, 13, 14]. However, the role of IL-10-secreting B cells in the progression of DCM has not been investi-gated. In this study, we compared the frequen-cies of different circulating Breg subtypes in DCM patients, HF controls, and healthy

[image:5.612.92.520.72.538.2]
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IL-10-secreting B cells in dilated cardiomyopathy

jects. We determined that the frequency of cir-culating Bregs, especially those producing IL- 10, was dramatically higher in DCM patients than in individuals from the control groups. Bregs are important suppressors of inflamma-tion and other autoimmune responses, produc-ing regulatory cytokines, such as IL-10, [6-8, 13, 15] TNF-α, lymphotoxin, IL-6, and IL-17 [16, 17] The rare IL-10-secreting B cells are distin-guished from other Bregs by their capacity to produce anti-inflammatory cytokine IL-10 [14, 18]. IL-10-secreting B cell development and function have not been completely elucidated; however, B cell antigen receptor- and toll-like receptor-related signals appear to be involved [19, 20]. Emerging evidence indicates that CD5+CD1d+ B cell subsets are rich in IL-10+ B cells, and that the regulation of CD5+CD1d+ B cells function depends on IL-10 [10, 21]. CD19+CD5+CD1dhiIL-10+ B cells play a critical role in the maintenance of immune homeosta-sis [22].

Increased levels of IL-10-secreting B cells were shown to regulate the immune responses in patients with human immunodeficiency virus infection, chronic hepatitis B virus infection, systemic lupus erythematosus, immune throm-bocytopenia, and experimental autoimmune encephalomyelitis, suggesting that these cells were involved in the immunological response elicited after viral infections and in T cell-medi-ated autoimmune diseases [5, 13, 23-26]. On the other hand, the level of IL-10-secreting B cells was reduced in rheumatoid arthritis pa- tients [27]. Clinical trials and animal experi-ments studying this B cell subset have provided inconsistent results, suggesting that IL-10-pro- ducing Bregs may be elevated in some but re- duced in other autoimmune diseases.

A number of patients with viral myocarditis (VMC) may gradually progress to DCM with sys-tolic dysfunction and ventricular dilation. An increasing number of studies suggest that myo-cardial tissue injury can occur not only by viral proliferation, but also as a result of inflamma-tion and other autoimmune responses [28, 29]. Some researchers perceive VMC and DCM as different stages of the same disease. Our unpublished data indicated that the level of IL-10-producing B cells was elevated in CVB3-induced AVMC mice. However, it was not clear

whether a similar increase in IL-10-producing B cells would be observed in DCM patients. Yu et al. [30] have demonstrated that the levels of B cell subsets secreting IL-1β, IL-6, IL-10, IL-17, and TNF-α were all elevated in DCM patients; however, only TNF-α-producing B cell frequencies were significantly higher in DCM patients than in healthy controls. Even though our present study is similar to the one abo- ve, important differences can be identified between the two. Our data showed that the frequencies of CD19+IL-10+-, CD19+CD5+-, CD19+CD5+CD1d+-, and CD19+CD5+CD1d+ IL10+-B cells were significantly higher in DCM patients than in HF control and HC individuals. However, no statistically significant differences in the levels of Breg subsets between HC and HF control patients, including those with isch-emic heart disease and valvular heart disease, were found (Figure 4).

Our study presents a number of limitations, including a small sample size and the unmet challenge of eliminating the confounding effects of heart failure etiological heterogene-ity, in spite of the inclusion of the HF control group. As part of future efforts, it will be impor-tant to study bigger patient cohorts, including DCM patients with normal cardiac function. Further prospective studies should also explore the relationship between B cells and myocardi-al fibrosis in order to elucidate the pathophysi-ological function of B cells in patients with DCM.

Conclusion

The results of the present study show, for the first time, that the levels of IL-10-secreting Bregs are significantly increased in DCM pa- tients, suggesting that this Breg subset may participate in the pathogenesis of DCM. How- ever, how B cells differentiate into IL-10-se- creting Bregs and what functions these cells play in DCM progression remain to be further investigated.

Acknowledgements

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Disclosure of conflict of interest

None.

Address correspondence to: Dr. Weifeng Wu, De-

partment of Cardiology, The First Affiliated Hospital

of Guangxi Medical University, Nanning 530021, Guangxi, China. Tel: 771-5350642; Fax: +86-771-5358562; E-mail: wucna65@163.com

References

[1] Luk A, Ahn E, Soor GS and Butany J. Dilated cardiomyopathy: a review. J Clin Pathol 2009; 62: 219-225.

[2] Jefferies JL and Towbin JA. Dilated cardiomy-opathy. Lancet 2010; 375: 752-762.

[3] Sagar S, Liu PP and Cooper LT Jr. Myocarditis. Lancet 2012; 379: 738-747.

[4] Mann MK, Ray A, Basu S, Karp CL and Dittel BN. Pathogenic and regulatory roles for B cells in experimental autoimmune encephalomyeli-tis. Autoimmunity 2012; 45: 388-399.

[5] Das A, Ellis G, Pallant C, Lopes AR, Khanna P, Peppa D, Chen A, Blair P, Dusheiko G, Gill U, Kennedy PT, Brunetto M, Lampertico P, Mauri C and Maini MK. IL-10-producing regulatory B cells in the pathogenesis of chronic hepatitis B virus infection. J Immunol 2012; 189: 3925-3935.

[6] Mauri C and Ehrenstein MR. The “short” histo-ry of regulatohisto-ry B cells. Trends Immunol 2008; 29: 34-40.

[7] Watanabe R, Ishiura N, Nakashima H, Kuwano Y, Okochi H, Tamaki K, Sato S, Tedder TF and Fujimoto M. Regulatory B cells (B10 cells) have a suppressive role in murine lupus: CD19 and

B10 cell deficiency exacerbates systemic auto -immunity. J Immunol 2010; 184: 4801-4809. [8] Ronet C, Hauyon-La Torre Y, Revaz-Breton M,

Mastelic B, Tacchini-Cottier F, Louis J and Launois P. Regulatory B cells shape the devel-opment of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production. J Immunol 2010; 184: 886-894.

[9] Yanaba K, Bouaziz JD, Haas KM, Poe JC, Fujimoto M and Tedder TF. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype

controls T cell-dependent inflammatory re -sponses. Immunity 2008; 28: 639-650. [10] Jeong YI, Hong SH, Cho SH, Lee WJ and Lee SE.

Induction of IL-10-producing CD1dhighCD5+ regulatory B cells following Babesia microti-in-fection. PLoS One 2012; 7: e46553.

[11] Cen Z, Guo Y, Kong Q, Zhou Q and Wu W. IL-10-producing B cells involved in the pathogenesis of Coxsackie virus B3-induced acute viral

myo-carditis. Int J Clin Exp Pathol 2015; 8: 830-835.

[12] Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I, Martin I and Nordet P. Report of the 1995 World Health Organization/ International Society and Federation of Cardio-

logy Task Force on the Definition and Classi-fication of cardiomyopathies. Circulation 1996;

93: 841-842.

[13] Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM, Szabolcs PM, Bernstein SH, Magro CM, Williams AD, Hall RP, St Clair EW and Tedder TF. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood 2011; 117: 530-541.

[14] Carter NA, Vasconcellos R, Rosser EC, Tulone C, Munoz-Suano A, Kamanaka M, Ehrenstein MR, Flavell RA and Mauri C. Mice lacking en-dogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a de-crease in regulatory T cells. J Immunol 2011; 186: 5569-5579.

[15] Bouaziz JD, Calbo S, Maho-Vaillant M, Saussine A, Bagot M, Bensussan A and Musette P. IL-10 produced by activated human B cells regulates CD4 (+) T-cell activation in vitro. Eur J Immunol 2010; 40: 2686-2691.

[16] Bar-Or A, Fawaz L, Fan B, Darlington PJ, Rieger A, Ghorayeb C, Calabresi PA, Waubant E, Hauser SL, Zhang J and Smith CH. Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS? Ann Neurol 2010; 67: 452-461.

[17] Vazquez-Tello A, Halwani R, Li R, Nadigel J, Bar-Or A, Mazer BD, Eidelman DH, Al-Muhsen S and Hamid Q. IL-17A and IL-17F expression in B lymphocytes. Int Arch Allergy Immunol 2012; 157: 406-416.

[18] Candando KM, Lykken JM and Tedder TF. B10 cell regulation of health and disease. Immunol Rev 2014; 259: 259-272.

[19] Yanaba K, Bouaziz JD, Matsushita T, Tsubata T and Tedder TF. The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals. J Immunol 2009; 182: 7459-7472.

[20] Yoshizaki A, Miyagaki T, DiLillo DJ, Matsushita T, Horikawa M, Kountikov EI, Spolski R, Poe JC, Leonard WJ and Tedder TF. Regulatory B cells control T-cell autoimmunity through IL-21-de- pendent cognate interactions. Nature 2012; 491: 264-268.

(8)

autoim-IL-10-secreting B cells in dilated cardiomyopathy

munity by provision of IL-10. Nat Immunol 2002; 3: 944-950.

[22] Xing C, Ma N, Xiao H, Wang X, Zheng M, Han G, Chen G, Hou C, Shen B, Li Y and Wang R. Critical role for thymic CD19+CD5+CD1dhiIL- 10+ regulatory B cells in immune homeosta-sis. J Leukoc Biol 2015; 97: 547-556.

[23] Liu J, Zhan W, Kim CJ, Clayton K, Zhao H, Lee E, Cao JC, Ziegler B, Gregor A, Yue FY, Huibner S, MacParland S, Schwartz J, Song HH, Benko E, Gyenes G, Kovacs C, Kaul R and Ostrowski M. IL-10-producing B cells are induced early in

HIV-1 infection and suppress HIV-1-specific T

cell responses. PLoS One 2014; 9: e89236. [24] Yang X, Yang J, Chu Y, Wang J, Guan M, Zhu X,

Xue Y and Zou H. T follicular helper cells medi-ate expansion of regulatory B cells via IL-21 in Lupus-prone MRL/lpr mice. PLoS One 2013; 8: e62855.

[25] Matsushita T, Horikawa M, Iwata Y and Tedder TF. Regulatory B cells (B10 cells) and regula-tory T cells have independent roles in control-ling experimental autoimmune encephalomy-elitis initiation and late-phase immunopatho-genesis. J Immunol 2010; 185: 2240-2252.

[26] Hua F, Ji L, Zhan Y, Li F, Zou S, Chen L, Gao S, Li Y, Chen H and Cheng Y. Aberrant frequency of IL-10-producing B cells and its association with Treg/Th17 in adult primary immune thrombocytopenia patients. Biomed Res Int 2014; 2014: 571302.

[27] Liang MH, Couto MC, Duarte CC, Gall V, White P, Naides S, Schumacher HR Jr, Hwang AS, Holers VM, Deane KD, Gupta S, Ho IC, Finckh A, Kopec J, Choi CB and Sayre EC. An

Internet-based technique for the identification of per

-sons with symptoms of inflammatory polyar -thritis of less than 12 weeks. Clin Rheumatol 2015; 34: 465-470.

[28] Reddy J, Massilamany C, Buskiewicz I and Huber SA. Autoimmunity in viral myocarditis. Curr Opin Rheumatol 2013; 25: 502-508. [29] Yajima T and Knowlton KU. Viral myocarditis:

from the perspective of the virus. Circulation 2009; 119: 2615-2624.

[30] Yu M, Wen S, Wang M, Liang W, Li HH, Long Q, Guo HP, Liao YH and Yuan J.

Figure

Table 1. Clinical characteristics of DCM patients and control individu-als
Figure 2. Serum brain natriuretic peptide (BNP) in DCM patients and controls. Brain natriuretic peptide (BNP) was measured in serum from individuals in the DCM, HF control, and HC groups
Figure 3. Fluorescence-activated cell sorting (FACS) of Bregs in peripheral blood from DCM patients and controls
Figure 4. Graphic representation of Bregs FACS data from DCM patients and controls. CD19+IL-10+- (A), CD19+CD5+- (B), CD19+CD5+CD1d+- (C), and CD19+CD5+CD1d+IL-10+-B cell (D) frequencies in DCM patients, HF controls, and HC subjects

References

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