CCR4 Controls the Suppressive Effects of Regulatory T Cells on Early and Late Events during Severe Sepsis

RESEARCH ARTICLE CCR4 Controls the Suppressive Effects of Regulatory T Cells on Early and Late Events during Severe Sepsis Raphael Molinaro1, Cyntia Pecli1, Rafael F. Guilherme1, José Carlos Alves-Filho2, Fernando Q. Cunha2, Claudio Canetti3, Steven L. Kunkel4, Marcelo T. Bozza5, Claudia F. Benjamim1* 1 Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 2 Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil, 3 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 4 Department of Pathology, University of Michigan, Ann Arbor, Michigan, 48109, United States of America, 5 Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil * Abstract OPEN ACCESS Citation: Molinaro R, Pecli C, Guilherme RF, AlvesFilho JC, Cunha FQ, Canetti C, et al. (2015) CCR4 Controls the Suppressive Effects of Regulatory T Cells on Early and Late Events during Severe Sepsis. PLoS ONE 10(7): e0133227. doi:10.1371/journal. pone.0133227 Editor: Ari Waisman, University Medical Center of the Johannes Gutenberg University of Mainz, GERMANY Received: December 18, 2012 Accepted: June 25, 2015 Published: July 21, 2015 Copyright: © 2015 Molinaro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and the activation of different types of cells. This altered state of cell activation, termed leukocyte reprogramming, contributes to patient outcome. However, the understanding of the process underlying sepsis and the role of regulatory T cells (Tregs) in sepsis remains to be elucidated. In this study, we investigated the role of CCR4, the CCL17/ CCL22 chemokine receptor, in the innate and acquired immune responses during severe sepsis and the role of Tregs in effecting the outcome. In contrast with wild-type (WT) mice subjected to cecal ligation and puncture (CLP) sepsis, CCR4-deficient (CCR4-/-) septic mice presented an increased survival rate, significant neutrophil migration toward the infection site, a low bacterial count in the peritoneum, and reduced lung inflammation and serum cytokine levels. Thus, a better early host response may favor an adequate long-term response. Consequently, the CCR4-/- septic mice were not susceptible to secondary fungal infection, in contrast with the WT septic mice. Furthermore, Tregs cells from the CCR4-/septic mice showed reduced suppressive effects on neutrophil migration (both in vivo and in vitro), lymphocyte proliferation and ROS production from activated neutrophils, in contrast with what was observed for Tregs from the WT septic mice. These data show that CCR4 is involved in immunosuppression after severe sepsis and suggest that CCR4+ Tregs negatively modulate the short and long-term immune responses. Funding: Funding provided by Universal- CNPq, APQ1- FAPERJ and Fellowship - CAPES. Introduction Competing Interests: The authors have declared that no competing interests exist. More than 750,000 cases of sepsis occur annually in the United States. Septic shock is undoubtedly the most common lethal illness, accounting for approximately 10% of intensive care unit PLOS ONE | DOI:10.1371/journal.pone.0133227 July 21, 2015 1 / 21 CCR4 Modulates Treg Effects in Severe Sepsis admissions. In 2008, the mortality rates after severe sepsis and septic shock were approximately 27% and 45%, respectively [1]. Despite the availability of an increasing array of potent antibiotics and intensive medical care, sepsis mortality remains high [2]. One of the main consequences of severe sepsis is a profound state of immunosuppression, which is dependent on diverse cellular and molecular mechanisms [3]. Previous studies have shown that 80% of post-sepsis patients die within 8 years due to non-septic causes. The causes of death for these patients have included cancer, pneumonia and cardiovascular disease, which have all been correlated with the first insult [4]. In most cases, the post-sepsis state is clinically correlated with high risks of secondary infection and mortality [5,6]. The basis for this immunosuppression remains poorly understood. However, several lines of evidence have established that death from septic shock may be due to distinct mechanisms that occur over time, including enhanced leukocyte apoptosis, lymphocyte anergy, epigenetic changes and cellular reprogramming [7,8]. The latter mechanism is characterized by different activation profiles of cells such as dendritic cells [9], macrophages [10] and neutrophils [11–13] after severe sepsis. Patients after sepsis are clearly immunosuppressed, as evidenced by their development of immune anergy, frequent inability to eradicate primary infection, reduced expression of antigen-presenting molecules, shift to anti-inflammatory cytokines and toward a Th2 response and propensity to acquire secondary nosocomial infections [14]. CCR4 is a high-affinity receptor for CCL17 and CCL22 found on dendritic cells, macrophages, NK cells, platelets and basophils, but it is predominantly known for its expression on T cells, especially those with the Th2 phenotype [15]. Increased expression of CCR4 and its ligands occurs in several diseases, including pulmonary fibrosis [16,17], eosinophilic pneumonia [18], hepatic inflammation [19], granuloma development [20], and diabetes [21]. Furthermore, colon ascendens stent peritonitis (CASP)-induced sepsis in mice has been shown to increase CCL17 and CCL22 mRNA levels in the spleen and liver [22]. Additionally, a high CCL17 level has been detected in supernatant from resident peritoneal macrophages treated with LPS [23]. The majority of CCR4+ lymphocytes appear to be Th2-polarized CD4+ T cells that also exert suppressive functions via the production of immunosuppressive cytokines [24]. The chemokine receptors CCR4 and CCR8 are functionally expressed on human peripheral blood [25] and murine CD4+CD25+ T regulatory cells (Tregs) [26]. In patients with ovarian carcinoma, the host response to the tumor has been shown to be inhibited by Foxp3+CCR4+ Tregs recruited to the tumor by CCL17 and CCL22 [27]. In a mouse cardiac allotransplantation model, CCR4 and CCL22 have been shown to be up-regulated in tolerized allografts, and tolerance induction has not been achieved in CCR4-deficient (CCR4−/−) mice, indicating an important role of CCR4 in the generation and/or recruitment of Foxp3+ Tregs to cardiac allografts [28]. We have previously described post-inflammatory immunosuppression in a mouse model of sepsis induced by the cecal ligation and puncture (CLP) procedure and challenged with in (...truncated)