Hindawi Publishing Corporation Mediators of Inflammation Volume 2016, Article ID 3635809, 16 pages http://dx.doi.org/10.1155/2016/3635809 Research Article CCR9 Is a Key Regulator of Early Phases of Allergic Airway Inflammation C. López-Pacheco,1,2 G. Soldevila,2 G. Du Pont,1,2 R. Hernández-Pando,3 and E. A. García-Zepeda1,2 1 CBRL, Ciudad de Mexico,´ Mexico 2Departamento de Inmunolog´ıa, Instituto de Investigaciones Biomedicas,´ Universidad Nacional Autonoma´ de Mexico,´ 04510 Ciudad de Mexico,´ Mexico 3Departamento de Patolog´ıa Experimental, Instituto Nacional de Ciencias Medicas´ y Nutricion´ “Salvador Zubiran”,´ Tlalpan, 14080 Ciudad de Mexico,´ Mexico Correspondence should be addressed to E. A. Garc´ıa-Zepeda; [email protected] Received 9 May 2016; Accepted 7 August 2016 Academic Editor: Carolina T. Pineiro˜ Copyright © 2016 C. Lopez-Pacheco´ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Airway inflammation is the most common hallmark of allergic asthma. Chemokine receptors involved in leukocyte recruitment are closely related to the pathology in asthma. CCR9 has been described as a homeostatic and inflammatory chemokine receptor, but its role and that of its ligand CCL25 during lung inflammation remain unknown. To investigate the role of CCR9 as a modulator of airway inflammation, we established an OVA-induced allergic inflammation model in CCR9-deficient mice. Here, we report the expression of CCR9 and CCL25 as early as 6 hours post-OVA challenge in eosinophils and T-lymphocytes. Moreover, in challenged CCR9-deficient mice, cell recruitment was impaired at peribronchial and perivenular levels. OVA-administration in CCR9-deficient mice leads to a less inflammatory cell recruitment, which modifies the expression of IL-10, CCL11, and CCL25 at 24 hours afterOVA challenge. In contrast, the secretion of IL-4 and IL-5 was not affected in CCR9-deficient mice compared to WT mice. These results demonstrate for the first time that CCR9 and CCL25 expressions are induced in the early stages of airway inflammation and they have an important role modulating eosinophils and lymphocytes recruitment at the first stages of inflammatory process, suggesting that they might be a potential target to regulate inflammation in asthma. 1. Introduction and are regulated by Th2 cells-derived cytokines that are expressed by epithelial and inflammatory cells [7]. Allergic asthma is a chronic disease that affects more than 300 The complexity of cell trafficking during lung inflam- million people worldwide [1]. Its prevalence and mortality mation is tightly regulated by chemokines [8]. In this con- have been more common in the recent decades and it became text, it has been established that overexpression of certain an important health issue due to its increasing medical chemokine receptors is correlated with the localization and care expenses and a reduction in worker productivity. It is activation of inflammatory cells during and after an allergen estimated that there will be more than 100 million of new challenge. Increasing evidence has supported the role of asthmatics in the next decade [2–5]. chemokine receptors in allergic airway inflammation; how- Allergen-triggered airway inflammation mediated by ever, the involvement of CCR9 in asthma remains unclear. specific IgE is the most common feature of asthma [2]. CCR9 and its unique ligand, CCL25 (thymus-expressed Airway inflammation is mediated by the recruitment of chemokine, TECK), were originally described in thymus granulocytes and Th2 lymphocytes [6]; both are described where they were shown to play a role in thymocyte devel- as the main cellular effectors of the inflammatory process opment [9–11]. Also, their homeostatic expression in small 2 Mediators of Inflammation intestine is related with cell homing to the gut. Moreover, 2.3. Measurement of OVA-Specific Mouse IgE by ELISA. For the involvement of CCR9 under inflammatory conditions detection of serum OVA-specific IgE, 96-well ELISA plates has been widely described in the gut [10, 12]. However, its (Santa Cruz, TX, USA) were coated with 20 gofOVAin expression, function, and regulation in the lungs are unclear. 0.1 M carbonate buffer (pH 8.3). Plates were blocked one hour The expression of CCR9 in inflammatory cells that are with PBS-SFB 2%. Samples were diluted 10-fold and then recruited to the lungs has been described. After in vitro stimu- added to the wells. Antibodies were detected with rat anti- lation with proinflammatory mediators, human eosinophils- mouse IgE specific antibody (Zymed, CA, USA), followed derived cell lines upregulate the expression of CCR9 and by the addition of a secondary anti-rat biotinylated antibody respond to CCL25 in chemotaxis assays [13]. Also, CCR9 (BioLegend, CA, USA). Avidin-HRP (BD OptEIA, USA) was is upregulated in peripheral blood eosinophils of asthmatic added and reaction was developed with tetramethylbenzidine subjects [14]. Moreover, inflammatory macrophages which substrate (KPL, MD, USA) and stopped by adding H2SO4 2N. are crucial cells in allergic inflammation upregulate CCR9 Optical density of samples was measured at 450 nm (OD450) expression in the inflammatory microenvironment [15]. Den- using a Modulus II microplate reader (Promega, WI, USA). dritic cells are some of the most important effectors in early stages of airway sensitization. In these cells, CCR9 expression 2.4. Bronchoalveolar Lavage Fluid (BALF) Cell Analysis. is upregulated by IL-4 [16]; however, the effects of other BALF obtained from challenged mice was collected at 2, Th2-derived cytokines in this induction and its impact on 6, 24, and 48 h after the last OVA administration as indi- the regulation of the inflammatory process have not been cated. Briefly, 800 LofPBS/EDTA0.05Msolutionwas characterized. repeatedly introduced into the mice lungs and collected We have previously shown that a subpopulation of CD4+ until recovered volume was 4 mL. BALF was centrifuged ∘ CD25+ FoxP3+ T-lymphocytes have regulatory functions at 1500 rpm for 10 min at 4 C. Cells were suspended in and depend on CCR9 expression to control pathogen- 1 mL of PBS and total number of cells were quantified induced inflammation in the gut [17]. Therefore, the aim using standard staining protocols and differential cell types of this work was to analyse the role of CCR9 in regulating weredeterminedoncytospuncellsstainedwithDiffQuick cell recruitment and modulating the inflammatory process (Dade Behring Inc., Newark, DE) and then examined by during airway sensitization. Consistent with previous stud- microscopy. Morphological analysis of cells distinguished ies, we report that CCR9 is expressed by eosinophils and lymphocytes, macrophages, eosinophils, and neutrophils. T-lymphocytes. In addition, we demonstrate that CCR9- Remaining cells were processed for flow cytometry analysis. deficient mice (KO) sensitized with OVAshowed an impaired recruitment of granulocytes during the first stages of inflam- 2.5. Cytokines Determination in BALF. Levels of IL-4, IL- mation resulting in a diminished inflammatory response. 5, TNF-, and IL-10 cytokines were measured in BAL These data suggest that CCR9 expression plays a key role in supernatants using commercial ELISA kits (ELISA Max6, the modulation of cell recruitment into the airways during BioLegend, San Diego, CA, USA) according to the manu- pulmonary inflammation in asthma. facturer’s instructions. Additionally, a murine MultiAnalyte ELISArray kit (Qiagen Inc., Valencia, CA) was used to 2. Materials and Methods quantitate the levels of 12 proinflammatory cytokines and performed according to the manufacturer’s instructions. 2.1. Animals. Six- to eight-week-old C57BL/6 female mice were used in all experiments and maintained under 2.6. Flow Cytometry Analysis. To analyse chemokine receptor pathogen-free conditions. Water and food were supplied ad expression by FACS, cells were incubated first with blocking libitum. CCR9-deficient mice were kindly provided by Dr. M. solution (mouse serum in PBS) during 20 minutes, followed A. Wurbel (Boston Children’s Hospital, Boston, MA, USA). by a second incubation with a biotinylated mouse anti-CCR9 6 All experimental procedures involving animals were handled antibody (1 g/10 cells) (eBioscience, CA, USA) in FACS in strict accordance with good animal practices as defined buffer (PBS containing 2% fetal bovine serum and 0.1% ∘ andapprovedbytheAnimalExperimentalBio-Ethics NaN3)for30minat4C. Finally cells were incubated with Guidelines of the Instituto de Investigaciones Biomedicas,´ a solution of streptavidin-APC or streptavidin-PerCP during ∘ Universidad Nacional Autonoma´ de Mexico.´ 30 min at 4 Cinthedark. For phenotypic detection of other cell populations, single 2.2. Allergic Airway Inflammation Murine Model. To induce cell suspensions were preincubated with a mixture of anti- airway inflammation we followed a previously reported CD16/CD32 antibodies (BioLegend, CA, USA) during 10 min protocol with minor modifications [18, 19]. Briefly, mice at room temperature. Following first incubation and several were treated by intraperitoneal administration with 10 gof washes, cells were incubated with either anti-CD4-PeCy7, ovalbumin (OVA, Sigma Chemical, MO, USA), adsorbed to anti-CD8-PE, anti-Gr1-PeCy5 (BioLegend, CA, USA), or 10 mg of alum (Pierce, NJ, USA), and suspended in saline anti-Siglec (BD, CA, USA)