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Circulating innate lymphoid cells are differentially regulated in allergic and nonallergic subjects

2016; Elsevier BV; Volume: 138; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2015.12.1325

1097-6825

Vincent Lombardi, Chloé Beuraud, Catherine Neukirch, Hélène Moussu, Lise Morizur, S. Horiot, Sonia Luce, Erik Wambre, Peter S. Linsley, Sylvie Chollet‐Martin, Véronique Baron‐Bodo, Michel Aubier, Philippe Moingeon,

Asthma and respiratory diseases

A new family of lymphocyte-like cells, termed innate lymphoid cells (ILCs), lacking a specific antigen receptor, belonging to the innate immune system, and with a likely proinflammatory role in allergic rhinoconjunctivitis and asthma has been described recently.1Artis D. Spits H. The biology of innate lymphoid cells.Nature. 2015; 517: 293-301Crossref PubMed Scopus (1057) Google Scholar, 2Bartemes K.R. Kephart G.M. Fox S.J. Kita H. Enhanced innate type 2 immune response in peripheral blood from patients with asthma.J Allergy Clin Immunol. 2014; 134: 671-678Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar ILCs are currently subdivided into 3 subsets, ILC1s, ILC2s, and ILC3s, based on specific patterns of cytokine production matching those of polarized CD4+ T lymphocytes.1Artis D. Spits H. The biology of innate lymphoid cells.Nature. 2015; 517: 293-301Crossref PubMed Scopus (1057) Google Scholar To better understand the dynamics of circulating ILCs in human subjects, we studied the frequencies, phenotypes, and functions of ILC1s, ILC2s, and ILC3s in the peripheral blood of allergic patients with rhinoconjunctivitis (associated or not with asthma), as well as in samples from nonallergic control subjects. We also monitored ILC subsets in patients with grass pollen allergy receiving allergen immunotherapy (AIT). PBMCs were obtained from 63 adult patients with allergic rhinoconjunctivitis and 18 nonallergic subjects, as described in Table E1 in this article's Online Repository at www.jacionline.org. Freshly isolated PBMCs were stained with fluorescently labeled anti-CD45, anti-CD117, anti-CD127, and anti–chemoattractant receptor–homologous molecule expressed on TH2 lymphocyte (CRTH2) antibodies and a lineage cocktail comprising anti-CD3, anti-CD11c, anti-CD14, anti-CD16, anti-CD20, anti-CD34, anti-CD56, anti-CD123, and anti–T-cell receptor α/β antibodies (see the Methods section in this article's Online Repository at www.jacionline.org). Total ILCs were identified as CD45+ cells negative for lineage markers and positive for CD127. ILC2s are CRTH2+, whereas ILC1s and ILC3s are defined as CRTH2−CD117− and CRTH2−CD117+ cells, respectively (Fig 1, A).1Artis D. Spits H. The biology of innate lymphoid cells.Nature. 2015; 517: 293-301Crossref PubMed Scopus (1057) Google Scholar, 3Bernink J.H. Peters C.P. Munneke M. te Velde A.A. Meijer S.L. Weijer K. et al.Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues.Nat Immunol. 2013; 14: 221-229Crossref PubMed Scopus (726) Google Scholar In a first set of experiments, we observed that frequencies of ILC subsets are similar in PBMCs of allergic subjects when compared with those of nonallergic control subjects (Fig 1, B). The presence of high numbers of ILC2s in the blood of nonallergic subjects is intriguing but consistent with the notion that such cells mediate some beneficial activities, including regulation of adipose function4Brestoff J.R. Kim B.S. Saenz S.A. Stine R.R. Monticelli L.A. Sonnenberg G.F. et al.Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity.Nature. 2015; 519: 242-246Crossref PubMed Scopus (657) Google Scholar and repair of damaged tissues.5Rak G.D. Osborne L.C. Siracusa M.C. Kim B.S. Wang K. Bayat A. et al.IL-33-dependent group 2 innate lymphoid cells promote cutaneous wound healing.J Invest Dermatol. 2016; 136: 487-496Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar However, stratifying allergic patients according to their asthma status revealed that higher numbers of circulating ILC2s and ILC3s (P < .05) are found in patients with asthma (Global Initiative for Asthma [GINA] step 1) compared with those without asthma (Fig 1, B). The higher frequencies of ILC2s and ILC3s in the blood of asthmatic patients relative to nonasthmatic subjects might reflect the expansion of those cells before homing to the inflamed lower airways. In contrast, frequencies of ILC1s were not statistically different between allergic subjects with or without asthma and nonallergic control subjects (see Fig E1, A, in this article's Online Repository at www.jacionline.org). The analysis of cytokine production after in vitro stimulation with phorbol 12-myristate 13-acetate and ionomycin revealed that ILC1s from patients with allergic rhinoconjunctivitis produce less IFN-γ compared with cells from nonallergic subjects (Fig 1, C). No differences were observed between groups in terms of IL-4 and IL-13 production by ILC2s or IL-22 production by ILC3s (see Fig E1, B). To further explore ILC2 functions, we performed a whole-transcriptome analysis using total RNA sequencing of blood ILC2s sorted from 3 allergic patients and 3 nonallergic subjects (see Table E2 in this article's Online Repository at www.jacionline.org). Interestingly, we observed an upregulation of FOS, FOSB, and JUN genes (whose products assemble to form the proinflammatory activator protein 1 molecule) in ILC2s from allergic patients (Fig 1, D). In contrast, ILC2s from nonallergic subjects express higher levels of the NKG7, SOCS1, and TBX21 genes (Fig 1, D), which are known to downregulate TH2-associated transcriptional programs6Zhu J. Jankovic D. Oler A.J. Wei G. Sharma S. Hu G. et al.The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses.Immunity. 2012; 37: 660-673Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar and, more generally, inflammatory responses. Collectively, these results establish that blood ILC1s and ILC2s from allergic and nonallergic subjects exhibit distinct functional properties. To compare the effect of allergen exposure on blood ILC2 frequencies7Doherty T.A. Scott D. Walford H.H. Khorram N. Lund S. Baum R. et al.Allergen challenge in allergic rhinitis rapidly induces increased peripheral blood type 2 innate lymphoid cells that express CD84.J Allergy Clin Immunol. 2014; 133: 1203-1205Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar in allergic and nonallergic subjects, we measured peripheral ILC counts during and out of the grass pollen season in those 2 populations. ILC2 and ILC3 frequencies were significantly increased (P < .05) by 2-fold during the grass pollen season only in patients sensitized to grass pollen allergens (Fig 1, E, GP IgE+) but not in those unsensitized to grass pollen allergens (Fig 1, E, GP IgE−) or nonallergic control subjects. It is noteworthy that ILC1 blood counts were not modulated by seasonal allergen exposure (see Fig E1, C), and furthermore, no significant alterations in patterns of cytokine production by ILC1s, ILC2s, or ILC3s were noticed during the pollen season (see Fig E1, D). We subsequently investigated potential differences in surface expression of receptors controlling the homing of blood ILCs to tissues. To this aim, we compared the expression of several chemokine receptors, including CCR4 (involved in the trafficking of TH2 cells8Rivino L. Messi M. Jarrossay D. Lanzavecchia A. Sallusto F. Geginat J. Chemokine receptor expression identifies Pre-T helper (Th)1, Pre-Th2, and nonpolarized cells among human CD4+ central memory T cells.J Exp Med. 2004; 200: 725-735Crossref PubMed Scopus (234) Google Scholar) and CCR10 (a skin-homing receptor overexpressed by CD4+ T cells of patients with atopic dermatitis9Homey B. Alenius H. Muller A. Soto H. Bowman E.P. Yuan W. et al.CCL27-CCR10 interactions regulate T cell-mediated skin inflammation.Nat Med. 2002; 8: 157-165Crossref PubMed Scopus (647) Google Scholar) on circulating ILC subsets from nonallergic subjects (n = 7), as well as allergic patients with either no (n = 10), mild (GINA step 1, n = 14), or severe asthma (GINA step 4, n = 9). When compared with nonallergic subjects, CCR10 expression was significantly higher at the surface of ILC2s from allergic patients (P < .0001), including those with mild asthma (P < .01) and even further in those with more severe asthma (P < .001; Fig 1, F). In contrast, CCR4 expression in ILC2s was similar in all groups (Fig 1, F). The expression of CCR4 and CCR10 by ILC1s and ILC3s was comparable between allergic and nonallergic subjects (see Fig E2 in this article's Online Repository at www.jacionline.org). Altogether, these observations might reflect enhanced capacities of ILC2s from allergic patients to migrate to tissues. Specifically, in asthmatic patients, blood CCR10+ ILC2s could represent a subset of cells with a higher propensity to migrate to the lungs. This hypothesis is consistent with recent evidence that blood ILCs replenish the pool of tissue-resident ILCs in a mouse model of helminth infection.10Gasteiger G. Fan X. Dikiy S. Lee S.Y. Rudensky A.Y. Tissue residency of innate lymphoid cells in lymphoid and non-lymphoid organs.Science. 2015; 350: 981-985Crossref PubMed Scopus (520) Google Scholar Subsequently, we investigated ILC subset frequencies in the blood of patients with grass pollen–induced rhinoconjunctivitis receiving sublingual AIT for 4 months in the context of a double-blind, placebo-controlled study performed in a challenge chamber.11Horak F. Zieglmayer P. Zieglmayer R. Lemell P. Devillier P. Montagut A. et al.Early onset of action of a 5-grass-pollen 300-IR sublingual immunotherapy tablet evaluated in an allergen challenge chamber.J Allergy Clin Immunol. 2009; 124: 471-477Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar In this trial circulating ILCs were monitored in 11 and 13 subjects who received active or placebo tablets, respectively, before and after 2 and 4 months of AIT. Both patients undergoing active and those undergoing placebo treatments exhibited a minor progressive decrease in circulating ILC2 counts in the 4-month timeframe, likely because of the absence of sustained exposure to grass pollen in the airways due to the study being conducted out of the pollen season (Fig 1, G). Frequencies of blood ILCs were never significantly different before or after 2 or 4 months of AIT between patients from the active and placebo groups (Fig 1, G). As a confirmation, no significant correlations were observed during AIT between changes in ILC2 frequencies and improvement in clinical scores at an individual patient level (see Fig E3, A, in this article's Online Repository at www.jacionline.org). The ability of ILC2s to produce IL-4 and IL-13 was not modified by AIT (see Fig E3, B). Similarly, we did not detect any significant changes in ILC1 and ILC3 blood frequencies or function in the course of AIT (see Fig E3, C). We conclude that a 4-month AIT has no significant effect on circulating ILCs, despite clinical benefit. Obviously, our results do not preclude that a longer course of AIT could modulate ILC blood frequencies. Because our study was performed outside of the pollen season, our data do not oppose the recent observation that AIT can blunt the upregulation of blood ILC2s induced by seasonal allergen exposure.12Lao-Araya M. Steveling E. Scadding G.W. Durham S.R. Shamji M.H. Seasonal increases in peripheral innate lymphoid type 2 cells are inhibited by subcutaneous grass pollen immunotherapy.J Allergy Clin Immunol. 2014; 134: 1193-1195Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Altogether, we provide evidence for multiple differences in the regulation of circulating ILCs between allergic and nonallergic subjects. In the context of allergy, the regulation of circulating ILCs appears complex because it relates to both allergic disease severity and natural allergen exposure, thereby likely reflecting changes in the balance between distinct pools of ILCs in the blood and in tissues in response to the allergen. Given the growing evidence for a role of ILCs in allergic airway inflammation, it will be of great interest to further assess the frequencies, patterns of gene expression, functions, and trafficking of those cells, most particularly CCR10+ ILC2s, in patients with severe asthma. We thank Valérie Vignali and Albane Laparra from the Clinical Investigation Center of the Bichat-Claude Bernard Hospital for excellent sample management. Download .docx (.07 MB) Help with docx files Online Repository Data Download .pdf (.15 MB) Help with pdf files Fig E1 Download .pdf (.05 MB) Help with pdf files Fig E2 Download .pdf (.04 MB) Help with pdf files Fig E3 Download .docx (.01 MB) Help with docx files Table E1 Download .pdf (1.42 MB) Help with pdf files Table E2