| Abstract |
Asthma is a heterogeneous disease, characterized by airway hyperresponsiveness and chronic inflammation of the small-conducting and the large-conducting airways. It is defined by the history of recurrent respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough which vary over time and in intensity, accompanied by variable expiratory airflow limitation. Recognizable clusters of genetic, demographic, clinical and pathophysiological characteristics are often called “asthma phenotypes”. GINA (Global Initiative for Asthma) 2022 guidelines report the five most common “phenotypes” of asthma: 1. Allergic asthma. 2. Non-allergic asthma. 3. Adult-onset (late-onset) asthma. 4. Asthma with persistent airflow limitation. 5. Asthma with obesity. On the other hand, a pathogenetic classification of asthma in two endotypes is based on the type of the immune cell response that mediates disease development and progression. Hence, asthma is classified as: (a) type 2/allergic asthma, characterized predominantly by T helper type 2 (Th2) cell-mediated inflammation and eosinophilia, and (b) non-type 2 asthma associated mainly with Th1 and/or Th17 cell-mediated inflammation and increased numbers of neutrophils. Our research was focused on the most frequent asthma endotype/phenotype, the type 2/allergic asthma.
Allergic asthma represents the most common disorder of the airways, affecting more than 300 million people worldwide. The significantly increased prevalence of the disease, especially in the children of industrialized countries the last decades, makes asthma a substantial global social-economic burden. Many cell types (epithelial cells, antigen presenting cells, eosinophils, basophils, mast cells, B and T cells e.t.c.) are involved in the pathogenesis of allergic asthma while several studies depict the importance of the interaction between bronchial epithelial cells and immune cells following inhaled allergen encounter during the initiation of asthmatic responses. Main pathogenetic factor of the disease is the generation of aberrant Th2 immune responses against allergens. Th2 cells produce specific proteins, named cytokines that are critically involved in the development of allergic airway inflammation. The major Th2-type cytokines are the interleukins 4, 5, 9 and 13 (IL-4, IL-5, IL-9, IL-13). Dysregulated allergen-specific Th2 responses in the airways lead to pulmonary eosinophilic infiltration, mucus hypersecretion, reversible airway obstruction and airway hyperresponsiveness (AHR). Most of the current treatments only ameliorate certain clinical features of the disease without providing a cure. Of note, a group of patients has severe, treatment-refractory asthma, requires regular hospitalization and represents a major health-care and socioeconomic problem. Therefore, factors that can induce and/or enhance immunosuppression represent essential therapeutic targets for human allergic asthma.
Immunoregulatory mechanisms, including suppressive cytokines and regulatory T cells (Tregs), attenuate aberrant Th2-mediated allergic responses in experimental asthma mouse models. A large body of evidence has uncovered as key regulators of aberrant allergic Th2 responses two subtypes of Tregs, the thymus-derived natural Tregs (nTregs), that express the transcription factor Foxp3 and the peripherally-induced by naive CD4+ T cells, type 1 regulatory T cells (Tr1 cells) that secrete the cytokine IL-10. Moreover, immunoregulatory cytokines, such as IL-10, IL-35 and transforming growth factor- β (TGF-β), are critical for the induction and suppressive function of Tregs. Nevertheless, accumulating evidence supports the notion that the numbers and function of Tregs and immunosuppressive mediators are severely impaired in individuals with allergic asthma.
Activin-A is a cytokine, member of the TGF-β superfamily that is critically implicated in a variety of essential biological processes. It is increased in the sera of asthmatics and in mice during acute and chronic allergic airway inflammation. Studies by Dr. Xanthou’s group have revealed that activin-A is an immunosuppressive cytokine as it inhibits allergen-specific Th2 responses and protects against the development of AHR and allergic airway disease in mice. Activin-A exerts its regulatory functions through the induction of allergen-specific Treg cells that suppress Th2 responses in vitro and upon adoptive transfer in vivo. However, the exact role of activin-A in human allergic asthma remained elusive.
In the present study we hypothesized that activin-A can induce the differentiation of human regulatory CD4+ T cells and can suppress human Th2-mediated allergic responses. Based on previously published findings by Dr. Xanthou’s group, we hypothesized that activin-A represents a key cytokine for the regulation of allergic airway inflammation and human allergic asthma.Our specific aims were:
a) To investigate the effects of activin-A on the suppression of human Th2 cell-mediated allergic responses and
b) To delineate the role of activin-A in the induction of human Treg cells and their effects on the suppression of allergic Th2 responses.
Our data reveal activin-A as a critical immunosuppressive agent for human allergic asthma.
More specifically, we demonstrate that:
1) Activin-A drives the generation of human CD4+ T cells that produce copious amounts of the immunoregulatory cytokine IL-10 and express the surface molecules ICOS, LAG-3 and CD49b. Hence activin-A-induced human CD4+ T cells possess key feature characteristics of human Tr1 cells.
2) Activin-A-induced human Tr1-like cells (act-A-iTr1 cells) display strong suppressive functions toward allergen-driven responses induced by naive and in vivo-primed human Th2 cells. Act-A-iTr1 cells exert their regulatory functions through IL-10-, TGF-β- and ICOS-mediated pathways.
3) Activin-A signalling induces the activation of the transcription factors IRF4 and AhR in human CD4+ T cells, which bind in ICOS promoter elements and control gene expression. Furthermore, IRF4 along with AhR and its binding partner, ARNT, form a transcription factor complex that is essential for effector molecule expression by human act-A-iTr1 cells.
4) In vivo adoptive transfer of human act-A-iTr1 cells in humanized mouse models of experimental asthma, can prevent, and even reverse, established allergic airway inflammation and confer protection against cardinal asthma manifestations in an IL-10–dependent manner. 5) Addition of activin-A in in vitro stimulatory cultures with a clinically-relevant allergen of human CD4+ T cells isolated from atopic and asthmatic individuals with distinct disease severities, leads to the significant reduction of CD4+ T cell proliferation and effector cytokine release.
Collectively, our studies uncover for the first time activin-A as a novel inducer of human IL-10-producing regulatory CD4+ T cells that express the Tr1-cell-associated markers ICOS, LAG-3 and CD49b. Human act-A-iTr1 cells display robust suppressive functions against naive and effector T-cell responses to allergen through IL-10–, TGF-β–, and ICOS– mediated mechanisms. Regarding the underlying molecular mechanism, we reveal that the transcription factors IRF4 and AhR play pivotal role in the generation of human act-A-iTr1 cells since they form a transcriptional complex essential for the expression of IL10 and ICOS genes. Using a humanized mouse model of experimental asthma, we demonstrate that human act-A–iTr1 cells retain their immunosuppressive properties upon adoptive transfer in vivo and can prevent, and even reverse, established allergic airway inflammation and confer significant protection against cardinal asthma manifestations in an IL-10–dependent pathway. Of clinical relevance, our research illustrates that activin-A is able to restrain allergen-driven responses by CD4+ effector T cells obtained from asthmatic individuals across the spectrum of disease severity.
Hence our studies may mark activin-A as a potentially attractive new therapeutic target for human asthma. Future studies could address the utilization of human act-A-iTr1 cells in adoptive-transfer cell therapy regimes aiming at re-establishing tolerance in the human asthmatic airways.
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