17-β Estradiol (E2), the active form of female hormone estrogen is essential for bone homeostasis, cholesterol and glucose metabolism, smooth muscle cell function, body temperature regulation, differentiation of axons and dendrites in females and males [1]. These biological functions of E2 are mediated by two members of steroid receptors belonging to nuclear receptor subfamily 3: group A, NR3A1 (Estrogen Receptor α, ERα) and NR3A2 (Estrogen Receptor β, ERβ) that are encoded by ESR1 and ESR2, respectively. E2 elicited distinct phenotypes of E2-ER signalling based on multiple parameters including: (i) differential expression of ERα and ERβ in cell- and tissue-specific manner, (ii) dimerization of receptors, (iii) expression of distinct ER splice variant isoforms, (iv) association with co-activators/repressors, (v) physiological and pathophysiological conditions among others [2]. ER-mediated signalling can occur in either ligand-dependent or ligand-independent manner. Ligand-dependent action is initiated by binding of E2 to ER, followed by the formation of homo- or hetero-dimers of ERα and ERβ. These dimers then translocate into nuclei to bind to specific estrogen-response elements (ERE, a 5-bp palindrome with a 3-bp spacer; GGTCAnnnTGACC) and recruit various transcription factors to trigger trans-activation and/or repression of target genes.

Despite E2 being well studied for its traditional role in sexual dimorphism in mammals, emerging evidences suggest an active and crucial role of E2 in immune dimorphism as well [3]. Females are more susceptible towards pathological conditions such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE), yet the impact of E2 towards the pathophysiology of MS and SLE is protective and promoting, respectively [4], [5]. Impact of E2 towards immune system is mediated predominantly by ERα rather than ERβ [6]. E2-mediated inhibition of inflammatory biomolecules such as TNF-α, IFN-γ, IL-17, MCP-1 in experimental autoimmune encephalomyelitis (EAE) might be responsible for its protective role, which is dependent upon ERα signalling [7], [8]. E2-mediated induction of lupus phenotype in mice has been demonstrated to be dependent on pathways associated to ERα [9]. SLE patients also showed relatively higher expression of ERα than ERβ in PBMCs [10]. Furthermore, E2 is shown to upregulate neutrophils both in wild type and lupus-induced mice [11]. E2 has been shown to promote B-cell survival and induce lupus-related serology in non-autoimmune mice [12], [13]. E2 has also been shown to promote TNF-α-induced maturation of monocyte-derived dendritic cells and enhances their capacity to initiate Th2 cells [14]. In addition, ERα plays a crucial role in E2-mediated regulation of CD4 + helper T cells. The absence of ERα upregulates both the Tfh population as well as Tfh-dependent high affinity antibody class switching at the germinal centre (GC), and antibody production [15]. In the context of Th17 cell differentiation E2 has been shown to inhibit the expression of Rorγt by increasing the recruitment of repressor of estrogen receptor activity (REA) to the ERE sites of Rorγt promoter region in ERα-dependent manner [16]. In contrast, E2 promotes the induction of CD4 + CD25 + Tregs population as well as upregulate the expression of Foxp3 and IL-10 at a physiological concentration. This stimulatory impact of E2 on Tregs is inhibited in the presence of ICI 182, 780, an ERα receptor antagonist [17]. Interestingly, low dose of E2 treatment to ovariectomized mice resulted in an increased response of antigen-specific CD4 + T cell and expansion of IFN-γ-producing population [6]. Furthermore, it was shown that ERα but not ERβ was necessary for E2-mediated enhanced response of Th1 cells [6]. The role of E2 in the regulation of Th2 response is, however, not clearly elucidated. One of the reports suggests that pregnancy levels of E2 causes decreased antigen-specific IFN-γ response and increased production of Th2 cytokines [18]. It is highly opinionated that the endogenous level of E2 promotes Th1 to Th2 ratio whereas, pregnancy levels of E2 supports increased Th2 to Th1 ratio [19], [20].

In this study, we have elucidated that physiological level of E2 is sufficient to impair the differentiation of naïve T cell into IL-4-secreting Th2 cells. This downmodulation is associated with decreased expression of Th2-specific transcription factors including GATA3, BATF, IRF4 and Th2-specific cytokines such as IL-4, IL-13 and IL-10. Interestingly, we observed an increased expression of Sfpi1, a transcription factor known to regulate Th2 cells heterogeneity [21]. The ectopic expression of PU.1 attenuated the expression of Th2-specific cytokines including as IL-4, IL-5 and IL-13. Mechanistically, we have verified that E2 treatment decreased the recruitment of GATA3 and increased the recruit of PU.1 to various regulatory regions of Il4 promoters by directly interacting with both GATA3 and PU.1. E2 treatment also altered the recruitment of GATA3 and PU.1 at Il13 promoter albeit to a lesser extent as compared to Il4. Epigenetically, we observed that the regulatory impact of E2 might be related to altered acetylation pattern of H3K9 in response to comparative recruitment of P300. In contrast, there is a decreased acetylation of H4K5 across all locus of Il4 gene upon E2 treatment. We also observed differential recruitment of HDAC1 on Il4 gene; however, this activity could not be observed at Il13 locus. Hence, our study suggests a novel mechanism by which E2 regulates the expression of Il4 and Il13 which might involve the intergenic regulatory region CNS-1 of Il4 and Il13.