In a recent study published in Cells, researchers evaluated idiopathic pulmonary fibrosis (IPF) pathophysiological differences by the intestinal microbiome and by sex.
Previous studies have reported that pro-fibrotic cytokine substances, like tumor growth factor-beta 1 (TGF-β1) and interleukin-17 A (IL-17A), are involved in interstitial lung disease (ILD) pathogenesis. However, the interplay of intestinal dysbiosis, gonadal hormones, and the molecular regulators of profibrotic chemokine and cytokines, such as phosphorylated signal transducer and activator of transcription 3 (p-STAT-3), are not well-characterized.
About the study
In the present study, researchers investigated the influence of estrogen and intestinal dysbiosis on IPF pathophysiology.
The study comprised four human groups: healthy controls (n=25), individuals with sarcoidosis (n=31), IPF (n=45), and scleroderma (n=11). IPF patients were identified using the American thoracic society (ATS) criteria, and those with systemic sclerosis were identified using the 2013 American college of rheumatology (ACR) guidelines.
Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of patients, following which chromatin immunoprecipitation sequencing (ChIP-seq) analysis was performed to assess the primary human cluster of differentiation 4+ (CD4+) T lymphocytes. Pulmonary function tests (PFTs) were performed when required.
PF was assessed among menstruating females in different rearing environments. Additionally, estrogen receptor -1-negative (ESR-1-/-) or wild-type mice with bleomycin-induced PF were used, and the extent of PF in the murine tissues was determined using Ashcroft scores. Collagen content was determined using Sircol assays. T helper 17 (Th17) lymphocytes were detected by flow cytometry using main transcriptional factors, such as STAT-3.
Hormone pellets were implanted in ovariectomized C57BL/6J mice in vivo. Metagenomic sequencing and analysis of the intestinal microbiome were performed. To identify factors that might modulate STAT-3 levels during PF, the ChIP-seq datasets in the encyclopedia of deoxyribonucleic acid elements (ENCODE) 3 repository were interrogated. To investigate whether estrogen receptor-alpha (ER-α) interacted with the STAT-3 locus among CD4+ T lymphocytes via deoxyribonucleic acid binding activity, genome-wide ChIP-seq for ER-α-bound sites was performed.
Sites in the STAT-3 locus were enriched significantly for binding by ER-α. Significantly elevated counts of regulatory T (Treg) lymphocytes, compared to Th17 lymphocytes, were observed in the lungs of females. Ovariectomy or the lack of ESR-1 in mice significantly elevated IL-17A and p-STAT-3 levels among pulmonary CD4+ T lymphocytes, which were reduced following female hormone repletion. No statistically significant reductions in PF were observed in either of the conditions, indicative of the probable contribution of factors other than ovarian hormones.
Environments that favored intestinal dysbiosis and post-ovariectomy hormone repletion augmented PF, indicating that the pathologic interplay between gonadotrophic hormones and the intestinal microbiome is related to PF severity. Among female sarcoidosis patients, significantly lowered IL-17A and p-STAT-3 levels and concomitantly elevated TGF-β1 levels were observed in CD4+ T lymphocytes, in comparison to their male counterparts.
Human females who experienced pulmonary function losses due to progressive PF, and female mice with bleomycin-induced PF demonstrated elevated Treg counts and increased immunosuppressive TGF-β1 levels in the PF microenvironment. Low estrogen conditions among males and ovariectomized females showed higher IL-17A levels due to increased counts of pro-inflammatory Th17 lymphocytes in the lungs. Estrogen-induced PF was further increased by low intestinal microbial diversity, indicating sex-based differences in STAT-3 signaling among CD4+ T lymphocytes.
ER-α was bound to the STAT-3 locus among CD4+ T lymphocytes, particularly at established sites of chromatin accessibility shared with several types of cells. ESR-1 was critical in inducing IL-23R and IL-6 expression and repressing IL-17A and p-STAT-3 expression among CD4+ T lymphocytes during PF in females. The loss of gonadal hormones via ovariectomy decreased IL-6 levels, augmenting IL-17A and p-STAT-3 expression in CD4+ T lymphocytes.
Female hormones suppressed the expression of inflammatory and pro-fibrotic cytokines by inhibiting the p-STAT-3 pathway and IL-17A production in the pulmonary CD4+ T lymphocytes of bleomycin-induced PF mice. Pulmonary quantification following ovariectomy or ESR-1 loss showed less collagen in the tissues. A synergistic association between estrogen levels and intestinal dysbiosis, regarding PF severity, was observed.
Female intestinal microbiomes changed according to the rearing environment, with significantly less diversity in the ABSL-2 housing conditions. Progressive LF patients displayed sex-specific pro-fibrotic cytokine expression. Even though the female gonadal hormones were profibrotic, the hormones reduced IL-17A-induced inflammation among CD4+ T lymphocytes via ER-α binding of the STAT-3 locus. The subsequent lowering of inflammation could contribute to the mortality benefit among premenopausal female ILD patients.
Overall, the study findings highlighted the pro-fibrotic nature of estrogen and the augmentation of PF severity by intestinal dysbiosis among menstruating females, indicating a critical interplay of gonadal hormones and intestinal microflora in PF pathogenesis.
The findings could inform the development of sex-based and personalized immunological therapeutics for chronic pulmonary inflammation.
- Chioma, O.S.; Mallott, E.; Shah-Gandhi, B.; Wiggins, Z.; Langford, M.; Lancaster, A.W.; Gelbard, A.; Wu, H.; Johnson, J.E.; Lancaster, L.; et al. (2023). Low Gut Microbial Diversity Augments Estrogen-Driven Pulmonary Fibrosis in Female-Predominant Interstitial Lung Disease. Cells. doi: https://doi.org/10.3390/cells12050766 https://www.mdpi.com/2073-4409/12/5/766