Pulmonary fibrosis (PF) is a male-dominant disease developing between ages 50-70, coinciding with the onset of menopause in women and reduced testosterone levels in men, implying a role for sex hormones in disease onset. Animal models have shown contradictory results. Thus, a human model is required. Type 2 alveolar epithelial (AT2) cells are implicated in disease initiation but are difficult to isolate from patients.

We hypothesize that sex hormones modulate PF onset and AT2 cells derived from induced pluripotent stem cells (iAT2s) can be used to model this process.

Male and female-derived iAT2 cells were treated with estrogen or testosterone and subsequently with a pro-fibrotic cocktail (PC). Fibrotic response in combination with sex hormone treatment was investigated at the protein level using ELISA and the RNA level using qPCR, bulk, and single-cell RNA sequencing (scRNA-seq).

Surfactant proteins and sex hormone receptors were significantly downregulated in PC-treated iAT2s. Gene expression of TGF? and AREG was reduced while SLPI was increased in cells pre-treated with both sex hormones compared to control. ScRNA-seq showed increased expression of GPR87, ITGB6, and HIF-1? in PC-treated cells following estrogen, but decreased after testosterone treatment compared to control. Cluster analysis showed that in PC-treated cells testosterone led to an increased proportion of aberrant basaloid cells, while estrogen favoured senescence markers. Pathway analysis showed that HIF1 signalling is downregulated by estrogen while TNF and MAPK signalling are downregulated by testosterone.

This study demonstrates for the first time the effects of sex hormones on fibrosis onset using a human in vitro model.