PARTIALLY DIFFERENTIATED ILEAL AND RECTAL HUMAN CFTR-F508DEL ENTEROIDS SECRETE FLUID IN RESPONSE TO CAMP AND CGMP

Introduction: Chronic constipation is a continuing medical issue for patients with Cystic Fibrosis (CF). Approaches to managing constipation include increasing intestinal fluid secretion that involves inhibiting intestinal NaCl absorption and/or stimulating intestinal Cl secretion. We have recently reported an enterocyte population in the human ileal and colon that is present between the crypt Cl^- secretory cells and the mature villus or colonic surface Na⁺ absorptive cells that contain CFTR together with DRA. This population is called partially differentiated (PD) enterocytes. This study was undertaken to derive a strategy to stimulate intestinal fluid secretion from PD enterocytes in the F508del-CF-patient population by enhancing residual CFTR F508del activity using drugs that stimulate cAMP and cGMP. Methods: Ileal and rectal enteroids from F508del CF patients and healthy subjects (HS) were studied. Enteroids were maintained in growth media containing Wnt3A, R-spondin, and Noggin (undifferentiated/UD); induced to partially differentiate (PD) or fully differentiate (DF) by removal of growth factors (Wnt3A and R-spondin) for 3 or 6 days, respectively. Forskolin-induced swelling (FIS) assay and live cell microscopy were used to determine fluid secretion in 3d-DF enteroids. mRNA expression was determined using qRT-PCR. Results: Compared to HS enteroids, F508del enteroids had significantly higher mRNA expression of LGR5, and Ki67 in the UD state. The goblet cell marker MUC2 and enteroendocrine (EEC) marker ChgA increased with differentiation in both HS and F508del, while DF F508del enteroids had a significantly higher ChgA compared to DF HS enteroids. UD F508del enteroids had significantly higher expression of phosphodiesterase-3a (PDE3a) than HS enteroids, which further increased with differentiation, but did not change in HS. While UD and DF F508del rectal and ileal enteroids did not swell when exposed to cAMP (forskolin) and cGMP (linaclotide), 3d-DF F508del enteroids showed a swelling response when exposed to cAMP (forskolin) and cGMP (linaclotide). Fluid secretion from 3d-DF F508del enteroids was ~50% (cAMP) and ~67% (cGMP) of the response of intestinal enteroids from HS enteroids. Combinations of CFTR correctors and potentiators mimicking the makeup of Trikafta only partially enhanced fluid secretion in F508del CF enteroids as compared to HS. However, cGMP (linaclotide) additively enhanced Trikafta-induced fluid secretion from 3d-DF F508del enteroids. While PDE3a inhibitor additively enhanced Trikafta-induced fluid secretion from UD and 3d-DF F508del enteroids. Conclusions: CFTR F508del can be stimulated by cAMP and cGMP in PD enterocyte populations. Our findings identify a novel role of the partially differentiated ileal and rectal enterocyte populations which behave in a way that offers the potential to improve treatment for CF constipation.