Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice

Abstract

Background & aims: Sjögren's syndrome and autoimmune pancreatitis are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. Nonobese diabetic/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren's syndrome and chronic pancreatitis and MRL/Mp mice are models of autoimmune pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a ductal Cl^- channel essential for ductal fluid and HCO₃^- secretion. We used these models to ask the following questions: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function?

Methods: We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal, and acinar cells damage, infiltration, immune cells and function were measured in vivo and in isolated duct/acini.

Results: In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular, C18 restored salivation, rescued CFTR expression and localization, and nearly eliminated the inflammation and tissue damage. Transgenic overexpression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca²⁺ signaling, Orai1, inositol triphosphate receptors, Aquaporin 5 expression, and fluid secretion were restored by rescuing ductal CFTR.

Conclusions: Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren's syndrome and pancreatitis.

Keywords: CFTR; Duct; Pancreatitis; Sjögren's syndrome.

Figure 1. Treating NOD mice with the potentiator VX770 and the corrector C18 restore salivation and ductal fluid secretion

Wild-type and NOD mice were treated daily by I.P. injection of the solvent DMSO or 2 mg/kg VX770 (a) or 2 mg/kg C18 (b) for the indicated days and salivation in response to pilocarpine stimulation was measured every 3 days for 20 min. At day 9 (VX770) or 15 (C18) injection was stopped in 3 mice to determine the rate of return to pretreatment state. The numbers indicate the p values with respect to pretreatment (horizontal) and to wild-type (vertical). In (c, d) mice were treated with DMSO or VX770 (c) or C18 (d) for 14 days. The ducts were micro-dissected from the parotid glands, cultured for 24–36 hrs with the respective drugs to obtain sealed ducts and used to measure ductal fluid secretion. Here and in all other Figures the results are shown as mean±s.e.m of the indicated number of experiments. When error bars are not visible, they are within the size of the symbols.

Figure 2. Effect of treating NOD mice with VX770 and C18 on CFTR expression and inflammation

Wild-type and NOD mice were treated with VX770 (a, b) or C18 (c–e) for 14 days and expression of CFTR was analyzed by immunostaining and protein level (a, c). Protein levels were normalized to actin and then averaged. Inflammation was analyzed by viewing infiltration in H&E stained sections and analysis of the number and size of the inflammatory foci (b, d). In (e) SMG sections were stained for CD3 (upper, T cells) and CD68 (lower, Neutrophils) to assay infiltration.

Figure 3. Treating NOD mice with C18 inhibits inflammatory mediators

Inflammatory mediators were measured in extracts prepared from submandibular glands of wild-type mice injected with DMSO (black) or C18 (blue) and NOD mice treated with DMSO (red) or C18 (green) for 14 days. Each group included 4 mice. * denote p<0.05 or better relative to wild-type and # denote p<0.05 or better relative to DMSO treated NOD mice.

Figure 4. Treating NOD mice with the VX770 and C18 and MRL/Mp and MRL/Mp-Fas mice with C18 improved ductal function and reduced inflammation

Wild-type and NOD mice treated with VX770 or C18 for 14 days were used to evaluate number of foci (a), infiltration of T cells (CD3), macrophages (CD68) and B cells (B220) (b), and inflammation score according to Kanno et. al. (c). In (d), the pancreatic intralobular ducts were micro-dissected from mice treated with DMSO, VX770 or C18, cultured for 24–36 hrs and used to measure fluid secretion. In (e, f) MRL/Mp (e) or MRL/Mp-Fas mice (f) treated with and without Poly IC were treated with DMSO or C18 for 14 days and were used to evaluate tissue damage by histological score, T cells (CD3) and macrophages (CD68) infiltration.

Figure 5. Treating NOD mice with C18 restore acinar cells AQP5 expression

AQP5 in acinar cells was evaluated by immunostaining (a) and protein level (c) in wild-type and NOD mice treated with C18. The immunostaining was analyzed for fragmentation by measuring the intensity and length of the stains (b). Expression level of NKCC1 and ANO1 were also analyzed (d, e).

Figure 6. Effect of C18 treatment on Ca²⁺ signaling and fluid secretion by salivary glands and pancreatic acini

Dispersed acini prepared from the parotid glands of NOD mice (a, b) or the pancreas of NOD and MRL/Mp mice (d, e) treated with DMSO or C18and were used to measure [Ca²⁺]_i in response to stimulation with 2.5 μM carbachol in Ca²⁺-containing media (a), or 100 μM carbachol (b) in Ca²⁺-free media and then perfusing with solution containing 2 mM Ca²⁺ to evaluate Ca²⁺ influx (d). Pancreatic acini in Ca²⁺-free media were stimulated with 10 nM CCK8 or 25 μM CPA (d, MRL/Mp mice) and after return of [Ca²⁺]_i to basline they were perfused with a solution containing 5 or 2 mM Ca²⁺ (d). Acini from NOD mice were also stimulated with 2 pM CCK8 to evaluate Ca²⁺ oscillations (e). Parotid (c) and pancreatic (f) acini loaded with the cell volume reporter calcein were stimulated with 2.5 (c) or 100 μM carbachol (f). Note the different cell volume scale for parotid and pancreatic acini.

Figure 7. Expression of hCFTR in NOD submandibular gland ducts and treating BMP6 expressing glands with C18 restore salivation, AQP5 and CFTR expression

Ad5-GFP (control) or Ad5-GFP-hCFTR were infused into the SMG and after 7 days the mice were used to probe expressed of GFP-hCFTR (a) measure salivation (b) and expression of AQP5, ANO1 and NKCC1 (c). The salivary glands of wild-type mice were infused with AAV5 vector carrying GFP (control) or BMP6 to over-express BMP6 in the SMG. After six months the mice were treated with C18 for 14 days and used to measure salivary secretion (d), native CFTR (e) and AQP5 expression (f).