TNF-like weak inducer of apoptosis (TWEAK) promotes beta cell neogenesis from pancreatic ductal epithelium in adult mice

Abstract

Aim/hypothesis: The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes β-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice.

Methods: C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER(TM): R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px.

Results: TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or β-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic β-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion.

Conclusions/interpretation: We conclude that TWEAK is a novel factor mediating pancreatic β-cell neogenesis from ductal epithelium in normal adult mice.

Figure 1. Fn14 expression by human pancreatic ductal epithelium.

Variable amounts of Fn14 were seen in human pancreas with fairly strong intensity seen in tissue from a 16-year-old female donor (A) anti-Fn14 or (B) isotype control, and faint staining in a 57-year-old donor (C) anti-Fn14 and (D) isotype control. Specific staining was seen in islets as well as ducts. The islets were labeled as “i”. Scale bar  = 50 µm.

Figure 2. TWEAK treatment promotes proliferation of pancreatic duct and duct adjacent cells through its receptor Fn14.

Pancreatic tissue of normal adult mice stained for Ki-67 (black) on day 3 after injection of (A) control Ig or (B) Fc-TWEAK, and costained with Ki-67 (red) and ductal epithelial marker CK (CK) (green) on day 4 after (C) control Ig or (D) Fc-TWEAK acute treatment. Scale bar  = 10 µm. (E–F) Quantification of the % Ki-67⁺ pancreatic (E) duct and (F) duct adjacent cells in mice treated with control Ig or Fc-TWEAK twice weekly. Data are shown as mean±SEM (n = 4). * p<0.05 for Fc-TWEAK vs control Ig treatment. (G–H) Quantification of the % Ki-67⁺ (G) duct and (H) duct adjacent cells in normal adult WT or Fn14 KO mice treated with control Ig or Fc-TWEAK twice per week. Data are shown as mean±SEM (n = 4) for percentage of Ki-67+ duct cells (E&G) or Ki-67+ duct adjacent cells per total duct cells (F&H); * p<0.05 for Fc-TWEAK treatment vs control Ig.

Figure 3. TWEAK treatment induces Ngn3 expression in pancreatic duct cells.

Immunostaining for (A) Ngn3 (yellow), (B) CK (green) and (C) Ngn3/CK (shown as merge) on day 5 of chronic TWEAK treatment. Scale bar  = 10 µm. (D) Quantification of Ngn3⁺ cells on day 5 after twice weekly control or TWEAK treatment. Each point represents an individual animal, with horizontal bar the mean of 4 mice/group.

Figure 4. Chronic TWEAK treatment mimics the response to the partial pancreatectomy.

Ductal structures with characteristics of regenerative foci were identified by H&E staining (A) at day 18 after TWEAK treatment twice weekly in all 4 treated animals. Inset of (A) is shown on serial sections stained with Ki-67 (B), CK (C), insulin (D) and glucagon (E). (F–H) Serial sections of area from another representative animal were stained with H&E (F), CD3 (G) F4/80 (H); isotype control (I) and Fn14 (J). The islets were labeled as “i”. Scale bar  = 50 µm in all panels.

Figure 5. TWEAK promotes pancreatic regeneration through activation of pancreatic duct-derived progenitor cells.

(A) CAII-CreER^TM:R26R-eYFP (bigenic) mice were implanted with 3-week slow-release TM pellets at 4 weeks of age, followed by a 1 week washout period, and received treatment at 8 weeks of age twice weekly for 2 weeks, and killed at 18 days after start of treatment. (B) In sections from TM-treated mice immunostained for YFP (green) and insulin (red), YFP-marked insulin+ (coexpression, yellow) cells could be found in small aggregates and within islets. In control bigenic animals without tamoxifen, control Ig or Fc-TWEAK treatment, no labeled cells were found. Scale bar  = 50 µm in left two panels and 20 µm in far right panel. Quantification of labeled islets and aggregates is presented in Table 1.

Figure 6. Fn14 is transiently expressed in the regenerating foci after Px and its deficiency delays pancreatic regeneration.

(A–D) Representative images from serial sections immunostained for Fn14 (green) (A, C) and CK (red)/insulin (green)/DAPI (blue) (B, D) show transiently enhanced expression of Fn14 in immature regenerating foci (A, B) but not in mature foci (C, D). E. Representative stages of the maturation of the regenerating foci as previously defined . (F–G) Quantification of (F) total foci number and (G) total foci area per section of Fn14 KO mice or WT mice at day 4 and day 7 post surgery. (H–I) Distribution of foci at different stages of differentiation in Fn14KO and WT mice at (H) day 4 or (I) day 7 post surgery. Number and stage of regenerating foci were assessed on at least two sections/animal (≥ 200 µm apart) in WT and Fn14 KO mice (n ≥ 5 per group). No regenerating foci were identified in sham-operated mice. Data are shown as mean±SEM; * P<0.05 for Fn14 KO vs WT mice.

Figure 7. Proposed Model for TWEAK’s effect on pancreatic regeneration.

TWEAK signals through Fn14, which is normally expressed at low levels in adult pancreatic ducts and upregulated after injury, resulting in proliferation of duct epithelium followed by differentiation to mature pancreatic cell types, including β-cells. In addition, TWEAK/Fn14 signaling induces a localized leukocyte infiltrate that may be beneficial to the tissue regeneration, thereby creating a positive feedback loop. Although not addressed in the current study, this likely occurs through induction of chemokines or cytokines that promote inflammatory cell recruitment. We speculate that the proliferating duct epithelial cells regress to a less differentiated state with progenitor potential, giving rise to Ngn3+ endocrine progenitors that then differentiate to new endocrine cells.