Aberrant innate immune activation following tissue injury impairs pancreatic regeneration

Abstract

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic β-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury.

Figure 1. Pancreatic regeneration following acinar injury is impaired in Rag1^−/− mice.

(A) Caerulein injection regime, time of tissue harvesting and schematic depiction of acinar de-differentiation and regeneration following caerulein-induced injury in WT mice. (B and C) Representative H&E staining of pancreas from WT and Rag1^−/− mice treated with PBS control or 2- and 7-days following caerulein-induced injury. (D) Pancreatic immuno-fluorescent staining for CK19, β-catenin and amylase from WT and Rag1^−/− mice at CaeD7. (E and F) Combined and individual scoring of pancreatic histological parameters from WT and Rag1^−/− mice at CaeD2 and CaeD7 (n = 5–20, *P<0.05, **P<0.01, t test).

Figure 2. Macrophages are the predominant immune cell infiltrating the pancreas following caerulein-induced injury.

(A) Representative FACS plot showing the percentage of CD45⁺ cells that are F4/80⁺ compared to F4/80⁻CD11c⁺ dendritic cells and F4/80⁻Gr1⁺ myeloid cells in the pancreatic immune infiltrate at CaeD1. (B) Immuno-histochemical staining of F4/80⁺ cells from WT and Rag1^−/− treated with PBS or caerulein. (C and D) Immuno-fluorescent staining of the pancreas from WT and Rag1^−/− mice showing F4/80⁺ cells are associated with areas of the de-differentiated epithelium (visualized by β-catenin and Ecadherin) present only in Rag1^−/− mice 7-days after caerulein treatment and completely surrounding the lobular epithelium within 14-days. (E and F) Representative H&E and corresponding areas stained with F4/80 from the pancreas of Rag1^−/− mice treated with liposomes filled with either PBS or clodronate during caerulein treatment and harvested 2-days after the last caerulein injection.

Figure 3. Inflammatory activation of macrophages is increased in Rag1^−/− mice before and after injury.

(A and B) Representative FACS plots characterizing expression of the CD45⁺ immune cells that are F4/80⁺ and CD206⁺ in the pancreatic immune infiltrate at CaeD1 and CaeD2 (n = 8). (C) Representative immuno-histochemical staining of CD301⁺ and CD206⁺ cells in Rag1^−/− mice treated with caerulein at designated time points. (D) Representative FACS plots characterizing F4/80⁺ macrophages isolated from the pancreas of WT and Rag1^−/− mice at CaeD2 (n = 4–8). (E) Representative FACS plots of iNOS expression from macrophages isolated from the pancreas of WT and Rag1^−/− mice in untreated and CaeD2 treated mice (n = 4–8).

Figure 4. γPMNs are present during caerulein-induced injury in WT mice and are elevated in Rag1^−/− mice.

(A) IFNγ levels in serum isolated from WT and Rag1^−/− mice with and without caerulein treatment (n = 6–8, *P<0.05, **P≤0.01, t test). (B) FACS analysis of the percentage of F4/80⁻Thy1.2⁻ cells expressing Ly6G⁺ and IFNγ⁺ in the pancreas of WT and Rag1^−/− mice with or without caerulein treatment (n = 5–8, *P<0.05, **P<0.01, t test). (C) Percentage of Ly6G⁺IFNγ⁺ (CD45⁺F4/80⁻Thy1.2⁻) amongst live CD45⁺ cells infiltrating the pancreas with or without caerulein treatment as indicated. (D) Sort strategy isolating indicated populations from the pancreas of WT and Rag1^−/− mice and cytospin preparation visualizing May-Grünwald-Giemsa staining.

Figure 5. Decreasing inflammatory macrophage activation improves pancreatic injury.

(A) H&E staining of pancreas from Rag1^−/− mice treated with control or the iNOS inhibitor 1400 W 2-days following caerulein-induced injury. Injection regime of 1400 W (W*) is indicated in adjacent cartoon. (B) Combined and individual pancreatic histology scores from Rag1^−/− mice treated with control or iNOS inhibitor 1400 W (n = 6–9). (C) Representative FACS analysis of macrophages expressing iNOS isolated from the pancreas of Rag1^−/− mice treated with α-IFNγR and IgG2 isotype control at CaeD2 (n = 4–8). (D) H&E staining of pancreas from Rag1^−/− mice treated with α-IFNγR and IgG2 isotype control at CaeD2. Injection regime of α-IFNγR (Y) is indicated in adjacent cartoon. (E) Combined and individual pancreatic histology scores from Rag1^−/− mice treated with α-IFNγR and IgG2 isotype control (n = 8–11). (F) Schematic depicting how increased innate inflammation impacts exocrine de-diferentiation.

Figure 6. Inflammatory factors that influence effective versus ineffective pancreatic regeneration.

Schematic depicting the impact increased innate inflammation has on exocrine de-differentiation and regeneration in (A) the presence (WT mice) and absence (B) of adaptive immune cells (Rag1^−/− mice).