Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Background: Intestinal inflammation elicited by environmental determinants including dietary proteins and microbes is implicated in type 1 diabetes (T1D) pathogenesis. Also, intrinsic pancreatic abnormalities could precede classic insulitis, contributing to T1D. Materials and Methods: Spontaneous rat T1D models were used for in situ analyses of gut and pancreas to explore novel disease pathways using immunohistochemistry and detailed morphometry, gene expression studies, and molecular screening analyses. Results: In BBdp rats, feeding a cereal diet stimulated T1D under germ-free or specific pathogen-free (SPF) conditions compared with a protective hydrolyzed casein (HC) diet. Cereal-induced T1D was paralleled by increased gut T cell infiltration and TH1-associated pro-inflammatory transcription. HC-fed rats displayed an increased number of anti-inflammatory CD163+ M2 macrophages compared with cereal-fed rats. Cereal-associated promotion of T1D in Lewis diabetes-prone (LEW-DP) rats, a different rat model, similarly featured gut T cell infiltration in conjunction with decreased immunoregulation. The Camp gene was induced in diet-protected HC-fed BBdp rats. Camp encodes the cathelicidin antimicrobial peptide (CAMP), a pleiotropic immunomodulatory host defence factor. Intestinal CAMP was enriched in CD163+ M2 macrophages and could represent a novel marker of these tolerogenic innate immune cells. CAMP expression was also discovered in pancreatic lymph nodes (PLN) and islets, indicating a novel role for this factor in target tissue homeostasis. There was a positive correlation between pancreatic CAMP and total islet number. Also, islet-associated CAMP+ cells were increased in rats with islet inflammation, suggesting upregulation in parallel with insulitis. Exogenous CAMP/LL-37 injections increased the abundance of T1D-protective probiotic bacteria and promoted islet neogenesis in BBdp rats. A prospective partial pancreatectomy (PPx) study was performed to obtain pre-diabetic pancreas biopsies from iii pre-insulitic BBdp rats. The number of endothelium-associated CD68+ macrophages was increased in pre-diabetic pancreata, indicating that perivascular inflammation was an early lesion in the animals. In addition, pre-diabetic pancreata featured enhanced regenerative Reg3a and Reg3b gene expression, indicating abnormal islet expansion preceding insulitis. Conclusions: Small intestinal inflammation paired with deficits in local immunoregulation parallels T1D development. CAMP represents a novel factor in T1D that could have several pleiotropic functions including regulation of commensal microbes, intestinal homeostasis, and pancreatic homeostasis. In addition, target tissue abnormalities precede insulitis and T1D. This research focused on the integrative biology of T1D pathogenesis in spontaneous rat models. This work provides a novel working model that incorporates key roles for gut lumen antigens, intestinal immunity, and the role of islets and altered regenerative capacity in T1D. This research could lead to new therapeutic opportunities for T1D treatment.