Autoimmune Diabetes

THE AUTOIMMUNE DIABETES RESEARCH PROGRAM

 

Beta cell interaction with the islet APCs.

An important component driving the autoimmune process is the transfer of beta cell granules to the islet APC. This process is constitutive, but it has important implications. It is the conduit of immunological information from beta cell to APC, the process that brings the CD4+ T cells into operation. How are the beta cells donating the diabetogenic antigens, i.e., insulin in our studies, to the resident APCs? Beta cells normally are in close contact with the resident APCs. In this close contact interaction, the secretory granules, as well as peptide-bearing granules, are transferred to the macrophage and to the CD103+ DC.

We study the passage of secretory granules from beta cells to APC in an assay in which primary beta cells are cultured with APC (either DC or macrophages), after which we probe the presentation of the insulin pMHC-II complex with either of the two T cells directed two epitopes of insulin . Both T cells reacted, indicating that the APC was presenting both of the insulin peptide registers. The transfer requires a close interaction between beta cells and APC, separating them by a cell impermeable membrane blocked presentation. The transfer also requires viable beta cells.. Beta cells from NOD.Rag1-/- mice, the non-diabetic C57BL/6 mouse or human all transferred the granules to APCs. In the case of the latter two, the APCs were from NOD. Extrapolating these results to the autoimmune situation suggests that active inflammation or cell death in islets is not a requirement for the initial presentation to take place.

The process whereby the secretory granules are passed to beta cells is modulated by glucose concentration (6). Increasing glucose levels from the basal level of 2.5/5 mM, to 25 mM, resulted three times higher insulin presentation. We also found a requirement for intracellular calcium in the process. Live imaging analysis and electron microscope studies of islets confirm the results of the assay just described.

Electron microscopy of islets of non-diabetic mice and from NOD mice at the ~6 week stage of diabetes revealed the presence of typical macrophages containing beta cell granules. Some of the vesicles were the typical dense core granules surrounded by only one membrane, an indication that the whole granule was internalized. However, a second type of granule within a double membrane vesicle containing a proteinaceous-like material was also seen. We need to distinguish the macrophages from the DC and substantiate whether the latter also captures granules in vivo.

In a project led by Bernd Zinselmeyer, we have live images of APC interacting with beta cells and capturing the entire granules. We made use of an insulinoma cell line bearing granules expressing the ZnT8 granule proteins fused with GFP. This allowed us to track the movement of the secretory granule in real-time. Soon after putting the ZnT8+ insulinoma with DC, we saw a polarization of the granules to the site of contact. Then, one can see the movement of the intact GFP+ material from the insulinoma to the DC.

We hypothesize that the transfer of the intact granules to the APC has several important features to the diabetogenic process. First, the granules contain potential modulatory substrates, such as ATP, which enhance the activation of pattern recognition receptors on the APC. This activation may be physiologically important to prevent infection of beta cells by circulating pathogens, but may have the unintended consequence of creating a “poised” APC that is ready to prime diabetogenic T cell responses. Second, the granules are a depot of very concentrated insulin protein or peptides, as is the case of the crinophagic vesicles. This material may be the substrate that may be presented to T cells by the APC leading to the initiation of autoimmune diabetes.

 

 

The resident APC of the islets: the macrophage

Macrophages were perceived as a phagocytic cell derived from blood monocytes and involved in the cleaning of tissues. This view has changes from investigations made by many in the last few years. Our interest in islet macrophages started when we examined APCs in isolated islets.  Boris Calderon examined the islet phagocytes in C57BL/6 (B6) mice as a base to compare with those in the islets of NOD mice. Initial studies that we and others had reported in NOD mice had made a point of calling the intra-islet APC a DC based on their expression of CD11c. But, both in B6 and in NOD mice, the major intra islet phagocytes are macrophages, identified not only by their composite of different surface markers but also by their gene expression pattern.

The islet macrophages are represented by one set based on flow cytometric evaluation. In striking contrast, the macrophages in the interacinar stroma are different and made of two sets based mainly on their expression of the mannose receptor, CD206, and CLEC10A, CD301. The islet macrophages are found in the islets since birth and are derived from definitive hematopoiesis. The CD206/CD301 negative stromal set derives from blood monocytes and is constantly dividing. In contrast, the CD206/CD301+ population derives from yolk-sac hematopoiesis and shows slow turnover. Both sets of stromal macrophages actually have a different anatomical representation. The pancreatic ducts are surrounded by the CD206/CD301+ macrophages while the CD206/CD301 negative macrophages are interspersed throughout the acinar stroma. These resident macrophages, under steady state, do not exchange with monocytes but are maintained by a low level of replication.

 

We identified that the stromal and islet macrophages differed in their gene expression profiles and function. The islet-resident displayed an M1-like activation pattern as well as high levels of MHC-II on their surface. The stromal macrophages expressed an M2-like pattern of transcripts, and also high levels of MHC-II. Expression of different genes in macrophages is complex, so the M1/M2 pattern is an oversimplification as others have stated. Yet our point stands that the gene expression patterns of islets and stromal macrophages are distinct and compatible with the M1/M2 patterns, albeit with the qualification stated above.

 

The pattern of gene expressions in islet and stromal macrophages was maintained during adulthood and, importantly, after irradiation and replacement with stem cells. We attributed such difference to the particular anatomy of the pancreas. In unknown ways, the islet promotes the activated M1-like macrophage while the stroma favors the M2-like. Future studies are required to determine how the tissue microenvironment conditions the biology of macrophages.

 

There is a symbiotic relationship between beta cells and macrophages and both influence each other. The islet macrophages have a homeostatic or trophic role first reported by Jeffrey Pollard’s group’s examination of the osteopetrotic op/op mouse. The op mutation inactivates the Csf1 gene leading to a reduction or absence of macrophages at many anatomical locations. The islets of the op/op mouse are diminished in size and number. This mouse also showed impaired glucose tolerance. Calderon in our group showed that most of the islets of the op/op mouse lacked macrophages or contained no more than two per islet. In normal mice, we saw an average of 10 macrophages per islet. The range was dependent on the size of the islets, with bigger islets containing up 20 macrophages, while smaller islets contained fewer (78, 80).

 

How the macrophage and the islet endocrine cells are interacting is unknown. We hypothesize that the endocrine cells secrete signals that make the macrophages convert to a trophic supportive role. Additionally, all the islet resident macrophages are closely associated with the vascular endothelium. Live images of islets carried out by Bernd Zinselmeyer showed macrophages (labeled with CX3CR1-GFP) extending long cytoplasmic projections in between beta cells. These cytoplasmic projections can be seen crossing to the lumen of the blood vessel. Calderon did an ingenious experiment injecting B10.BR or NOD mice with 1um latex bead coated with either anti I-Ak or anti I-Ag7 and then isolating the islets a few minutes later: in the islets, the specific antibody coated beads were localized to blood vessels, always next to a macrophage. It is a telling result that suggests the intra-islet macrophage communicates with the circulating blood cells.

Concerning antigen presentation, the islet resident macrophages take up beta cell secretory granules and can present diabetogenic antigens. In our first series of studies, we isolated the islet macrophages from NOD mice and found them to spontaneously present diabetogenic antigens including insulin. These studies are complex in the sense that NOD mice, as we discuss below, also contain a set of CD103+ DCs that can present antigen. For other strains, where the only cell in islet is a macrophage, the conclusion points to macrophages as the APC. For example, we tested macrophages isolated from B10.BR mice (H-2k) that expressed HEL as a transgene under the control of the insulin promoter. These islet resident macrophages presented antigen to HEL-specific CD4+ T cells. In contrast, the stromal macrophages did not present the HEL peptides indicating a clear compartmentalization. Thus, the islet macrophages took up the immunogens from beta cells, processed and presented them. In the pLN of such mice, there is proliferation of HEL-reactive CD4+ T cells as measured by CFSE dilution. This indicates that the islet feeds beta cell derived antigens to the pLN. In this case, the most likely transport from islets to pLN is via the resident macrophages. It is known that islets lack lymphatics at steady state; instead the drainage lies in the surrounding acinar stroma. If the transport is by way of the APC, as we believe, it indicates that these move out of islets into the stroma to be able to reach the lymphatics.

 

We have also tried to determine if the islet macrophages contain beta cell material that can be visualized and/or quantified. We used an antibody that only detects insulin peptides and not insulin protein to find out if the islet macrophage, the only islet APC at steady state, contained insulin catabolic products. On average, there were about 10 insulin-peptide positive granules per macrophage. Electron micrographs of islets from various strains of mice also disclosed the presence of granules in APC. The granules in the APCs had the same morphology as the dense core granules, the typical morphology of the insulin containing granule, an indication that such granules have been transferred. We discuss these issues in more detail in a later section, but suffice to say here that definitely the islet phagocytes captures and present beta cell antigens. It is likely that such transferLi) is responsible for the activated biology of the islet macrophages, but, (ii) importantly in the context of autoimmune diabetes, is the conduit of immunological information to the T cell system, which activates fully when all the genetic components responsible for autoimmunity are in place, as happens with the NOD mouse.

 

Selected Publications:

 

 

1: Calderon B, Carrero JA, Ferris ST, Sojka DK, Moore L, Epelman S, Murphy KM, Yokoyama WM, Randolph GJ, Unanue ER. The pancreas anatomy conditions the origin and properties of resident macrophages. J Exp Med. 2015 Sep 21;212(10):1497-512. doi: 10.1084/jem.20150496. Epub 2015 Sep 7. PubMed PMID: 26347472; PubMed Central PMCID: PMC4577842.

  

2: Vomund AN, Zinselmeyer BH, Hughes J, Calderon B, Valderrama C, Ferris ST, Wan X, Kanekura K, Carrero JA, Urano F, Unanue ER. Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells. Proc Natl Acad Sci U S A. 2015 Oct 6;112(40):E5496-502. doi: 10.1073/pnas.1515954112. Epub 2015 Aug 31. PubMed PMID: 26324934; PubMed Central PMCID: PMC4603448.

  

3: Ferris ST, Carrero JA, Mohan JF, Calderon B, Murphy KM, Unanue ER. A minor subset of Batf3-dependent antigen-presenting cells in islets of Langerhans is essential for the development of autoimmune diabetes. Immunity. 2014 Oct 16;41(4):657-69. doi: 10.1016/j.immuni.2014.09.012. PubMed PMID: 25367577; PubMed Central PMCID: PMC4220295.

  

4: Unanue ER. Antigen presentation in the autoimmune diabetes of the NOD mouse. Annu Rev Immunol. 2014;32:579-608. doi: 10.1146/annurev-immunol-032712-095941. Epub 2014 Feb 5. Review. PubMed PMID: 24499272.

  

5: Calderon B, Carrero JA, Unanue ER. The central role of antigen presentation in islets of Langerhans in autoimmune diabetes. Curr Opin Immunol. 2014 Feb;26:32-40. doi: 10.1016/j.coi.2013.10.011. Epub 2013 Nov 16. Review. PubMed PMID: 24556398; PubMed Central PMCID: PMC4118295.

  

6: Carrero JA, Calderon B, Towfic F, Artyomov MN, Unanue ER. Defining the transcriptional and cellular landscape of type 1 diabetes in the NOD mouse. PLoS One. 2013;8(3):e59701. doi: 10.1371/journal.pone.0059701. Epub 2013 Mar 26.Erratum in: PLoS One. 2014;9(1). doi:10.1371/annotation/f277b29e-361b-4e56-b55b-612ebaca0432. PubMed PMID: 23555752; PubMed Central PMCID: PMC3608568.

  

7: Mohan JF, Petzold SJ, Unanue ER. Register shifting of an insulin peptide-MHC complex allows diabetogenic T cells to escape thymic deletion. J Exp Med. 2011 Nov 21;208(12):2375-83. doi: 10.1084/jem.20111502. Epub 2011 Nov 7. PubMed PMID: 22065673; PubMed Central PMCID: PMC3256971.

  

8: Mohan JF, Levisetti MG, Calderon B, Herzog JW, Petzold SJ, Unanue ER. Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes. Nat Immunol. 2010 Apr;11(4):350-4. doi:10.1038/ni.1850. Epub 2010 Feb 28. PubMed PMID: 20190756; PubMed Central PMCID: PMC3080751.