Several steps are crucial for induction of type 1 diabetes (T1D); the first is extravasation of CD4+ T lymphocytes from blood vessels into the pancreatic tissue, the second is penetration of the peri-islet basement membrane (BM) surrounding the β-islets, and third the β-cell destruction which leads to appearance of disease symptoms.

BMs act to separate tissue compartments and represent barriers to the movement of both soluble molecules and cells. Hence, cells penetrating such protein barriers must employ specialized mechanisms (1,2). The main question addressed in our nPOD project is what is the mechanism used by leukocytes to penetrate the peri-islet BM and, thereby, reach the insulin producing β-cells during the development of T1D, in the non-obese diabetic (NOD) mouse model and in recently diagnosed T1D patients.

We recently published a comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet BM and subjacent interstitial matrix, in development of T1D in NOD mice and in human T1D (3), for which nPOD samples were crucial. Our data demonstrated global loss of peri-islet BM components only at sites of leukocyte infiltration into the islet in both mouse and man. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step in disease development. We identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human T1D (3). Interestingly, the peri-islet BM is reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies (3).

We are now investigating how the cathepsins are involved in loss of the peri-islet BM: Studies are underway to detect and image cathepsin activity in mouse NOD and nPOD samples using cathepsin activity-based probes, which could lead to the development of novel diagnostic tools and therapeutic targets. In addition, we are examining the BM of the pancreas from different stages of human T1D and islet transplantation, with the aim of identifying ECM molecules that could promote islet survival.

Reference:

1) Korpos, E. et al, 2010. Cell Tissue Res. 339:47-57.

2) Wu, C. et al, 2009. Nat Med. 15:519-27.

3) Korpos et al, 2013. Diabetes 62:531-42.

 

Investigation of tertiary lymphoid organ formation in nPOD samples with ‘’typical insulitis’’

Lymphoid neogenesis is associated with chronic inflammatory diseases such as type 1 diabetes (T1D) and leads to formation of tertiary lymphoid organs (TLO). TLOs, like secondary lymphoid organs (lymph nodes and the spleen), are compartmentalized into T- and B-cell zones and contain reticular fiber networks composed of reticular fibroblast cells (RFCs) and reticular fibers (RFs), the latter of which are unique extracellular matrix (ECM) structures that have been characterized in our lab (Sixt et al, 2005; Song et al, 2013). The RFs of lymph nodes constitute the structural backbone of the organ, but also support the migration of T- and B-cells (Bajénoff et al, 2008) and conduct fluid transport of small molecules and soluble antigens (<70kDa), (Sixt et, 2005). Although TLOs have been described in many chronically inflamed tissues, their characterization from the ECM point of view is limited and their contribution to autoimmune diseases such as diabetes is not clear.

Using whole mount stainings and confocal microscopy we have characterized the cellular and ECM composition of RFs in TLOs of the inflamed pancreas of non-obese diabetic (NOD) mice, where the autoimmune destruction of insulin producing β-cells leads to replacement of pancreatic islets by TLOs. Our data suggest a role for the RFs in the conduction of chemokines from the infiltrating front to the high endothelial venules (HEVs) to propagate immune cell recruitment to the inflamed islet as well as antigen presentation to dendritic cells bound to the RFs, thereby, accelerating and promoting the inflammatory response. To be able to stem this acceleration step by interfering with TLO formation represents a potential mode of at least slowing T1D progression. However, it is first necessary to define whether the structure of the TLO is the same in mouse and in human T1D pancreatic samples with “typical” insulitis.

To investigate whether the presence of TLO in nPOD samples is correlated with insulitis we will perform immunofluorescence staining using antibodies specific for

1) different ECM molecules to identify the structure of the RFs;

2) HEVs, immune cell compartments and reticular fibroblasts to define the interconnection between the infiltrating front and the HEVs; and

3) auto-antigen (insulin) and chemokines to define whether the RFs also have a conduit function.

Reference

1) Sixt M, et al, 2005. Immunity, 22(1):19-29.

2) Bajénoff M, et al, 2008. J Immunol, 181(6):3947–3954.

3) Song J, et al, 2013. Proc Natl Acad Sci, 110(31):E2915-24.