Perforin Positive Regulatory DCs In Immune Tolerance: Novel Anti-Inflammatory Players In Metabolic Syndrome

Immature dendritic cells (imDCs) can have a tolerizing effect in the steady state or following transplantation. However, due to the significant heterogeneity of this cell population it is difficult to study the mechanisms of their tolerance induction. We previously described the generation of a highly defined population of imDCs expressing perforin and granzyme A (Perf-DCs) from hematopoietic progenitors; using TCR transgenic T cells we monitored their ability to delete cognate CD4 and CD8 T cells. While the former are deleted via an MHC-independent mechanism through the nitric oxide system, CD8⁺ T cell deletion occurs through a unique MHC-dependent perforin-based killing mechanism. This involves activation of Toll-like receptors 7, and signaling through Triggering Receptor Expressed on Myeloid cells -1. Importantly, this novel subpopulation of Perf-DCs was also detected in various lymphoid tissues in normal animals, and its frequency is markedly enhanced upon GM-CSF administration (Zangi et al, Blood 2012).

Here, we investigated the potential regulatory role of Perf-DCs in steady state in-vivo by selectively knocking out the expression of perforin in these cells. To this end, we generated BM chimeras using a 1:1 mixture of BM from perforin KO mice and from BM of mice ablated of CD11c^high DCs using diphtheria toxin expression under the CD11c promoter (Birnberg et al, Immunity 2008). In the resulting PKO-DTA chimeras, perforin expression was completely lost in conventional CD11c+ DCs, while 50% of the T and NK cell populations still expressed perforin. At 6 months post transplant, DTA-PKO chimeric mice spontaneously gained more weight than chimeras created using a mixture of normal BM with BM from perforin KO mice (WT-PKO). The increased weight gain observed in DTA-PKO mice prompted us to test whether this phenomenon was accompanied by other metabolic alterations. Indeed, DTA-PKO mice exhibited elevated serum cholesterol and triglyceride levels compared to control WT-PKO chimeras (140±3.5 vs. 115±8.6, 125±31vs. 88±9.8 mg/dl, N≥5). Total body fat percent as measured by body composition MRI was significantly higher in DTA-PKO mice (30.3%±2.2 vs. 14.5%±2.3), along with highly elevated levels of leptin (37±10.5 vs. 9.8±3 ng/ml).

In addition, DTA-PKO chimeric mice exhibited glucose intolerance (p=0.034) and reduced insulin sensitivity (p=6.07x10^-6). Immunohistological evaluation revealed a significant reduction in the percentage of insulin expressing pancreatic β cell- tissue (2.2%±0.54 vs. 5.75%±1.98). Importantly, the visceral adipose tissue (VAT) of DTA-PKO chimeras contained more crown-like structures typically formed when macrophages within inflamed AT surround dead adipocytes.

Based on these characteristics of metabolic syndrome that develop in DTA-PKO chimeras over 6 months, we tested whether high-fat diet (HFD) enhances the rate of disease development. Indeed, DTA-PKO chimeras maintained on HFD displayed more pronounced weight gain compared to their HFD-maintained WT counterparts when tested 6 weeks after HFD initiation. Likewise, cholesterol and triglycerides as well as leptin and IL-1b in the serum, and TNF-α and IL-6 in the AT were elevated in DTA-PKO mice compared to the WT-PKO animals. Importantly, analysis of immune cell populations in collagenase-digested VAT revealed more CD8+ and CD4+ T cells in DTA-PKO mice compared to control chimeras (78.3x10³±17.5x10³ vs. 24.9x10³±3.2x10³ and 113x10³±21x10³ vs. 43x10³±4.4x10³respectively). Thus, triggering of inflammation in the AT previously shown to be mediated by T cells (Winer et al, Nat.Med 2009; Nishimura et al, Nat.Med 2009), is not effectively regulated in mice lacking Perf-DCs. Interestingly, a similar enhanced rate of metabolic imbalance was found in regular diet-fed DTA-PKO chimeras using RIP-mOVA mice expressing ovalbumin in the thymus, pancreas and kidneys, and known to be more prone to autoimmunity. Moreover, a significantly larger fraction of dividing cells was observed when CD8 T cells, isolated from AT of DTA-PKO chimeric RIP-mOVA mice were stimulated against splenocytes of mice expressing ovalbumin in all tissues (wOVA mice). Taken together, our results demonstrate that Perf-DCs have a unique immune regulatory role under steady state, controlling unwanted inflammatory processes in adipose tissue. Further studies of the role of Perf-DCs in other models of autoimmunity are warranted.

Disclosures:

No relevant conflicts of interest to declare.

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