Physiologic Doses of Bilirubin Contribute to Tolerance of Islet Transplants by Suppressing the Innate Immune Response.

Bilirubin has been recognized as a powerful cytoprotectant when used at physiologic doses and was recently shown to have immunomodulatory effects in islet allograft transplantation, conveying donor-specific tolerance in a murine model. We hypothesized that bilirubin, an antioxidant, acts to suppress the innate immune response to islet allografts through two mechanisms: 1) by suppressing graft release of damage-associated molecular patterns (DAMPs) and inflammatory cytokines, and 2) by producing a tolerogenic phenotype in antigen-presenting cells. Bilirubin was administered intraperitoneally before pancreatic procurement or was added to culture media after islet isolation in AJ mice. Islets were exposed to transplant-associated nutrient deprivation and hypoxia. Bilirubin significantly decreased islet cell death after isolation and hypoxic stress. Bilirubin supplementation of islet media also decreased the release of DAMPs (HMGB1), inflammatory cytokines (IL-1ß and IL-6), and chemokines (MCP-1). Cytoprotection was mediated by the antioxidant effects of bilirubin. Treatment of macrophages with bilirubin induced a regulatory phenotype, with increased expression of PD-L1. Coculture of these macrophages with splenocytes led to expansion of Foxp3+ Tregs. In conclusion, exogenous bilirubin supplementation showed cytoprotective and antioxidant effects in a relevant model of islet isolation and hypoxic stress. Suppression of DAMP release, alterations in cytokine profiles, and tolerogenic effects on macrophages suggest that the use of this natural antioxidant may provide a method of preconditioning to improve outcomes after allograft transplantation.

Effect of disrupted mitochondria as a source of damage-associated molecular patterns on the production of tumor necrosis factor α by splenocytes from dogs.

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells. SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes. PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow-differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay. RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system. CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.

Continuous retinoic acid induces the differentiation of mature regulatory monocytes but fails to induce regulatory dendritic cells.

BACKGROUND: Myeloid cells (MC) have potent immunoregulatory abilities that can be therapeutically useful to treat inflammatory disease. However, the factors which promote regulatory myeloid cell differentiation remain poorly understood. We have previously shown that estriol (E3) induces mature regulatory dendritic cells in vivo. To determine whether additional steroid hormones could induce mature regulatory myeloid cells, we investigated the effects of retinoic acid (RA) on MCs. Retinoic acid is a steroid hormone important in regulating mucosal immunity in the gut and promoting myeloid differentiation. We hypothesized that the presence of RA during differentiation would promote the formation of mature regulatory myeloid cells (MCregs). METHODS: To determine RA's ability to induce regulatory myeloid cells, we differentiated bone marrow progenitor cells with granulocytic-macrophage colony-stimulating factor (GM-CSF) under the influence of RA. We found that day 7 MCs differentiated in the presence of RA had an increase in the percent positive and relative expression levels of both maturation (CD80, CD86, and MHCII) and inhibitory (PD-L1 and PD-L2) markers compared to control cells. Functionally, these day 7 RA MCs expressed increased intracellular IL-10, induced regulatory T cells in vitro compared to controls and suppressed the proliferation of responder immune cells even after inflammatory challenge with LPS. CONCLUSION: RA induced mature regulatory myeloid cells that were suppressive and had a CD11b+ CD11c-Ly6C low/intermediate monocyte phenotype. Surprisingly, RA CD11c+ dendritic cells were not suppressive and could contribute to enhanced proliferation. These results suggest that continuous RA has unique effects on different myeloid populations during monopoeisis and dendropoiesis and promotes a population of regulatory monocytes.

Mitochondrial transcription factor A, an endogenous danger signal, promotes TNFα release via RAGE- and TLR9-responsive plasmacytoid dendritic cells.

OBJECTIVE: Mitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNFα release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE. MATERIALS AND METHODS: Murine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNFα release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-κB. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition. MAIN RESULTS: TFAM augmented splenocyte TNFα release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-κB, were essential for splenocyte TNFα release in response to TFAM+CpGA DNA. Interestingly, TNFα release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNFα release. CONCLUSIONS: TFAM promoted TNFα release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNFα release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals.

Mitochondrial transcription factor A serves as a danger signal by augmenting plasmacytoid dendritic cell responses to DNA.

Plasmacytoid dendritic cells (pDC) are potent APCs known to regulate immune responses to self-Ags, particularly DNA. The mitochondrial fraction of necrotic cells was found to most potently promote human pDC activation, as reflected by type I IFN release, which was dependent upon the presence of mitochondrial DNA and involved TLR9 and receptors for advanced glycation end products. Mitochondrial transcription factor A (TFAM), a highly abundant mitochondrial protein that is functionally and structurally homologous to high mobility group box protein 1, was observed to synergize with CpG-containing oligonucleotide, type A, DNA to promote human pDC activation. pDC type I IFN responses to TFAM and CpG-containing oligonucleotide, type A, DNA indicated their engagement with receptors for advanced glycation end products and TLR9, respectively, and were dependent upon endosomal processing and PI3K, ERK, and NF-κB signaling. Taken together, these results indicate that pDC contribute to sterile immune responses by recognizing the mitochondrial component of necrotic cells and further incriminate TFAM and mitochondrial DNA as likely mediators of pDC activation under these circumstances.