Innate and Adaptive Immune Responses in Intestinal Transplant Rejection: Through the Lens of Inflammatory Bowel and Intestinal Graft-Versus-Host Diseases.

Intestinal transplantation is a life-saving procedure utilized for patients failing total parenteral nutrition. However, intestinal transplantattion remains plagued with low survival rates and high risk of allograft rejection. The authors explore roles of innate (macrophages, natural killer cells, innate lymphoid cells) and adaptive immune cells (Th1, Th2, Th17, Tregs) in inflammatory responses, particularly inflammatory bowel disease and graft versus host disease, and correlate these findings to intestinal allograft rejection, highlighting which effectors exacerbate or suppress intestinal rejection. Better understanding of this immunology can open further investigation into potential biomolecular targets to develop improved therapeutic treatment options and immunomonitoring techniques to combat allograft rejection and enhance patient lives.

Circulating, cell-free methylated DNA indicates cellular sources of allograft injury after liver transplant.

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here, we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to patterns established to identify cell types in different organs. For liver cell types DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and regulatory regions functionally important for the respective cell types. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However, sustained elevation of hepatocyte and biliary epithelial cfDNA beyond the first week indicates early-onset allograft injury. Further, cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and timely intervention.

Type 1 Innate Lymphoid Cells Are Proinflammatory Effector Cells in Ischemia-Reperfusion Injury of Steatotic Livers.

Innate lymphoid cells (ILCs), the most recently described family of lymphoid cells, play fundamental roles in tissue homeostasis through the production of key cytokine. Group 1 ILCs, comprised of conventional natural killer cells (cNKs) and type 1 ILCs (ILC1s), have been implicated in regulating immune-mediated inflammatory diseases. However, the role of ILC1s in nonalcoholic fatty liver disease (NAFLD) and ischemia-reperfusion injury (IRI) is unclear. Here, we investigated the role of ILC1 and cNK cells in a high-fat diet (HFD) murine model of partial warm IRI. We demonstrated that hepatic steatosis results in more severe IRI compared to non-steatotic livers. We further elicited that HFD-IRI mice show a significant increase in the ILC1 population, whereas the cNK population was unchanged. Since ILC1 and cNK are major sources of IFN-γ and TNF-α, we measured the level of ex vivo cytokine expression in normal diet (ND)-IRI and HFD-IRI conditions. We found that ILC1s in HFD-IRI mice produce significantly more IFN-γ and TNF-α when compared to ND-IRI. To further assess whether ILC1s are key proinflammatory effector cells in hepatic IRI of fatty livers, we studied both Rag1-/- mice, which possess cNK cells, and a substantial population of ILC1s versus the newly generated Rag1-/-Tbx21-/- double knockout (Rag1-Tbet DKO) mice, which lack type 1 ILCs, under HFD IRI conditions. Importantly, HFD Rag1-Tbet DKO mice showed significant protection from hepatic injury upon IRI when compared to Rag1-/- mice, suggesting that T-bet-expressing ILC1s play a role, at least in part, as proinflammatory effector cells in hepatic IRI under steatotic conditions.

Oral N-acetylcysteine decreases IFN-γ production and ameliorates ischemia-reperfusion injury in steatotic livers.

Type 1 Natural Killer T-cells (NKT1 cells) play a critical role in mediating hepatic ischemia-reperfusion injury (IRI). Although hepatic steatosis is a major risk factor for preservation type injury, how NKT cells impact this is understudied. Given NKT1 cell activation by phospholipid ligands recognized presented by CD1d, we hypothesized that NKT1 cells are key modulators of hepatic IRI because of the increased frequency of activating ligands in the setting of hepatic steatosis. We first demonstrate that IRI is exacerbated by a high-fat diet (HFD) in experimental murine models of warm partial ischemia. This is evident in the evaluation of ALT levels and Phasor-Fluorescence Lifetime (Phasor-FLIM) Imaging for glycolytic stress. Polychromatic flow cytometry identified pronounced increases in CD45+CD3+NK1.1+NKT1 cells in HFD fed mice when compared to mice fed a normal diet (ND). This observation is further extended to IRI, measuring ex vivo cytokine expression in the HFD and ND. Much higher interferon-gamma (IFN-γ) expression is noted in the HFD mice after IRI. We further tested our hypothesis by performing a lipidomic analysis of hepatic tissue and compared this to Phasor-FLIM imaging using "long lifetime species", a byproduct of lipid oxidation. There are higher levels of triacylglycerols and phospholipids in HFD mice. Since N-acetylcysteine (NAC) is able to limit hepatic steatosis, we tested how oral NAC supplementation in HFD mice impacted IRI. Interestingly, oral NAC supplementation in HFD mice results in improved hepatic enhancement using contrast-enhanced magnetic resonance imaging (MRI) compared to HFD control mice and normalization of glycolysis demonstrated by Phasor-FLIM imaging. This correlated with improved biochemical serum levels and a decrease in IFN-γ expression at a tissue level and from CD45+CD3+CD1d+ cells. Lipidomic evaluation of tissue in the HFD+NAC mice demonstrated a drastic decrease in triacylglycerol, suggesting downregulation of the PPAR-γ pathway.

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.