Adoptive transfer of pre-activated nTreg does not protect animals from IRI.

<p>nTreg were obtained from FoxP3 GFP mouse spleens by flow sorting followed by expansion <i>in vitro</i> for 21 days and further flow-sorting for purity [a–c]. Flow cytometry of spleen and liver confirmed successful transfer [d,e]. There was no difference in injury severity between nTreg supplemented and control animals at 3 hours of reperfusion (n = 4 per group) [f].</p

Treg depletion in the FoxP3.LuciDTR mouse does not increase susceptibility to IRI.

<p>FoxP3.LuciDTR mice or wildtype control (n = 5–10 per group) received 25 ng/g DT 24 hours prior to ischemic insult. ALT release [a] and histological injury score [b] did not differ between groups. DT treatment of DTR animals effected almost total depletion of Treg from the circulation [c,d], spleen [e] and liver [f].</p

Treg antibody depletion does not increase susceptibility to IRI.

<p>Wildtype mice received the CD25 depleting antibody pc61 7 and 2 days or PBS (n = 5–8 per group) prior to ischemia reperfusion injury. Injury severity was no different between Treg intact and Treg depleted animals in terms of ALT [a] or histological injury score [b]. PC61 treatment resulted in significant depletion of CD4+CD25+[c] and CD4+FoxP3+[d] cells. FlowCytomix was used to profile circulating chemo/cytokines. No differences were detected in post-operative rises in CXCL-10/IP-10 [e], KC/CXCL-1 [f], IL-6 [g] and GM-CSF [h] between Treg intact and depleted animals. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and IL-10) were not detected.</p

<i>In vivo</i> expansion of Treg does not protect animals from IRI.

<p>Mice received IL-2/JES6-A12 complexes or PBS for three consecutive days prior to hepatic ischemia reperfusion insults (n = 8 per group). Animals were culled at 24 hours of reperfusion, and tissues analysed for Treg frequency. There was significant expansion of splenic [a], circulating [b] and hepatic [c] Treg. Injury severity was similar between Treg expanded and control animals, in terms of ALT and histological injury [d,e]. FlowCytomix was used to profile circulating chemo/cytokines. Levels of IL-2 [f], CXCL-10/IP-10 [g] and IL-6 [h] were elevated. Rises in KC/CXCL-1 [i], and GM-CSF [j] did not differ between groups. Other analytes (IL-1α, IL-1β, IL-10, IL-17, IL-17F, IFNγ, MIP-1α) were not detected.</p

Treg mobilization during reperfusion is not enhanced by IPC.

<p>Mice (n = 5–8 per group) were subjected to ischemia reperfusion injury with or without ischemic preconditioning and killed at 3 or 24 hours. Ischemic preconditioning protected the liver from injury both in terms of ALT release [a] and histological injury score [b] measured at 24 hours. CD4+FoxP3+cells were mobilized into the circulation during reperfusion [c]. Hepatic CD4+FoxP3+cells increased over the same time period [d]. Total CD3+lymphocytes were stable throughout reperfusion [e]. FlowCytomix was used to profile circulating chemo/cytokines. Rises were detected in CXCL-10/IP-10 [f], CXCL-1/KC [g], IL-6 [h] and GMCSF [i]. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and the Treg cytokine IL-10) were not detected.</p

Adoptive transfer of pre-activated iTreg does not protect animals from IRI.

<p>iTreg were generated <i>in vitro</i> from sorted CD62L high, FoxP3- naïve T cells cultured in the presence of TGFβ and IL-2 for 5 days before flow sorting to maximize purity [a-d]. Successful transfer was confirmed by detection of FoxP3 GFP+cells in spleen [e] and liver [f]. No difference was detected in injury severity between iTreg supplemented and PBS treated control animals at 3 or 24 hours of reperfusion (n = 4–5 per group) [g].</p

IgM and B cells are not critical to resolution of injury in the liver.

<p>Given the apparent lack of pathogenicity of IgM or B cells in this model of hepatic IRI, it was decided to see if they had a significant role in the resolution from injury. The recovery from injury was compared between WT and μMT mice. Representative H&E sections (x25 magnification), demonstrate similar injury at 24 hours, which resolved almost completely by day 7 post-IRI in both WT and μMT mice (residual necrosis<2% all mice [WT (n = 3), μMT (n = 4)]).</p

The role of B and T cells in hepatic IRI.

<p>To investigate whether the protection seen in RAG1-/- mice resulted from B and/or T cells, WT controls and mice deficient respectively in mature B cells (μMT) and T cells (CD3εKO) underwent 40 minutes left hepatic lobe ischemia (n = 12/group). Following 24 hours, reperfusion a significant reduction (Kruskall-Wallis p = 0.0037) was seen in mice lacking T, but not B cells.</p

IgM deposition in acute sterile liver injury.

<p>Tissue sections of explanted livers from patients transplanted for fulminant liver failure secondary to paracetamol overdose were stained looking for evidence of IgM deposition; extensive deposition of IgM is demonstrated within areas of parenchymal injury (<b>A</b>). Tissue sections from male WT mice, with demonstrable sterile liver injury on sections stained with H&E following injection of 200mg/kg paracetamol or 10mg/kg con A also showed extensive IgM staining upon immunohistochemical staining (<b>B</b>). Male mice underwent 40 minutes left lobe ischemia and were then reperfused for 0 hours to 7 days (n = 2–6 per timepoint). The extent of IgM deposition was compared with the histological injury seen on slides stained with H&E; x25 magnification slides are shown at selected time points (<b>C</b>). IgM deposition was calculated as a percentage of total section area across the time-course (median + Standard Error of Mean (SEM)) (<b>D</b>); this deposition of IgM was rapid (seen within 1 hour) and disappeared with macroscopic resolution of injury.</p