P6303Developmental origin of cardiac macrophages in steady state and myocardial infarction

Background Macrophages are the most abundant immune cells in the myocardial tissue in steady state. The sterile inflammation caused by myocardial infarction triggers a massive immune reaction, which leads to a profound influx of neutrophils and monocytes. In the postacute phase of infarction macrophages play an essential role in reparative processes. Recently, it has become clear that macrophages in the heart have a dual developmental origin from embryonic and bone marrow (BM) hematopoiesis. In this study, we sought to investigate the contribution of embryonic derived macrophages to the cardiac macrophage pool in steady state as well as the acute and chronic phase after ischemia/reperfusion injury. Methods/Results To address the origin of macrophages in steady state we used different models of lineage tracing to determine the developmental origin of cardiac macrophages. Using FLT3-Cre mice and radiation-independent CD45.1/.2 bone marrow chimera, we found that the resident macrophage population in the heart is mainly independent of definitive hematopoiesis (approximately 70–80% of cardiac macrophages). The BM-dependent population on the other hand is replenished by blood-derived monocytes. Further we used the radiation-independent CD45.1/.2 bone marrow chimera to characterize the origin of macrophages at different time points after I/R-injury. In the acute phase after myocardial infarction we observed a profound influx of BM-derived macrophages in the infarct region and also in the remote area. 30 days after I/R-injury the composition of the resident macrophage pool was mainly comprised of BM-independent macrophages, similar to steady state conditions. To address the role of BM-derived macrophages we used CCR2-ko mice, which have low numbers of inflammatory monocytes in peripheral blood. CCR2-ko mice showed reduced macrophage numbers in the acute phase after myocardial infarction. Using positron emission tomography we investigated the influence of CCR2-deficiency on cardiac function after I/R-injury. In comparison to WT mice, CCR2-ko mice showed a significantly increased infarct size. Cardiac remodeling, determined by end-diastolic volume, on the other hand was improved in CCR2-ko mice. The ejection fraction was similar in both groups. Conclusion The cardiac macrophage pool is mainly comprised of BM-independent macrophages. In response to I/R-injury monocyte-derived macrophages transiently enter the myocardium but do not persist in significant numbers over time. The influx of BM-derived macrophages after I/R-injury was reduced using CCR2-ko mice, which led to improved cardiac remodeling. Our findings are of potential importance for understanding the cardiac immune response and for the therapeutic targeting of macrophages in inflammatory conditions. Acknowledgement/Funding German Society of Cardiology, German Centre for Cardiovascular Research, LMU Excellence, SFB 914

Stat2 loss disrupts damage signalling and is protective in acute pancreatitis

ABSTRACTSeverity of sterile inflammation, as seen in acute pancreatitis, is determined by damage-sensing receptors, signalling cascades and cytokine production. Stat2 is a type I interferon signalling mediator that also has interferon-independent roles in murine lipopolysaccharide-induced NF-κB-mediated sepsis. However its role in sterile inflammation is unknown. We hypothesised that Stat2 determines severity of non-infective inflammation in the pancreas.Wild type (WT) and Stat2−/− mice were injected intraperitoneally with cerulein or L-arginine. Specific cytokine-blocking antibodies were used in some experiments. Pancreata and blood were harvested 1h and 24h after the final dose of cerulein and up to 96h post L-arginine. Whole-tissue phosphoproteomic changes were assessed using label-free mass spectrometry. Tissue-specific Stat2 effects were studied in WT/Stat2−/− bone-marrow chimera and using Cre-lox recombination to delete Stat2 in pancreatic and duodenal homeobox 1(Pdx1)-expressing cells.Stat2−/− mice were protected from cerulein- and L-arginine-induced pancreatitis. Protection was independent of type I interferon signalling. Stat2−/− mice had lower cytokine levels including TNFα and IL-10 and reduced NF-kB nuclear localisation in pancreatic tissue compared to WT. Inhibition of TNFα improved (inhibition of IL-10 worsened) cerulein-induced pancreatitis in WT but not Stat2−/− mice. Phosphoproteomics showed down-regulation of mitogen-activated protein kinase (MAPK) mediators but accumulation of Ser412-phosphorylated Tak1. Stat2 deletion in Pdx1-expressing acinar cells (Stat2flox/Pdx1-cre) reduced pancreatic TNFα expression, but not histological injury or serum amylase. WT/Stat2−/− bone-marrow chimera were protected from pancreatitis irrespective of host or recipient genotype.Stat2 loss results in disrupted signalling in pancreatitis, upstream of NF-κB in non-acinar and/or bone marrow derived cells.

IL-27 confers a protumorigenic activity of regulatory T cells via CD39

Expression of ectonucleotidase CD39 contributes to the suppressive activity of Foxp3+ regulatory T cells (Tregs) by hydrolyzing immunogenic ATP into AMP. The molecular mechanism that drives CD39 expression on Tregs remains elusive. We found that tumor-infiltrating Tregs (Ti-Tregs) failed to up-regulate CD39 in mice lacking EBI3 subunit of IL-27 or IL-27Ra. Mixed bone marrow chimera and in vitro studies showed that IL-27 signaling in Tregs directly drives CD39 expression on Ti-Tregs in a STAT1-dependent, but STAT3- and T-bet–independent, manner. Tregs stimulated with IL-27 showed enhanced suppressive activities against CD8+ T cell responses in vitro. Moreover, IL-27Ra–deficient Tregs and STAT1-deficient Tregs were less efficient than WT Tregs in suppressing antitumor immunity in vivo. CD39 inhibition significantly abolished IL-27–induced suppressive activities of Tregs. Thus, IL-27 signaling in Tregs critically contributes to protumorigenic properties of Tregs via up-regulation of CD39.

Distinct requirements for T-bet in gut innate lymphoid cells

Interleukin (IL)-22–producing innate lymphoid cells (ILCs; ILC22) comprise a heterogeneous population of cells that are dependent on the transcription factor retinoid-related orphan γt (RORγt) and are critical for barrier function of the intestinal mucosa. A distinct ILC22 subset expresses the natural cytotoxicity receptor NKp46 (NKp46+ ILC22); however, the factors that contribute to the generation of this population versus other subsets are largely unknown. Herein, we show that T-bet (encoded by Tbx21) was highly expressed in NKp46+ ILC22, a feature shared by all NKp46+ cells present in the intestine but not by other IL-22–producing populations. Accordingly, the absence of T-bet resulted in loss of NKp46+ ILC22 in the intestinal lamina propria. The residual NKp46+ ILC22 present in Tbx21−/− mice showed a marked reduction of Rorγt expression and impairment in IL-22 production. Generation and functions of gut NK1.1+ cells were also altered. Bone marrow chimera experiments revealed a cell-intrinsic requirement for T-bet in these subsets and competitive reconstitution experiments revealed roles for T-bet in multiple ILC subsets. Thus, T-bet has a general importance for ILC in the gut and plays a selective and critical role in the generation of NKp46+ ILC22.

TCR-dependent differentiation of thymic Foxp3+ cells is limited to small clonal sizes

Numerous studies have highlighted the importance of high-affinity interactions between T cell receptors (TCRs) and their ligands in the selection of Foxp3+ regulatory T cells (T reg cells). To determine the role of the TCR in directing T cells into the Foxp3+ lineage, we generated transgenic (Tg) mice expressing TCRs from Foxp3+ cells. Initial analyses of the TCR Tg mice crossed with RAG-deficient mice showed that the percentage of Foxp3+ cells was very low. However, intrathymic injection and bone marrow chimera experiments showed a saturable increase of the Foxp3+ population when T reg TCR Tg cells were present in low numbers. Furthermore, when analyzing whole thymi of T reg TCR Tg RAG-deficient mice, we found significantly more Foxp3+ cells than in conventional T cell TCR Tg mice. Our results indicate that although the TCR has an instructive role in determining Foxp3 expression, selection of Foxp3+ individual clones in the thymus is limited by a very small niche.