Stefan M. Brunner

Interleukin-33 prolongs allograft survival during chronic cardiac rejection

Interleukin‐33 (IL‐33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL‐33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C‐H2bm12/KhEg hearts were transplanted into MHC class II‐mismatched C57Bl/6J mice; IL‐33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL‐33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft‐infiltrating cells. Proinflammatory IL‐17A production was decreased with IL‐33 treatment, while increased levels of IL‐5, IL‐10, and IL‐13 were observed. After IL‐33 therapy, flow cytometry showed a direct induction of CD4+ Foxp3+ Treg, whereas the number of B220+ CD19+ B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL‐33 treatment, a significant decrease in graft‐infiltrating CD11bhighGr1high granulocytes coincided with a significant increase in CD11bhighGr1intermediate myeloid‐derived suppressor cells (MDSC). In conclusion, IL‐33 treatment in the setting of chronic rejection promotes the development of a Th2‐type immune response that favors MDSC and Treg expansion, reduces antibody‐mediated rejection (AMR), and ultimately, prolongs allograft survival.

Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation

BACKGROUND & AIMS The aim of this study was to examine the development of biliary epithelial damage between organ retrieval and transplantation and its clinical relevance for patients. METHODS Common bile duct samples during donor hepatectomy, after cold storage, and after reperfusion were compared to healthy controls by hematoxylin and eosin (H&E) staining and immunofluorescence for tight junction protein 1 and Claudin-1. A bile duct damage score to quantify biliary epithelial injury was developed and correlated with recipient and donor data and patient outcome. RESULTS Control (N=16) and donor hepatectomy bile ducts (N=10) showed regular epithelial morphology and tight junction architecture. After cold storage (N=37; p=0.0119), and even more after reperfusion (N=62; p=0.0002), epithelial damage, as quantified by the bile duct damage score, was markedly increased, and both tight junction proteins were detected with inappropriate morphology. Patients with major bile duct damage after cold storage had a significantly increased risk of biliary complications (relative risk 18.75; p<0.0001) and graft loss (p=0.0004). CONCLUSIONS In many cases, the common bile duct epithelium shows considerable damage after cold ischemia with further damage occurring after reperfusion. The extent of epithelial damage can be quantified by our newly developed bile duct damage score and is a prognostic parameter for biliary complications and graft loss. Possibly, in an intraoperative histological examination, this bile duct damage score may influence decision-making in transplantation surgery.

Tumor‐infiltrating, interleukin‐33–producing effector‐memory CD8+ T cells in resected hepatocellular carcinoma prolong patient survival

Interleukin‐33 (IL‐33), a cytokine with pleiotropic functions, is elevated in serum of patients with hepatocellular carcinoma (HCC). This study investigated the effects of local IL‐33 expression in resected HCC on patient survival and on the immunological and molecular tumor microenvironment. Tissue of resected HCCs was stained for hematoxylin and eosin, Masson trichrome, alpha‐smooth muscle actin, IL‐33, CD8, and IL‐13 and analyzed by flow cytometry. Besides histomorphologic evaluation, the immunohistochemical stainings were analyzed for the respective cell numbers separately for tumor area, infiltrative margin, and distant liver stroma. These findings were correlated with clinical data and patient outcome. Further, gene expression of different HCC risk groups was compared using microarrays. In multivariable analysis, infiltration of HCCs by IL‐33+ cells (P = 0.032) and CD8+ cells (P = 0.014) independently was associated with prolonged patient survival. Flow cytometry demonstrated that cytotoxically active subpopulations of CD8+ cells, in particular CD8+CD62L–KLRG1+CD107a+ effector‐memory cells, are the main producers of IL‐33 in these HCC patients. Using infiltration by IL‐33+ and CD8+ cells as two separate factors, an HCC immune score was designed and evaluated that stratified patient survival (P = 0.0004). This HCC immune score identified high‐ and low‐risk patients who differ in gene expression profiles (P < 0.001). Conclusion: Infiltration of HCCs by IL‐33+ and CD8+ cells is independently associated with prolonged patient survival. We suggest that this is due to an induction of highly effective, cytotoxically active CD8+CD62L–KLRG1+CD107a+ effector‐memory cells producing IL‐33. Based on these two independent factors, we established an HCC immune score that provides risk stratification for HCC patients and can be used in the clinical setting. (Hepatology 2015;61:1957‐1967)

IRAK-M Expression in Tumor Cells Supports Colorectal Cancer Progression through Reduction of Antimicrobial Defense and Stabilization of STAT3

Colorectal cancer (CRC) is associated with loss of epithelial barrier integrity, which facilitates the interaction of the immunological microenvironment with the luminal microbiome, eliciting tumor-supportive inflammation. An important regulator of intestinal inflammatory responses is IRAK-M, a negative regulator of TLR signaling. Here we investigate the compartment-specific impact of IRAK-M on colorectal carcinogenesis using a mouse model. We demonstrate that IRAK-M is expressed in tumor cells due to combined TLR and Wnt activation. Tumor cell-intrinsic IRAK-M is responsible for regulation of microbial colonization of tumors and STAT3 protein stability in tumor cells, leading to tumor cell proliferation. IRAK-M expression in human CRCs is associated with poor prognosis. These results suggest that IRAK-M may be a potential therapeutic target for CRC treatment.

IL-13 signaling via IL-13Rα2 triggers TGF-β1-dependent allograft fibrosis

BackgroundAllograft fibrosis still remains a critical problem in transplantation, including heart transplantation. The IL-13/TGF-β1 interaction has previously been identified as a key pathway orchestrating fibrosis in different inflammatory immune disorders. Here we investigate if this pathway is also responsible for allograft fibrosis and if interference with the IL-13/TGF-β1 interaction prevents allograft fibrosis.MethodsFVB or control DBA/1 donor hearts were transplanted heterotopically into DBA/1 recipient mice and hearts were explanted at day 60 and 100 post-transplantation. Cardiac tissue was examined by Masson’s trichrome staining and immunohistochemistry for CD4, CD8, CD11b, IL-13, Fas ligand, matrix metalloproteinase (MMP)-1, MMP-13, β2-microglobulin, and Gremlin-1. Graft-infiltrating cells were isolated and analyzed by flow cytometry. IL-13 and TGF-β1 levels were determined by enzyme-linked immunosorbent assay (ELISA) and the amount of collagen was quantified using a Sircol assay; IL-13Rα2 expression was detected by Western blotting. In some experiments IL-13/ TGF-β1 signaling was blocked with specific IL-13Rα2 siRNA. Additionally, a PCR array of RNA isolated from the allografts was performed to analyze expression of multiple genes involved in fibrosis.ResultsBoth groups survived long-term (>100 days). The allogeneic grafts were infiltrated by significantly increased numbers of CD4+ (P <0.0001), CD8+ (P <0.0001), and CD11b+ cells (P = 0.0065) by day 100. Furthermore, elevated IL-13 levels (P = 0.0003) and numbers of infiltrating IL-13+ cells (P = 0.0037), together with an expression of IL-13Rα2, were detected only within allografts. The expression of IL-13 and IL-13Rα2 resulted in significantly increased TGF-β1 levels (P <0.0001), higher numbers of CD11bhighGr1intermediateTGF-β1+ cells, and elevated cardiac collagen deposition (P = 0.0094). The allograft fibrosis found in these experiments was accompanied by upregulation of multiple profibrotic genes, which was confirmed by immunohistochemical stainings of allograft tissue. Blockage of the IL-13/TGF-β1 interaction by IL-13Rα2 siRNA led to lower numbers of CD11bhighGr1intermediateTGF-β1+, CD4+, CD8+, and CD11b+ cells, and prevented collagen deposition (P = 0.0018) within these allografts.ConclusionsIL-13 signaling via IL-13Rα2 induces TGF-β1 and causes allograft fibrosis in a murine model of chronic transplant rejection. Blockage of this IL-13/TGF-β1 interaction by IL-13Rα2 siRNA prevents cardiac allograft fibrosis. Thus, IL-13Rα2 may be exploitable as a future target to reduce allograft fibrosis in organ transplantation.

The complement receptors CD46, CD55 and CD59 are regulated by the tumour microenvironment of head and neck cancer to facilitate escape of complement attack

BACKGROUND Membrane-bound complement restriction proteins (mCRPs) CD46, CD55 and CD59 enable tumour cells to evade complement dependent cytotoxicity and antibody-dependent killing mechanisms. But less is known about the role of these mCRPs in head and neck cancer. METHODS In this study we determined the expression of the mCRPs on head and neck squamous cell carcinoma (HNSCC) cell lines, on tumour tissue and TDLNs (tumour-draining lymph nodes) as well as on lymphocytes from HNSCC patients. The influence of the HNSCC microenvironment on the mCRP regulation was analysed using Flow Cytometry, Western blotting and small interfering RNAs (siRNA) transfection studies. RESULTS We examined the effects of the HNSCC tumour milieu on the expression levels of CD46, CD55 and CD59. We investigated the susceptibility of HNSCC cells to CDC (complement-dependent cytotoxicity) while silencing the mCRPs. Our results demonstrate a huge influence of the HNSCC tumour microenvironment on the regulation of mCRP expression and show a reciprocal regulation between the different mCRPs themselves. CONCLUSIONS In summary, our data indicate that HNSCC has evolved different strategies to evade complement attacks and that the tumour microenvironment leads to the enhancement of complement resistance of the surrounding tissue.

IL-13 Orchestrates Resolution of Chronic Intestinal Inflammation via Phosphorylation of Glycogen Synthase Kinase-3β

Spontaneous amelioration of inflammation (often accompanied by fibrosis) is a well-known, but poorly understood, outcome of many chronic inflammatory processes. We studied this phenomenon in a chronic trinitrobenzene sulfonic acid–induced colitis model, an experimental colitis in mice that we showed to ultimately undergo spontaneous resolution, despite continued trinitrobenzene sulfonic acid stimulation. Analysis of the mechanism of this resolution revealed that it was critically dependent on IL-13 activation of STAT6, followed by phosphorylation (inactivation) of glycogen synthase kinase-3β, at least in part via STAT6 induction of p38 MAPK. Such glycogen synthase kinase-3β inactivation causes changes in CREB and p65 DNA-binding activity that favors decreased proinflammatory IL-17 production and increased anti-inflammatory IL-10 production. Thus, in this case, IL-13 acts as a molecular switch that leads to resolution of inflammation.

RORγt+ hematopoietic cells are necessary for tumor cell proliferation during colitis-associated tumorigenesis in mice

Colorectal cancer (CRC) is one of the most common tumor entities. In patients with inflammatory bowel diseases, the development of colitis‐associated colon cancer is considered a dangerous long‐term complication. IL‐17A and the transcription factor retinoic acid receptor‐related orphan receptor γt (RORγt) play fundamental roles in the pathogenesis of inflammatory bowel diseases; in human studies, we detected a dense infiltration of RORγt‐dependent CD4+IL17A+ T helper (Th)17 cells in specimens of CRC, ulcerative colitis, and ulcerative colitis‐associated colorectal cancer. However, the mechanistic role of RORγt+ hematopoietic cells in colitis‐associated tumorigenesis remains unclear. To investigate colitis‐associated colon tumorigenesis, we conducted studies in the AOM+DSS mouse model that revealed the importance of RORγt for colon tumor progression. In the absence of RORγt‐dependent Th17 lymphocytes, mice showed signs of intense chronic colitis, but developed significantly fewer macroscopic tumor nodules. The reduction of tumor development in RORγt−/− mice was not due to reduced colon tumor initiation. However, the proliferation rate of tumor cells was reduced in the absence of RORγt‐dependent Th17 cells and tumor cells showed pronounced signs of senescence‐associated epigenetic and lysosomal changes. These results indicate an important role for the immunological milieu in colitis‐associated cancer, which is shaped in‐part by RORγt‐dependent Th17 lymphocytes that support CRC growth.

Tumor-infiltrating B cells producing antitumor active immunoglobulins in resected HCC prolong patient survival

Background & Aims The immunological microenvironment of HCC influences patient outcome, however, the role of B cells remains unclear. This study investigated effects of local B-cell infiltration in HCC cohorts on patient survival and immunological and molecular tumor microenvironment. Results Unsupervised gene expression analysis of full cancer transcriptomes (N=2158) revealed a highly co-regulated immunological cluster in HCC that mainly contained immunoglobulin fragments. More specifically, in an independent patient cohort (N=242) that compares HCC with non tumorous liver tissue high expression of these B-cell associated genes was associated with better patient outcome (P=0.0149). Conclusively, the immunohistochemical analysis of another independent cohort of resected HCCs (N=119) demonstrated that infiltration of HCCs by CD20+ cells (P=0.004) and CD79a+ cells (P=0.038) at the infiltrative margin were associated with prolonged patient survival. Further, the immunoglobulin fragments that were identified in the gene expression analysis were detected at high levels in patients with dense B-cell infiltration. Methods Gene expression of 2 independent HCC tissue databases was compared using microarrays. Additionally, tissue of resected HCCs was stained for CD20, CD79a and immunoglobulins and analysed for the respective cell numbers separately for tumor, infiltrative margin and distant liver stroma. These findings were correlated with clinical data and patient outcome. Conclusions Infiltration of HCCs by B cells is associated with prolonged patient survival. Further, a distinct B-cell like immunoglobulin profile of HCCs was identified that goes along with better patient outcome. We suggest that B cells contribute to local tumor control by secreting increased levels of immunoglobulins with antitumor activity.

Peritoneal carcinomatosis of colorectal cancer is characterized by structural and functional reorganization of the tumor microenvironment inducing senescence and proliferation arrest in cancer cells

GRAPHICAL ABSTRACTIllustration 1. Overview of immune response in primary CRC and PC. ABSTRACT Background: Peritoneal carcinomatosis (PC) is a terminal evolution from primary colorectal cancer (pCRC) associated with poor patient survival. Impact of the immune cell infiltrate on PC pathogenesis is unknown. Therefore, we characterized the immunological tumor microenvironment regarding proliferation, senescence and neovascularization. Methods: Formalin-fixed and paraffin-embedded (FFPE) tissue of PC and pCRC was examined by immunohistochemistry. Cells infiltrating resected tissue were isolated and analyzed by flow cytometry. PCR arrays detected the expression of genes relevant for helper T (TH) cell responses, like TH1, TH2 and TH17 response. Results: PC tumor cells demonstrate significantly lower proliferation rates than pCRC, but show significantly more senescence. PC is surrounded by significantly increased numbers of cytotoxic active Natural Killer (NK) cells, follicular helper T cells (TFH) and B cells, whereas pCRC shows more CD4+ TH cells, CD8+ cytotoxic T (TC) cells, eosinophilic granulocytes, TH17 and regulatory T (Treg) cells. PC is characterized by significantly increased interferon-γ (IFNγ), an upregulation of tumor necrosis factor (TNF) and the NK cell-regulating cytokine interleukin-15 (IL-15). An upregulation of angiogenesis-related genes, like vascular endothelial growth factor-A (VEGF-A), leads to severe neovascularization in PC. Correlations of PC results reveal that elevated numbers of interleukin-17 (IL-17) positive cells are associated with high cancer cell proliferation, whereas high numbers of IFNγ positive cells correlate with more tumor cells in senescence. Conclusion: The cellular immune reaction is modified during metastasis, inducing senescence in PC tumor cells. Immune surveillance in PC is facilitated by NK cells and high levels of IFNγ and TNF. Counteracting this effect, TFH and B cells combined with VEGF-A enhancement promote neovascularization in PC (Illustration 1). During metastasis from primary CRC to PC the immune cell infiltrate changes, accompanied by the induction of senescence in PC cancer cells (marked red): In pCRC, the antitumor immune response is facilitated by CD4+TH cells, CD8+TC cells and PRG2+ eosinophilic granulocytes. The premetastatic niche development is promoted by Treg cells and TH17 cells producing systemic factors like VEGF-A, TGF-β and TNF. Along with TFH and B cells, as with a pro-tumor immune response, they support metastatic formation and lead to severe neovascularization in PC. This is counterbalanced by the IL-15-induced activation and proliferation of NK cells. The secreted cytokines IFNγ and TNF mediate immunosurveillance.