Protective and Aggravating Effects of Nlrp3 Inflammasome Activation in IBD Models: Influence of Genetic and Environmental Factors

Background: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1 beta and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3(-/-) mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3(-/-) mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. Methods: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we in-vestigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3(-/-) mice, as well as by antibiotic treatment. Results: Nlrp3(-/-) mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3(-/-) mice in steady state conditions. Interestingly, after cohousing, Nlrp3(-/-) mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3(-/-) mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. Conclusions: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3(-/-) mice. This observation may - at least in part - explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD. Copyright (C) 2012 S. Karger AG, Base

To Protect Fatty Livers from Ischemia Reperfusion Injury: Role of Ischemic Postconditioning

BACKGROUND The benefit of ischemic postconditioning (IPostC) might be the throttled inflow following cold ischemia. The current study investigated advantage and mechanisms of IPostC in healthy and fatty rat livers. METHODS Male SD rats received a high-fat diet to induce fatty livers. Isolated liver perfusion was performed after 24 h ischemia at 4°C as well as in vivo experiments after 90 min warm ischemia. The so-called follow-up perfusions served to investigate the hypothesis that medium from IPostC experiments is less harmful. Lactate dehydrogenase (LDH), transaminases, different cytokines, and gene expressions, respectively, were measured. RESULTS Fatty livers showed histologically mild inflammation and moderate to severe fat storage. IPostC reduced LDH and TXB2 in healthy and fatty livers and increased bile flow. LDH, TNF-\textgreeka, and IL-6 levels in serum decreased after warm ischemia + IPostC. The gene expressions of Tnf, IL-6, Ccl2, and Ripk3 were downregulated in vivo after IPostC. CONCLUSIONS IPostC showed protective effects after ischemia in situ and in vivo in healthy and fatty livers. Restricted cyclic inflow was an important mechanism and further suggested involvement of necroptosis. IPostC represents a promising and easy intervention to improve outcomes after transplantation

Ischemic Postconditioning (IPostC) Protects Fibrotic and Cirrhotic Rat Livers after Warm Ischemia

Background. Decreased organ function following liver resection is a major clinical issue. The practical method of ischemic postconditioning (IPostC) has been studied in heart diseases, but no data exist regarding fibrotic livers. Aims. We aimed to determine whether IPostC could protect healthy, fibrotic, and cirrhotic livers from ischemia reperfusion injury (IRI). Methods. Fibrosis was induced in male SD rats using bile duct ligation (BDL, 4 weeks), and cirrhosis was induced using thioacetamide (TAA, 18 weeks). Fibrosis and cirrhosis were histologically confirmed using HE and EvG staining. For healthy, fibrotic, and cirrhotic livers, isolated liver perfusion with 90 min of warm ischemia was performed in three groups (each with n=8): control, IPostC 8x20 sec, and IPostC 4x60 sec. additionally, healthy livers were investigated during a follow-up study. Lactate dehydrogenase (LDH) and thromboxane B-2 (TXB2) in the perfusate, as well as bile flow (healthy/TAA) and portal perfusion pressure, were measured. Results. LDH and TXB2 were reduced, and bile flow was increased by IPostC, mainly in total and in the late phase of reperfusion. The follow-up study showed that the perfusate derived from a postconditioned group had much less damaging potential than perfusate derived from the nonpostconditioned group. Conclusion. IPostC following warm ischemia protects healthy, fibrotic, and cirrhotic livers against IRI. Reduced efflux of TXB2 is one possible mechanism for this effect of IPostC and increases sinusoidal microcirculation. These findings may help to improve organ function and recovery of patients after liver resection

Dying cells expose a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies

Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply

Interleukin-22 Is Frequently Expressed in Small- and Large-Cell Lung Cancer and Promotes Growth in Chemotherapy-Resistant Cancer Cells

Introduction: In lung cancer, interleukin-22 (IL-22) expression within primary tissue has been demonstrated, but the frequency and the functional consequence of IL-22 signaling have not been addressed. This study aims at analyzing the cellular effects of IL-22 on lung carcinoma cell lines and the prognostic impact of IL-22 tissue expression in lung cancer patients. Methods: Biological effects of IL-22 signaling were investigated in seven lung cancer cell lines by Western blot, flow cytometry, real-time polymerase chain reaction, and proliferation assays. Tumor tissue specimens of two cohorts with a total of 2300 lung cancer patients were tested for IL-22 expression by immunohistochemistry. IL-22 serum concentrations were analyzed in 103 additional patients by enzyme-linked immunosorbent assay. Results: We found the IL-22 receptor 1 (IL-22-R1) to be expressed in six of seven lung cancer cell lines. However IL-22 signaling was functional in only four cell lines, where IL-22 induced signal transducer activator of transcription 3 phosphorylation and increased cell proliferation. Furthermore, IL-22 induced the expression of antiapoptotic B-cell lymphoma 2, but did not rescue tumor cells from carboplatin-induced apoptosis. Cisplatin-resistant cell lines showed a significant up-regulation of IL-22-R1 along with a stronger proliferative response to IL-22 stimulation. IL-22 was preferentially expressed in small- and large-cell lung carcinoma (58% and 46% of cases, respectively). However, no correlation between IL-22 expression by immunohistochemistry and prognosis was observed. Conclusion: IL-22 is frequently expressed in lung cancer tissue. Enhanced IL-22-R1 expression and signaling in chemotherapy-refractory cell lines are indicative of a protumorigenic function of IL-22 and may contribute to a more aggressive phenotype

IFN regulatory factor 4 controls post-ischemic inflammation and prevents chronic kidney disease

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)–mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4−/− mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo, a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4−/− mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies

Selective Bispecific T Cell Recruiting Antibody and Antitumor Activity of Adoptive T Cell Transfer

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. Methods: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40+ and EpCAM+). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met+ human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase+ melanoma cells by TCR-, as well as of CEA+ colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of AC

Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer

Background: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy.Methods: SV40 T antigen–specific T cells from T cell receptor (TCR)-I–transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR–anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40+ and EpCAM+). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met+ human tumor cell lines. Differences between experimental conditions were analyzed using the Student’s t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided.Results: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR–transduced T cells to immobilized c-Met and recognition of tyrosinase+ melanoma cells by TCR-, as well as of CEA+ colon cancer cells by chimeric antigen receptor (CAR)–modified T cells.Conclusions: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT