Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT.
Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury.
The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.
VEGF-A directly contributes to the formation of a reperfusion edema, which might be reduced by its blockade. However, the α-VEGF effect on the endothelial integrity might also favor arterial thrombosis formation.
Post chemotherapy relapsing breast cancer is often characterized by an increased aggressiveness and potential to metastasize. The mechanisms by which cancer cells escape chemotherapy are yet not completely understood. Toll-like receptor (TLR)-3, activated by RNAs, as well as NOD-1 and NOD-2, two newly described regulators of the innate immune system, play a role in tumor growth by regulating apoptosis or macrophage invasion into tumors. We hypothesize that chemotherapy leads to release of nucleotides from apoptotic cells which then in turn are capable of activating the pro-inflammatory TLR-3 pathway. The parallel activation of NOD-1/-2 via TRAF6 then leads to an amplification of the metastasizing potential of post chemotherapy breast cancer cells by increasing the recruitment of macrophages. Balb/c mice bearing 4T1 syngeneic breast cancer tumors were treated with a combination chemotherapy (CMF; cyclophosphamide 50 mg/kg, methotrexate 25 mg/kg, 5-fluoro-uracil 50 mg/kg; n=8) cycled twice i.p., solvent (NaCl; n=8) cycled twice i.p. or without therapy (n=8). After 21 days animals were sacrificed, tumor weight was measured, metastases were detected histologically, cell proliferation rate was analyzed by Ki-67 expression, macrophage count was evaluated immunohistologically and tissue mRNA expression was examined by RT-PCR. CMF treatment (376.0 ± 61.14 mg) significantly retarded tumor growth vs. untreated (620.6 ± 58.95 mg) or NaCl (665.8 ± 45.46 mg) treated controls (P<0.05 respectively P<0.01). Interestingly, pulmonary metastases occurred only in the CMF group. Proliferation as measured by Ki-67 expression however was not decreased in CMF treated animals and was even significantly higher as compared to untreated animals (34.76 ± 3.475 vs. 18.58 ± 2.666; P<0.05). Moreover macrophage invasion into CMF treated tumors was significantly increased vs. untreated animals (35.25 ± 4.131 vs. 22.86 ± 2.511 cells/field; P<0.05). TLR-3 (1471 ± 278.3 vs. 291.3 ± 33.4 or 512.7 ± 88.4; P<0.001) mRNA expression as well as TLR-3 signaling members’ TRAF6 (1207 ± 145.0 vs. 418.1 ± 78.3 or 239.0 ± 32.8; P<0.001) and Interferon beta1 (190.2 ± 25.5 vs. 54.50 ± 15.7 or 63.43 ± 33.4; P<0.05) mRNA expression was significantly upregulated in CMF vs. untreated or NaCl treated mice. Increased cellular turnover upon chemotherapy leads to NOD-1 (74.54 ± 14.59 vs. 34.43 ± 6.46 or 26.11 ± 7.39; P<0.05) activation vs. controls. Together with macrophage invasion, NOD-2 (82.61 ± 14.30 vs. 33.35 ± 5.33 or 42.17 ± 5.87; P<0.01) expression is significantly upregulated vs. controls. TLR-3 activation in 4T1 breast cancer tumors might lead to increased NOD-1/ -2 activation and simultaneously to increased macrophage invasion representing an independent indicator for the aggressiveness of breast tumors. Supported by a grant from the Grants4Targets initiative from Bayer Pharma AG to P.P. Citation Format: Patrick Paulus, Peter Ellinghaus, Anja Urbschat, Karin Zins, Pia Ockelmann, Christin Reissig, Kai Zacharowski. Post chemotherapy aggressiveness of breast cancer cells is related to TLR-3 / NOD-1 -2 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4957. doi:10.1158/1538-7445.AM2013-4957
HCC is the third leading cause of cancer-related mortality worldwide. Metabolic syndrome diseases are shown to facilitate the progression of HCC in industrial countries. Recent studies unraveled pivotal roles of long non-coding RNAs (lncRNAs) in carcinogenesis and cancer metastasis. The controversy that the non-translated H19 mRNA harbors pro- as well as anti-tumorigenic potential shows that its function is poorly characterized. TLR3 plays a key role in the recognition of exogenous pathogen-associated dsRNA and even more importantly TLR3 is activated by endogenous injury-associated ligands. There is increasing evidence that the implication of TLR3 in HCC development might be associated with tumor size, aggressiveness and survival. To study the relationship between the H19 mRNA and TLR3 we first looked at the TLR3 signaling in livers from TLR3-/- mice in comparison to WT mice. By performing real-time RT-PCR experiments, we could demonstrate that TLR3-/- mice showed a significantly enhanced H19 mRNA expression. In order to find out whether H19 mRNA acts as a regulator of TLR3 signaling, we used RNA interference (RNAi) and added the synthetic TLR3 ligand poly I:C on the human hepatoma cell line HepG2. TLR3 knockdown was followed by a significant increase in H19 mRNA expression levels, whereas poly I:C treatment resulted in significantly decreased H19 levels. Vice versa, H19 mRNA knockdown verified a significant reduction in TLR3 mRNA expression. Determination of free RNA in the cell culture supernatants revealed a significant increase of free RNA only after hypoxic incubation whereas no changes could be detected after poly I:C nor RNAi application. Treatment based on H19 inhibition may be a specific approach to target tumor cells, as it is already known that silencing of H19 significantly reduces cell viability. Due to the implication of TLR3 in mRNA stabilization, we addressed to question of a reciprocal relation between both genes by actinomycin D treatment. Indeed, H19 expression was shown to depend on TLR3 expression and a reduced H19 stability was detected after RNAi of TLR3. Since caspase-3 is a key executor of apoptosis and due to the fact that TLRs are known to be involved in the regulation of the apoptotic machinery, we performed caspase-3-like activity assay. Whereas no significant changes in basal caspase-3 like activity could be detected, a strong increase in caspase-3 like activity was seen after siRNA-mediated TLR3 knockdown under normoxic and hypoxic conditions. This indicates that TLR3 may play a crucial role in mediating the signaling through non-coding RNAs, which function is so far poorly understood. We show for the first time the direct relation between H19 and TLR3. Changes in expressions of either of these molecules results in an altered cell response. The knowledge of this interplay might lead to novel strategies for treating HCC. Citation Format: Patrick Paulus, Anja Urbschat, Christin Reissig, Kai Zacharowski, Stefan Dröse, Bertram Scheller, Elisabeth Tybl. Toll-like receptor (TLR3) signaling in human hepatocellular carcinoma (HCC) depends on the tumor-associated long-noncoding RNA H19. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2014-537
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