Cathepsin S Contributes to Lung Inflammation in Acute Respiratory Distress Syndrome.

Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.

C/EBP-Family Redundancy Determines Patient Survival and Lymph Node Involvement in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPß or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan-Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPß and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPß as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPß and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBß and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression.

Preclinical Assessment of ADAM9-Responsive Mesoporous Silica Nanoparticles for the Treatment of Pancreatic Cancer.

Pancreatic adenocarcinoma (PDAC) remains largely refractory to chemotherapeutic treatment regimens and, consequently, has the worst survival rate of all cancers. The low efficacy of current treatments results largely from toxicity-dependent dose limitations and premature cessation of therapy. Recently, targeted delivery approaches that may reduce off-target toxicities have been developed. In this paper, we present a preclinical evaluation of a PDAC-specific drug delivery system based on mesoporous silica nanoparticles (MSNs) functionalized with a protease linker that is specifically cleaved by PDAC cells. Our previous work demonstrated that ADAM9 is a PDAC-enriched protease and that paclitaxel-loaded ADAM9-responsive MSNs effectively kill PDAC cells in vitro. Here, we show that paclitaxel-loaded ADAM9-MSNs result in off-target cytotoxicity in clinically relevant models, which spurred the development of optimized ADAM9-responsive MSNs (OPT-MSNs). We found that these OPT-MSNs still efficiently kill PDAC cells but, as opposed to free paclitaxel, do not induce death in neuronal or bone marrow cells. In line with these in vitro data, paclitaxel-loaded OPT-MSNs showed reduced organ damage and leukopenia in a preclinical PDAC xenograft model. However, no antitumor response was observed upon OPT-MSN administration in vivo. The poor in vivo antitumor activity of OPT-MSNs despite efficient antitumor effects in vitro highlights that although MSN-based tumor-targeting strategies may hold therapeutic potential, clinical translation does not seem as straightforward as anticipated.

Myeloid DNA methyltransferase3b deficiency aggravates pulmonary fibrosis by enhancing profibrotic macrophage activation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and severe disease characterized by excessive matrix deposition in the lungs. Macrophages play crucial roles in maintaining lung homeostasis but are also central in the pathogenesis of lung diseases like pulmonary fibrosis. Especially, macrophage polarization/activation seems to play a crucial role in pathology and epigenetic reprograming is well-known to regulate macrophage polarization. DNA methylation alterations in IPF lungs have been well documented, but the role of DNA methylation in specific cell types, especially macrophages, is poorly defined. METHODS: In order to determine the role of DNA methylation in macrophages during pulmonary fibrosis, we subjected macrophage specific DNA methyltransferase (DNMT)3B, which mediates the de novo DNA methylation, deficient mice to the bleomycin-induced pulmonary fibrosis model. Macrophage polarization and fibrotic parameters were evaluated at 21 days after bleomycin administration. Dnmt3b knockout and wild type bone marrow-derived macrophages were stimulated with either interleukin (IL)4 or transforming growth factor beta 1 (TGFB1) in vitro, after which profibrotic gene expression and DNA methylation at the Arg1 promotor were determined. RESULTS: We show that DNMT3B deficiency promotes alternative macrophage polarization induced by IL4 and TGFB1 in vitro and also enhances profibrotic macrophage polarization in the alveolar space during pulmonary fibrosis in vivo. Moreover, myeloid specific deletion of DNMT3B promoted the development of experimental pulmonary fibrosis. CONCLUSIONS: In summary, these data suggest that myeloid DNMT3B represses fibrotic macrophage polarization and protects against bleomycin induced pulmonary fibrosis.

Alveolar epithelial TET2 is not involved in the development of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology with minimal treatment options. Repetitive alveolar epithelial injury has been suggested as one of the causative mechanisms of this disease. Type 2 alveolar epithelial cells (AEC2) play a crucial role during fibrosis by functioning as stem cells able to repair epithelial damage. The DNA demethylase Tet methylcytosine dioxygenase 2 (TET2) regulates the stemness of multiple types of stem cells, but whether it also affects the stemness of AEC2 during fibrosis remains elusive. To study the role of TET2 in AEC2 during fibrosis, we first determined TET2 protein levels in the lungs of IPF patients and compared TET2 expression in AEC2 of IPF patients and controls using publicly available data sets. Subsequently, pulmonary fibrosis was induced by the intranasal administration of bleomycin to wild-type and AEC2-specific TET2 knockout mice to determine the role of TET2 in vivo. Fibrosis was assessed by hydroxyproline analysis and fibrotic gene expression. Additionally, macrophage recruitment and activation, and epithelial injury were analyzed. TET2 protein levels and gene expression were downregulated in IPF lungs and AEC2, respectively. Bleomycin inoculation induced a robust fibrotic response as indicated by increased hydroxyproline levels and increased expression of pro-fibrotic genes. Additionally, increased macrophage recruitment and both M1 and M2 activation were observed. None of these parameters were, however, affected by AEC2-specific TET2 deficiency. TET2 expression is reduced in IPF, but the absence of TET2 in AEC2 cells does not affect the development of bleomycin-induced pulmonary fibrosis.

Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling.

Hedgehog proteins are pivotal morphogens acting through a canonical pathway involving first activation of ligand binding to Patched followed by alleviation of Smoothened receptor inhibition, leading to activation of Gli transcription factors. Noncanonical Hedgehog signaling remains poorly characterized but is thought to be mainly dependent on Smoothened. However, Smoothened inhibitors have yielded only partial success in combating Hedgehog signal transduction-dependent cancer, suggesting that noncanonical Smoothened-independent pathways also are clinically relevant. Moreover, several Smoothened-dependent effects (e.g. neurite projection) do not require transcriptional activation, further suggesting biological importance of noncanonical Smoothened-dependent pathways. We comprehensively characterized the cellular kinome in Hedgehog-challenged murine WT and Smoothened-/- fibroblasts as well as Smoothened agonist-stimulated cells. A peptide assay-based kinome analysis (in which cell lysates are used to phosphorylate specific kinase substrates), along with endocytosis, Lucifer Yellow-based, and immunoblotting assays, identified an elaborate signaling network of both Smoothened-dependent and -independent pathways that mediates actin reorganization through Src-like kinases, activates various proinflammatory signaling cascades, and concomitantly stimulates Wnt and Notch signaling while suppressing bone morphogenetic protein (BMP) signaling. The contribution of noncanonical Smoothened-independent signaling to the overall effects of Hedgehog on cellular physiology appears to be much larger than previously envisioned and may explain the transcriptionally independent effects of Hedgehog signaling on cytoskeleton. The observation that Patched-dependent, Smoothened-independent, noncanonical Hedgehog signaling increases Wnt/Notch signaling provides a possible explanation for the failure of Smoothened antagonists in combating Hedgehog-dependent but Smoothened inhibitor-resistant cancer. Our findings suggest that inhibiting Hedgehog-Patched interaction could result in more effective therapies as compared with conventional Smoothened-directed therapies.

Early macrophage infiltrates impair pancreatic cancer cell growth by TNF-α secretion.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a grim disease with high mortality rates. Increased macrophage influx in PDAC is a common hallmark and associated with poor prognosis. Macrophages have high cellular plasticity, which can differentiate into both anti- and pro-tumorigenic properties. Here, we investigated how naïve (M0) macrophages differ from other macrophages in their anti-tumorigenic activities. METHODS: In vitro BrdU proliferation and Annexin V cell death analyses were performed on PANC-1 and MIA PaCa-2 PDAC cell lines exposed to conditioned medium of different macrophage subsets. Macrophage secreted factors were measured by transcript analysis and ELISA. Therapeutic antibodies were used to functionally establish the impact of the identified cytokine on PDAC proliferation. RESULTS: Proliferation and cell death assays revealed that only M0 macrophages harbor anti-tumorigenic activities and that M1, M2, and TAMs do not. mRNA analysis and ELISA results suggested TNF-α as a potential candidate to mediate M0 macrophage induced cell death. To demonstrate the importance of TNF-α in M0 macrophage-induced cell death, PANC-1 and MIA PaCa-2 cell-lines were exposed to M0 macrophage conditioned medium in the presence of the TNF-α inhibitor Infliximab, which effectively diminished the anti-tumor activities of M0 macrophages. CONCLUSION: Newly tumor-infiltrated naive M0 macrophages exert anti-tumorigenic activities via TNF-α secretion. Their subsequent differentiation into either M1, M2, or TAM subsets reduces TNF-α levels, thereby abolishing their cytotoxic activity on PDAC cells. These data suggest that reestablishing TNF-α secretion in differentiated macrophages might yield a therapeutic benefit.

Pharmacological PAR-1 inhibition reduces blood glucose levels but does not improve kidney function in experimental type 2 diabetic nephropathy.

Vorapaxar-dependent protease-activated receptor (PAR)-1 inhibition diminishes diabetic nephropathy in experimental type 1 diabetes. As most patients with diabetic nephropathy suffer from type 2 diabetes, the aim of this study was to investigate whether PAR-1 inhibition also limits diabetic nephropathy in experimental type 2 diabetes. Consequently, leptin-deficient black and tan brachyuric (BTBRob/ob) mice were randomly assigned to vorapaxar (1.75 mg/kg; twice weekly via oral gavage) or vehicle treatment, whereas matched wild-type (WT) BTBR (BTBRWT) mice served as nondiabetic controls. Weight and (nonfasting) blood glucose levels were monitored for up to 18 wk, after which kidney function and histologic damage was evaluated postmortem. We show that blood glucose levels and body weight increased in diabetic BTBRob/ob mice compared with nondiabetic BTBRWT controls. Vorapaxar-dependent PAR-1 inhibition reduced but did not normalize blood glucose levels in BTBRob/ob mice, whereas it potentiated the increase in body weight. Vorapaxar did not, however, preserve kidney function, whereas it only minimally reduced histopathological signs of kidney injury. Overall, we thus show that PAR-1 inhibition reduces blood glucose levels during the progression of diabetic nephropathy in experimental type 2 diabetes but does not improve renal function. This is in contrast to the therapeutic potential of vorapaxar in type 1 diabetes-induced nephropathy, highlighting the importance of disease-dependent treatment modalities.-Waasdorp, M., Florquin, S., Duitman, J., Spek, C. A. Pharmacological PAR-1 inhibition reduces blood glucose levels but does not improve kidney function in experimental type 2 diabetic nephropathy.

Action and clinical significance of CCAAT/enhancer-binding protein delta in hepatocellular carcinoma.

CCAAT/enhancer-binding protein delta (CEBPD) is associated with the regulation of apoptosis and cell proliferation and is a candidate tumor suppressor gene. Here, we investigated its role in hepatocellular carcinoma (HCC). We observe that CEBPD mRNA expression is significantly downregulated in HCC tumors as compared with adjacent tissues. Protein levels of CEBPD are also lower in tumors relative to adjacent tissues. Reduced expression of CEBPD in the tumor correlates with worse clinical outcome. In both Huh7 and HepG2 cells, shRNA-mediated CEBPD knockdown significantly reduces cell proliferation, single cell colony formation and arrests cells in the G0/G1 phase. Subcutaneous xenografting of Huh7 in nude mice show that CEBPD knockdown results in smaller tumors. Gene expression analysis shows that CEBPD modulates interleukin-1 signaling. We conclude that CEBPD expression uncouples cancer compartment expansion and clinical outcome in HCC, potentially by modulating interleukin-1 signaling. Thus, although our results support the notion that CEBPD acts as a tumor suppressor in HCC, its action does not involve impairing compartment expansion per se but more likely acts through improving anticancer immunity.

Protease-activated receptor-1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy.

End-stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease-activated receptor (PAR)-1, which recently emerged as an inducer of epithelial-to-mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR-1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR-1-deficient mice during unilateral ureter obstruction (UUO) and that PAR-1-deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild-type and PAR-1-deficient mice suggesting that diminished fibrosis in PAR-1-deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR-1-deficient animals which were accompanied by diminished production of MCP-1 and TGF-ß. Overall, we thus show that PAR-1 drives EMT of TECs in vitro and aggravates UUO-induced renal fibrosis although this is likely due to PAR-1-dependent pro-fibrotic cytokine production rather than EMT.

Protease-Activated Receptor 2 Facilitates Bacterial Dissemination in Pneumococcal Pneumonia.

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor 2 (PAR2) is expressed by different cell types in the lungs and can mediate inflammatory responses. We sought to determine the role of PAR2 during pneumococcal pneumonia. Pneumococcal pneumonia or sepsis was induced in wild-type and PAR2 knock-out (Par2-/-) mice by infection with viable S. pneumoniae. Par2-/- mice demonstrated improved host defense, a largely preserved lung barrier integrity, and reduced mortality during pneumococcal pneumonia. PAR2 deficiency did not influence bacterial growth after intravenous infection. Inhibition of the endogenous PAR2 activating proteases tissue factor/factor VIIa or tryptase did not impact on bacterial burdens during pneumonia. In a PAR2 reporter cell line it was demonstrated that S. pneumoniae-derived proteases are able to cleave PAR2. These results show that S. pneumoniae is able to cleave and exploit PAR2 to disseminate systemically from the airways.

Dabigatran potentiates gemcitabine-induced growth inhibition of pancreatic cancer in mice.

Pancreatic cancer is one of the most lethal solid malignancies with little treatment options. We have recently shown that expression of protease activated receptor (PAR)-1 in the tumor microenvironment drives progression and induces chemoresistance of pancreatic cancer. As thrombin is the prototypical PAR-1 agonist, here we addressed the effect of the direct thrombin inhibitor dabigatran on pancreatic cancer growth and drug resistance in an orthotropic pancreatic cancer model. We show that dabigatran treatment did not affect primary tumor growth whereas it significantly increased tumor dissemination throughout the peritoneal cavity. Increased dissemination was accompanied by intratumoral bleeding and increased numbers of aberrant and/or collapsed blood vessels in the primary tumors. In combination with gemcitabine, dabigatran treatment limited primary tumor growth, did not induce bleeding complications and prevented tumor cell dissemination. Dabigatran was however not as efficient as genetic ablation of PAR-1 in our previous study suggesting that thrombin is not the main PAR-1 agonist in the setting of pancreatic cancer. Overall, we show that dabigatran potentiates gemcitabine-induced growth inhibition of pancreatic cancer but does not affect primary tumor growth when used as a monotherapy.

Potential importance of protease activated receptor (PAR)-1 expression in the tumor stroma of non-small-cell lung cancer.

BACKGROUND: Protease activated receptor (PAR)-1 expression is increased in a variety of tumor cells. In preclinical models, tumor cell PAR-1 appeared to be involved in the regulation of lung tumor growth and metastasis; however the role of PAR-1 in the lung tumor microenvironment, which is emerging as a key compartment in driving cancer progression, remained to be explored. METHODS: In the present study, PAR-1 gene expression was determined in lung tissue from patients with non-small-cell lung cancer (NSCLC) using a combination of publicly available RNA microarray datasets and in house-made tissue microarrays including tumor biopsies of 94 patients with NSCLC (40 cases of adenocarcinoma, 42 cases of squamous cell carcinoma and 12 cases of other type of NSCLC at different stages). RESULTS: PAR-1 gene expression strongly correlated with tumor stromal markers (i.e. macrophage, endothelial cells and (myo) fibroblast markers) but not with epithelial cell markers. Immunohistochemical analysis confirmed the presence of PAR-1 in the tumor stroma and showed that PAR-1 expression was significantly upregulated in malignant tissue compared with normal lung tissue. The overexpression of PAR-1 in tumor stroma of NSCLC appeared to be independent from tumor type, tumor stage, histopathological differentiation status, disease progression and patient survival. CONCLUSION: Overall, our data provide evidence that PAR-1 in NSCLC is mainly expressed on cells that constitute the pulmonary tumor microenvironment, including vascular endothelial cells, macrophages and stromal fibroblasts.

Detrimental role for CCAAT/enhancer binding protein δ in blood-borne brain infection.

BACKGROUND: The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus (S.) pneumoniae. CCAAT/enhancer binding protein δ is a transcription factor that has recently been hypothesized to play a detrimental role in outcome of meningitis caused by S. pneumoniae. Here, we studied the role of C/EBPδ prior to the development of pneumococcal meningitis. METHODS: Wild-type and C/EBPδ-deficient mice (C/EBPδ-/-) were intraveneously infected with S. pneumoniae and sacrificed after 24 or 48 h. cebpδ expression, bacterial loads, inflammatory response and pathology in the brain were assessed. RESULTS: S. pneumoniae induces cebpδ expression in the brain during blood-borne brain infection. In comparison to wild-type mice, C/EBPδ-/- animals showed decreased bacterial loads in blood and brain 48 h after inoculation. In the blood compartment, the host inflammatory response was significantly lower upon infection in C/EBPδ-/- mice as compared to wild-type mice. CONCLUSION: C/EBPδ facilitates bacterial dissemination to the brain and enhances the immune response in the blood compartment. Our study suggests that C/EBPδ plays a detrimental role during the initial development of blood-borne brain infection.

Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis.

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2.

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2 deficiency persistently reduces bleomycin-induced pulmonary fibrosis or merely delays disease progression and whether pharmacological PAR-2 inhibition limits experimental pulmonary fibrosis. Bleomycin was instilled intranasally into wild-type or PAR-2-deficient mice in the presence/absence of a specific PAR-2 antagonist (P2pal-18S). Pulmonary fibrosis was consistently reduced in PAR-2-deficient mice throughout the fibrotic phase, as evident from reduced Ashcroft scores (29%) and hydroxyproline levels (26%) at d 28. Moreover, P2pal-18S inhibited PAR-2-induced profibrotic responses in both murine and primary human pulmonary fibroblasts (p < 0.05). Once daily treatment with P2pal-18S reduced the severity and extent of fibrotic lesions in lungs of bleomycin-treated wild-type mice but did not further reduce fibrosis in PAR-2-deficient mice. Importantly, P2pal-18S treatment starting even 7 d after the onset of fibrosis limits pulmonary fibrosis as effectively as when treatment was started together with bleomycin instillation. Overall, PAR-2 contributes to the progression of pulmonary fibrosis, and targeting PAR-2 may be a promising therapeutic strategy for treating pulmonary fibrosis.

CCAAT/enhancer-binding protein δ (C/EBPδ) aggravates inflammation and bacterial dissemination during pneumococcal meningitis.

BACKGROUND: The prognosis of bacterial meningitis largely depends on the severity of the inflammatory response. The transcription factor CAAT/enhancer-binding protein δ (C/EBPδ) plays a key role in the regulation of the inflammatory response during bacterial infections. Consequently, we assessed the role of C/EBPδ during experimental meningitis. METHODS: Wild-type and C/EBPδ-deficient mice (C/EBPδ(-/-)) were intracisternally infected with Streptococcus pneumoniae and sacrificed after 6 or 30 h, or followed in a survival study. RESULTS: In comparison to wild-type mice, C/EBPδ(-/-) mice showed decreased bacterial loads at the primary site of infection and decreased bacterial dissemination to lung and spleen 30 h after inoculation. Expression levels of the inflammatory mediators IL-10 and KC were lower in C/EBPδ(-/-) brain homogenates, whereas IL-6, TNF-α, IL-1ß, and MIP-2 levels were not significantly different between the two genotypes. Moreover, C/EBPδ(-/-) mice demonstrated an attenuated systemic response as reflected by lower IL-10, IL-6, KC, and MIP-2 plasma levels. No differences in clinical symptoms or in survival were observed between wild-type and C/EBPδ(-/-) mice. CONCLUSION: C/EBPδ in the brain drives the inflammatory response and contributes to bacterial dissemination during pneumococcal meningitis. C/EBPδ does, however, not affect clinical parameters of the disease and does not confer a survival benefit.

Protease-activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that functions as a cell-surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR-2 and activation of PAR-2 may induce their proliferation and migration. Interestingly, however, PAR-2 expression is increased in stroma-rich pancreatic cancer regions, suggesting a potential role of PAR-2 in the tumour microenvironment. Here, we assessed the importance of PAR-2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR-2-positive, whereas stromal cells are PAR-2-negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR-2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR-2-deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube-formation assays show that PAR-2 does not inhibit the intrinsic tube-forming capacity of lymphatic endothelial cells, but that PAR-2 actually inhibits cancer cell-induced tube formation. Overall, stromal PAR-2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR-2 in the tumour microenvironment and pinpoint PAR-2 as a negative regulator of lymphangiogenesis.