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Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a low 5-year survival rate of only 13%. Despite intense research efforts, PDAC remains insufficiently understood. In part, this is attributed to opposing effects of key players being unraveled, including the stroma but also molecules that act in a context-dependent manner. One such molecule is the transcription factor C/EBPδ, where we recently showed that C/EBPδ exerts tumor-suppressive effects in PDAC cells in vitro. To better understand the role of C/EBPδ in different contexts and the development of PDAC, we here build on these findings and assess the effect of C/EBPδ in a PDAC model in mice. We establish that the lack of oxygen in vivo-hypoxia-counteracts the tumor-suppressive effects of C/EBPδ, and identify a reciprocal feedback loop between C/EBPδ and HIF-1α. RNA sequencing of C/EBPδ-induced cells under hypoxia also suggests that the growth-limiting effects of C/EBPδ decrease with oxygen tension. Consequently, in vitro proliferation assays reveal that the tumor-suppressive activities of C/EBPδ are abrogated due to hypoxia. This study demonstrates the importance of considering major physiological parameters in preclinical approaches.

COVID-19-related acute respiratory distress syndrome (ARDS) can lead to long-term pulmonary fibrotic lesions. Alveolar fibroproliferative response (FPR) is a key factor in the development of pulmonary fibrosis. N-terminal peptide of procollagen III (NT-PCP-III) is a validated biomarker for activated FPR in ARDS. This study aimed to assess the association between dynamic changes in alveolar FPR and long-term outcomes, as well as mortality in COVID-19 ARDS patients. We conducted a prospective cohort study of 154 COVID-19 ARDS patients. We collected bronchoalveolar lavage (BAL) and blood samples for measurement of 17 pulmonary fibrosis biomarkers, including NT-PCP-III. We assessed pulmonary function and chest computed tomography (CT) at 3 and 12 mo after hospital discharge. We performed joint modeling to assess the association between longitudinal changes in biomarker levels and mortality at day 90 after starting mechanical ventilation. 154 patients with 284 BAL samples were analyzed. Of all patients, 40% survived to day 90, of whom 54 completed the follow-up procedure. A longitudinal increase in NT-PCP-III was associated with increased mortality (HR 2.89, 95% CI: 2.55-3.28; P < 0.001). Forced vital capacity and diffusion for carbon monoxide were impaired at 3 mo but improved significantly at one year after hospital discharge ( P = 0.03 and P = 0.004, respectively). There was no strong evidence linking alveolar FPR during hospitalization and signs of pulmonary fibrosis in pulmonary function or chest CT images during 1-yr follow-up. In COVID-19 ARDS patients, alveolar FPR during hospitalization was associated with higher mortality but not with the presence of long-term fibrotic lung sequelae within survivors. NEW & NOTEWORTHY This is the first prospective study on the longitudinal alveolar fibroproliferative response in COVID-19 ARDS and its relationship with mortality and long-term follow-up. We used the largest cohort of COVID-19 ARDS patients who had consecutive bronchoalveolar lavages and measured 17 pulmonary fibroproliferative biomarkers. We found that a higher fibroproliferative response during admission was associated with increased mortality, but not correlated with long-term fibrotic lung sequelae in survivors.

Treatment of pancreatic ductal adenocarcinoma (PDAC), a dismal disease with poor survival rates, is hampered by the high prevalence of chemotherapy resistance. Resistance is accompanied by macrophage infiltration into the tumor microenvironment, and infiltrated macrophages are key players in chemotherapy resistance. In the current manuscript, we identify CCAAT/enhancer-binding protein delta (C/EBPδ) as an important transcription factor driving macrophage-dependent gemcitabine resistance. We show that conditioned medium obtained from wild type macrophages largely diminishes gemcitabine-induced cytotoxicity of PDAC cells, whereas conditioned medium obtained from C/EBPδ-deficient macrophages only minimally affects gemcitabine-induced PDAC cell death. Subsequent analysis of RNA-Seq data identified the pyrimidine metabolism pathway amongst the most significant pathways down-regulated in C/EBPδ-deficient macrophages and size filtration experiments indeed showed that the low molecular weight and free metabolite fraction most effectively induced gemcitabine resistance. In line with a role for pyrimidines, we next show that supplementing macrophage conditioned medium with deoxycytidine overruled the effect of macrophage conditioned media on gemcitabine resistance. Consistently, macrophage C/EBPδ-dependent resistance is specific for gemcitabine and does not affect paclitaxel or 5-FU-induced cytotoxicity. Overall, we thus show that C/EBPδ-dependent deoxycytidine biosynthesis in macrophages induces gemcitabine resistance of pancreatic cancer cells.

CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.

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.

Purpose: Targeting tumor-infiltrating macrophages limits progression and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma (PDAC). Protease-activated receptor (PAR)1 drives monocyte/macrophage recruitment, and stromal ablation of PAR1 limits cancer growth and enhances gemcitabine sensitivity in experimental PDAC. However, the functional interplay between PAR1, macrophages and tumor cells remains unexplored. Here we address the PAR1-macrophage-tumor cell crosstalk and assess its contributions to tumor progression., Methods: PAR1 expression and macrophage infiltration were correlated in primary PDAC biopsies using gene expression datasets and tissue microarrays. Medium transfer experiments were used to evaluate the functional consequences of macrophage-tumor cell crosstalk and to assess the contribution of PAR1 to the observed responses. PAR1 cleavage assays were used to identify a macrophage-secreted PAR1 agonist, and the effects of candidate proteases were assessed in medium transfer experiments with specific inhibitors and/or recombinant agonist., Results: PAR1 expression correlates with macrophage infiltration in primary PDACs, and macrophages induce mesenchymal transition of PDAC cells through PAR1 activation. Protease profiling identified macrophage-secreted matrix metalloprotease 9 (MMP9) as the relevant PAR1 agonist in PDAC. PAR1 and/or MMP9 inhibition limited macrophage-driven mesenchymal transition. Likewise, preventing mesenchymal transition by silencing ZEB1 or by pharmacological inhibition of the MMP9/PAR1 axis significantly reduced the ability of tumor cells to survive the anti-tumor activities of macrophages., Conclusion: Macrophages secrete MMP9, which acts upon PDAC cell PAR1 to induce mesenchymal transition. This macrophage-induced mesenchymal transition supports the tumor-promoting role of macrophage influx, explaining the dichotomous contributions of these immune cells to tumor growth.

CCAAT/enhancer-binding protein δ (C/EBPδ) is a transcription factor involved in growth arrest and differentiation, which has consequently been suggested to harbor tumor suppressive activities. However, C/EBPδ over-expression correlates with poor prognosis in glioblastoma and promotes genomic instability in cervical cancer, hinting at an oncogenic role of C/EBPδ in these contexts. Here, we explore the role of C/EBPδ in pancreatic cancer. We determined C/EBPδ expression in biopsies from pancreatic cancer patients using public gene-expression datasets and in-house tissue microarrays. We found that C/EBPδ is highly expressed in healthy pancreatic ductal cells but lost in pancreatic ductal adenocarcinoma. Furthermore, loss of C/EBPδ correlated with increased lymph node involvement and shorter overall survival in pancreatic ductal adenocarcinoma patients. In accordance with this, in vitro experiments showed reduced clonogenic capacity and proliferation of pancreatic ductal adenocarcinoma cells following C/EBPδ re-expression, concurrent with decreased sphere formation capacity in soft agar assays. We thus report a previously unrecognized but important tumor suppressor role of C/EBPδ in pancreatic ductal adenocarcinoma. This is of particular interest since only few tumor suppressors have been identified in the context of pancreatic cancer. Moreover, our findings suggest that restoration of C/EBPδ activity could hold therapeutic value in pancreatic ductal adenocarcinoma, although the latter claim needs to be substantiated in future studies.

Protease activated receptor (PAR)-1 expression in tumor cells is associated with disease progression and overall survival in a variety of cancers of epithelial origin; however, the importance of PAR-1 in the tumor microenvironment remains unexplored. Utilizing an orthotopic pancreatic cancer model in which tumor cells are PAR-1 positive whereas stromal cells are PAR-1 negative, we show that PAR-1 expression in the microenvironment drives progression and induces chemoresistance of pancreatic cancer. PAR-1 enhances monocyte recruitment into the tumor microenvironment by regulating monocyte migration and fibroblast dependent chemokine production thereby inducing chemoresistance. Overall, our data identify a novel role of PAR-1 in the pancreatic tumor microenvironment and suggest that PAR-1 may be an attractive target to reduce drug resistance in pancreatic cancer., (© 2014 UICC.)

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.

The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. There is still limited insight into the molecular mechanisms that maintain the chemotherapy-resistant phenotype, despite the obvious clinical relevance that such knowledge would have. In this study, we show that the chemotherapy-resistant phenotype of myeloid leukemia cells correlates with activation of the Hedgehog (Hh) pathway, whereas in chemosensitive cells, such activation is less pronounced. Importantly, the overexpression of Hh pathway components induces chemoprotection and inhibition of the pathway reverts chemoresistance of Lucena-1 cells, apparently by interfering with P-glycoprotein-dependent drug resistance. Our data thus identify the Hh pathway as an essential component of multidrug resistance (MDR) myeloid leukemia and suggest that targeting the Hh pathway might be an interesting therapeutic avenue for overcoming MDR resistance in myeloid leukemia.

Objective: In spite of major advances in reperfusion therapy for patients presenting with acute coronary syndrome, long-term morbidity is still substantial. A limitation of initial treatment of myocardial ischemia is the lack of prevention of ischemia/reperfusion (I/R) injury. Activated protein C (APC), a crucial mediator in the coagulation process, plays a prominent role in the crosstalk between coagulation and inflammation and provides cytoprotective effects via inhibition of apoptosis and inflammation in several human and animal studies., Methods and Results: APC was administered in an animal model for myocardial I/R. APC largely inhibited early myocardial I/R injury after varying reperfusion times, an effect that was absent on administration of heparin, a nonspecific anticoagulant agent. The protective effects of APC were absent in case of absence or blockade of protease activated receptor-1 (PAR-1), indicating a critical role for PAR-1 in this process. Furthermore, we showed a strong antiapoptotic effect of APC in the early phase of reperfusion combined with an antiinflammatory effect at an early stage (IL-6), as well as at a later stage (leukocyte infiltration)., Conclusions: APC exerts strong protective effects on early myocardial I/R injury, primarily via inhibition of apoptosis and inflammation, which are regulated via PAR-1.

Background: Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored., Objectives: To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury., Methods: We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion., Results: ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IkappaBalpha upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-kappaB (NF-kappaB) mediated cell signaling. Levels of nuclear activated NF-kappaB and proteins influenced by the NF-kappaB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis)., Conclusions: We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-kappaB mechanism.

The current interest in expression of groups of functionally related genes creates a demand for novel experimental tools. We describe a multiplex ligation-dependent amplification procedure (RT-MLPA), which accurately quantifies up to 45 transcripts of interest in a one-tube assay. The output, a set of fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. The procedure is highly sensitive and reproducible over a 100-fold range of input RNA, with excellent compatibility with RT-PCR and microarrays. We targeted two comprehensive sets of human genes: 35 apoptosis regulators and 30 genes involved in inflammation. Both probe sets accurately assessed specific changes in gene expression in two relevant model systems. Stimulation of lymphocytes with various Toll-like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated with cytostatic drugs showed as primary response strong up-regulation of the apoptogenic p53-inducible PUMA transcript. Suppression by RNAi validated that indeed Puma expression was responsible for apoptosis induction. Thus, RT-MLPA enables relevant changes in transcription patterns to be quickly pinpointed and guide further experiments. This can be an advantage compared to hypothesis-free whole genome screens where large numbers of differentially expressed genes can obscure functional interpretation.