JanWillem Duitman

CCAAT/enhancer-binding protein δ facilitates bacterial dissemination during pneumococcal pneumonia in a platelet-activating factor receptor-dependent manner

CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ−/− mice are relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection. Moreover, expression of platelet-activating factor receptor (PAFR), which is known to potentiate bacterial translocation of Gram-positive bacteria, was significantly reduced during infection in C/EBPδ−/− mice compared with WT controls. Importantly, cell stimulation experiments revealed that C/EBPδ potentiates PAFR expression induced by lipoteichoic acid and pneumococci. Thus, C/EBPδ exaggerates bacterial dissemination during Streptococcus pneumoniae-induced pulmonary infection, suggesting an important role for PAFR-dependent bacterial translocation.

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis

Background Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical signalling pathways in pathology and physiology. Bleomycin-induced lung fibrosis has been shown to be diminished in PAR-1-deficient mice. The purpose of this study is to investigate whether pharmacological PAR-1 inhibition is a potential therapeutic option to combat pulmonary fibrosis. Methods Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type mice with or without a specific PAR-1 antagonist (ie, P1pal-12, a pepducin that blocks the PAR-1/G-protein interaction). Fibrosis was assessed by hydroxyproline analysis, immunohistochemistry, quantitative PCR and western blot for fibrotic markers expression. Results We first show that P1pal-12 effectively inhibits PAR-1-induced profibrotic responses in fibroblasts. Next, we show that once daily treatment with 0.5, 2.5 or 10 mg/kg P1pal-12 reduced the severity and extent of fibrotic lesions in a dose-dependent manner. These findings correlated with significant decreases in fibronectin, collagen and α smooth muscle actin expression at the mRNA and protein level in treated mice. To further establish the potential clinical applicability of PAR-1 inhibition, we analysed fibrosis in mice treated with P1pal-12 1 or 7 days after bleomycin instillation. Interestingly, when administered 7 days after the induction of fibrosis, P1pal-12 was as effective in limiting the development of pulmonary fibrosis as when administration was started before bleomycin instillation. Conclusions Overall, targeting PAR-1 may be a promising treatment for pulmonary fibrosis.

R-roscovitine reduces lung inflammation induced by lipoteichoic acid and Streptococcus pneumoniae.

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniaein vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-α and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense.

Protease‐activated receptor‐1 drives pancreatic cancer progression and chemoresistance

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.

Expression and Function of Transforming Growth Factor β in Melioidosis

ABSTRACT Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast Asia and northern Australia. An important controller of the immune system is the pleiotropic cytokine transforming growth factor β (TGF-β), of which Smad2 and Smad3 are the major signal transducers. In this study, we aimed to characterize TGF-β expression and function in experimental melioidosis. TGF-β expression was determined in 33 patients with culture-proven infection with B. pseudomallei and 30 healthy controls. We found that plasma TGF-β concentrations were strongly elevated during melioidosis. In line with this finding, TGF-β expression in C57BL/6 mice intranasally inoculated with B. pseudomallei was enhanced as well. To assess the role of TGF-β, we inhibited TGF-β using a selective murine TGF-β antibody. Treatment of mice with anti-TGF-β antibody resulted in decreased lung Smad2 phosphorylation. TGF-β blockade appeared to be protective: mice treated with anti-TGF-β antibody and subsequently infected with B. pseudomallei showed diminished bacterial loads. Moreover, less distant organ injury was observed in anti-TGF-β treated mice as shown by reduced blood urea nitrogen (BUN) and aspartate transaminase (AST) values. However, anti-TGF-β treatment did not have an effect on survival. In conclusion, TGF-β is upregulated during B. pseudomallei infection and plays a limited but proinflammatory role during experimental melioidosis.

Tissue Factor-Dependent Chemokine Production Aggravates Experimental Colitis

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.

CCAAT-enhancer binding protein delta (C/EBPδ) attenuates tubular injury and tubulointerstitial fibrogenesis during chronic obstructive nephropathy

CCAAT-enhancer-binding protein delta (C/EBPδ) is a transcription factor mainly known for its role in inflammation and apoptosis/proliferation. Considering that these are key processes in renal fibrosis, we hypothesized that C/EBPδ would potentiate renal fibrosis. In line with this hypothesis, C/EBPδ has recently been suggested to regulate the fibrotic response during glomerulonephritis. Here we determined the importance of C/EBPδ in the development of renal tubulointerstitial fibrosis by subjecting 8- to 12-week-old C/EBPδ-deficient mice and age- and sex-matched wild-type controls to the unilateral ureteral obstruction model. Mice were killed at 1, 3, or 7 days post surgery, and renal tissues were obtained for RNA, protein, and immunohistochemical analysis. We show that C/EBPδ deficiency resulted in a more profound fibrotic response as evident from enhanced tubular injury, collagen deposition in the interstitial area, and higher expression of transforming growth factor-β. Moreover, we show that the increase in renal fibrosis in C/EBPδ-deficient mice does not depend on an altered proliferation/apoptosis balance or on a differential inflammatory response in the obstructed kidney. In conclusion, our study provides direct evidence that C/EBPδ is a novel mediator of renal fibrosis. Modulating C/EBPδ expression could consequently be a potential antifibrotic strategy in patients with chronic kidney disease.

Protease-activated receptor-1 deficiency protects against streptozotocin-induced diabetic nephropathy in mice.

Endogenously administered activated protein C ameliorates diabetic nephropathy (DN) in a protease-activated receptor-1 (PAR-1)-dependent manner, suggesting that PAR-1 activation limits the progression of DN. Activation of PAR-1 in fibroblast-like cells, however, induces proliferation and extracellular matrix production, thereby driving fibrotic disease. Considering the key role of mesangial proliferation and extracellular matrix production during DN, PAR-1 may in fact potentiate diabetes-induced kidney injury. To determine the net effect of PAR-1 in DN, streptozotocin-induced DN was studied in wild type and PAR-1 deficient mice. Subsequent mechanistic insight was obtained by assessing profibrotic responses of mesangial and tubular epithelial cells in vitro, following PAR-1 stimulation and inhibition. Despite having similar glucose levels, PAR-1 deficient mice developed less kidney damage after induction of diabetes, as evidenced by diminished proteinuria, plasma cystatin C levels, expansion of the mesangial area, and tubular atrophy. In vitro, PAR-1 signaling in mesangial cells led to increased proliferation and expression of matrix proteins fibronectin and collagen IV. Conversely, a reduction in both proliferation and fibronectin deposition was observed in diabetic PAR-1 deficient mice. Overall, we show that PAR-1 plays an important role in the development of DN and PAR-1 might therefore be an attractive therapeutic target to pursue in DN.

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

BackgroundThe 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.MethodsWild-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.ResultsIn 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-1β, 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.ConclusionC/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.

Human airway trypsin-like protease exerts potent, antifibrotic action in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by the deposition of excessive extracellular matrix and the destruction of lung parenchyma, resulting from an aberrant wound‐healing response. Although IPF is often associated with an imbalance in protease activity, the mechanisms underlying the sustained repair mechanisms are not fully understood. Here, we addressed the role of the recently identified, membrane‐anchored serine protease human airway trypsin‐like protease (HAT). In the present study, we show that both HAT expression and activity were up‐regulated in human IPF specimens. Next, adenoviral overexpression of HAT before bleomycin challenge attenuated lung injury as well as extracellular matrix deposition in the bleomycin‐induced pulmonary fibrosis model. In vitro, HAT prevented specific fibrosis‐associated responses in primary human pulmonary fibroblasts and induced the expression of mediators associated with the prostaglandin E2 pathway. Altogether, our findings suggested that HAT could have a protective role in IPF and other fibrotic lung disorders.—Menou, A., Flajolet, P., Duitmen, J., Justet, A., Moog, S., Jaillet, M., Tabèze, L., Solhonne, B., Garnier, M., Mal, H., Mordant, P., Castier, Y., Cazes, A., Sallenave, J.‐M., Mailleux, A. A., Crestani, B. Human airway trypsin‐like protease exerts potent, anti‐fibrotic action in pulmonary fibrosis. FASEB J. 32, 1250‐1264 (2018). www.fasebj.org