Identification and Biosynthesis of an N-Glucuronide Metabolite of Camonsertib.

Camonsertib is a novel ATR kinase inhibitor in clinical development for advanced cancers targeting sensitizing mutations. This article describes the identification and biosynthesis of an N-glucuronide metabolite of camonsertib. This metabolite was first observed in human hepatocyte incubations and was subsequently isolated to determine the structure, evaluate its stability as part of bioanalytical method development and for use as a standard for estimating its concentration in Phase I samples. The N-glucuronide was scaled-up using a purified bacterial culture preparation and was subsequently isolated using preparative chromatography. The bacterial culture generated sufficient material of the glucuronide to allow for one- and two-dimensional 1H and 13C NMR structural elucidation and further bioanalytical characterization. The NOE data combined with the gradient HMBC experiment and molecular modeling, strongly suggests that the point of attachment of the glucuronide results in the formation of (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(5-(4-((1R,3r,5S)-3-hydroxy-8-oxabicyclo[3.2.1]octan-3-yl)-6-((R)-3-methylmorpholino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-1H-pyrazol-1-yl)tetrahydro-2H-pyran-2-carboxylic acid. Significance Statement This is the first report of a glucuronide metabolite of camonsertib formed by human hepatocyte incubations. This study reveals the structure of an N-glucuronide metabolite of camonsertib using detailed elucidation by one- and two-dimensional NMR after scale-up using a novel bacterial culture approach yielding significant quantities of a purified metabolite.

Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin.

[Figure: see text].

Mechanical Feed-Forward Loops Contribute to Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a progressive scarring disease characterized by extracellular matrix accumulation and altered mechanical properties of lung tissue. Recent studies support the hypothesis that these compositional and mechanical changes create a progressive feed-forward loop in which enhanced matrix deposition and tissue stiffening contribute to fibroblast and myofibroblast differentiation and activation, which further perpetuates matrix production and stiffening. The biomechanical properties of tissues are sensed and responded to by mechanotransduction pathways that facilitate sensing of changes in mechanical cues by tissue resident cells and convert the mechanical signals into downstream biochemical signals. Although our understanding of mechanotransduction pathways associated with pulmonary fibrosis remains incomplete, recent progress has allowed us to begin to elucidate the specific mechanisms supporting fibrotic feed-forward loops. The mechanosensors discussed here include integrins, Piezo channels, transient receptor potential channels, and nonselective ion channels. Also discussed are downstream transcription factors, including myocardin-related transcription factor and Yes-associated protein/transcriptional coactivator with PDZ-binding motif. This review describes mechanosensors and mechanotransduction pathways associated with fibrosis progression and highlights promising therapeutic insights.

Dung biomass smoke exposure impairs resolution of inflammatory responses to influenza infection.

Epidemiological studies associate biomass smoke with an increased risk for respiratory infections in children and adults in the developing world, with 500,000 premature deaths each year attributed to biomass smoke-related acute respiratory infections including infections caused by respiratory viruses. Animal dung is a biomass fuel of particular concern because it generates more toxic compounds per amount burned than wood, and is a fuel of last resort for the poorest households. Currently, there is little biological evidence on the effects of dung biomass smoke exposure on immune responses to respiratory viral infections. Here, we investigated the impact of dung biomass exposure on respiratory infection using a mouse model of dung biomass smoke and cultured primary human small airway epithelial cells (SAECs). Mice infected with influenza A virus (IAV) after dung biomass smoke exposure had increased mortality, lung inflammation and virus mRNA levels, and suppressed expression of innate anti-viral mediators compared to air exposed mice. Importantly, there was still significant tissue inflammation 14 days after infection in dung biomass smoke-exposed mice even after inflammation had resolved in air-exposed mice. Dung biomass smoke exposure also suppressed the production of anti-viral cytokines and interferons in cultured SAECs treated with poly(I:C) or IAV. This study shows that dung biomass smoke exposure impairs the immune response to respiratory viruses and contributes to biomass smoke-related susceptibility to respiratory viral infections, likely due to a failure to resolve the inflammatory effects of biomass smoke exposure.

Aryl hydrocarbon receptor deficiency causes the development of chronic obstructive pulmonary disease through the integration of multiple pathogenic mechanisms.

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease.

Inflammation resolution: a dual-pronged approach to averting cytokine storms in COVID-19?

Severe coronavirus disease (COVID-19) is characterized by pulmonary hyper-inflammation and potentially life-threatening "cytokine storms". Controlling the local and systemic inflammatory response in COVID-19 may be as important as anti-viral therapies. Endogenous lipid autacoid mediators, referred to as eicosanoids, play a critical role in the induction of inflammation and pro-inflammatory cytokine production. SARS-CoV-2 may trigger a cell death ("debris")-induced "eicosanoid storm", including prostaglandins and leukotrienes, which in turn initiates a robust inflammatory response. A paradigm shift is emerging in our understanding of the resolution of inflammation as an active biochemical process with the discovery of novel endogenous specialized pro-resolving lipid autacoid mediators (SPMs), such as resolvins. Resolvins and other SPMs stimulate macrophage-mediated clearance of debris and counter pro-inflammatory cytokine production, a process called inflammation resolution. SPMs and their lipid precursors exhibit anti-viral activity at nanogram doses in the setting of influenza without being immunosuppressive. SPMs also promote anti-viral B cell antibodies and lymphocyte activity, highlighting their potential use in the treatment of COVID-19. Soluble epoxide hydrolase (sEH) inhibitors stabilize arachidonic acid-derived epoxyeicosatrienoic acids (EETs), which also stimulate inflammation resolution by promoting the production of pro-resolution mediators, activating anti-inflammatory processes, and preventing the cytokine storm. Both resolvins and EETs also attenuate pathological thrombosis and promote clot removal, which is emerging as a key pathology of COVID-19 infection. Thus, both SPMs and sEH inhibitors may promote the resolution of inflammation in COVID-19, thereby reducing acute respiratory distress syndrome (ARDS) and other life-threatening complications associated with robust viral-induced inflammation. While most COVID-19 clinical trials focus on "anti-viral" and "anti-inflammatory" strategies, stimulating inflammation resolution is a novel host-centric therapeutic avenue. Importantly, SPMs and sEH inhibitors are currently in clinical trials for other inflammatory diseases and could be rapidly translated for the management of COVID-19 via debris clearance and inflammatory cytokine suppression. Here, we discuss using pro-resolution mediators as a potential complement to current anti-viral strategies for COVID-19.

Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis.

The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities, such as diabetes, cardiovascular disease, and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1-knockout mice are prone to diet-induced obesity. Although the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation has a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3-L1 preadipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment prevented that phenomenon. Methylation-sensitive pyrosequencing analysis showed that CpG sites at the Thy1 locus have increased methylation during adipogenesis, as well as increased methylation in adipose tissue from diet-induced obese mice. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity.-Flores, E. M., Woeller, C. F., Falsetta, M. L., Susiarjo, M., Phipps, R. P. Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis.

Evaluation of the procoagulant properties of a newly developed platelet modified lysate product.

BACKGROUND: Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. METHODS: Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. RESULTS: PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. CONCLUSIONS: The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.

Lipoxin B4 Enhances Human Memory B Cell Antibody Production via Upregulating Cyclooxygenase-2 Expression.

Vaccination has been the most effective way to prevent or reduce infectious diseases; examples include the eradication of smallpox and attenuation of tetanus and measles. However, there is a large segment of the population that responds poorly to vaccines, in part because they are immunocompromised because of disease, age, or pharmacologic therapy and are unable to generate long-term protection. Specialized proresolving mediators are endogenously produced lipids that have potent proresolving and anti-inflammatory activities. Lipoxin B4 (LXB4) is a member of the lipoxin family, with its proresolving effects shown in allergic airway inflammation. However, its effects on the adaptive immune system, especially on human B cells, are not known. In this study, we investigated the effects of LXB4 on human B cells using cells from healthy donors and donors vaccinated against influenza virus in vitro. LXB4 promoted IgG Ab production in memory B cells and also increased the number of IgG-secreting B cells. LXB4 enhanced expression of two key transcription factors involved in plasma cell differentiation, BLIMP1 and XBP1. Interestingly, LXB4 increased expression of cyclooxygenase-2 (COX2), an enzyme that is required for efficient B cell Ab production. The effects of LXB4 are at least partially COX2-dependent as COX2 inhibitors attenuated LXB4-stimulated BLIMP1 and Xpb-1 expression as well as IgG production. Thus, our study reveals for the first time, to our knowledge, that LXB4 boosts memory B cell activation through COX2 and suggests that LXB4 can serve as a new vaccine adjuvant.

Secondhand Smoke Induces Inflammation and Impairs Immunity to Respiratory Infections.

Despite advocacy to reduce smoking-related diseases, >1 billion people worldwide continue to smoke. Smoking is immunosuppressive and an important etiological factor in the development of several human disorders including respiratory diseases like chronic obstructive pulmonary disease. However, there is a critical gap in the knowledge of the role of secondhand smoke (SHS) in inflammation and immunity. We therefore studied the influence of SHS on pulmonary inflammation and immune responses to respiratory infection by nontypeable Haemophilus influenzae (NTHI) recurrently found in chronic obstructive pulmonary disease patients. Chronic SHS-exposed mice were chronically infected with NTHI and pulmonary inflammation was evaluated by histology. Immune cell numbers and cytokines were measured by flow cytometry and ELISA, respectively. Chronic SHS exposure impaired NTHI P6 Ag-specific B and T cell responses following chronic NTHI infection as measured by ELISPOT assays, reduced the production of Abs in serum and bronchoalveolar lavage, and enhanced albumin leak into the bronchoalveolar lavage as determined by ELISA. Histopathological examination of lungs revealed lymphocytic accumulation surrounding airways and bronchovasculature following chronic SHS exposure and chronic infection. Chronic SHS exposure enhanced the levels of inflammatory cytokines IL-17A, IL-6, IL-1ß, and TNF-α in the lungs, and impaired the generation of adaptive immunity following either chronic infection or P6 vaccination. Chronic SHS exposure diminished bacterial clearance from the lungs after acute NTHI challenge, whereas P6 vaccination improved clearance equivalent to the level seen in air-exposed, non-vaccinated mice. Our study provides unequivocal evidence that SHS exposure has long-term detrimental effects on the pulmonary inflammatory microenvironment and immunity to infection and vaccination.

Activated Human Lung Fibroblasts Produce Extracellular Vesicles with Antifibrotic Prostaglandins.

The differentiation of interstitial lung fibroblasts into contractile myofibroblasts that proliferate and secrete excessive extracellular matrix is critical for the pathogenesis of pulmonary fibrosis. Certain lipid signaling molecules, such as prostaglandins (PGs), can inhibit myofibroblast differentiation. However, the sources and delivery mechanisms of endogenous PGs are undefined. Activated primary human lung fibroblasts (HLFs) produce PGs such as PGE2. We report that activation of primary HLFs with IL-1ß inhibited transforming growth factor ß-induced myofibroblast differentiation in both the IL-1ß-treated cells themselves (autocrine signal) and adjacent naive HLFs in cocultures (paracrine signal). Additionally, we demonstrate for the first time that at least some of the antifibrotic effect of activated fibroblasts on nearby naive fibroblasts is carried by exosomes and other extracellular vesicles that contain several PGs, including high levels of the antifibrotic PGE2. Thus, activated fibroblasts communicate with surrounding cells to limit myofibroblast differentiation and maintain homeostasis. This work opens the way for future research into extracellular vesicle-mediated intercellular signaling in the lung and may inform the development of novel therapies for fibrotic lung diseases.

Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.

Thy1 (CD90), a glycosylated, glycophosphatidylinositol-anchored membrane protein highly expressed by subsets of mesenchymal stem cells and fibroblasts, inhibits adipogenesis. The role of Thy1 on bone structure and function has been poorly studied and represents a major knowledge gap. Therefore, we analyzed the long bones of wild-type (WT) and Thy1 knockout (KO) mice with micro-computed tomography (micro-CT) and histomorphometry to compare changes in bone architecture and overall bone structure. micro-CT analysis of long bones revealed Thy1 KO and WT mice fed a high-fat diet demonstrated bone structural parameters at 4 mo that differed significantly between WT and KO mice. A significant reduction in trabecular bone volume was noted in Thy1 KO mice. The most prominent differences were observed in trabecular bone volume ratio and trabecular bone connectivity density. Consistent with micro-CT measurements, histomorphometric analysis also showed decreased bone volume in the obese Thy1 KO mice compared to obese WT mice. In vitro assays revealed that osteogenic conditions increased Thy1 expression during OB differentiation and absence of Thy1 attenuated osteoblastogenesis. Together, these findings support the concept that Thy1 serves as a major mechanistic link to regulate bone formation and negatively regulate adipogenesis.-Paine, A., Woeller, C. F., Zhang, H., Garcia-Hernandez, M. L., Huertas, N., Xing, L., Phipps, R. P., Ritchlin, C. T. Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.

Total plasma heme concentration increases after red blood cell transfusion and predicts mortality in critically ill medical patients.

BACKGROUND: Relationships between red blood cell (RBC) transfusion, circulating cell-free heme, and clinical outcomes in critically ill transfusion recipients are incompletely understood. The goal of this study was to determine whether total plasma heme increases after RBC transfusion and predicts mortality in critically ill patients. STUDY DESIGN AND METHODS: This was a prospective cohort study of 111 consecutive medical intensive care patients requiring RBC transfusion. Cell-free heme was measured in RBC units before transfusion and in the patients' plasma before and after transfusion. RESULTS: Total plasma heme levels increased in response to transfusion, from a median (interquartile range [IQR]) of 35 (26-76) µmol/L to 47 (35-73) µmol/L (p < 0.001). Posttransfusion total plasma heme was higher in nonsurvivors (54 [35-136] µmol/L) versus survivors (44 [31-65] µmol/L, p = 0.03). Posttransfusion total plasma heme predicted hospital mortality (odds ratio [95% confidence interval] per quartile increase in posttransfusion plasma heme, 1.76 [1.17-2.66]; p = 0.007). Posttransfusion total plasma heme was not correlated with RBC unit storage duration and weakly correlated with RBC unit cell-free heme concentration. CONCLUSIONS: Total plasma heme concentration increases in critically ill patients after RBC transfusion and is independently associated with mortality. This transfusion-associated increase in total plasma heme is not fully explained by RBC unit storage age or cell-free heme content. Additional studies are warranted to define mechanisms of transfusion-related plasma heme accumulation and test prevention strategies.

Sickle red blood cells are more susceptible to in vitro haemolysis when exposed to normal saline versus Plasma-Lyte A.

BACKGROUND: Normal saline has been the fluid of choice for resuscitation, rehydration and fluid replacement during plasma or red cell exchange/cytapheresis. There are increased concerns about its clinical effects and data showing it causes more haemolysis in vitro than buffered solutions such as Plasma-Lyte A. METHODS: We investigated whether normal saline or Plasma-Lyte A was associated with greater haemolysis during hours of in vitro incubation with both normal red cells and samples from patients with sickle cell anaemia. RESULTS: Sickle red cells haemolysed more than normal red cells did in both crystalloid solutions. The results of 24-hour exposure to saline were particularly striking (median of 163 mg/dl (IQ range 105-247) for sickle red cells vs. 53 (48-92) for normal red cells (P < 0·0001). In patient samples containing variable quantities of haemoglobin S red cells, increased haemoglobin S was associated with increased haemolysis. This effect was greater for normal saline than Plasma-Lyte A (P = 0·12). CONCLUSIONS: These in vitro models demonstrate that short-term ex vivo exposure of sickle red cells to normal saline leads to greater haemolysis than short-term exposure of normal red cells, and this effect is exacerbated by normal saline. Whether use of normal saline causes increased haemolysis in vivo is unknown. Given recent evidence that normal saline increases renal failure and mortality in critically ill patients, further studies are urgently needed.

Placental Chorionic Cyst Fluid Has Prothrombotic Properties and Differs From Amniotic Fluid.

INTRODUCTION: Chorionic cysts of the chorion laeve, fetal chorionic plate, septum, and free membranes have been associated with placental hypoxia, but they have no clear clinical significance. Although immunohistochemistry has identified fibronectin and collagen IV in cyst fluid, the contents have yet to be fully characterized. METHODS: Placental chorionic cysts (N = 10) were sampled by fluid extraction and hemotoxylin and eosin-stained sections. Amniotic fluid samples (N = 8) were obtained from pregnant women who had cytogenetic evaluation. The content of the cysts was tested for thrombogenicity using thromboelastography. The cyst content was tested by Luminex multiplex and ELISA assays and for known prothrombotic and proinflammatory factors. RESULTS: We identified cysts, especially those in the chorionic plate, adjacent to intervillous thrombi with apparent cyst rupture. Thromboelastography revealed a significantly shorter R time compared to whole blood control samples. Concentration of creatinine, α-fetoprotein, and surfactant D in the cyst fluid differed significantly from amniotic fluid. Cyst fluids had a significantly higher expression of all prothrombotic and some proinflammatory factors. DISCUSSION: Our data provide the first evidence that chorionic cyst fluid is prothrombotic and different from amniotic fluid. The association of ruptured cysts with adjacent thrombi and the prothrombotic properties of cyst fluid suggest a causal relationship; however, further studies are needed.

Transfusion medicine: A research agenda for the coming years.

The important scientific and clinical advances of the last century in transfusion medicine include methods for avoiding hemolytic transfusion reactions and preventing transmission of viral infectious diseases. The next great clinical advances will require improving the efficacy and safety of transfusions, as well as acknowledgement of the now proven serious complications of transfusion, including nosocomial infection, thrombosis, inflammation and multi-organ failure. Possible strategies include (1) universal leukoreduction to mitigate transfusion immunomodulation effects and improve storage conditions, (2) minimizing transfusion of ABO incompatible antibodies and cellular/soluble antigens, (3) substituting use of safer solutions for normal saline during apheresis, component infusion and washing (4) new techniques to improve the efficacy and safety of blood components, including improved storage solutions/conditions, supernatant removal by washing, and rejuvenation and (5) maximizing the risk to benefit ratio of transfusions by employing more restrictive and physiologic indications for transfusion (including patient blood management) and improving clinical decision making through novel laboratory and bedside tests such as thromboelastography.

Microbial biotransformation - an important tool for the study of drug metabolism.

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials. Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes. A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies. Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.

Cigarette smoke dampens antiviral signaling in small airway epithelial cells by disrupting TLR3 cleavage.

Cigarette smokers and people exposed to second-hand smoke are at an increased risk for pulmonary viral infections, and yet the mechanism responsible for this heightened susceptibility is not understood. To understand the effect of cigarette smoke on susceptibility to viral infection, we used an air-liquid interface culture system and exposed primary human small airway epithelial cells (SAEC) to whole cigarette smoke, followed by treatment with the viral mimetic polyinosinic polycytidylic acid (poly I:C) or influenza A virus (IAV). We found that prior smoke exposure strongly inhibited production of proinflammatory (interleukin-6 and interleukin-8) and antiviral [interferon-γ-induced protein 10 (IP-10) and interferons] mediators in SAECs in response to poly I:C and IAV infection. Impaired antiviral responses corresponded to increased infection with IAV. This was associated with a decrease in phosphorylation of the key antiviral transcription factor interferon response factor 3 (IRF3). Here, we found that cigarette smoke exposure inhibited activation of Toll-like receptor 3 (TLR3) by impairing TLR3 cleavage, which was required for downstream phosphorylation of IRF3 and production of IP-10. These results identify a novel mechanism by which cigarette smoke exposure impairs antiviral responses in lung epithelial cells, which may contribute to increased susceptibility to respiratory infections.

Activated human T lymphocytes inhibit TGFß-induced fibroblast to myofibroblast differentiation via prostaglandins D2 and E2.

In pulmonary fibrosis (PF), fibroblasts and myofibroblasts proliferate and deposit excessive extracellular matrix in the interstitium, impairing normal lung function. Because most forms of PF have a poor prognosis and limited treatment options, PF represents an urgent unmet need for novel, effective therapeutics. Although the role of immune cells in lung fibrosis is unclear, recent studies suggest that T lymphocyte (T cell) activation may be impaired in PF patients. Furthermore, we have previously shown that activated T cells can produce prostaglandins with anti-scarring potential. Here, we test the hypothesis that activated T cells directly inhibit myofibroblast differentiation using a coculture system. Coculture with activated primary blood-derived T cells, from both healthy human donors and PF patients, inhibited transforming growth factor ß-induced myofibroblast differentiation in primary human lung fibroblasts isolated from either normal or PF lung tissue. Coculture supernatants contained anti-fibrotic prostaglandins D2 and E2, and the inhibitory effect of coculture on myofibroblast differentiation was largely reversed when prostaglandin production was abrogated either by resting the T cells before coculture or via specific pharmacological inhibitors. Moreover, coculture conditions induced COX-2 in HLFs but not in T cells, suggesting that T cells deliver an activating signal to HLFs, which in turn produce anti-fibrotic prostaglandins. We show for the first time that coculture with activated primary human T lymphocytes strongly inhibits myofibroblast differentiation, revealing a novel cell-to-cell communication network with therapeutic implications for fibrotic lung diseases.

Antifibrotic Actions of Peroxisome Proliferator-Activated Receptor γ Ligands in Corneal Fibroblasts Are Mediated by ß-Catenin-Regulated Pathways.

Wound healing after corneal injury typically involves fibrosis, with transforming growth factor ß1 (TGF-ß1) as one of its strongest mediators. A class of small molecules-peroxisome proliferator-activated receptor γ (PPARγ) ligands-exert potent antifibrotic effects in the cornea by blocking phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, why this blocks fibrosis remains unknown. Herein, we show that PPARγ ligands (rosiglitazone, troglitazone, and 15-deoxy-Δ12,14-prostaglandin J2) decrease levels of ß-catenin. We also show that ß-catenin siRNA and the Wingless/integrated (Wnt) inhibitor pyrvinium block the ability of corneal fibroblasts to up-regulate synthesis of α-smooth muscle actin (α-SMA), collagen 1 (COL1), and fibronectin (FN) in response to TGF-ß1. Activation of TGF-ß receptors and p38 MAPK increased glycogen synthase kinase 3ß (GSK3ß) phosphorylation, whereas a chemical inhibitor of p38 MAPK (SB203580) reduced the phosphorylation of GSK3ß, decreasing active ß-catenin levels in both cytoplasmic and nuclear fractions. Finally, lithium chloride, a GSK3 inhibitor, also attenuated the TGF-ß1-induced increase in α-SMA, COL1, and FN expression. All in all, our results suggest that TGF-ß1 stimulation increases active ß-catenin concentration in cultured corneal fibroblasts through p38 MAPK regulation of canonical Wnt/ß-catenin signaling, increasing α-SMA, COL1, and FN synthesis. Thus, PPARγ ligands, by blocking TGF-ß1-induced p38 MAPK phosphorylation, prevent increases in both total and active ß-catenin through p38 MAPK-GSK3ß signaling.