Translational Clinical Strategies for the Prevention of Gastrointestinal Tract Graft Versus Host Disease.

Graft versus host disease (GVHD) is the major non-relapse complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, GVHD occurs in roughly half of patients following this therapy and can induce severe life-threatening side effects and premature mortality. The pathophysiology of GVHD is driven by alloreactive donor T cells that induce a proinflammatory environment to cause pathological damage in the skin, gastrointestinal (GI) tract, lung, and liver during the acute phase of this disease. Recent work has demonstrated that the GI tract is a pivotal target organ and a primary driver of morbidity and mortality in patients. Prevention of this complication has therefore emerged as an important goal of prophylaxis strategies given the primacy of this tissue site in GVHD pathophysiology. In this review, we summarize foundational pre-clinical studies that have been conducted in animal models to prevent GI tract GVHD and examine the efficacy of these approaches upon subsequent translation into the clinic. Specifically, we focus on therapies designed to block inflammatory cytokine pathways, inhibit cellular trafficking of alloreactive donor T cells to the GI tract, and reconstitute impaired regulatory networks for the prevention of GVHD in the GI tract.

Effects of propofol and its formulation components on macrophages and neutrophils in obese and lean animals.

We hypothesized whether propofol or active propofol component (2,6-diisopropylphenol [DIPPH] and lipid excipient [LIP-EXC]) separately may alter inflammatory mediators expressed by macrophages and neutrophils in lean and obese rats. Male Wistar rats (n = 10) were randomly assigned to receive a standard (lean) or obesity-inducing diet (obese) for 12 weeks. Animals were euthanized, and alveolar macrophages and neutrophils from lean and obese animals were exposed to propofol (50 µM), active propofol component (50 µM, 2,6-DIPPH), and lipid excipient (soybean oil, purified egg phospholipid, and glycerol) for 1 h. The primary outcome was IL-6 expression after propofol and its components exposure by alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were the production of mediators released by macrophages from adipose tissue, and neutrophils from lung and adipose tissues, and neutrophil migration. IL-6 increased after the exposure to both propofol (median [interquartile range] 4.14[1.95-5.20]; p = .04) and its active component (2,6-DIPPH) (4.09[1.67-5.91]; p = .04) in alveolar macrophages from obese animals. However, only 2,6-DIPPH increased IL-10 expression (7.59[6.28-12.95]; p = .001) in adipose tissue-derived macrophages. Additionally, 2,6-DIPPH increased C-X-C chemokine receptor 2 and 4 (CXCR2 and CXCR4, respectively) in lung (10.08[8.23-29.01]; p = .02; 1.55[1.49-3.43]; p = .02) and adipose tissues (8.78[4.15-11.57]; p = .03; 2.86[2.17-3.71]; p = .01), as well as improved lung-derived neutrophil migration (28.00[-3.42 to 45.07]; p = .001). In obesity, the active component of propofol affected both the M1 and M2 markers as well as neutrophils in both alveolar and adipose tissue cells, suggesting that lipid excipient may hinder the effects of active propofol.

Anti-inflammatory activity of caffeine (1,3,7-trimethylxanthine) after experimental challenge with virulent Listeria monocytogenes in Swiss mice.

BACKGROUND: Immunomodulatory therapies are claimed to enhance antimicrobial immunity and counterbalance antimicrobial resistance mechanisms of pathogenic bacteria. PURPOSE: To investigate whether caffeine can be useful for control of inflammation derived from experimental systemic infection with Listeria monocytogenes. METHODS: Peritoneal macrophages (pMØ) from Swiss mice were cultured with caffeine in 96-well plates, and then infected with virulent L. monocytogenes 619. In another experiment, the pMØ were first infected with the bacterium and then treated with caffeine. Swiss mice were inoculated intraperitoneally with L. monocytogenes and then treated intravenously with caffeine (0.05; 0.5 or 5 mg/Kg). RESULTS: Caffeine did not exert direct antibacterial activity in vitro against L. monocytogenes. Macrophages exposed to caffeine before or after infection with L. monocytogenes had increased cell viability, although the intracellular bacterial loads were similar to the control groups. Caffeine treatments of Swiss mice reduced leukocyte infiltration into the peritoneal cavity after L. monocytogenes infection. However, the bacterial burden was reduced in the spleen and liver. The mRNA expressions of IL-1ß, IL-6 and the enzyme inducible nitric oxide synthase (iNOS) were reduced whereas IL-10 was increased. CONCLUSION: Caffeine has an anti-infectious potential and ameliorated infection-derived inflammation following experimental infection with L. monocytogenes.

Unrestrained Gαi2 Signaling Disrupts Neutrophil Trafficking, Aging, and Clearance.

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gαißγ signaling, whose magnitude and kinetics are governed by RGS protein/Gαi interactions. RGS proteins typically limit Gαi signaling by reducing the duration that Gαi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gαi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gαi2/RGS protein interactions both limit and facilitate Gαi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Anti-inflammatory activity of citrus pectin on chicken monocytes' immune response.

Pectin is a dietary fibre composed of galacturonic acid, primarily found in the citrus fruits' cell walls. Citrus pectin (CP) has demonstrated antioxidative, anticancer, and anti-inflammatory properties in humans and animals. In broilers, CP supplementation improves energy utilization and nutrient digestibility, but limited information on its effects on chicken immunity is available so far. This study aimed to assess the in vitro impact of CP on chicken monocytes' immune response. Cells were purified from whole blood of healthy chickens and incubated with increasing concentrations (0, 0.25, 0.5, 0.75, 1 mg/mL) of CP to determine CP working concentration. The effects of different CP concentrations on cells' apoptosis and viability were assessed by measuring caspase-3 and -7 and the cells' metabolic activity (MTT assay), respectively. CP had no dose-dependent effect on monocyte apoptosis and viability.Then, the effects of CP (0.5 mg/mL) on chicken monocytes' chemotaxis and phagocytosis were assessed by measuring transwell migration and fluorescein-labelled E. coli incorporation, respectively. CP inhibited both monocytes' chemotaxis and phagocytosis.These data demonstrate that CP exerts an immunomodulatory role in chicken monocytes, supporting its integration in nutrition strategies that might be beneficial for the animal's immunity and health.

Targeting cancer homing into the lymph node with a novel anti-CCR7 therapeutic antibody: the paradigm of CLL.

Lymph node (LN) is a key tissue in the pathophysiology of mature blood cancers, especially for chronic lymphocytic leukemia (CLL). Within the multiple de-regulated pathways affecting CLL homeostasis, the CC-chemokine receptor 7 (CCR7) grants homing of CLL cells into the LN where protective environments foster tumor progression. To cover the lack of specific therapies targeting the CCR7-dependence of CLL to enter into the LN, and aiming to displace the disease from LN, we generated CAP-100, an antibody that specifically binds to hCCR7 and neutralizes its ligand-binding site and signaling. In various in vitro and in vivo preclinical models CAP-100 strongly inhibited CCR7-induced migration, extravasation, homing, and survival in CLL samples. Moreover, it triggered potent tumor cell killing, mediated by host immune mechanisms, and was effective in xenograft models of high-risk disease. Additionally, CAP-100 showed a favorable toxicity profile on relevant hematopoietic subsets. Our results validated CAP-100 as a novel therapeutic tool to prevent the access of CLL cells, and other neoplasia with nodal-dependence, into the LN niches, thus hitting a central hub in the pathogenesis of cancer. The first-in-human clinical trial (NCT04704323), which will evaluate this novel therapeutic approach in CLL patients, is pending.

Methamphetamine facilitates pulmonary and splenic tissue injury and reduces T cell infiltration in C57BL/6 mice after antigenic challenge.

Methamphetamine (METH) is a strong addictive central nervous system stimulant. METH abuse can alter biological processes and immune functions necessary for host defense. The acquisition and transmission of HIV, hepatitis, and other communicable diseases are possible serious infectious consequences of METH use. METH also accumulates extensively in major organs. Despite METH being a major public health and safety problem globally, there are limited studies addressing the impact of this popular recreational psychostimulant on tissue adaptive immune responses after exposure to T cell dependent [ovalbumin (OVA)] and independent [lipopolysaccharide (LPS)] antigens. We hypothesized that METH administration causes pulmonary and splenic tissue alterations and reduces T cell responses to OVA and LPS in vivo, suggesting the increased susceptibility of users to infection. Using a murine model of METH administration, we showed that METH causes tissue injury, apoptosis, and alters helper and cytotoxic T cell recruitment in antigen challenged mice. METH also reduces the expression and distribution of CD3 and CD28 molecules on the surface of human Jurkat T cells. In addition, METH decreases the production of IL-2 in these T-like cells, suggesting a negative impact on T lymphocyte activation and proliferation. Our findings demonstrate the pleotropic effects of METH on cell-mediated immunity. These alterations have notable implications on tissue homeostasis and the capacity of the host to respond to infection.

Artemisinin Attenuates Transplant Rejection by Inhibiting Multiple Lymphocytes and Prolongs Cardiac Allograft Survival.

Immunological rejection is an important factor resulting in allograft dysfunction, and more valid therapeutic methods need to be explored to improve allograft outcomes. Many researches have indicated that artemisinin and its derivative exhibits immunosuppressive functions, apart from serving as a traditional anti-malarial drug. In this assay, we further explored the therapeutic effects of artemisinin for transplant rejection in a rat cardiac transplantation model. We found that it markedly attenuated allograft rejection and histological injury and significantly prolonged the survival of allograft. Upon further exploring the mechanism, we demonstrated that artemisinin not only attenuated T cell-mediated rejection (TCMR) by reducing effector T cell infiltration and inflammatory cytokine secretion and increasing regulatory T cell infiltration and immunoregulatory cytokine levels, but also attenuated antibody-mediated rejection (ABMR) through inhibition of B cells activation and antibody production. Furthermore, artemisinin also reduced macrophage infiltration in allografts, which was determined to be important for TCMR and ABMR. Moreover, we demonstrated that artemisinin significantly inhibited the function of pure T cells, B cells, and macrophages in vitro. All in all, this study provide evidence that artemisinin significantly attenuates TCMR and ABMR by targeting multiple effectors. Therefore, this agent might have potential for use in clinical settings to protect against transplant rejection.

Toxicological and anti-inflammatory profile of Synadenium grantii Hook. f. in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Synadenium grantii Hook. f., popularly known as "janaúba" or "leiterinha", is used in the folk medicine to treat gastric disorders, some types of neoplasias and inflammatory diseases. AIM OF THE STUDY: The aim of this study was to show the anti-inflammatory activity of the methanol extract obtained from S. grantii stems and also certify the safety of the extract performing toxicological analysis. MATERIAL AND METHODS: The anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally pre-treated with the S. grantii extract (1, 3 or 10 mg/kg). The leukocyte influx (optical microscopy) and secretion of chemical mediators (TNF, IL-6 and IL-1ß, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. The toxicity was investigated using the dose-fixed procedure (acute toxicity) and repeated dose 28-day (subacute toxicity) in mice orally treated with S. grantii extract. The open field and rota-rod test were used to evaluate possible interference of adverse effect of S. grantii on motor coordination, locomotor and exploratory activity. RESULTS: The analysis of the inflammatory exudate of S. grantii-treated mice demonstrated reduction in the polymorphonuclear cells (PMN) migration to the inflamed tissue, as well as the reduction of the pro-inflammatory cytokines TNF and IL-1ß. Furthermore, the acute and sub-acute toxicity studies did not show significant changes in body weight, general behaviour, biochemical parameters, organ weight and liver and kidney histopathological analysis. However, animals acutely treated with S. grantii presented reduction in the number of crosses in relation to the vehicle group, without significant difference in the number of elevations and latency time between the groups in rota-rod test. The obtained results allow to set the NOAEL (Non-observed-adverse-effect level) in 100 mg/kg for this specie of rodent. CONCLUSIONS: Together, the results herein obtained show that S. grantii extract presented anti-inflammatory activity by decreasing the influx of PMN to the inflamed tissue, as well as the cytokines TNF and IL-1ß levels. In addition, S. grantii extract seemed not to present significant acute or subacute toxicity when administered to mice, demonstrating for the first time the safety of this extract, when orally administered.

17ß-Estradiol Treatment Protects Lungs Against Brain Death Effects in Female Rat Donor.

BACKGROUND: Brain death (BD) affects the viability of lungs for transplantation. A correlation exists between high-lung inflammation after BD and the decrease in female sex hormones, especially estradiol. Therefore, we investigated the effects of 17ß-estradiol (E2) treatment on the lungs of female brain dead rats. METHODS: Female Wistar rats were divided into 4 groups: BD (submitted to BD for 6 h), sham (false operated), E2-T0 (treated with E2 immediately after BD; 50 µg/mL, 2 mL/h), and E2-T3 (treated with E2 after 3 h of BD; 50 µg/mL, 2 mL/h). Lung edema, hemorrhage, and leukocyte infiltration were analyzed. Adhesion molecules were evaluated, and analysis of NO synthase gene and protein expression was performed using real-time PCR and immunohistochemistry, respectively. Release of chemokines and matrix degradation in the lungs was analyzed. RESULTS: BD increased leukocyte infiltration, as shown by intravital microscopy (P = 0.017), bronchoalveolar lavage cell count (P = 0.016), the release of inflammatory mediators (P = 0.02), and expression of adhesion molecules. BD also increased microvascular permeability and the expression and activity of matrix metalloproteinase-9 in the lungs. E2 treatment reduced leukocyte infiltration, especially in the E2-T3 group, release of inflammatory mediators, adhesion molecules, and matrix metalloproteinase activity in the lungs. CONCLUSIONS: E2 treatment was successful in controlling the lung inflammatory response in females submitted to BD. Our results suggest that E2 directly decreases the release of chemokines, restraining cell traffic into the lungs. Thus, E2 has a therapeutic potential, and its role in improving donor lung quality should be explored further.

Anti-Inflammatory Effect of KW-2449 on Autoimmune Encephalomyelitis: An Experimental Study on Mice.

BACKGROUND: The KW-2449 is a novel multikinase inhibitor that inhibits FLT3, ABL, ABL-T315I, and Aurora A. FLT3 and Aurora A kinases play an important role in the pathogenesis of multiple sclerosis (MS). KW-2449 could modulate immune cells, but the immunomodulatory effects of KW-2449 on experimental autoimmune encephalomyelitis (EAE) have not been investigated yet. The aim of the present study is to investigate the effects of KW-2449 on EAE mouse model. METHODS: In this study, C57BL/6 EAE mice were orally treated with (10 mg/kg/day) KW-2449 solution and compared with EAE and control mice. Following the treatment, histological analyses were performed on the brain and cerebellum to evaluate the pathological score. The gene expression levels of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) were measured using qRT-PCR. The serum levels of TNF-α, IL-6, CCL-2 and MMP-2 were determined by using quantitative enzyme-linked immunosorbent assay (ELISA). RESULTS: The results indicated that the clinical score, the infiltration of inflammatory cells and the demyelination in EAE mice treated with KW-2449 decreased significantly compared to control groups. KW-2449 also decreased TNF-α, IL-6, CCL-2 inflammatory cytokines, and MMP-2 in both brain mRNA expressions and serum levels of EAE mice. CONCLUSION: The KW-2449, aging as a multi-kinase inhibitor, modulates the inflammatory responses of cytokine cascades either in the brain or in plasma and reduces EAE pathogenesis manifestation.

Antioxidant and anti-inflammatory action (in vivo and in vitro) from the trunk barks of Cabreúva (Myrocarpus frondosus Allemao, Fabaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Myrocarpus frondosus, known as cabreúva, is a tree whose trunk barks are used in folk medicine as tea, syrup, ointments, and tinctures for the treatment of inflammation. However, there is no scientific evidence demonstrating this activity. AIM OF THE STUDY: The present investigation was focused on evaluating the antioxidant and anti-inflammatory activities of M. frondosus, using the in vitro model of RAW 264.7 macrophages induced by LPS and the in vivo model of mouse pleurisy induced by carrageenan. MATERIALS AND METHODS: M. frondosus trunk barks were dried at room temperature for seven days and subjected to exhaustive maceration with ethanol (70%) to obtain its crude extract (CE). CE was subjected to UPLC-HRMS analysis to establish its chemical profile. Its antioxidant activity was evaluated using the DPPH method, reducing power by the iron (III) to iron (II) reduction assay and the ß-carotene-linoleic acid bleaching assay. The RAW 264.7 macrophages were pretreated with the CE in a non-cytotoxic concentration and induced by LPS (1 µg/mL). After 24 h, using the supernatant, we evaluated the nitric oxide (NOx) and interleukin-6 (IL-6) levels. The anti-inflammatory effects of CE (at doses of 30, 100 and 300 mg/kg) were evaluated on leukocyte migration (total and differential), exudate concentrations, myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities, NOx, tumor necrosis factor-α (TNF-α), and IL-6 levels, by using a murine model of neutrophilic inflammation. RESULTS: The UPLC-HRMS of CE revealed the presence of isoflavonones, including biochanin A and formononetin. CE exhibited good antioxidant activity by quenching and decreasing free radicals, as well as reducing pro-oxidant metals. CE did not show cytotoxicity at a concentration below 11 µg/mL and reduced the secretion of the pro-inflammatory NOx in the inflamed macrophages. In vivo assay revealed that CE caused a pronounced inhibition on leukocyte migration, and this inhibition was due to its ability to reduce neutrophil migration. Moreover, CE was also able to reduce the release of critical pro-inflammatory mediators such as MPO, NOx, TNF-α, and IL-6. CONCLUSIONS: All these findings indicate that M. frondosus exhibited antioxidant activity and anti-inflammatory effect.

Pharmacological blockade of TNFα prevents sarcopenia and prolongs survival in aging mice.

Sarcopenia is a hallmark of aging. Inflammation due to increased generation of cytokines such as TNFα, IL-1ß and IL-6 has been implicated in the pathogenesis of sarcopenia. In skeletal muscle of C57BL/6 mice from 12 until 28 months of age, we observed a progressive reduction of myofiber cross sectional area, loss of type II fibers and infiltration by inflammatory cells. Muscle strength decreased in parallel. Pharmacological TNFα blockade by weekly subcutaneous injection of Etanercept from 16 to 28 months of age prevented atrophy and loss of type II fibers, with significant improvements in muscle function and mice lifespan. The effects on leukocyte recruitment were limited. These results provide a proof of principle that endogenous TNFα is sufficient to cause sarcopenia and to reduce animal survival, and open a novel perspective on novel potential pharmacological treatment strategies based on TNFα blockade to prevent the noxious events associated with aging.

Role of the Intracellular Sodium Homeostasis in Chemotaxis of Activated Murine Neutrophils.

The importance of the intracellular Ca2+ concentration ([Ca2+]i) in neutrophil function has been intensely studied. However, the role of the intracellular Na+ concentration ([Na+]i) which is closely linked to the intracellular Ca2+ regulation has been largely overlooked. The [Na+]i is regulated by Na+ transport proteins such as the Na+/Ca2+-exchanger (NCX1), Na+/K+-ATPase, and Na+-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na+]i. fMLF induces a sustained increase of [Na+]i, surprisingly, reaching higher values in TRPM2-/- neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na+]i. The difference in [Na+]i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na+ concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2-/- neutrophils. Increasing the [Na+]i by inhibiting the Na+/K+-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na+ homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na+ concentration gradients. Increasing the [Na+]i by a few mmol/l may suffice to switch its transport mode from forward (Ca2+-efflux) to reverse (Ca2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.

The effect of pamidronate delivery in bisphosphonate-naïve patients on neutrophil chemotaxis and oxidative burst.

The pathogenesis of medication-related osteonecrosis of the jaw (MRONJ), a morbid condition associated with bisphosphonate administration, has not been fully elucidated. Recent research utilizing a murine model has revealed that the neutrophil becomes dysfunctional following exposure to bisphosphonates. Accordingly, the impairment of neutrophil function could play an important role in the pathogenesis of MRONJ via an infectious mechanism mediated by the suppression of the innate immune system. Currently, the existing human data are insufficient to substantiate this theory. To investigate, we isolated neutrophils from blood and oral rinse samples from bisphosphonate-naïve patients who were recently diagnosed with multiple myeloma both prior to and one month following their initial infusion of pamidronate, an intravenous bisphosphonate agent. Stimulated blood and oral neutrophil superoxide production and chemotactic capabilities were found to be impaired relative to baseline values. These results suggest that impaired neutrophil function may partially contribute to the aetiology underlying the pathophysiological processes linked to the development of MRONJ. Further, as the functional status of circulating neutrophils was reflected in the oral cavity where sampling can be accomplished in a non-invasive fashion, it is conceivable that neutrophil function could serve as a potential biomarker for MRONJ prognostication.

Inflammatory and lipid regulation by cholinergic activity in epicardial stromal cells from patients who underwent open-heart surgery.

The modulation of acetylcholine (ACh) release by botulinum toxin injection into epicardial fat diminishes atrial fibrillation (AF) recurrence. These results suggest an interaction between autonomic imbalance and epicardial fat as risk factors of AF. Our aim was to study the inflammatory, lipidic and fibroblastic profile of epicardial stroma from patients who underwent open-heart surgery, their regulation by cholinergic activity and its association with AF. We performed in vitro and ex vivo assays from paired subcutaneous and epicardial stromal cells or explants from 33 patients. Acute ACh effects in inflammation and lipid-related genes were analysed by qPCR, in intracellular calcium mobilization were performed by Fluo-4 AM staining and in neutrophil migration by trans-well assays. Chronic ACh effects on lipid accumulation were visualized by AdipoRed. Plasma protein regulation by parasympathetic denervation was studied in vagotomized rats. Our results showed a higher pro-inflammatory profile in epicardial regarding subcutaneous stromal cells. Acute ACh treatment up-regulated monocyte chemoattractant protein 1 levels. Chronic ACh treatment improved lipid accumulation within epicardial stromal cells (60.50% [22.82-85.13] vs 13.85% [6.17-23.16], P < .001). Additionally, patients with AF had higher levels of fatty acid-binding protein 4 (1.54 ± 0.01 vs 1.47 ± 0.01, P = .005). Its plasma levels were pronouncedly declined in vagotomized rats (2.02 ± 0.21 ng/mL vs 0.65 ± 0.23 ng/mL, P < .001). Our findings support the characterization of acute or chronic cholinergic activity on epicardial stroma and its association with AF.

PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury.

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

Nintedanib Reduces Neutrophil Chemotaxis via Activating GRK2 in Bleomycin-Induced Pulmonary Fibrosis.

Neutrophils are involved in the alveolitis of idiopathic pulmonary fibrosis (IPF). However, their pathogenic mechanisms are still poorly understood. Nintedanib has antifibrotic and anti-inflammatory activity in IPF. This study aimed to investigate the regulatory mechanism of nintedanib on neutrophil chemotaxis in bleomycin (BLM)-induced pulmonary fibrosis. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h after a bleomycin intratracheal injection (1.5 U/kg). Lung histopathological findings, the expression of cytokines, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed. The effect of nintedanib was also investigated in a mouse model with adoptive neutrophil transfer in vivo. Nintedanib significantly decreased the histopathological changes and neutrophil recruitment in BLM-induced pulmonary fibrosis. Nintedanib mediated a downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and very late antigen 4 (VLA-4) expression, as well as an upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in BLM-induced pulmonary fibrosis. Nintedanib also decreased the activation of endothelial cells by the decreased expression of vascular cell adhesion molecule 1 (VCAM-1). The effect of nintedanib on regulating neutrophil chemotaxis was also confirmed by a mouse model with adoptive neutrophil transfer in vivo. In conclusion, nintedanib reduces neutrophil chemotaxis and endothelial cell activation to regulate the severity of BLM-induced pulmonary fibrosis. These effects are associated with an enhancement of GRK2 activity and a reduction in CXCR2 and VLA-4 expression on neutrophils and a decrease in VCAM-1 expression on endothelial cells.

Synergistic effects of low­dose chemotherapy and T cells in renal cell carcinoma.

Renal cell carcinoma (RCC) is not sensitive to conventional radiotherapy and chemotherapy, and the effectiveness rate of molecular targeted therapy is low. Therefore, it is urgent to identify new treatment methods. Recently, adoptive T­cell therapy has provided a new option for cancer treatment. Furthermore, low­dose chemotherapy not only has no evident side effects and inhibitory effects on the human immune system, but can also enhance the immune activity of some effector cells. Therefore, it is surmised that the combination of different mechanisms of chemotherapy and immunotherapy could be a new treatment concept. In the present study, the effects of low­dose chemotherapy combined with T cells in the treatment of renal cell carcinoma were explored using cytotoxicity assays, enzyme­linked immunosorbent assay (ELISA), western blot analysis and flow cytometric analysis. The results revealed that low­dose chemotherapy and T cells had synergistic effects on tumor cell elimination in vitro. The transforming growth factor (TGF)­ß signaling pathway may be involved in the inhibition of T­cell functions. The targeted inhibition of TGF­ß signals may be a promising therapeutic strategy for the treatment of renal cancer. The present results provided a novel strategy for the combination of low­dose chemotherapy and T cells to enhance the therapeutic efficacy of RCC treatment.