Leukocyte Cytokine Production Research Articles

Effects of calcitriol on oxidative burst, phagocytic function, and leukocyte cytokine production in shelter dogs

BackgroundThe active metabolite of vitamin D, calcitriol, has been shown across many different species to augment innate immune responses and dampen aberrant proinflammatory cytokine production. Community acquired infections are common in shelters and consume limited shelter resources, impact adoption rates, and can result in unnecessary euthanasia. Prophylactic oral vitamin D supplementation decreases the incidence and severity of upper and lower respiratory tract infections in humans. Before a clinical trial investigating the clinical benefit of oral vitamin D supplementation in shelter dogs can be pursued, an in vitro study evaluating the immunomodulatory effects of calcitriol in blood from shelter dogs is warranted. Therefore, the objective of this study was to determine if incubation of whole blood obtained from apparently healthy dogs housed in a shelter for ≥7 days with calcitriol would alter granulocyte/monocyte (GM) oxidative burst and phagocytic function as well as pathogen-associated molecular pattern (PAMP)-stimulated leukocyte production of tumor necrosis factor (TNF)-α, and interleukin (IL)-6, and IL-10.ResultsTen dogs housed in a shelter for ≥7 days were enrolled in a prospective cohort study. Whole blood from these dogs was incubated with calcitriol (10− 7 M) or diluent (control) for 24 h. Subsequent to this incubation, phagocytosis of opsonized-Escherichia coli (E. coli) and E. coli-induced oxidative burst were evaluated via flow cytometry. In addition, leukocyte production of TNF-α, IL-6, and IL-10 were measured using a canine-specific multiplex bead assay. Calcitriol significantly decreased leukocyte TNF-α production (p = 0.009) and increased IL-10 production (p = 0.002). Tumor necrosis factor-α-to-IL-10 ratio was significantly decreased with calcitriol (p = 0.017), while IL-6 production as well as GM oxidative burst and phagocytic function were not significantly affected.ConclusionsThese data indicate that calcitriol attenuates proinflammatory immune responses without affecting GM oxidative burst or phagocytic function in vitro in whole blood obtained from apparently healthy shelter dogs.

Immunomodulation by an Omega-6 Fatty Acid Reduced Mixed Lipid Emulsion in Murine Acute Respiratory Distress Syndrome.

Background: Acute respiratory distress syndrome (ARDS) is associated with both high morbidity and mortality in intensive care units worldwide. Patients with ARDS often require parenteral nutrition with lipid emulsions as essential components. In the present study, we assessed the immunomodulatory and apoptotic effects of a modern, n-6-reduced lipid emulsion mixture in murine ARDS. Methods: Mice received an infusion of either normal saline solution, pure long-chain triglyceride (LCT) emulsion, or SMOF (soybean oil, medium-chain triglycerides, olive oil, and fish oil) before a lipopolysaccharide (LPS) challenge. Mice were sacrificed at different time points (0, 24, or 72 h) after ARDS induction, and an analysis of inflammatory cytokines, protein concentrations, and the cellular composition of the alveolar and interstitial compartments was performed with special focus on alveolar apoptosis and necrosis. Results: Mice infused with SMOF showed decreased leukocyte invasion, protein leakage, myeloperoxidase activity, and cytokine production in alveolar spaces after LPS challenge compared to animals that received LCT. There were fewer cells in the lung interstitium of the SMOF group compared to the LCT group. Both lipid emulsions exerted pro-apoptotic and pro-necrotic properties on alveolar immune cells, with significantly increased necrosis in mice infused with LCT compared to SMOF. Conclusion: SMOF has both anti-inflammatory and pro-resolving influences in murine ARDS. Partial replacement of n-6 fatty acids with n-3/n-9 fatty acids may therefore benefit critically ill patients at risk for ARDS who require parenteral nutrition.

Local administration of 4-Thiouridine, a novel molecule with potent anti-inflammatory properties, protects against experimental colitis and arthritis

Previous studies in a rat model of Sephadex induced lung inflammation showed that 4-Thiouridine (4SU), a thiol substituted nucleoside, was very effective in reducing edema, leukocyte influx and TNF levels in bronchoalvelolar lavage fluid. However, little is known about the factors and mechanisms underlying these effects. In the present study, we have used two separate mouse models of chronic inflammation, a model of dextran sulphate sodium (DSS) induced colitis and a model of antigen induced arthritis, to evaluate the anti-inflammatory effect of 4-thiouridine. We have analyzed a broad spectrum of inflammatory mediators in order to delineate the mechanisms behind a potential anti-inflammatory effect of 4SU. Colitis was induced in C57BL/6 mice by administration of 3.5% DSS in drinking water for 5 days and the potential anti-colitic effect of 4SU was assessed by monitoring the disease activity index (DAI), measurement of colon length and histopathological analysis of colon tissue. We analyzed tissue myeloperoxidase (MPO) activity, serum pro-inflammatory cytokines (IL-1β, IL-6 and TNF), mRNA and protein expression of pro-inflammatory cytokines, COX-2, and NF-κB activity in colitis tissue. Intracolonic administration of 4SU (5 mg/kg & 10 mg/kg.) significantly inhibited MPO activity and reduced the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF) as well as COX-2. Further, NF-κB activation was also blocked by attenuating the phosphorylation of IkB kinase (IKK α/β) in DSS-induced colitis tissues. Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA and 4SU was administered locally by direct injection into the knee joint. The antiarthritic potential of 4SU was calculated by histopathological scores and histochemical analysis of joint tissue. Further, immunohistochemistry was used to study inflammatory cell infiltration and expression of cytokines and adhesion molecules in the synovium. Local administration of 50–100 mg/kg 4SU at the time of arthritis onset clearly prevented development of joint inflammation and efficiently inhibited synovial expression of CD18, local cytokine production and recruitment of leukocytes to the synovium. Taken together, our data clearly demonstrates a potent anti-inflammatory effect of 4SU in two experimental models. In conclusion 4SU could be a new promising candidate for therapeutic modulation of chronic inflammatory diseases like ulcerative colitis and arthritis.

Bioinformatics analysis of vascular RNA-seq data revealed hub genes and pathways in a novel Tibetan minipig atherosclerosis model induced by a high fat/cholesterol diet

BackgroundAtherosclerosis is a major contributor to cardiovascular events, however, its molecular mechanism remains poorly known. Animal models of atherosclerosis can be a valuable tool to provide insights into the etiology, pathophysiology, and complications of atherosclerosis. In particular, Tibetan minipigs are a feasible model for studying diet-related metabolic and atherosclerotic diseases.MethodsWe used vascular transcriptomics to identify differentially expressed genes (DEGs) in high fat/cholesterol (HFC) diet-fed Tibetan minipig atherosclerosis models, analyzed the DEGs gene ontology (GO) terms, pathways and protein-protein interactions (PPI) networks, and identified hub genes and key modules using molecular complex detection (MCODE), Centiscape and CytoHubba plugin. The identified genes were validated using the human carotid atherosclerosis database (GSEA 43292) and RT-PCR methods.ResultsOur results showed that minipigs displayed obvious dyslipidemia, oxidative stress, inflammatory response, atherosclerotic plaques, as well as increased low-density lipoprotein (LDL) and leukocyte recruitment after 24 weeks of HFC diet feeding compared to those under a regular diet. Our RNA-seq results revealed 1716 DEGs in the atherosclerotic/NC group, of which 1468 genes were up-regulated and 248 genes were down-regulated. Functional enrichment analysis of DEGs showed that the HFC diet-induced changes are related to vascular immune-inflammatory responses, lipid metabolism and muscle contraction, indicating that hypercholesterolemia caused by HFC diet can activate innate and adaptive immune responses to drive atherosclerosis development. Furthermore, we identified four modules from the major PPI network, which are implicated in cell chemotaxis, myeloid leukocyte activation, cytokine production, and lymphocyte activation. Fifteen hub genes were discovered, including TNF, PTPRC, ITGB2, ITGAM, VCAM1, CXCR4, TYROBP, TLR4, LCP2, C5AR1, CD86, MMP9, PTPN6, C3, and CXCL10, as well as two transcription factors (TF), i.e. NF-ĸB1 and SPI1. These results are consistent with the expression patterns in human carotid plaque and were validated by RT-PCR.ConclusionsThe identified DEGs and their enriched pathways provide references for the development and progression mechanism of Tibetan minipig atherosclerosis model induced by the HFC diet.

Effects of Orally Administered Resveratrol on TNF, IL-1β, Leukocyte Phagocytic Activity and Oxidative Burst Function in Horses: A Prospective, Randomized, Double-Blinded, Placebo-Controlled Study.

Resveratrol, a phytophenol, is a commonly used equine nutraceutical supplement touted to exert anti-inflammatory effects. The effect of orally administered resveratrol on tumor necrosis factor (TNF), interleukin-1β (IL-1β), leukocyte phagocytic activity or oxidative burst function have not been reported in horses. The objective of this study was to determine the effects of a commercially available, orally administered resveratrol product on innate immune functions in healthy adult horses. Whole blood was collected from 12 horses prior to and following 3 weeks of treatment with either the manufacturer’s recommended dose of resveratrol or placebo. Phagocytosis, oxidative burst and pathogen associated molecular pattern (PAMP) motif-stimulated leukocyte production of TNF and IL-1β were compared pre- and post-treatment between treatment groups. Phagocytosis and oxidative burst capacity were evaluated via flow cytometry. Tumor necrosis factor and IL-1β were measured using cytotoxicity and ELISA assays, respectively. There were no significant differences in phagocytosis, oxidative burst or stimulated TNF or IL-1β production between resveratrol and placebo treatment groups. Orally administered resveratrol at a routinely recommended dose for a duration of 3 weeks did not significantly affect phagocytic activity, oxidative burst function or PAMP-stimulated leukocyte cytokine production.

Chronic Endometritis: Old Problem, Novel Insights and Future Challenges.

Chronic endometritis (CE) is a poorly investigated pathology which has been related to adverse reproductive out- comes, such as implantation failure and recurrent miscarriage. In this paper, we aim to provide an overview of diag- nosis, etiology, pathophysiology and treatment of CE, its impact on endometrial microenvironment and its associa- tion with infertility. We present a narrative review of the current literatures, synthesizing the findings retrieved from searches of computerized databases. CE is more prevalent in infertile patients. Effective antibiotic treatment of CE seems to improve the pregnancy and live birth rate in patients with unexplained recurrent pregnancy loss (RPL), and increase ongoing pregnancy rate in patients with recurrent implantation failure. In order to increase the diagnostic ac- curacy, immunohistochemistry is recommended besides the conventional histology. In addition, hysteroscopy could be considered as gold standard tool for diagnosis, considering its high correlation with histological findings. CE, as the chronic inflammation of endometrium, is usually asymptomatic and probably underestimated. Interaction of bac- teria with endometrial microenvironment promotes changes in leukocyte population, cytokine production and growth factors which support its negative impact on endometrial receptivity. Nevertheless, standardization of the criteria for histopathological diagnosis and immunohistochemistry technique needs to be defined.

Abstract # 2065 Markers of peripheral immune cells are increased in regions of blood-brain barrier disruption after repetitive blast injury

Cycles of blood-brain barrier (BBB) disruption have been observed in a variety of rodent brain injury models. Changes in BBB permeability are correlated with pro-inflammatory cytokine production and brain infiltration of peripheral leukocytes. Whether the influx of peripheral leukocytes through a permeable BBB affords prolonged neuroprotection, or promotes detrimental changes to the anatomical site of entry remains to be elucidated. In a mouse model of blast-induced traumatic brain injury (TBI), we observed brain-region specific BBB permeability to radiolabeled albumin at 72 h. Moreover, we measured an increase in pro-inflammatory tumor necrosis factor alpha (TNF α ) protein expression and mRNA abundance in brain regions vulnerable to BBB disruption. The nitric oxide synthase inhibitor N(G)-nitro- l -arginine methyl ester (L-NAME) attenuated TNF α levels and abrogated albumin permeability. We hypothesize that leukocyte infiltration into vulnerable brain regions after TBI may also be mediated by nitric oxide dependent mechanisms. Using flow cytometry, we measured an increase of blood-derived monocytes (Ly6C/G-high and CD45-high) and T-cells (CD3+ and CD4+) in brain regions vulnerable to BBB disruption at 72 h post-TBI. The physiological process of leukocyte infiltration could be driven by nitric oxide dependent mechanisms, which could be exploited, at the right time, to assist in chronic neurotrauma resolution.

Innate Immunity Provides Biomarkers of Health for Teleosts Exposed to Nanoparticles.

In recent years, the unique properties of nanoparticles have fostered novel applications in various fields such as biology, pharmaceuticals, agriculture, and others. Unfortunately, their rapid integration into daily life has also led to environmental concerns due to uncontrolled release of nanoparticles into the aquatic environment. Despite increasing awareness of nanoparticle bioaccumulation in the aquatic environment, much remains to be learned about their impact on aquatic organisms and how to best monitor these effects. Herein, we provide the first review of innate immunity as an emerging tool to assess the health of fish following nanoparticle exposure. Fish are widely used as sentinels for aquatic ecosystem pollution and innate immune parameters offer sensitive and reliable tools that can be harnessed for evaluation of contamination events. The most frequent biomarkers highlighted in literature to date include, but are not limited to, parameters associated with leukocyte dynamics, oxidative stress, and cytokine production. Taken together, innate immunity offers finite and sensitive biomarkers for assessment of the impact of nanoparticles on fish health.

Effects of calcitriol on phagocytic function, toll-like receptor 4 expression, and cytokine production of canine leukocytes.

OBJECTIVE To determine the in vitro effects of calcitriol on indicators of immune system function in blood samples collected from healthy dogs. SAMPLE Blood samples from 8 healthy adult dogs. PROCEDURES Blood samples were incubated with calcitriol (10-7M) or control substance for 24 hours. Afterward, lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate (MDP)-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) were measured with a canine-specific multiplex assay. Phagocytosis of opsonized Escherichia coli and leukocyte expression of constitutive toll-like receptor 4 (TLR4) were evaluated via flow cytometry. Blood samples from 3 dogs were used to create a concentration-response curve to evaluate whether the observed cytokine modulation was concentration dependent. RESULTS Incubation of canine blood samples with calcitriol resulted in significant decreases in LPS-, LTA-, and MDP-stimulated leukocyte production of TNF but not IL10. Blunting of TNF production was concentration dependent. Leukocyte calcitriol exposure had no significant effect on phagocytosis and TLR4 expression. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in canine leukocytes exposed to LPS, LTA, and MDP in vitro, without altering phagocytosis or TLR4 expression. Thus, calcitriol could represent a novel candidate immunomodulatory treatment for dogs.

Effects of calcitriol on apoptosis, toll-like receptor 4 expression, and cytokine production of endotoxin-primed canine leukocytes.

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs. SAMPLE Blood samples from 6 healthy adult dogs. PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10-7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry. RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

Glucocorticoids, Sex Hormones, and Immunity.

Glucocorticoid hormones regulate essential body functions in mammals, control cell metabolism, growth, differentiation, and apoptosis. Importantly, they are potent suppressors of inflammation, and multiple immune-modulatory mechanisms involving leukocyte apoptosis, differentiation, and cytokine production have been described. Due to their potent anti-inflammatory and immune-suppressive activity, synthetic glucocorticoids (GCs) are the most prescribed drugs used for treatment of autoimmune and inflammatory diseases. It is long been noted that males and females exhibit differences in the prevalence in several autoimmune diseases (AD). This can be due to the role of sexual hormones in regulation of the immune responses, acting through their endogenous nuclear receptors to mediate gene expression and generate unique gender-specific cellular environments. Given the fact that GCs are the primary physiological anti-inflammatory hormones, and that sex hormones may also exert immune-modulatory functions, the link between GCs and sex hormones may exist. Understanding the nature of this possible crosstalk is important to unravel the reason of sexual disparity in AD and to carefully prescribe these drugs for the treatment of inflammatory diseases. In this review, we discuss similarities and differences between the effects of sex hormones and GCs on the immune system, to highlight possible axes of functional interaction.

Chikungunya Virus-Induced Arthritis: Role of Host and Viral Factors in the Pathogenesis.

Chikungunya virus (CHIKV), a member of Alphavirus genus, is responsible for chikungunya fever (CHIKF), which is characterized by the presence of fever, rash, myalgia, and arthralgia. Reemergence of CHIKV has become a significant public health concern in Asian and African countries and is newly emerging in the Middle East, Pacific, American, and European countries. Cytokines, innate (monocytes, natural killer cells) and adaptive immune response (role of B cells and T cells i.e. CD4+ and CD8+), and/or viral factors contribute to CHIKV-induced arthritis. Vector factors such as vector competence (that includes extrinsic and intrinsic factors) and effect of genome mutations on viral replication and fitness in mosquitoes are responsible for the spread of virus, although they are not directly responsible for CHIKV-induced arthritis. CHIKV-induced arthritis mimics arthritis by involving joints and a common pattern of leukocyte infiltrate, cytokine production, and complement activation. Successful establishment of CHIKV infection and induction of arthritis depends on its ability to manipulate host cellular processes or host factors. CHIKV-induced joint damage is due to host inflammatory response mediated by macrophages, T cells, and antibodies, as well as the possible persistence of the virus in hidden sites. This review provides insight into mechanisms of CHIKV-induced arthritis. Understanding the pathogenesis of CHIKV-induced arthritis will help in developing novel strategies to predict and prevent the disease in virus-infected subjects and combat the disease, thereby decreasing the worldwide burden of the disease.

Effects of parenteral fish oil on plasma nonesterified fatty acids and systemic inflammatory mediators in dogs following ovariohysterectomy.

To evaluate the effects of intravenous (IV) infusion of fish oil (FO) emulsion following ovariohysterectomy (OVH) on inflammatory mediators and plasma omega-3 nonesterified fatty acids (NEFA) concentrations in dogs. Prospective clinical study. University teaching hospital. Twenty-nine privately owned dogs undergoing routine OVH. Postoperative 3-hour IV infusion of saline (n = 9), FO (Omegaven, n = 10), or soybean oil (SO, intralipid, n = 10) emulsion and blood collected before, 5 and 24 hours following OVH for plasma NEFA and RBC membrane fatty acids (FAs) concentrations, leukocyte cytokine production capacity, and C-reactive protein (CRP) measurement. Plasma omega-3 NEFA, eicosapentaenoic acid, docosahexaenoic acid, and total long-chain omega-3 FA significantly increased shortly after FO infusion (8.8±3.3 μM, 13.6±5.6 μM, and 25.1±9.6 μM, respectively) compared to SO (0.7±0.9, 2.3±1.8, and 4.2±3.0 μM, respectively) and saline infusion (1.6±2.5, 2.6±3.1, and 5.9±6.4 μM, respectively). Plasma CRP concentration significantly increased after OVH, but with no significant group differences. A weak negative correlation occurred between post-OVH CRP and postinfusion total long-chain omega-3 FA concentrations (r2 = 0.21, P = 0.014). Stimulated leukocyte interleukin (IL) 6 production capacity increased (P = 0.001) after OVH in all groups; SO infusion resulted in reduced leukocyte IL-6 production capacity (1048.1±277.7 pg/mL) compared to FO (1299.9±302.1 pg/mL, P = 0.048) and saline infusions (1499.0±363.1 pg/mL, P = 0.01). No significant group difference was observed in leukocyte IL-10 and tumor necrosis factor α production capacities. Postoperative administration of FO emulsion increases plasma omega-3 NEFA concentrations promptly, but does not significantly attenuate CRP production or leukocyte cytokine production capacity. FO infusion at the dosage used in the present study can be safely used in dogs, but it was not clearly beneficial in decreasing post-OVH indices of inflammation.

Probucol attenuates lipopolysaccharide-induced leukocyte recruitment and inflammatory hyperalgesia: effect on NF-кB activation and cytokine production

Probucol 4,4′- (Isopropylidenedithio)bis(2,6-di-tert-butylphenol) is a synthetic molecule clinically used for prevention and treatment of hypercholesterolemia and atherosclerosis. Recent studies have shown that the beneficial effects of probucol mainly derive from its anti-inflammatory and antioxidant properties. Gram-negative bacteria are common infectious agents and their wall components, e.g. lipopolysaccharide (LPS), are important elicitors of inflammation. LPS is sensed by tissue resident cells and it triggers a Toll-like receptor 4/MyD88-dependent signaling cascade resulting in endothelial activation, leukocyte recruitment and nociception. Therefore the present study aimed to investigate the anti-inflammatory and analgesic effects of probucol in models of LPS-induced acute inflammation. Probucol at 0.3–30mg/kg was administrated to male Swiss mice per oral 1h before intraplantar or intraperitoneal lipopolysaccharide stimulus. Probucol at 3mg/kg reduced lipopolysaccharide-induced mechanical and thermal hyperalgesia. These effects were accompanied by reduced leukocyte influx and cytokine production in both paw skin and peritoneum exudate. Unexpectedly, probucol did not alter lipopolysaccharide-induced tissue oxidative stress at anti-inflammatory /analgesic dose. On the other hand, probucol inhibited lipopolysaccharide-induced nuclear factor kappa B (NF-кB) activation in paw tissue as well as NF-кB activity in cultured macrophages in vitro, reinforcing the inhibitory effect of probucol over the NF-кB signaling pathway. In this sense, we propose that probucol acts on resident immune cells, such as macrophages, targeting the NF-кB pathway. As a result, it prevents the amplification and persistence of the inflammatory response by attenuating NF-кB-dependent cytokine production and leukocyte recruitment explaining its analgesic effects as well.

Bilirubin Prevents Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor-Deficient Mice by Inhibiting Endothelial VCAM-1 and ICAM-1 Signaling.

BackgroundNumerous epidemiological studies support an inverse association between serum bilirubin levels and the incidence of cardiovascular disease; however, the mechanism(s) by which bilirubin may protect against atherosclerosis is undefined. The goals of the present investigations were to assess the ability of bilirubin to prevent atherosclerotic plaque formation in low‐density lipoprotein receptor‐deficient (Ldlr −/−) mice and elucidate the molecular processes underlying this effect.Methods and ResultsBilirubin, at physiological concentrations (≤20 μmol/L), dose‐dependently inhibits THP‐1 monocyte migration across tumor necrosis factor α–activated human umbilical vein endothelial cell monolayers without altering leukocyte binding or cytokine production. A potent antioxidant, bilirubin effectively blocks the generation of cellular reactive oxygen species induced by the cross‐linking of endothelial vascular cell adhesion molecule 1 (VCAM‐1) or intercellular adhesion molecule 1 (ICAM‐1). These findings were validated by treating cells with blocking antibodies or with specific inhibitors of VCAM‐1 and ICAM‐1 signaling. When administered to Ldlr −/− mice on a Western diet, bilirubin (30 mg/kg intraperitoneally) prevents atherosclerotic plaque formation, but does not alter circulating cholesterol or chemokine levels. Aortic roots from bilirubin‐treated animals exhibit reduced lipid and collagen deposition, decreased infiltration of monocytes and lymphocytes, fewer smooth muscle cells, and diminished levels of chlorotyrosine and nitrotyrosine, without changes in VCAM‐1 or ICAM‐1 expression.ConclusionsBilirubin suppresses atherosclerotic plaque formation in Ldlr −/− mice by disrupting endothelial VCAM‐1‐ and ICAM‐1‐mediated leukocyte migration through the scavenging of reactive oxygen species signaling intermediaries. These findings suggest a potential mechanism for the apparent cardioprotective effects of bilirubin.

Dectin-3 Is Not Required for Protection against Cryptococcus neoformans Infection.

C-type lectin receptors (CLRs) are diverse, trans-membrane proteins that function as pattern recognition receptors (PRRs) which are necessary for orchestrating immune responses against pathogens. CLRs have been shown to play a major role in recognition and protection against fungal pathogens. Dectin-3 (also known as MCL, Clecsf8, or Clec4d) is a myeloid cell-specific CLR that recognizes mycobacterial trehalose 6,6’-dimycolate (TDM) as well as α-mannans present in the cell wall of fungal pathogens. To date, a potential role for Dectin-3 in the mediation of protective immune responses against C. neoformans has yet to be determined. Consequently, we evaluated the impact of Dectin-3 deficiency on the development of protective immune responses against C. neoformans using an experimental murine model of pulmonary cryptococcosis. Dectin-3 deficiency did not lead to increased susceptibility of mice to experimental pulmonary C. neoformans infection. Also, no significant differences in pulmonary leukocyte recruitment and cytokine production were observed in Dectin-3 deficient mice compared to wild type infected mice. In addition, we observed no differences in uptake and anti-cryptococcal activity of Dectin-3 deficient dendritic cells and macrophages. Altogether, our studies show that Dectin-3 is dispensable for mediating protective immune responses against pulmonary C. neoformans infection.

A Mouse Model of Post-Stroke Pneumonia Induced by Intra-Tracheal Inoculation with Streptococcus pneumoniae

Background: Stroke-induced immunodeficiency increases the risk of infectious complications, which adversely affects neurological outcome. Among those, pneumonia affects as many as one third of stroke patients and is the main contributor to mortality in the post-acute phase of stroke. Experimental findings on post-stroke susceptibility to spontaneous pneumonia in mice are contradictory. Here, we established a mouse model inducing standardized bacterial pneumonia and characterized the impaired pulmonary cellular and humoral immune responses after experimental stroke. Methods: Bacterial pneumonia was induced by intra-tracheal inoculation with Streptococcus pneumoniae at different time points after transient middle cerebral artery occlusion (MCAO). Bacterial counts in lungs and blood, histological changes, and cytokine production in the lungs were assessed. Furthermore, we investigated the effect of pneumonia on stroke outcome. Results: Intra-tracheal inoculation resulted in reproducible pneumonia and bacteraemia, and demonstrated post-stroke susceptibility to streptococcal pneumonia developing with a delay of at least 24 h after MCAO. Higher bacterial counts in mice infected 3 days after stroke induction correlated with reduced neutrophil and macrophage infiltration in the lungs and lower levels of pro-inflammatory cytokines in the broncho-alveolar lavage compared to sham-operated animals. Pneumonia increased mortality without affecting brain-infiltrating leukocytes. Conclusions: In this standardized mouse model of post-stroke pneumonia, we describe attenuated leukocyte infiltration and cytokine production in response to bacterial infection in the lungs that has a profound effect on outcome.

Cold-inducible RNA-binding protein causes endothelial dysfunction via activation of Nlrp3 inflammasome.

Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern (DAMP) molecule which stimulates proinflammatory cytokine release in hemorrhage and sepsis. Under these medical conditions, disruption of endothelial homeostasis and barrier integrity, typically induced by proinflammatory cytokines, is an important factor contributing to morbidity and mortality. However, the role of CIRP in causing endothelial dysfunction has not been investigated. In this study, we show that intravenous injection of recombinant murine CIRP (rmCIRP) in C57BL/6 mice causes lung injury, evidenced by vascular leakage, edema, increased leukocyte infiltration and cytokine production in the lung tissue. The CIRP-induced lung damage is accompanied with endothelial cell (EC) activation marked by upregulation of cell-surface adhesion molecules E-selectin and ICAM-1. Using in vitro primary mouse lung vascular ECs (MLVECs), we demonstrate that rmCIRP treatment directly increases the ICAM-1 protein expression and activates NAD(P)H oxidase in MLVECs. Importantly, CIRP stimulates the assembly and activation of Nlrp3 inflammasome in MLVECs accompanied with caspase-1 activation, IL-1β release and induction of proinflammatory cell death pyroptosis. Finally, our study demonstrates CIRP-induced EC pyroptosis in the lungs of C57BL/6 mice for the first time. Taken together, the released CIRP in shock can directly activate ECs and induce EC pyroptosis to cause lung injury.

Local but Not Systemic Administration of Uridine Prevents Development of Antigen-Induced Arthritis.

ObjectiveUridine has earlier been show to down modulate inflammation in models of lung inflammation. The aim of this study was to evaluate the anti-inflammatory effect of uridine in arthritis.MethodsArthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA. Uridine was either administered locally by direct injection into the knee joint or systemically. Systemic treatment included repeated injections or implantation of a pellet continuously releasing uridine during the entire experimental procedure. Anti-mBSA specific immune responses were determined by ELISA and cell proliferation and serum cytokine levels were determined by Luminex. Immunohistochemistry was used to identify cells, study expression of cytokines and adhesion molecules in the joint.ResultsLocal administration of 25–100 mg/kg uridine at the time of arthritis onset clearly prevented development of joint inflammation. In contrast, systemic administration of uridine (max 1.5 mg uridine per day) did not prevent development of arthritis. Protection against arthritis by local administration of uridine did not affect the anti-mBSA specific immune response and did not prevent the rise in serum levels of pro-inflammatory cytokines associated with the triggering of arthritis. In contrast, local uridine treatment efficiently inhibited synovial expression of ICAM-1 and CD18, local cytokine production and recruitment of leukocytes to the synovium.ConclusionLocal, but not systemic administration of uridine efficiently prevented development of antigen-induced arthritis. The protective effect did not involve alteration of systemic immunity to mBSA but clearly involved inhibition of synovial expression of adhesion molecules, decreased TNF and IL-6 production and prevention of leukocyte extravasation. Further, uridine is a small, inexpensive molecule and may thus be a new therapeutic option to treat joint inflammation in RA.

The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18).

BackgroundOpioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.ResultsIn this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.ConclusionsTogether, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0027-0) contains supplementary material, which is available to authorized users.