Anita Nishiyama

Differential effects of short-chain fatty acids on proliferation and production of pro- and anti-inflammatory cytokines by cultured lymphocytes

Short-chain fatty acids (SCFA) are produced by fermentation of water-soluble fiber by anaerobic bacteria in the large bowel. Fiber-rich diets decrease the risk of developing inflammatory bowel disease (IBD) and butyrate enemas are effective as a therapy in some patients. Crohns disease, one form of IBD, appears to involve an exagerated T helper-1 (Th1) lymphocyte phenotype, characterised by production of interleukin (IL)-2 and interferon (IFN)-gamma, that drives the inflammation. To examine whether SCFA influence pro- and anti-inflammatory cytokine production, rat mesenteric lymph node lymphocytes were cultured in the presence of acetate (10 mM), butyrate (1.5 mM) or propionate (2 mM) and the production of cytokines in response to concanavalin A determined. Butyrate, but not acetate or propionate, inhibited lymphocyte proliferation and IL-2 production. Acetate and propionate were able to partly prevent the inhibitory effect of butyrate on IL-2 production. Acetate and propionate increased IFN-gamma production, whereas butyrate inhibited it. Acetate and propionate in combination were able to prevent the inhibitory effect of butyrate on IFN-gamma production. IL-4 was not detected in any cultures. Acetate and propionate increased IL-10 production, which was not affected by butyrate. It is concluded that butyrate significantly inhibits Th1-type responses and that this might explain the therapeutic effect of butyrate in IBD patients. Acetate and propionate have less marked modulatory actions, and in some cases have effects that oppose those of butyrate. A combination of the three SCFA causes a shift in the T helper lymphocyte phenotype towards a more anti-inflammatory phenotype and this might explain the protective effects of fiber.

Soy lecithin supplementation alters macrophage phagocytosis and lymphocyte response to concanavalin A: a study in alloxan‐induced diabetic rats

Dietary soy lecithin supplementation decreases hyperlipidemia and influences lipid metabolism. Although this product is used by diabetic patients, there are no data about the effect of soy lecithin supplementation on the immune system. The addition of phosphatidylcholine, the main component of lecithin, to a culture of lymphocytes has been reported to alter their function. If phosphatidylcholine changes lymphocyte functions in vitro as previously shown, then it could also affect immune cells in vivo. In the present study, the effect of dietary soy lecithin on macrophage phagocytic capacity and on lymphocyte number in response to concanavalin A (ConA) stimulation was investigated in non‐diabetic and alloxan‐induced diabetic rats. Supplementation was carried out daily with 2 g kg−1 b.w. lecithin during 7 days. After that, blood was drawn from fasting rats and peritoneal macrophages and mesenteric lymph node lymphocytes were collected to determine the phospholipid content. Plasma triacylglycerol (TAG), total and HDL cholesterol and glucose levels were also determined. Lymphocytes were stimulated by ConA. The MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) dye reduction method and flow cytometry were employed to evaluate lymphocyte metabolism and cell number, respectively. Soy lecithin supplementation significantly increased both macrophage phagocytic capacity (+29%) in non‐diabetic rats and the lymphocyte number in diabetic rats (+92%). It is unlikely that plasma lipid levels indirectly affect immune cells, since plasma cholesterol, TAG, or phospholipid content was not modified by lecithin supplementation. In conclusion, lymphocyte and macrophage function were altered by lecithin supplementation, indicating an immunomodulatory effect of phosphatidylcholine. Copyright

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 microM. The treatment of peritoneal macrophages with 64 microM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 +/- 16.3 vs 100.0 +/- 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 microM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 +/- 6.8 vs 100.0 +/- 5.5%, N = 15), while both 32 and 64 microM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 +/- 2.6 vs 19.4 +/- 2.5 microM) and 46.4% (10.4 +/- 3.1 vs 19.4 +/- 2.5 microM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 microM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Pulmonary inflammation due to exercise-induced pulmonary haemorrhage in Thoroughbred colts during race training

This study investigated the putative roles of inflammation and platelet-activating factor (PAF) in exercise-induced pulmonary haemorrhage (EIPH). Two-year-old Thoroughbred colts (n=37) were exercised on a racetrack for 5months before commencement of the study. Each colt was then exercised at 15-16m/s over 800-1000m and broncho-alveolar lavage fluid (BALF) was collected 24h later. The colts were subsequently divided into two groups on the basis of BALF analysis; an EIPH-positive group (presence of haemosiderophages, n=23) and an EIPH-negative group (absence of haemosiderophages, n=14). BALF from the EIPH-positive group had a significantly higher protein concentration (0.39±0.28 vs. 0.19±0.12mg/mL, P=0.031), higher PAF bioactivity (0.18±0.12 vs. 0.043±0.05 340:380nm ratio, P=0.042) and a higher lipid hydroperoxide concentration compared to the EIPH-negative group. There was also a lower nitrite concentration and reduced production of superoxide anion and hydrogen peroxide by alveolar macrophages in the EIPH-positive group. There was evidence of pulmonary inflammation and a decreased innate immune response of alveolar macrophages in EIPH-positive colts compared with the EIPH-negative group.

Platelet‐Activating Factor and Evidence of Oxidative Stress in the Bronchoalveolar Fluid of Thoroughbred Colts during Race Training

BACKGROUND Inflammatory airway disease (IAD) is prevalent in young racehorses during training, being the 2nd most commonly diagnosed ailment interrupting training of 2-year-old Thoroughbred racehorses. HYPOTHESIS That stabling and exercise cause oxidative stress, release of platelet-activating factor (PAF) and inflammation in airways of Thoroughbred colts. ANIMALS Colts in breeding farms (NC, n = 45), stabled for 30 days (EC, n = 40), and race trained (EX, n = 34). METHODS Cytological profile and parameters of bronchoalveolar lavage fluid (BALF) related to oxidative stress, bioactivity of the proinflammatory mediator PAF, catalase activity, and alveolar macrophage function. RESULTS Percentages of neutrophils and eosinophils in the BALF of the EX group were higher (5.4 +/- 6.4% versus 0.9 +/- 1.2%) than the upper limits for normal horses (3-5%). BALF from the EX group (45.6 +/- 2.8 cells/microL of BALF) also displayed significantly (P = .017) higher total nucleated cell count. PAF bioactivity and the total protein concentration in the BALF were higher in the EX group (0.0683 +/- 0.076 versus 0.0056 +/- 0.007 340 : 380 nm ratio P = .0039, 0.36 +/- 0.30 versus 0.14 +/- 0.15 mg of proteins/mL of BALF P < .001). Concentration of BALF hydroperoxides was higher in the EC group (104.7 +/- 80.0 versus 35.2 +/- 28.0 nmol/mg of proteins, P = .013) and catalase activity was higher in the EX group (0.24 +/- 0.16 versus 0.06 +/- 0.02 micromol H2O2/min/mg of proteins, P = .0021). Alveolar macrophage phagocytosis (P = .048) as well as production of superoxide anion (P = .0014) and hydrogen peroxide (P = .0011) were significantly lower in EX group. CONCLUSIONS AND CLINICAL IMPORTANCE Further studies should be performed to elucidate the role of PAF in the pathophysiology of IAD. Its presence in bronchoalveolar fluid of young athletic horses makes it a potential therapeutic target to be investigated.

Dietary supplementation with soybean lecithin increases pulmonary PAF bioactivity in asthmatic rats

The prevalence of asthma has risen over the last few decades, and some studies correlate this with the greater consumption of polyunsaturated fatty acids (PUFAs). Dietary PUFAs are known to increase the susceptibility of biological structures to lipid peroxidation, a process by which platelet-activating factor (PAF)-like lipids can be generated. These lipids functionally mimic the bioactivity of PAF, a potent proinflammatory mediator that exerts several deleterious effects on asthma. Thus, this work aimed to investigate if dietary supplementation with soybean lecithin (SL), a source of PUFAs, increases lipid peroxidation and PAF bioactivity in lungs of asthmatic Wistar rats. Animals were separated into groups: control, supplemented, asthmatic, asthmatic supplemented with SL (2 g/kg body weight), asthmatic supplemented with SL (2 g/kg body weight) and DL-alpha-tocopheryl acetate (100 mg/kg body weight). Asthmatic inflammation increased pulmonary lipid peroxidation, PAF bioactivity, alveolar-capillary barrier permeability and production of nitric oxide. In asthmatics, dietary supplementation with SL promoted an increase in pulmonary lipid peroxidation and PAF bioactivity, and an increase in the permeability of the alveolar-capillary barrier. Moreover, the treatment of asthmatic rats with DL-alpha-tocopheryl acetate inhibited the lipid peroxidation and decreased the PAF bioactivity. Therefore, the increase in pulmonary PAF bioactivity in asthmatic individuals elicited by the dietary supplementation with SL probably involves the generation of PAF-like lipids. This finding suggests that PAF-like lipids may account for the deleterious effects of dietary PUFAs on asthma.

PAF increases phagocytic capacity and superoxide anion production in equine alveolar macrophages and blood neutrophils.

Phagocytosis exerted by alveolar macrophages and neutrophils is crucial in the clearance of exogenous particles deposited in the airways. Therefore, substances that activate these phagocytes in the airways can exert important effects on the particle clearance rate. PAF, particularly, was proved to be a potent activator of several immune cells and was shown to be present in the equine lower airways in specific conditions, such as after exercise. The present study aimed to investigate if PAF is able to increase the phagocytic capacity and the production of superoxide anion in equine alveolar macrophage and blood neutrophils. The results show that PAF increased these parameters in both phagocytes even in concentrations as low as 0.1 and 1.0 nM. On that ground, the present work suggests that PAF is involved in the process of particle clearance in equine lower airways.

Intratracheal therapy with autologous bone marrow-derived mononuclear cells reduces airway inflammation in horses with recurrent airway obstruction.

This research evaluated the effects of bone marrow-derived mononuclear cells (BMMCs) on the inflammatory process in the equine recurrent airway obstruction (RAO). Eight horses in RAO clinical score were divided into cell therapy group (Gcel) treated with a single intratracheal dose of BMMCs, and dexamethasone group (Gdex) treated with 21days of oral dexamethasone. The horses were clinically revaluated on days 7 and 21, together with cytological evaluation of the BALF, and detection of inflammatory markers (interleukins [IL]-10, -4, and -17, and interferon γ and α). There were decreases in respiratory effort and clinical score on days 7 and 21(p<0.05) for both groups. The percentage of neutrophils decreased and macrophages increased on days 7 and 21 (p<0.005) in both groups. IL-10 levels increased in the Gcel group on day 21 compared to days 0 and 7 (p<0.05), but this was not observed in the Gdex group. The quantification of IL-4, IL-17, IFN-γ, and IFN-α did not change between evaluations in both groups. These preliminary results suggest that BMMCs may ameliorate the inflammatory response of RAO.

Natural Killer Cell Assessment in Peripheral Circulation and Bronchoalveolar Lavage Fluid of Patients with Severe Sepsis: A Case Control Study

Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-γ). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56bright or dim) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis.