Joshua J. Malago

Anti-inflammatory properties of probiotic bacteria on Salmonella-induced IL-8 synthesis in enterocyte-like Caco-2 cells

Invasion of the gut by pathogenic Salmonella leads to production of IL-8 that initiates inflammatory reactions to combat the bacterium. However, its persistent production causes tissue damage and interventions that suppress IL-8 production prevent tissue damage. We hypothesised that probiotics could mediate their benefits via inhibition of IL-8 synthesis. Caco-2 cells were infected with probiotic Bifidobacterium infantis W52, Lactobacillus casei W56, Lactococcus lactis W58, Lactobacillus acidophilus W70, Bifidobacterium bifidum W23, or Lactobacillus salivarius W24 or pathogenic Salmonella enterica serovar Enteritidis 857 at 0, 0.2, 1, 2, 10, 20, 100 or 200 bacterial cells/Caco-2 cell for 1 hour. Cells were also exposed to a combination of one probiotic bacterium (200 bacterial cells/Caco-2 cell) and the graded numbers of Salmonella as either co-incubation (1 hour) or pre-incubation of the probiotic bacterium (1 hour) followed by Salmonella (1 hour). The cells recovered for 2 or 24 hours. IL-8 and Hsp70 were determined by ELISA and Western blot respectively. Both probiotics and Salmonella induced a dose- and time-dependent synthesis of IL-8 but probiotics induced far lower IL-8 levels than Salmonella. The Salmonella-induced IL-8 was significantly suppressed by B. infantis W52, L. casei W56 and L. lactis W58 at low numbers of Salmonella (0.2 to 20 bacterial cells/Caco-2 cell) and within 2 hours of recovery. The observed probiotic-mediated reduction in IL-8 secretion was transient, and lost after a few hours. In addition, these three probiotics induced a significant increase in Hsp70 expression while L. acidophilus W70, B. bifidum W23 and L. salivarius W24 induced a weak Hsp70 expression and could not suppress the Salmonella-induced IL-8 synthesis. We conclude that suppression of Salmonella-induced IL-8 synthesis by Caco-2 cells is exhibited by probiotics that induce expression of Hsp70, suggesting that the protective role of probiotics could be mediated, at least in part, via Hsp70 expression. This suppression is limited to a low number of infecting pathogenic Salmonella.

Microbial Products from Probiotic Bacteria Inhibit Salmonella enteritidis 857-Induced IL-8 Synthesis in Caco-2 Cells

Oral administration of Lactobacillus spp. as probiotics is gaining importance in the treatment of intestinal inflammations. However, their mechanism of action is unknown. We investigated whether nonspecific binding Lactobacillus casei Shirota (LcS) and mannose-specific Lactobacillus plantarum 299v (Lp) and their spent culture supernatant (SCS) affect Salmonella enteritidis 857 (Se) growth, IL-8 and Hsp70 syntheses. In one set of experiments human enterocyte-like Caco-2 cells were infected with LcS, Lp or Se at 1–500 bacteria per cell for 1 h. In another set, cells were exposed to Se (0–200 per cell, 1 h) after exposure to lactobacilli (LB) (500 per cell, 30 min) or by co-incubation of Se and LB (1 h). The third set of experiments involved exposure of cells for 1 h to SCS or Se (100 per cell) pretreated (1 h) in SCS. The effect of LB SCS on Se growth was evaluated by agar plate diffusion test. IL-8 and Hsp70 were assessed over 2–24 h using ELISA and Western blotting, respectively. Neither LcS nor Lp affected the Se growth and IL-8 production. In addition, they did not induce Hsp70 expression by Caco-2 cells. Instead, their SCS inhibited the Se growth and IL-8 production and induced the expression of Hsp70 by both crypt- and villus-like cells. The beneficial effect of Lactobacillus spp. to the intestinal inflammations might be associated with a decrease in IL-8 levels. This effect could be mediated, at least in part, via a secreted antimicrobial product(s) either directly against the pathogens or indirectly through the synthesis of Hsp70.

Sodium arsenite reduces severity of dextran sulfate sodium-induced ulcerative colitis in rats

The histopathological features and the associated clinical findings of ulcerative colitis (UC) are due to persistent inflammatory response in the colon mucosa. Interventions that suppress this response benefit UC patients. We tested whether sodium arsenite (SA) benefits rats with dextran sulfate sodium (DSS)-colitis. The DSS-colitis was induced by 5% DSS in drinking water. SA (10 mg/kg; intraperitoneally) was given 8 h before DSS treatment and then every 48 h for 3 cycles of 7, 14 or 21 d. At the end of each cycle rats were sacrificed and colon sections processed for histological examination. DSS induced diarrhea, loose stools, hemoccult positive stools, gross bleeding, loss of body weight, loss of epithelium, crypt damage, depletion of goblet cells and infiltration of inflammatory cells. The severity of these changes increased in the order of Cycles 1, 2 and 3. Treatment of rats with SA significantly reduced this severity and improved the weight gain.

Intraperitoneal administration of butyrate prevents the severity of acetic acid colitis in rats

Intrarectal infusion of butyrate improves colorectal disorders including ulcerative colitis (UC). However, it is not established whether systemically administered butyrate benefits such patients. The current study aimed at exploring and comparing the potential of intraperitoneally, intrarectally, and orally administered butyrate against acetic acid (AA)-induced UC in rats. Intrarectal administration of 2 ml of 50% AA was done after or without prior treatment of rats for 7 consecutive days with 100 mg/kg sodium butyrate (SB) intraperitoneally, intrarecfally, or orally. Rats were sacrificed after 48 h of AA-treatment. Subsequently, colon sections were processed routinely for histopathological examination. We clinically observed diarrhea, loose stools, and hemoccult-positive stools, and histologically, epithelial loss and ulceration, crypt damage, goblet cell depletion, hemorrhage, and mucosal infiltration of inflammatory cells. The changes were significantly reduced by intraperitoneal, intrarectal, or oral butyrate, with intraperitoneal butyrate exhibiting the highest potency. It is concluded that intraperitoneal administration of butyrate abrogates the lesions of AA-induced UC and its potency surpasses that of intrarectal or oral butyrate.摘要目的探索腹腔注射丁酸盐对防止乙酸性结肠炎的疗效。创新点首次对大鼠进行腹腔注射丁酸盐, 通过与直肠灌注和口服比较, 探索三种不同给药方式对预防乙酸性结肠炎的疗效差异。方法以40只Wistar大鼠为实验对象, 分组进行连续7天的腹腔注射、 直肠灌注和口服100 mg/kg丁酸钠(SB), 第8天进行乙酸(AA)直肠灌注, 48小时后处死。 记录实验大鼠的临床症状, 包括体重减少、 腹泻、 便血等。 对结肠切片进行组织病理学观察, 最后对试验数据进行统计分析。结论腹腔注射、 直肠灌注和口服丁酸盐均能明显缓解大鼠乙酸性结肠炎的炎症, 其中以腹腔注射疗效最佳。

Contribution of microbiota to the intestinal physicochemical barrier.

The large number of intestinal microorganisms, which exceeds the total number of human cells by ten folds, alludes to a significant contribution to human health. This is vivid in enteric and some systemic diseases emanating from disruption of the microbiota. As life style keeps shifting towards disruption of the microbiota in most societies worldwide, interest in the contribution of the microbiota to gut health has grown enormously. Many studies have been conducted to elucidate the exact contribution of the microbiota to human health. The knowledge gained from these studies indicates that the microbiota interacts with the intestinal milieu to maintain gut health. In this review, the crosstalk of microbiota with the intestinal physicochemical barrier pivotal to the gut innate immunity is highlighted. In particular, the review focuses on the role of the microbiota on competitive exclusion of pathogens, intestinal pH, epithelial mechanical barrier integrity, apical actin cytoskeleton, antimicrobial peptides, and the mucus layer. Understanding this microbe-host relationship will provide useful insight into overcoming some diseases related to the disruption of the host microbiota.

Probiotic-Pathogen Interactions and Enteric Cytoprotection

The intestinal epithelium forms a physicochemical barrier that impedes enteric pathogens from invading the epithelium and cause disease. In order for a particular pathogen to colonise the intestinal mucosa, it needs to break and cross this barrier. The barrier consists of a low pH area mainly resulting from carbohydrate fermentation, a mucus layer along the epithelial surface, an epithelial mechanical barrier maintained by intercellular tight junctions, an apical actin cytoskeleton, and the presence of stable microbiota. In addition, this barrier produces inflammatory mediators, mainly cytokines.

Factors Causing Disturbances of the Gut Microbiota

The gut microbiota is part and parcel of the innate immunity that protects the host against enteric pathogens. Soon after birth, its composition starts to establish in the infant depending upon the method of delivery and the environment. Factors like nutrition, antimicrobial usage, age, changes in intestinal motility, behavioural changes, and several others, subsequently affect the individual’s microbiotal composition throughout life. The effects are beneficial when the microbiotal population is dominated by protective bacteria, particularly Bifidobacteria and Lactobacillus and to a lesser extent, some Bacteroides species while the numbers of potentially pathogenic organisms like Clostridium species and Escherichia coli are low or undetectable. Noticeable effects that are of clinical significance are due to reduced number of beneficial bacteria with or without subsequent overgrowth of potentially pathogenic microbiota species and alterations in secreted proteins or metabolites and fermentation products. Since large numbers of particular beneficial bacteria and or their fermentation products are pivotal to host immunity, their disturbances often lead to enteric disorders such as diarrhoea and chronic inflammatory bowel disease. In this chapter the key factors that disturb or affect the gut microbiota and their potential in development of enteric disorders are discussed.

Modulation of Immune System by Probiotics to Protect Against Enteric Disorders

Emerging diseases of the immune system are increasing with socio-economic and sanitary conditions. This is evidenced by the high incidences of allergic and autoimmune diseases in Western countries than in developing countries. Evidence is accumulating that disruption of normal microbiota and their associated beneficial effects on intestinal mucosal immunity is pivotal to occurrence of these disorders. Their pathogeneses appear to revolve around impaired ability of the intestinal microbiota to effectively modulate and thus balance the body immune responses. As a result the immune system reacts excessively to antigens that would otherwise be tolerated and thus result in allergic reactions, or reacts to self antigens to cause autoimmune diseases. Some of these disorders are characterised by persistent production of inflammatory mediators leading to chronic inflammation and tissue damage. Several reports have indicated that oral administration of excessive numbers of selected members of microbiota, so called probiotic bacteria, prevents, treats, and improves the conditions of patients with immune-mediated disorders like allergy, inflammatory bowel diseases, and type 1 diabetes. Current knowledge suggests that probiotic bacteria mediate their beneficial effects via modulation of the immune system to balance its responses and institute protection. Herein the current knowledge on the pathogenesis of some of the immune mediated diseases and the beneficial effect of probiotic bacteria to such patients are described. The chapter also explores the role of probiotic bacteria in modulating the immune system and confer protection to the host.