Dietary administration of a commercial mixed-species probiotic improves growth performance and modulates the intestinal immunity of tilapia, Oreochromis niloticus.

The growth performance, immunological status, intestinal morphology and microbiology of tilapia, Oreochromis niloticus, were investigated after dietary administration of the commercial probiotic AquaStar(®) Growout. Tilapia (29.02 ± 0.33 g) were split into five treatments; control (CON), 1.5 g kg(-1) probiotic (PRO-1.5), 3 g kg(-1) probiotic (PRO-3), pulsed probiotic feeding (PRO-PULSE) or an initial probiotic feed followed by control feeding (PRO-INI). After six weeks of experimental feeding, fish fed PRO-3 displayed significantly higher final weight, weight gain and SGR compared to the CON or PRO-INI treatments. Supplementation of the probiotic at this dose induced an up-regulation of intestinal caspase-3, PCNA and HSP70 mRNA levels compared to the CON fed fish. Immuno-modulatory pathways were also affected; significantly higher expression of TLR2, pro-inflammatory genes TNFα and IL-1ß, and anti-inflammatory genes IL-10 and TGFß suggest that the probiotic may potentiate a higher state of mucosal tolerance and immuno-readiness. Histological appraisal revealed significantly higher numbers of intraepithelial leucocytes in the intestine of PRO-3 fed fish compared with treatments CON, PRO-PULSE and PRO-INI but not PRO-1.5. Additionally, fish receiving PRO-3 had a significantly higher abundance of goblet cells in their mid-intestine when compared with fish from all other treatments. Together, these data suggest that continuous provision of AquaStar(®) Growout at 3 g kg(-1) can improve tilapia growth and elevate the intestinal immunological status of the host.

Heat-killed probiotic bacteria differentially regulate colonic epithelial cell production of human ß-defensin-2: dependence on inflammatory cytokines.

The inducible antimicrobial peptide human ß-defensin-2 (hBD-2) stimulated by pro-inflammatory cytokines and bacterial products is essential to antipathogen responses of gut epithelial cells. Commensal and probiotic bacteria can augment such mucosal defences. Probiotic use in the treatment of inflammatory bowel disease, however, may have adverse effects, boosting inflammatory responses. The aim of this investigation was to determine the effect of selected probiotic strains on hBD-2 production by epithelial cells induced by pathologically relevant pro-inflammatory cytokines and the role of cytokine modulators in controlling hBD-2. Caco-2 colonic intestinal epithelial cells were pre-incubated with heat-killed probiotics, i.e. Lactobacillus casei strain Shirota (LcS) or Lactobacillus fermentum strain MS15 (LF), followed by stimulation of hBD-2 by interleukin (IL)-1ß and tumour necrosis factor alpha (TNF-α) in the absence or presence of exogenous IL-10 or anti-IL-10 neutralising antibody. Cytokines and hBD-2 mRNA and protein were analysed by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. LcS augmented IL-1ß-induced hBD-2, whereas LF enhanced TNF-α- and suppressed IL-1ß-induced hBD-2. LF enhanced TNF-α-induced TNF-α and suppressed IL-10, whereas augmented IL-1ß-induced IL-10. LcS upregulated IL-1ß-induced TNF-α mRNA and suppressed IL-10. Endogenous IL-10 differentially regulated hBD-2; neutralisation of IL-10 augmented TNF-α- and suppressed IL-1ß-induced hBD-2. Exogenous IL-10, however, suppressed both TNF-α- and IL-1ß-induced hBD-2; LcS partially rescued suppression in TNF-α- and IL-1ß-stimulation, whereas LF further suppressed IL-1ß-induced hBD-2. It can be concluded that probiotic strains differentially regulate hBD-2 mRNA expression and protein secretion, modulation being dictated by inflammatory stimulus and resulting cytokine environment.

Effects of dietary ß-(1,3)(1,6)-D-glucan supplementation on growth performance, intestinal morphology and haemato-immunological profile of mirror carp (Cyprinus carpio L.).

In recent years, aquaculture research has focused on probiotics, prebiotics, and ß-glucans, in order to improve health status and growth performance. Information regarding the effects of ß-glucan on growth performance and intestinal immunity of mirror carp (Cyprinus carpio L.) is scarce. An experiment was therefore conducted to investigate the effects of a yeast ß-glucan preparation (MacroGard(®) ) on growth performance, intestinal morphology and haemato-immunological indices of mirror carp. Carp (initial weight 11.1 ± 0.0 g) were fed highly purified diets supplemented with 0% (control), 0.1%, 1% or 2% MacroGard(®) for 8 weeks. Fish fed diets containing 1% and 2% MacroGard(®) showed significant improvements in weight gain, specific growth rate and feed conversion ratio compared to fish fed both the control and the 0.1% MacroGard(®) containing diet. Histological appraisal of the intestine showed a significantly higher infiltration of leucocytes into the epithelial layer of fish fed diets supplemented with 1% and 2% MacroGard(®) in the anterior intestine compared to fish fed the control and 0.1% MacroGard(®) diet. This effect was not observed in the posterior intestine. There were no significant differences in the intestinal absorptive surface area and number of goblet cells in either intestinal region. At the end of the experiment, the haematological status of the fish was examined. Compared to control fed fish, the haematocrit value was significantly elevated in fish fed the 2% MacroGard(®) diet. Furthermore, the blood monocyte fraction was significantly higher in fish fed the 1% and 2% MacroGard(®) diets. No significant changes were observed in the other blood parameters assessed. The present study shows that high dietary ß-glucan inclusion increases growth performance without detrimental effects on the health indicators assessed. Increased intraepithelial leucocytes in the anterior intestine may indicate a localized immune response; no detrimental effects on intestinal morphology were observed.

Probiotic Pediococcus acidilactici modulates both localised intestinal- and peripheral-immunity in tilapia (Oreochromis niloticus).

The application of probiotics in aquaculture has received concerted research efforts but the localised intestinal immunological response of fish to probiotic bacteria is poorly understood. Therefore, a study was conducted to evaluate the probiotic effect of Pediococcus acidilactici on Nile tilapia (Oreochromis niloticus) with specific emphasis on intestinal health and probiotic levels as well as system level responses such as growth performance, feed utilization and haemato-immunological parameters under non-challenged conditions. Fish (9.19 ± 0.04 g) were fed either a control diet or a P. acidilactici supplemented diet (at 2.81 × 10(6) CFU g(-)(1)) for six weeks. At the end of the study the probiotic was observed to populate the intestine, accounting for ca. 3% (1.59 × 10(5) CFU g(-)(1)) of the cultivable intestinal bacterial load. Real-time PCR indicated that the probiotic treatment may potentiate the immune-responsiveness of the intestine as up-regulation of the gene expression of the pro-inflammatory cytokine TNFα was observed in the probiotic fed fish (P < 0.05). Light microscopy observations revealed elevated intraepithelial leucocyte (IEL) levels in the intestine of P. acidilactici fed tilapia after six weeks (P < 0.05) of feeding and a trend towards elevated goblet cells was also observed after six weeks feeding (P = 0.08). Concomitantly at week six, along with elevated IELs and elevated TNFα mRNA levels in the intestine, an increased abundance of circulating neutrophils and monocytes were observed in fish fed the probiotic supplemented diet (P < 0.05). This haemopoietic expansion of innate immune cells could be reflective of an elevated state of immuno-readiness. Together these results suggest that the probiotic has a protective action on the intestinal mucosal cells, stimulating the innate immune response after feeding for a period of six weeks. These immunological modulations did not impair growth performance or the remaining haematological and zootechnical parameters compared to the control group (P > 0.05).

Probiotic bacterial strains differentially modulate macrophage cytokine production in a strain-dependent and cell subset-specific manner.

Gut mucosal macrophages play a pivotal role in driving mucosal immune responses, resulting in either activation of inflammatory immune responses to pathogenic challenge or tolerance to beneficial luminal contents such as food and commensal bacteria. Macrophage responses elicited are dependent on tissue environment and the resulting cell subset, where homeostatic macrophages resemble the M2 macrophage subset and inflammatory macrophages resemble M1s. Probiotics can modulate macrophage function with outcome dependent on subset present. Using a THP-1 monocyte cell line-derived model of CD14high/low M1 and M2 macrophages, the aim of this study was to investigate the immunomodulatory effects of a panel of heat-killed probiotic bacteria and their secreted proteins on the subset-specific inflammatory marker profile of TNFα, IL-6 and NFκB. M1 and M2 cells were generated by differentiation of monocyte stable transfectants for high and low CD14 expression with phorbol 12-myristate 13-acetate and vitamin D3, respectively, where the resulting CD14lo M2 and CD14hi M1s mimicked homeostatic and inflammatory mucosal macrophages. Subsets were stimulated by enteropathic lipopolysaccharides in the presence or absence of heat-killed (HK) or secreted proteins (SP) from a panel of probiotic bacteria. Regulation of cytokine expression was measured by ELISA and NFκB activity by reporter assay. HK probiotics suppress CD14lo and augment CD14hi M1 and M2 production of TNFα whereas SPs augmented CD14hi M1 TNFα and were generally suppressive in the other subtypes. M2 macrophage IL-6 production was suppressed by both HK and SPs and differentially regulated in CD14lo and CD14hi M1s. NFκB activation failed to parallel the regulatory profiles for TNFα and IL-6 which is suggestive of probiotic bacteria exerting their regulatory effects on these cytokines in an NFκB-independent manner. In conclusion, HK and SP probiotics differentially regulate macrophage cytokines and NFκB activation in a subset-dependent manner and suggest a cautionary approach to probiotic treatment of mucosal inflammation.

The importance of T cell interactions with macrophages in rheumatoid cytokine production.

The analysis of suppression of cytokines in rheumatoid synovial tissue and fluid pioneered the studies of human cytokines in diseased tissue due to the relative ease of staining samples, even at the height of the inflammatory process. These studies led to the study of synovial cytokine regulation, and the identification of TNF as a therapeutic target, which has been amply validated in clinical trials and now routine therapy. The next key question was how is TNF disregulated in synovium. Are there differences between the mechanisms of synovial TNF production compared to the production of protective TNF during an immune response? Are there differences between the induction of the pro-inflammatory TNF and the anti inflammatory IL-10? The analysis of the interaction of the two most abundant synovial cells, T lymphocytes and macrophages has provided interesting clues to new therapeutic approaches based on disrupting T-macrophage interaction.

CD40 ligation induces macrophage IL-10 and TNF-alpha production: differential use of the PI3K and p42/44 MAPK-pathways.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine produced in the rheumatoid arthritis (RA) joint by macrophages/monocytes and infiltrating peripheral blood derived lymphocytes. Recent data suggest a role for physical cell-to-cell interactions in the production of IL-10. In this report, we have investigated the signalling mechanisms involved in IL-10 production by peripheral blood-derived macrophages upon interaction with fixed CD40L transfectants. IL-10 and tumour necrosis factor alpha (TNF-alpha) are produced by macrophage colony-stimulating factor (M-CSF)-primed monocytes/macrophages in response to CD40 ligation. The utilization of the inhibitors, wortmannin and LY294002, demonstrated a role for phosphatidylinositol 3-kinase (PI3K) whereas rapamycin demonstrated p70 S6-kinase (p70S6K) involvement in the production of IL-10 by these monocytes. The production of TNF-alpha was enhanced by wortmannin and LY294002, suggesting negative regulation by PI3K; however, it was dependent on p70S6K suggesting a PI3K-independent mechanism of p70S6K activation. One alternative pathway that activates p70S6K independently of PI3K and also differentiates between IL-10 and TNF-alpha is the p42/44 mitogen-activated protein kinase (MAPK), which regulates TNF-alpha production in a PI3K-independent manner. These observations suggest that CD40 ligation induces macrophage IL-10 and TNF-alpha production, the mechanism of which is p70S6K-dependent yet bifurcates at the level of PI3K and p42/44 MAPK.

Route of monocyte differentiation determines their cytokine production profile: CD40 ligation induces interleukin 10 expression.

Interleukin 10 is a potent anti-inflammatory and immunomodulatory cytokine. Little is known regarding its induction in monocytes/macrophages, however LPS, a reproducible trigger of IL-10, is augmented by direct contact with T cells. In this context, the role of CD40-ligation is investigated. In the rheumatoid synovium, IL-10 is produced by tissue macrophages. Monocytes primed with M-CSF, a cytokine present in rheumatoid joints, produced IL-1beta, TNF-alpha and IL-10 upon CD40-ligation at an IL-1: TNF-alpha: IL-10 ratio of 10:0.5:1. IFN-gamma-primed monocytes, however, predominantly produced TNF-alpha and IL-1beta. Both differentiated monocytes display an endogenous IL-10 activity regulatable by CD40 stimulation. Additionally, these monocytes display differential control by exogenous and endogenous IL-1 and TNF-alpha. M-CSF-primed monocyte IL-10 production was dependent on endogenous TNF-alpha and, to a lesser extent, IL-1, whereas IFN-gamma-primed monocytes were partially dependent on endogenous IL-1. The addition of exogenous IL-1 augments CD40 induced IL-10 production by IFN-gamma-primed monocytes. These data indicate that CD40 ligation regulates cell contact mediated macrophage IL-10 and that the route of differentiation determines the cytokine profile.

Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases.

IL-10 is an anti-inflammatory cytokine with potent immunomodulatory effects, including inhibition of cytokine production. However, regulation of monocyte IL-10 production is poorly understood. In this report we have investigated the mechanisms of LPS-induced IL-10 production by human peripheral blood monocytes and demonstrate that IL-10 synthesis is uniquely dependent on the endogenous proinflammatory cytokines IL-1 and/or TNF-alpha. LPS signal transduction in monocytes has been shown to involve activation of the p38 and p42 mitogen-activated protein kinase (MAPK) cascades. The results in this paper indicate that inhibition of p38 MAPK potently inhibited the production of IL-10, IL-1beta, and TNF-alpha, whereas blockade of the p42/44 MAPK pathway, while partially inhibiting TNF-alpha and IL-1beta production, had no effect on monocyte secretion of IL-10. Furthermore, neither the inhibition of monocyte TNF-alpha induced by IL-10 nor the stimulation of soluble TNF receptor production was affected by inhibition of the p42/44 MAPK pathway, suggesting that this signaling event is not involved in either monocyte production of or anti-inflammatory responses to IL-10. These data raise the interesting possibility that proinflammatory TNF-alpha-mediated effects may be selectively blocked without modulating the induction or the response to IL-10, whereas the signaling events associated with the anti-inflammatory events induced by IL-10 remain to be elucidated.

Modulation of cytokine production by human mononuclear cells following impairment of Na, K-ATPase activity.

Cytokines, including TNF alpha and IL-l beta, are central to the chronic inflammatory process and tissue damage that characterises diseases such as rheumatoid arthritis. The mechanisms responsible for long-term generation of these molecules are poorly understood. We have previously demonstrated impaired activity of Na, K-ATPase, a key enzyme regulating intracellular cation levels, on rheumatoid mononuclear cells. Mimicking this 'defect' on normal mononuclear cells with ouabain has been shown to induce TNF alpha and, in particular, IL-l beta production, whereas IL-6 synthesis was suppressed. A similar pattern of cytokine generation was noted when mononuclear cells were treated with the sodium ionophore, monensin. Induction of cytokine production was related to up-regulation of the appropriate mRNA, although enhanced secretion of processed IL-l beta was also observed. The mechanism underlying these cellular responses appears to involve sodium/calcium exchange across the cell membrane. Impaired Na,K-ATPase activity might promote pro-inflammatory cytokine secretion in patients with rheumatoid arthritis.

Impaired activity of thiol-dependent ATPases in rheumatoid mononuclear cell membranes.

Ion-motive ATPase play an essential role in many aspects of cell biology, including mononuclear cell (MNC) functions relevant to chronic inflammation. For example, ouabain, a specific inhibitor of Na+, K+ ATPase, suppresses both T and B cell proliferation but induces synthesis of IL-1. Using a cytochemical assay quantified by microdensitometry, total and ouabain-sensitive ATPase activities have been compared in MNC from rheumatoid and control subjects. The sensitivity of these enzymes to inactivation by thiol-blocking reagents has been studied by preincubation with an impermeant SH blocker p-hydroxymercuriphenylsulphonate (pHMPSA). The results show that rheumatoid MNC have significantly impaired ATPase activity compared to healthy cells and that both total and ouabain-sensitive ATPase activities are readily inhibited by pHMPSA. The depressed ATPase activity in rheumatoid MNC could thus be due to blockade/oxidation of a reactive surface thiol, and could contribute to perpetuation of the chronic inflammatory process in these patients.