Andrew D. Foey

Probiotics, Prebiotics and Immunomodulation of Gut Mucosal Defences: Homeostasis and Immunopathology

Probiotics are beneficial microbes that confer a realistic health benefit on the host, which in combination with prebiotics, (indigestible dietary fibre/carbohydrate), also confer a health benefit on the host via products resulting from anaerobic fermentation. There is a growing body of evidence documenting the immune-modulatory ability of probiotic bacteria, it is therefore reasonable to suggest that this is potentiated via a combination of prebiotics and probiotics as a symbiotic mix. The need for probiotic formulations has been appreciated for the health benefits in “topping up your good bacteria” or indeed in an attempt to normalise the dysbiotic microbiota associated with immunopathology. This review will focus on the immunomodulatory role of probiotics and prebiotics on the cells, molecules and immune responses in the gut mucosae, from epithelial barrier to priming of adaptive responses by antigen presenting cells: immune fate decision—tolerance or activation? Modulation of normal homeostatic mechanisms, coupled with findings from probiotic and prebiotic delivery in pathological studies, will highlight the role for these xenobiotics in dysbiosis associated with immunopathology in the context of inflammatory bowel disease, colorectal cancer and hypersensitivity.

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).

Impact of VIP and cAMP on the regulation of TNF-α and IL-10 production: implications for rheumatoid arthritis

Vasoactive intestinal peptide (VIP) is an anti-inflammatory immunomodulatory neuropeptide with therapeutic potential demonstrated for collagen-induced arthritis. The aim of this study was to characterise its potential anti-arthritic effect on human monocytes, macrophages, T cells, and rheumatoid arthritis synovial membrane cells. Monocytes, macrophages, and T cells derived from human peripheral blood were treated with VIP and compared with other cAMP-elevating drugs for a range of activating stimuli. Cytokine production was assessed for cell cultures and, in addition, the ability of VIPs to activate cAMP response element binding protein. VIP partially suppressed monocyte- and macrophage-derived tumour necrosis factor α (TNF-α) with no effect on IL-10, whereas VIP fails to regulate IL-10 and TNF-α production by T lymphocytes. No such modulation of cytokine profile was observed for rheumatoid arthritis synovial membrane cells. Elevation of intracellular cAMP, on the other hand, potently suppressed macrophage TNF-α production and modulated T-cell response by inhibiting TNF-α and IFN-γ. VIPs lack of effect on IL-10 and its slight effect on TNF-α results from cAMP being rapidly degraded as the phosphodiesterase IV inhibitor, rolipram, rescues cAMP-dependent activation of cAMP response element binding protein. Interestingly, macrophages stimulated with phorbol 12-myristate 13-acetate/ionomycin displayed an augmented IL-10 response upon addition of dibutyryl cAMP, with corresponding downregulation in TNF-α, suggesting a complex interaction between protein kinase C and protein kinase A in cytokine regulation. In conclusion, VIP may represent an efficaceous anti-arthritic treatment modulating macrophage and T-cell cytokine profiles when used alongside a phosphodiesterase inhibitor.

Resting CD4+effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

BackgroundPreviously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.MethodsHere, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood.ResultsAfter stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.ConclusionTaken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

Effects of dietary β-(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 β-glucans, in order to improve health status and growth performance. Information regarding the effects of β-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 β-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 β-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.

Conventional protein kinase C and atypical protein kinase Cζ differentially regulate macrophage production of tumour necrosis factor-α and interleukin-10

In chronic inflammatory diseases such as rheumatoid arthritis, joint macrophages/monocytes are the major source of pro‐ and anti‐inflammatory cytokines. Little is understood regarding the signalling pathways which determine the production of the pro‐inflammatory cytokine, tumour necrosis factor‐α (TNF‐α) and the anti‐inflammatory cytokine, interleukin‐10 (IL‐10). Two pathways integral to macrophage function are the protein kinase C (PKC)‐ and the cAMP‐dependent pathways. In this report, we have investigated the involvement of PKC and cAMP in the production of TNF‐α and IL‐10 by peripheral blood monocyte‐derived macrophages. The utilization of the PKC inhibitors Go6983, Go6976 and RO‐32‐0432 demonstrated a role for conventional PKCs (α and β) in the production of TNF‐α in response to stimulation by lipopolysaccharide and phorbol 12‐myristate 13‐acetate (PMA)/ionomycin. PKC stimulation resulted in the downstream activation of the p42/44 mitogen‐activated protein kinase (MAPK) pathway which differentially regulates TNF‐α and IL‐10. The addition of cAMP however, suppressed activation of this MAPK and TNF‐α production. Cyclic‐AMP augmented IL‐10 production and cAMP response element binding protein activation upon stimulation by PMA/ionomycin. In addition, cAMP activated PKCζ; inhibition of which, by a dominant negative adenovirus construct, selectively suppressed IL‐10 production. These observations suggest that pro‐inflammatory and anti‐inflammatory cytokines are differentially regulated by PKC isoforms; TNF‐α being dependent on conventional PKCs (α and β) whereas IL‐10 is regulated by the cAMP‐regulated atypical PKCζ.

Cytokine regulation in RA synovial tissue: role of T cell/macrophage contact-dependent interactions.

Chapter summary Several groups have documented the expression of cytokines in rheumatoid arthritis synovial tissue over the past 15 years or so. These studies have indicated that most cytokines examined are expressed at the mRNA levels at least, and many other cytokines are found in abundance as proteins. Our attention has recently focused on the mechanisms that induce and regulate tumour necrosis factor and IL-10. Other workers and ourselves have found that cell–cell contact is an important signal for the induction of cytokines, and our work has demonstrated that tumour necrosis factor and IL-10 production in rheumatoid arthritis synovial joint cells cultures is dependent on T cell/macrophage interaction. In this chapter, we review recent advances in this area and also highlight areas where new therapeutic intervention opportunities arise.

Biparental mucus feeding: a unique example of parental care in an Amazonian cichlid

SUMMARY Vertebrates display a wide variety of parental care behaviours, including the guarding of offspring pre and post nutritional independence as well as the direct provision of nutrients during the early development period. The Amazonian cichlid Symphysodon spp. (discus fish) is unusual among fish species, in that both parents provide offspring with mucus secretions to feed from after hatching. This extensive provision of care, which can last up to a month, imposes a physiological demand on both parents and gives rise to conflict between the parent and offspring. Here, we investigated the relationship between parents and offspring during a breeding cycle, determining both mucus composition (total protein, cortisol, immunoglobulin, and Na+, K+ and Ca2+ concentrations) and the behavioural dynamics of the parent–offspring relationship. Over the course of a breeding cycle, a significant increase in offspring bite rate was recorded, with a concomitant increase in the frequency of turns the male and female parent took at caring for their young. A peak in mucus antibody provision was seen as offspring reached the free-swimming stage, suggesting a role analogous to colostrum provision in mammals. Mucus protein content was lowest during the second and third weeks of free swimming, and a weaning period, similar to that seen in mammalian parental care, occurred when the offspring had been free swimming for ∼3 weeks. In many ways, the parental behaviour of discus fish is more similar to mammalian and avian parental care than other fish species, and represents an exciting aquatic model for studying the parent–offspring conflict.

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.