An organotypic oral mucosal infection model to study host-pathogen interactions.

Early in vitro oral mucosal infection models (OMMs) failed to consider the suitability of the model environment to represent the host immune response. Denture stomatitis (DS) is mediated by Candida albicans, but the role of Staphylococcus aureus remains uncertain. A collagen hydrogel-based OMM containing HaCaT and HGF cell types was developed, characterised and employed to study of tissue invasion and pro-inflammatory cytokine production in response to pathogens. Models formed a robust epithelium. Despite their inflammatory baseline, 24-h infection with C. albicans, and/or S. aureus led to tissue invasion, and significantly upregulated IL-6 and IL-8 production by OMMs when compared to the unstimulated control. No significant difference in IL-6 or IL-8 production by OMMs was observed between single and dual infections. These attributes indicate that this newly developed OMM is suitable for the study of DS and could be implemented for the wider study of oral infection.

Prognostic Role of CD68+ and CD163+ Tumour-Associated Macrophages and PD-L1 Expression in Oral Squamous Cell Carcinoma: A Meta-Analysis.

Background: Oral squamous cell carcinoma (OSCC) is a common malignant cancer in humans. An abundance of tumour associated macrophages (TAMs) create an immunosuppressive tumour microenvironment (TME). TAM markers (CD163 and CD68) are seen to serve as prognostic factors in OSCC. PD-L1 has seen to widely modulate the TME but its prognostic significance remains controversial. The aim of this meta-analysis is to evaluate the prognostic role of CD163+, CD68+ TAMs and PD-L1 in OSCC patients. Methods: Searches in PubMed, Scopus and Web of Science were performed; 12 studies were included in this meta-analysis. Quality assessment of included studies was performed according to REMARK guidelines. Risk of bias across studies was investigated according to the rate of heterogeneity. Meta-analysis was performed to investigate the association of all three biomarkers with overall survival (OS). Results: High expression of CD163+ TAMs were associated with poor overall survival (HR = 2.64; 95% Cl: [1.65, 4.23]; p < 0.0001). Additionally, high stromal expression of CD163+ TAMs correlated with poor overall survival (HR = 3.56; 95% Cl: [2.33, 5.44]; p < 0.00001). Conversely, high CD68 and PD-L1 expression was not associated with overall survival (HR = 1.26; 95% Cl: [0.76, 2.07]; p = 0.37) (HR = 0.64; 95% Cl: [0.35, 1.18]; p = 0.15). Conclusion: In conclusion, our findings indicate CD163+ can provide prognostic utility in OSCC. However, our data suggests CD68+ TAMs were not associated with any prognostic relevance in OSCC patients, whereas PD-L1 expression may prove to be a differential prognostic marker dependent on tumour location and stage of progression.

Porphyromonas gingivalis-stimulated macrophage subsets exhibit differential induction and responsiveness to interleukin-10.

OBJECTIVES: Oral mucosal macrophages (Mϕs) determine immune responses; maintaining tolerance whilst retaining the capacity to activate defences against pathogens. Mϕ responses are determined by two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2-Mϕs. Tolerance induction is driven by M2 Mϕs, whereas M1-like Mϕs predominate in inflammation, such as that exhibited in chronic Porphyromonas gingivalis (PG) periodontal infection. Mϕ responses can be suppressed to benefit either the host or the pathogen. Chronic stimulation by pathogen associated molecular patterns (PAMPs), such as LPS, is well established to induce tolerance. The aim of this study was to investigate the P. gingivalis-driven induction of and responsiveness to the suppressive, anti-inflammatory cytokine, IL-10, by Mϕ subsets. METHODS: M1- and M2-like Mϕs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and Vitamin D3, respectively. Mϕ subsets were stimulated by PG-LPS in the presence or absence of IL-10. RESULTS: PG-LPS differentially induced IL-10 secretion and endogenous IL-10 activity in M1- and M2-like subsets. In addition, these subsets exhibited differential sensitivity to IL-10-mediated suppression of TNFα, where M2 Mϕs where sensitive to IL-10 and M1 Mϕs were refractory to suppression. In addition, this differential responsiveness to IL-10 was independent of IL-10-binding and expression of the IL-10 receptor signal transducing subunit, IL-10Rß, but was in fact dependent on activation of STAT-3. CONCLUSION: P.gingivalis selectively tolerises regulatory M2 Mϕs with little effect on pro-inflammatory M1 Mϕs; differential suppression facilitating immunopathology at the expense of immunity.

Macrophage subset sensitivity to endotoxin tolerisation by Porphyromonas gingivalis.

Macrophages (MΦs) determine oral mucosal responses; mediating tolerance to commensal microbes and food whilst maintaining the capacity to activate immune defences to pathogens. MΦ responses are determined by both differentiation and activation stimuli, giving rise to two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2- MΦs. M2-like subsets predominate tolerance induction whereas M1 MΦs predominate in inflammatory pathologies, mediating destructive inflammatory mechanisms, such as those in chronic P.gingivalis (PG) periodontal infection. MΦ responses can be suppressed to benefit either the host or the pathogen. Chronic stimulation by bacterial pathogen associated molecular patterns (PAMPs), such as LPS, is well established to induce tolerance. The aim of this study was to investigate the susceptibility of MΦ subsets to suppression by P. gingivalis. CD14(hi) and CD14(lo) M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively. MΦ subsets were pre-treated with heat-killed PG (HKPG) and PG-LPS prior to stimulation by bacterial PAMPs. Modulation of inflammation was measured by TNFα, IL-1ß, IL-6, IL-10 ELISA and NFκB activation by reporter gene assay. HKPG and PG-LPS differentially suppress PAMP-induced TNFα, IL-6 and IL-10 but fail to suppress IL-1ß expression in M1 and M2 MΦs. In addition, P.gingivalis suppressed NFκB activation in CD14(lo) and CD14(hi) M2 regulatory MΦs and CD14(lo) M1 MΦs whereas CD14(hi) M1 pro-inflammatory MΦs were refractory to suppression. In conclusion, P.gingivalis selectively tolerises regulatory M2 MΦs with little effect on pro-inflammatory CD14(hi) M1 MΦs; differential suppression facilitating immunopathology at the expense of immunity.

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.

Conventional protein kinase C and atypical protein kinase Czeta differentially regulate macrophage production of tumour necrosis factor-alpha 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-alpha (TNF-alpha) 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-alpha 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 (alpha and beta) in the production of TNF-alpha 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-alpha and IL-10. The addition of cAMP however, suppressed activation of this MAPK and TNF-alpha production. Cyclic-AMP augmented IL-10 production and cAMP response element binding protein activation upon stimulation by PMA/ionomycin. In addition, cAMP activated PKCzeta; 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-alpha being dependent on conventional PKCs (alpha and beta) whereas IL-10 is regulated by the cAMP-regulated atypical PKCzeta.

Impact of cAMP on the T-cell response to type II collagen.

There is considerable interest in the possible use of cAMP-elevating agents in the treatment of autoimmune diseases such as rheumatoid arthritis. The objective of this study was to evaluate the impact of different cAMP-elevating agents on the T-cell response to type II collagen within the context of collagen-induced arthritis, a murine model of rheumatoid arthritis. Spleen cells or lymph node cells from type-II-collagen-immunized DBA/1 mice were cultured in the presence of type II collagen plus one of five different cAMP-elevating agents: rolipram, forskolin, prostaglandin E2, 8-bromo-cAMP, or cholera toxin. Levels of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-5 were measured in culture supernatants by enzyme-linked immunosorbent assay. All of the cAMP-elevating agents tested were found to profoundly suppress IFN-gamma production in a dose-dependent manner. IL-4 and IL-5 production was slightly up-regulated at low concentrations of the cAMP-elevating agents and was modestly suppressed at the highest concentrations of cAMP-elevating agents. Experiments were then carried out to determine whether T cells were directly affected by cAMP-elevating agents or whether the immunomodulatory effects were mediated via antigen-presenting cells. Pulsing T cells alone for a brief period with cholera toxin produced an almost identical effect to pulsing antigen-presenting cells alone, i.e. down-regulation of proliferation, down-regulation of IFN-gamma production with little effect on IL-5 production. It was concluded that cAMP-elevating agents suppressed T helper type 1 responses to type II collagen to a greater extent than T helper type 2 responses. The cAMP-elevating agents could directly influence the activity of T cells but, in addition, influenced the ability of antigen-presenting cells to support T helper type 1 responses.

Impact of VIP and cAMP on the regulation of TNF-alpha 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 alpha (TNF-alpha) with no effect on IL-10, whereas VIP fails to regulate IL-10 and TNF-alpha 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-alpha production and modulated T-cell response by inhibiting TNF-alpha and IFN-gamma. VIP's lack of effect on IL-10 and its slight effect on TNF-alpha 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-alpha, 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.