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Macrophages (Mϕs) play a central role in mucosal immunity by pathogen sensing and instruction of adaptive immune responses. Prior challenge to endotoxin can render Mφs refractory to secondary exposure, suppressing the inflammatory response. Previous studies demonstrated a differential subset-specific sensitivity to endotoxin tolerance (ET), mediated by LPS from the oral pathogen, Porphyromonas gingivalis (PG). The aim of this study was to investigate ET mechanisms associated with Mφ subsets responding to entropathogenic E. coli K12-LPS. M1- and M2-like Mφs were generated in vitro from the THP-1 cell line by differentiation with PMA and Vitamin D3, respectively. This study investigated ET mechanisms induced in M1 and M2 Mφ subsets, by measuring modulation of expression by RT-PCR, secretion of cytokines by sandwich ELISA, LPS receptor, TLR4, as well as endogenous TLR inhibitors, IRAK-M and Tollip by Western blotting. In contrast to PG-LPS tolerisation, E. coli K12-LPS induced ET failed to exhibit a subset-specific response with respect to the pro-inflammatory cytokine, TNFα, whereas exhibited a differential response for IL-10 and IL-6. TNFα expression and secretion was significantly suppressed in both M1- and M2-like Mφs. IL-10 and IL-6, on the other hand, were suppressed in M1s and refractory to suppression in M2s. ET suppressed TLR4 mRNA, but not TLR4 protein, yet induced differential augmentation of the negative regulatory molecules, Tollip in M1 and IRAK-M in M2 Mφs. In conclusion, E. coli K12-LPS differentially tolerises Mφ subsets at the level of anti-inflammatory cytokines, associated with a subset-specific divergence in negative regulators and independent of TLR4 down-regulation. [ABSTRACT FROM AUTHOR]

Objectives: Oral mucosal macrophages (MØs) determine immune responses; maintaining tolerance whilst retaining the capacity to activate defences against pathogens. MØ responses are determined by two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2-MØs. Tolerance induction is driven by M2 MØs, whereas M1-like MØs predominate in inflammation, such as that exhibited in chronic Porphyromonas gingivalis (PG) periodontal infection. MØ 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, antiinflammatory cytokine, IL-10, by MØ subsets. Methods: M1- and M2-like MØs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and Vitamin D3, respectively. MØ 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 MØs where sensitive to IL-10 and M1 MØs 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-10Rβ, but was in fact dependent on activation of STAT-3. Conclusion: P.gingivalis selectively tolerises regulatory M2 MØs with little effect on pro-inflammatory M1 MØs; differential suppression facilitating immunopathology at the expense of immunity. [ABSTRACT FROM AUTHOR]