Abstract
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.
Highlights
Endotoxin tolerance (ET) is a phenomenon where cells become hypo-responsive to endotoxin/lipopolysaccharide (LPS), unable to respond to repeated LPS challenge
Mφ subsets have previously been shown to exhibit a differential sensitivity to ET induced in response to PG-LPS, derived from the oral keystone pathogen associated with chronic periodontitis, Porphyromonas gingivalis [5]
M1 Mφs were sensitive to suppression of IL-6 and IL-10 and in contrast, M2 Mφs were refractory to suppression of IL-6 and IL-10
Summary
Endotoxin tolerance (ET) is a phenomenon where cells become hypo-responsive to endotoxin/lipopolysaccharide (LPS), unable to respond to repeated LPS challenge. Macrophage tolerisation by E. coli LPS and detrimental to both the host and pathogen alike; immune suppression in general, will benefit the host by dampening down harmful host-derived inflammatory responses that result in tissue degradation, whereas this suppression will give the pathogen a reprieve from hostilities, enabling expansion of pathogen numbers. Tolerance induction and sensitivity to ET is fundamental to the homeostatic function of the gut mucosa; effectively allowing the gastrointestinal tract to determine immune fate, tolerating safe non-self, such as commensal microbes and food whereas maintaining the ability to be activated in inflammatory responses mounted to unsafe non-self, pathogenic material. Gut mucosal macrophages (Mφs) are essential to ET; their differentiation and activation status is indicative of whether the mucosal environment is harmful to the pathogen or host tissue. ET induced in Mφs results as a consequence of many different mechanisms, which include induction and responsiveness to anti-inflammatory cytokines (e.g. IL-10 and TGFβ), down-regulation of PRRs (e.g. TLR4), shedding of cytokine receptors and PRRs and induction of negative regulatory molecules, which have a functional role in inhibition of TLR4 signal transduction, such as Tollip, Myd88s, SARM, IRAK-M and SIGIRR (reviewed in [1])
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