Macrophage subsets exhibit distinct E. coli-LPS tolerisable cytokines associated with the negative regulators, IRAK-M and Tollip

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 D 3 , 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.

Analysis of interleukin-1 receptor associated kinase-3 (IRAK3) function in modulating expression of inflammatory markers in cell culture models: A systematic review and meta-analysis

“… Intervention was performed using LPS [ 17 , 23 – 25 , 27 , 28 , 46 , 47 , 55 , 61 , 76 , 77 , 84 , 88 , 92 , 93 ], L . donovani [ 13 ] or H .…”

“… In one-challenge intervention group, IRAK3 mRNA expression was measured at 1h [ 25 , 90 , 101 ], 3h [ 11 , 33 , 103 ], 4h [ 61 ], 6h [ 88 ], 12h [ 69 ], 18h [ 23 ] after one-challenge with heat-stable enterotoxigenic E . coli pathogen-associated molecular patterns [ 69 ] or LPS.…”

“… In one-challenge intervention group, TNF-α and IL-6 protein expression was measured at 3h [ 11 ], 4h [ 44 , 47 ], 6h [ 53 , 87 , 88 ], 8h [ 33 ], 12h [ 54 ], 18h [ 23 , 49 ], 24h [ 21 , 25 , 59 , 66 , 73 , 80 , 83 , 90 , 98 , 104 , 111 , 113 ] after one-challenge with peptidoglycan [ 21 ] or LPS (other studies included in the meta-analyses). In two-challenge intervention group, cells were first challenged with lipoteichoic acid [ 44 ], peptidoglycan [ 21 ] or LPS (other studies in the meta-analyses), and then received the second challenge with peptidoglycan [ 21 ] or LPS.…”

“…At an interval after the second challenge, TNF-α and IL-6 protein expression was measured. The first and second challenge of two-challenge interventions were respectively 24h and 18h [ 23 ]; 18h and 18h [ 49 ]; 18h and 4h [ 44 ]; 24h and 6h [ 53 ], 16h and 24h [ 21 ]; 24h and 24h [ 25 , 59 , 66 , 73 , 83 , 98 , 104 ]; 16h and 6h [ 87 ]; 20h and 24h [ 111 , 113 ]; 18h and 12h [ 54 ]; 5d and 24h [ 90 ]; 4h and 24h [ 47 ]; 8h and 24h [ 80 ]; 15h and 6h [ 88 ]; 8h and 8h [ 33 ]; 24h and 3h [ 11 ].…”

A Novel Lactic Acid Bacteria Mixture: Macrophage-Targeted Prophylactic Intervention in Colorectal Cancer Management

“…Pro-inflammatory (M1-like) macrophages and anti-inflammatory (M2-like) macrophages were generated by differentiation of these monocytes in the presence of 25 ng/mL phorbol 12-myristate 13-acetate (PMA) for three days or 10 nM 1,25-(OH) 2 -Vitamin D 3 (Sigma-Aldrich, Poole, UK), for seven days, respectively [33]. M1-like macrophages were then washed and allowed to rest in fresh R10 medium without PMA for an additional 24 h [34]. Macrophages were plated out to a final density of 5 × 10 5 cells/500 µL/well in R10 medium in 24 flat-bottomed well tissue culture plates.…”