Less Severe Lipopolysaccharide-Induced Inflammation in Conditional mgmt-Deleted Mice with LysM-Cre System: The Loss of DNA Repair in Macrophages

Abstract

Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of mgmt in the macrophage cell line (RAW264.7) and mgmt null (mgmtflox/flox; LysM-Crecre/−) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOS and IL-1β) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (mgmtflox/flox; LysM-Cre−/−). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both mgmt null and control mice. However, LPS upregulated mgmt only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the mgmt null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of mgmt in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.

Polymeric Particle BAM15 Targeting Macrophages Attenuates the Severity of LPS-Induced Sepsis: A Proof of Concept for Specific Immune Cell-Targeted Therapy

“…Here, BAM15, at a high dose of free-form or BAM15 particles, was not toxic to the mice ( Figure 7 ). Regarding the selective blockage only in macrophages, our particles might be an additional strategy to the conditional gene deletion (LyM-Cre) [ 24 , 32 ] and nanoparticle [ 25 , 26 ] strategies that have been previously described. With our previous protocol [ 20 ], PLGA particles were selectively delivered to macrophages but not DCs ( Figure 6 ), and BAM15 particles had neutralizing effects on LPS-induced inflammation only in macrophages but not neutrophils ( Figure 5 ).…”

“…The mortality rate for sepsis remains high, despite improved supportive treatment, which is partly due to the hyper-responses to microbial molecules of several immune cells, especially macrophages [ 23 ]. Indeed, the immunological blockage on macrophages using LysM-Cre mice [ 24 ] and nanomaterials targeting macrophages successfully attenuates hyper-inflammation in sepsis [ 25 , 26 ]. Here, several tests were performed to assess (i) the impacts of BAM15 on lipopolysaccharide (LPS)-induced M1 polarization or M2 polarization (using IL-4 and cancer cell supernatant) and (ii) the influence of BAM15-PLGA particles (BAM15 particles) on macrophages and in mice.…”