Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response

Abstract

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1β), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

More Prominent Inflammatory Response to Pachyman than to Whole-Glucan Particle and Oat-β-Glucans in Dextran Sulfate-Induced Mucositis Mice and Mouse Injection through Proinflammatory Macrophages

“…Some parts of BG in the serum of BG-administered DSS mice might be the BG that was orally administered because of the DSS-induced gut barrier defect [ 9 ], and these BG might have different effects in mice. To test the impacts of different glucans, BG, with or without LPS, were injected in mice.…”

“…The method of fecal microbiota analysis followed that of previous publications [ 9 , 27 , 32 , 109 , 110 ]. Briefly, feces (0.25 g per mouse) were used to extract total DNA by a power DNA isolation kit (MoBio, Carlsbad, CA, USA), and the metagenomic DNA quality was evaluated by agarose gel electrophoresis with nanodrop spectrophotometry.…”

“…(the most common fungus in mammal gut) and bacteria are mainly β-(1→3) backbones with different lengths, with higher and lessor β-(1→6) side chains present in the fungal vs. bacterial BG, respectively [ 7 , 8 ]. Due to the foreignness of these molecules to mammals, all BGs may be categorized as pathogen-associated molecular patterns (PAMPs) if they present to immune cells with appropriate pattern recognition receptors (PRRs) [ 9 , 10 , 11 , 12 ]. Although immune responses against PAMPs generally induce inflammatory responses, there are diverse directions of immune activation by different BGs described in the literature.…”

Neutrophil Extracellular Traps in Severe SARS-CoV-2 Infection: A Possible Impact of LPS and (1→3)-β-D-glucan in Blood from Gut Translocation

“…Nevertheless, leaky gut in COVID-19 infection is severe enough for the translocation of LPS, BG, and, perhaps, fragmented bacterial DNA, but not the viable organisms. Indeed, the presence of bacteria-free DNA in serum without positive conventional bacterial culture could be due to the death of the viable bacteria after gut translocation (DNA from lytic dead bacteria) or the transfer of only bacteria-free DNA (but not the whole viable bacteria) through the gut barrier [ 68 ]. Notably, intact bacteria-free DNA (6.5 × 10 4 –9.8 × 10 6 kDa) naturally breaks down into fragment-free DNA (less than 65 kDa) [ 69 , 70 , 71 , 72 ], similar to the size of LPS and BG (10–100 kDa) [ 73 , 74 , 75 ]; however, this could not pass through the healthy gut barrier, which only allows the passive transport of molecules smaller than 0.6 kDa [ 76 , 77 ].…”

“…Serum of patients with COVID-19 (moderate and severe cases) activated NETs in isolated neutrophils from healthy volunteers, implying the impact of some blood components, such as pathogen molecules (LPS, BG, and bacteria-free DNA) and/or cytokines, in the samples [ 68 , 84 , 85 ]. After incubation by the serum of severe COVID-19 cases, NETs formation was even higher than the activation by PMA, a potent NETs inducer [ 9 ].…”