Interference on Cytosolic DNA Activation Attenuates Sepsis Severity: Experiments on Cyclic GMP–AMP Synthase (cGAS) Deficient Mice

Abstract

Although the enhanced responses against serum cell-free DNA (cfDNA) in cases of sepsis—a life-threatening organ dysfunction due to systemic infection—are understood, the influence of the cytosolic DNA receptor cGAS (cyclic guanosine monophosphate–adenosine monophosphate (GMP–AMP) synthase) on sepsis is still unclear. Here, experiments on cGAS deficient (cGAS-/-) mice were conducted using cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection sepsis models and macrophages. Severity of CLP in cGAS-/- mice was less severe than in wildtype (WT) mice, as indicated by mortality, serum LPS, cfDNA, leukopenia, cytokines (TNF-α, IL-6 and IL-10), organ histology (lung, liver and kidney) and spleen apoptosis. With the LPS injection model, serum cytokines in cGAS-/- mice were lower than in WT mice, despite the similar serum cfDNA level. Likewise, in LPS-activated WT macrophages, the expression of several mitochondria-associated genes (as revealed by RNA sequencing analysis) and a profound reduction in mitochondrial parameters, including maximal respiration (determined by extracellular flux analysis), DNA (mtDNA) and mitochondrial abundance (revealed by fluorescent staining), were demonstrated. These data implied the impact of cfDNA resulting from LPS-induced cell injury. In parallel, an additive effect of bacterial DNA on LPS, seen in comparison with LPS alone, was demonstrated in WT macrophages, but not in cGAS-/- cells, as indicated by supernatant cytokines (TNF-α and IL-6), M1 proinflammatory polarization (iNOS and IL-1β), cGAS, IFN-γ and supernatant cyclic GMP–AMP (cGAMP). In conclusion, cGAS activation by cfDNA from hosts (especially mtDNA) and bacteria was found to induce an additive proinflammatory effect on LPS-activated macrophages which was perhaps responsible for the more pronounced sepsis hyperinflammation observed in WT mice compared with the cGAS-/- group.

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

“…The co-existence of LPS and bacterial DNA in the serum of sepsis mice (CLP and LPS models) may enhance systemic inflammation through macrophage M1 polarization (iNOS and IL-1β expression) [34,35] and NFκB upregulation. Indeed, in macrophages with DNA stimulation alone, there was less potent pro-inflammatory activation (low supernatant IL-10) with TLR-4 upregulation [75], while LPS + DNA exhibited a profound inflammatory response (Figure 9). Notably, the similar downregulation of TLR-4, perhaps from the TLR-4 internalization (endocytosis) [53] between LPS and LPS + DNA activation suggests that bacteria-free DNA did not activate TLR-4, despite a possible DNA-induced TLR-4 activation in some situations [39].…”

“…To test the impact of bacterial DNA on sepsis mice, bacterial DNA was administered with and without LPS in mice. Accordingly, DNA was extracted from Escherichia coli (ATCC 25922) (ATCC, Manassas, VA, USA) following a published protocol [75]. Briefly, E. coli in Luria-Bertani (LB) broth isolated DNA using the Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech, Wembley, WA, Australia) and quantified by a Nanodrop ND-100 (Thermo Scientific, Waltham, MA, USA).…”

“…The working hypothesis demonstrates a simultaneous stimulation of surface TLR-4 and endosomal TLR-9 by LPS and bacteria-free DNA, respectively, which induces inflammatory responses through MyD88, and non-MyD88 (TRIF), which induces TRAF-6[92,93]. The vigorous inflammatory stimulation, especially cytokine production, requires high cell energy, mostly by the glycolysis pathway[81], which might cause mitochondrial damage with higher inflammatory responses[75,94]. MyD88, myeloid differentiation primary response 88; TRIF, TIR-domain-containing adapter-inducing interferon-β; TRAF6, TNF receptor associated factor 6; IRF-3, interferon regulatory factor 3.Figure created using Biorender (https://biorender.com/, accessed on 29 January 2022).…”

Enhanced Bacteremia in Dextran Sulfate-Induced Colitis in Splenectomy Mice Correlates with Gut Dysbiosis and LPS Tolerance

“…Although the loss of microbial control due to asplenia is extensively mentioned, studies on the possible impact of LPS tolerance (due to chronic endotoxemia post-splenectomy) on organismal control by macrophages [63,64] are still less. As such, LPS tolerance of macrophages induced lower cytokines, upregulated anti-inflammatory genes (Arginase-1 and TGF-β), downregulated pro-inflammatory genes (iNOS and IL-1β), and reduced cell energy status (both mitochondria and glycolysis) when compared with the single LPS stimulation [65][66][67]. With proteomic analysis, LPS tolerance reduced enzymes for glycolysis energy production, a major energy source for macrophage cytokine production [68], as indicated by decreased phosphofructokinase (a rate-limiting enzyme of glycolysis), which was compensated by enhanced glucose utilization by increased hexokinase [69].…”

Uremia-Induced Gut Barrier Defect in 5/6 Nephrectomized Mice Is Worsened by Candida Administration through a Synergy of Uremic Toxin, Lipopolysaccharide, and (1➔3)-β-D-Glucan, but Is Attenuated by Lacticaseibacillus rhamnosus L34

“…Because of the importance of macrophages in recognizing pathogen molecules [ 50 , 51 ], bone marrow-derived cells were used in the experiments. As such, IS alone (≥2 mM) or with LPS (or BG) reduced macrophage viability, and the combination between IS with LPS or BG did not reduce the toxic concentration of IS ( Figure 6 A).…”