Mitochondrial DNA Pretreatment Suppresses Subsequent LPS Induced Pulmonaryinflammation

BackgroundTrauma is among the leading causes of death in young people in the United States. Even though some victims could survive the initial time period after injury, they are at high risk for pulmonary infection which could be a "second challenge" and cause acute respiratory disease syndrome (ARDS). Previous studies show that ventilator-associated pneumonia (VAP) after traumatic injury is associated with high mortality and longer ICU stays. Although severe injury is associated with disturbed immune, less is known about the causes of the immunosuppression caused by traumatic injury. Acute lung injury (ALI) often occurs in severe trauma, sepsis and hemorrhagic shock. Although the term acute lung injury has been abandoned in the 2012 Berlin classification of ARDS and this state is now called mild ARDS, it still widely used in fundamental research and clinical practices. It is the common critical diseases in respiratory medicine, and also has been the difficulties of this field. The mechanism of ALI is very complex. The pathogenesis of it involves multiple links. However, the molecular mechanisms which are responsible for the development of these conditionsare poorly understood, and there are currently no Food and Drug Administration-approved drug therapies. The core of the development of ALI is Systemic Inflammatory Response Syndrome (SIRS). To find out the upstream of SIRS will contribute to better understand of ALI. Recent research found that infection and non-infection was recognized by pattern-recognition receptors (PRRs) as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), respectively.Upon activation, caspase-1cleaves inactive proforms of IL-1β and IL-18 into bioactive cytokines that then initiate or amplify diverse downstream signalling pathways and drive proinflammatory processes in the airway.Lipopolysaccharide (LPS), a major pathogen associated molecular pattern (PAMP) stimulates macrophages. In some situations, prior exposure of macrophages to LPS could induce transienthyporesponsiveness to subsequent LPS stimulation, which is known asLPStolerance.The phenomenon that reproduces certain aspects of the immunosuppression observed frequently in patients during late-phase sepsis.In recent years, our knowledge of the mechanisms of LPS tolerance and macrophage desensitization by repeated LPS exposure has increased considerably as a result of novel insightsinto the LPS signalling pathways.It had been found that LPS signal transductionis initiated by Toll-like receptor 4 (TLR-4), which belongs to the group of pattern recognition receptors.These receptors mediate activation of the innate immune system by microbial compounds.The DAMPs/PAMPs was recognized by PRRs which plays important role in the maturation and release of IL-1β. The main PRRs that have been found mainly included three categories:Toll-like receptors (TLRs), retinoic acid-inducible gene-I [RIG-I]-likereceptors (RLRs) and nucleotide-binging oligomerization domain[NOD]-like receptors (NLRs). In lig ht of the finding that cellular responses to different microbial stimuli are mediated by different TLRs, studies were initiated to determine whether, in analogyto LPS tolerance, prestimulated with microbial non-LPSstimuli also induces hyporesponsiveness to subsequent restimulation. Indeed, it has been reported that stimulation with prototypical ligands for TLR-2, TLR-4, TLR-5and TLR-9also induces this state of hyporesponsiveness towards subsequent stimulation withthe same ligand. Moreover, stimuli signalling via TLR-2and TLR-4,as well as TLR-4 and TLR-9,can substitute for each other, mediating cross-tolerance in vitro and vivo. Among these TLRs, TLR9 has been shown to be activated by bacterial CpG-containing DNA, some double-stranded DNA viruses 9 andsynthetic CpG-oligodesoxynucleotides (ODN).Nuclear DNA and mitochondrial DNA (mtDNA) are thoughtto be of separate evolutionary origins, with mtDNA being derived from the circular genomes of bacteria.Like bDNA, unmethylated cytosine-phosphate-guanine (CpG) dinucleotides are common in mtDNA and elicit inflammatory and immunostimulatory responses. Therefore, mtDNA as the DAMPs, its mechanism attracted much attention. Previous results of our project team found that the concentration of circulate mtDNA was significantly higher in the traumatic ALI patients, which suggested the association between mtDNA and the development of traumatic ALI.Our previous and some other studies have revealed that mtDNA is released into blood circulation soon after traumatic injury and it active macrophages via TLR9.Moreover, it is indicated that mtDAMPs pretreatment is capable of suppressing pulmonary inflammation by subsequent pulmonary contusion. However, the exact role of preconditioned mtDNA in LPS tolerance was still unknown. Therefore, we conducted the study to explore the possible role.Previous studies have indicated that mtDNA would activate TLR9-p38 MAPK pathway in neutrophils and induce sterile inflammation and acute lung injury. Another study of our group also revealed that p38 MAPK pathway was responsible for mtDNA induced inflammation in macrophages and numbers of inflammatory factor gene expression. P38 MAPK was also a critical factor in LPS induced inflammation. The role of p38MAPK in mtDNA induced LPS tolerance is still unclear. We conducted this study to evaluate the role of mtDNA in subsequent LPS challenge and explore the role of p38MAPK played in the process.Methods and MaterialsMitochondrial DNA was extracted from the isolated mitochondria pellets from THP-1 cells and fresh mice liver. The A260/280 ratio of mtDNA was between 1.8 and 2.0 to exclude any significant protein contamination. Differentiated THP-1 macrophages were stimulated with different concentrations of mtDNA or 1640 for 2h or 24h. After that, cells were allowed to rest for 1h, and then re-challenged by LPS (100ng/ml) for another 3h. RNA from THP-1 cells was extracted by using RNA isolation kit. qRT-PCR was performed to test the mRNA expression level. Cell proteins were obtained from THP-1 macrophages and Western blot analysis of cellular lysates was performed to detect the role of p38MAPK in the mtDNA induced LPS tolerance.Healthy male C57BL/6 mice weighting 20-22g and aged 7-8weeks were randomly divided into four groups (group A, B, C and D) with 9 animals per group. Group A only received NS; Group B received intraperitoneal mtDNA; Group C received intratracheal instillation of LPS and Group D received both. Mices were sacrificed 3h after LPS challenge.The concentration of various cytokines in the mouse lung extracts were assessed by ELISA kits. Histopathology and Wet/Dry ratio of the lungs were investigated in the 4 groups’ mice.Results1. MtDNA pretreatment induced"cross-tolerance" to LPS in macrophage:The ELISA results of THP-1 supernatant shown that mtDNA pretreatment for 24h was capable of suppressing LPS induced TNF-α, IL-6 and IL-1β release, but a decrease of IL-8 was no statistical significance. The expression of above proinflammatory factors were also analyzed and it was shown that mtDNA pretreatment could suppress subsequent LPS induced TNF-α, IL-1β and IL-6 gene expression but got no effect on IL-8 (CXCL-8) mRNA.2. P38 MAPK in mtDNA induced LPS tolerance:The results of Westren blot indicated that single mtDNA or LPS exposure was capable of inducing significant up-regulation of phosphorylated p38 MAPK. Compared with no-mtDNA primed treatment, LPS induced phosphorylated p38 was significantly decreased in mtDNA primed treatment.3. In vivo study of mtDNA treatment on LPS induced pulmonary inflammation: Systemic mtDNA pretreatment also reduced LPS-induced pathological changes of pulmonary inflammation and pulmonary edema.TNF-α, IL-1β, IL-6 and IL-8 in lung extracts were analyzed and it was indicated that mtDNA pretreatment would significantly suppressTNF-α and IL-6 release by LPS re-challenge while no statistical changes of IL-1β and IL-8 were detected.ConclusionTo summarize, this present study demonstrated that mtDNA pretreatment was capable of inducing cell tolerance to subsequent LPS and decreased pulmonary inflammation. This should be partially due to impaired p38 MAPK activation and decreased inflammatory gene expression. This might be a cause of immunosuppression and increased risk to infection in traumatic patients.