The Mechanism Of Human Antimicrobial Peptide LL-37 Promoting Extracellular Mitochondrial DNA Clearance By Macrophages And Mitigating Lung Inflammation

Background and ObjectiveSystemic inflammatory response syndrome(SIRS)is a significant reason of common complications,such as acute lung injury(ALI),following severe trauma and hemorrhagic shock.The strategic link between the pathogen associated molecular patterns(PAMPs)or damage associated molecular patterns(DAMPs),pattern recognition receptors(PRRs),and inflammation is a main mechanism in the pathogenesis of SIRS.Extracellular free mitochondrial DNA(mtDNA)is one of the newly identified DAMPs.Since mitochondria evolve from saprophyric bacteria to endosymbionts to organelles,mtDNA contains unmethylated CpG motifs similar to bacterial DNA.Several investigations have discovered that mtDNA could be released to plasma quickly after injury,and activate the p38 MAPK signal pathway via TLR9 in neutrophils or macrophages,then potentially contribute to the development of an inflammatory response.TLR9 recognizes unmethylated CpG motifs that are frequently present in bacteria and viruses but are rare in mammalian cells.TLR9 is exclusively sequestered in the endoplasmic reticulum(ER)in unstimulated cells and rapidly traffic to endolysosomes after ligand stimulation.It was also demonstrated that TLR9 could recognize mtDNA based on low-methylated CpG repeats and activate macrophages.Our previous study also proved that TLR9-p38 MAPK pathway mediated the proinflammatory potential of mtDNA on THP-1 macrophages.These indicate that the traffic of TLR9 from ER to endolysosomes may play an immportant role in the mtDNA stimulation.Human antimicrobial peptide LL-37 is the singulare mature peptide of human cathelicidins family,which plays an important role in lung inflammation.Recent studies have found that LL-37 could form complex with extracellular DNA to induce DNA uptake.However,the effect of this complex on immune cells is controversial.Some studies found that LL-37 can form complexes with self-DNA to induce self-DNA uptake in plasmacytoid dendritic cells(pDCs),exhibit high activation of TLR9,and upregulate downstream expression of interferons.Other studies showed that LL-37 could interact with DNA in psoriatic skin,neutralize cytosolic DNA in keratinocytes and block AIM2 inflammasome activation.These indicate multiple functions of LL-37-DNA complex in different processes of disease.The influence of LL-37 on mtDNA is still indeterminate.Only one study demonstrated that LL-37-mtDNA complex activates TLR9-mediated inflammatory responses in pDCs through escaping from autophagic recognition,and promotes the formation of atherosclerotic lesion.However,there was no investigation about LL-37-mtDNA complex in other immune cells or tissues.In this study,we conducted in-vitro exposure of mtDNA in the presence or absence of LL-37 on THP-1 macrophage,a human monocytic and macrophage cell line,to explore the influence of LL-37 on mtDNA uptake and stimulation.Then we confirmed the results with intratracheal administration of mtDNA in the presence or absence of CRAMP(LL-37 mouse homologous protein)in mice.This study would provide theoretical basis for LL-37 as mtDNA inhibitor in pulmonary inflammation and lung injury.Methods and Materials1.THP-1 macrophages were exposed to Alexa Fluor 488 labeled mtDNA in the presence or absence of LL-37,and the fluorescence intensity was analysed using fluorescence microscope and flow cytometry.We also examined the inflammatory cytokines induced by LL-3 7 in different concertrations on THP-1 macrophages using ELISA to exclude the inflammatory effect of LL-37 itself.After exposure to mtDNA in the presence or absence of LL-37,the cytokine production by THP-1 macrophages was examined by qPCR,and the cytokine concentration in the cell supernatant was determined by ELISA.Meanwhile,the phosphorylation level of p38 MAPK protein was detected by Western blot.Immunofluorescence technique was applied to explore the localization of Alexa Fluor 488 labeled mtDNA and TLR9 or lysosomes in the presence or absence of LL-37.2.Intratracheal instillation of mtDNA in the presence or absence of CRAMP in a volume of 60?l was performed to establish the animal model of topical inflammation within lung tissue induced by mtDNA and explore the influence of CRAMP on mtDNA induced lung inflammation.Intratracheal instillation of CRAMP was also conducted to exclude the inflammatory effect of CRAMP itself.Lung tissue paraffin sections were stained with hematoxylin-eosin to observe the inflammatory morphological pathology induced by mtDNA or CRAMP exposure.Levels of CRAMP protein expression were examined in four lung inflammation models:LPS model,mtDNA model,CLP model and high tidal volume mechanical ventilation model,using Western blot.Several proinflammatory cytokine levels in mice bronchoalveolar lavage fluid were examined by ELISA.Meanwhile,the phosphorylation level of p38 protein in lung tissue following mtDNA exposure in the presence or absence of CRAMP was detected by Western blot.Results1.Human antimicrobial peptide LL-37 enhances extracellular mitochondrial DNA uptake by THP-1 macrophages without TLR9-p38 MAPK activation to mitigate inflammation induced by mtDNA.THP-1 macrophages could uptake a little percent of extracellular mtDNA,while with the existence of LL-37,a large amount of mtDNA enterd cells.ELISA assay demonstrated that inflammatory effect of LL-37 depended on concentration,LL-37 could not increase level of inflammatory cytokines at light concentration.LL-37 significantly attenuated the inflammatory cytokines concentration induced by mtDNA in the THP-1 macrophages supernatant.qPCR examination also indicated that LL-37 mitigated the up-regulated production of inflammatory cytokines induced by mtDNA.Meanwhile,Western blot assay showed that the phosphorylation level of p38 MAPK protein in THP-1 macrophages was dramatically down-regulated following mtDNA exposure in the presence of LL-37 compared with mtDNA alone.Immunofluorescence showed that when exposed to mtDNA alone,mtDNA which enterd cells was localized in the TLR9 positive region,while exposed to mtDNA in the presence of LL-37,mtDNA incorporated was not localized in the TLR9 strongly positive region.However,no matter in the presence or absence of LL-37,mtDNA incorporated was localized in the LAMP-1 positive region2.LL-37 mouse homologous protein CRAMP attenuates mtDNA induced lung inflammation and injury in mice.After intratracheal instillation of mtDNA,the histological score of H&E stained lung tissue paraffin sections significantly raise,demonstrating alveolar edema,vascular congestion,and inflammatory cells infiltration.Additionally,ELISA assay demonstrated that mtDNA exposure significantly increased the inflammatory cytokines expression in mice BALF.However,exposure to CRAMP alone did not induce up-regulated release of inflammatory cytokines.Levels of CRAMP protein expression were significantly up-regulated in four lung inflammation models:LPS model,mtDNA model,CLP model and mechanical ventilation model,analysed by Western blot.While intratracheal instillation of mtDNA preincubated with CRAMP,the histological score of H&E stained lung tissue paraffin sections fell significantly,and the inflammatory cytokines release in mice BALF was also down-regulated.Meanwhile,Western blot assay showed that after intratracheal instillation of mtDNA preincubated with CRAMP,the phosphorylation level of p38 MAPK protein in lung tissues was also lower than intratracheal instillation of mtDNA alone.Conclusion1.We proved that LL-37 could form complex with mtDNA and enhance its traffic into THP-1 macrophages.Previous studies investigating the complex of LL-37 and extracellular DNA were focused on the role of nuclear DNA,mtDNA was less studied.This study provides a new evidence for a more comprehensive understanding of LL-37 promoting extracellular DNA entry into cells.2.We proved that LL-37 could interact with and neutralize mtDNA,inhibit mtDNA recognition by TLR9,block TLR9-p38 MAPK activation and mitigate inflammation induced by mtDNA.This uncovers a new effect of LL-37 on mtDNA induced endogenous inflammation,and provides a new evidence for LL-37 inhibitting mtDNA induced macrophages inflammation.3.We proved that LL-37 mouse homologous protein CRAMP attenuated mtDNA induced lung inflammation and injury in mice.It may provides theoretical basis for future use of LL-37 as mtDNA inhibitor in lung inflammation and lung injury.