Monocyte exocytosis of mitochondrial danger-associated molecular patterns in sepsis suppresses neutrophil chemotaxis

Abstract

BACKGROUND Trauma and sepsis both increase the risk for secondary infections. Injury mobilizes mitochondrial (MT) danger-associated molecular patterns (mtDAMPs) directly from cellular necrosis. It is unknown, however, whether sepsis can cause active MT release and whether mtDAMPs released by sepsis might affect innate immunity. METHODS Mitochondrial release from human monocytes (Mo) was studied after LPS stimulation using electron microscopy and using fluorescent video-microscopy of adherent Mo using Mito-Tracker Green (MTG) dye. Release of MTG+ microparticles was studied using flow cytometry after bacterial stimulation by size exclusion chromatography of supernatants with polymerase chain reaction (PCR) for mitochondrial DNA (mtDNA). Human neutrophil (PMN), chemotaxis, and respiratory burst were studied after PMN incubation with mtDNA. RESULTS LPS caused Mo to release mtDAMPs. Electron microscopy showed microparticles containing MT. mtDNA was present both in microvesicles and exosomes as shown by PCR of the relevant size exclusion chromatography bands. In functional studies, PMN incubation with mtDNA suppressed chemotaxis in a dose-dependent manner, which was reversed by chloroquine, suggesting an endosomal, toll-like receptor-9–dependent mechanism. In contrast, PMN respiratory burst was unaffected by mtDNA. CONCLUSION In addition to passive release of mtDAMPs by traumatic cellular disruption, inflammatory and infectious stimuli cause active mtDAMP release via microparticles. mtDNA thus released can have effects on PMN that may suppress antimicrobial function. mtDAMP-mediated “feed-forward” mechanisms may modulate immune responses and potentially be generalizable to other forms of inflammation. Where they cause immune dysfunction the effects can be mitigated if the pathways by which the mtDAMPs act are defined. In this case, the endosomal inhibitor chloroquine is benign and well tolerated. Thus, it may warrant study as a prophylactic antiinfective after injury or prior sepsis.

Trauma-induced heme release increases susceptibility to bacterial infection

“…PMN, isolated from the BM of naïve mice negatively selected against Ly6G + cells were then cultured with naïve mouse serum or trauma serum for 90 minutes. PMN were then exposed to S. aureus and ROS were measured by chemiluminescence as previously reported (69). PMN incubated with trauma serum showed reduced ROS generation compared to naïve-serum treated PMN suggesting on potential mechanism that would explain, in part, the lack of bacterial clearance (Figure 5K) PMN from trauma patients show reduced expression of CD16, TLR2 and TLR4.…”

Circulating mitochondrialN-formyl peptides contribute to secondary nosocomial infection in patients with septic shock

“…These data indicate that human mtFPs were not released from bacteria but from the human PBMCs. Our recent study also showed that monocytes stimulated by infectious stimuli actively released mtDAMPs into the circulation via microparticles ( 28 ).…”

“…ND6 is likely to be both the most potent FPR1 agonist and a marker for the other mtFPs. However, other mtDAMPs like mtDNA released during septic shock may act differently, such as modulating toll-like receptor-9–related pathways ( 28 ). The SOFA scores at the onset of secondary infection were significantly higher in patients who developed secondary infections (10.7 ± 1.5) than in secondary infection–negative patients (4.3 ± 1.2) ( SI Appendix , Table S8 ).…”

“…Consistently high circulating mtFP levels might be attributed to ongoing tissue injuries. Furthermore, active mtDAMP release via microparticle extrusion from monocytes stimulated by secondarily infected bacteria might also increase circulating mtFP levels ( 28 ). Exposure to the plasma of patients who developed secondary infections, that contained large amounts of ND6, significantly suppressed the chemotaxis of PMNs obtained from healthy volunteers and FPR1 expression on the PMN membrane.…”

Exosome: The Regulator of the Immune System in Sepsis

“…Monocytes stimulated by Inflammation and infection release exosomes containing mitochondrial damage-associated molecular patterns (mt-DAMPs), which reduce the chemotaxis and sterilization of neutrophils through TLR9 inhibition mediated by endosomal acidification ( Itagaki et al, 2011 ; Konecna et al, 2021 ). LPS can stimulate endothelial cells to express adhesion molecule ICAM-1, which can bind to monocytes and trigger inflammatory response ( Wang et al, 2015a ).…”