Organic dust-induced mitochondrial dysfunction could be targeted via cGAS-STING or cytoplasmic NOX-2 inhibition using microglial cells and brain slice culture models

Abstract

Organic dust (OD) exposure in animal production industries poses serious respiratory and other health risks. OD consists of microbial products and particulate matter and OD exposure induced respiratory inflammation is under intense investigation. However, the effect of OD exposure on brain largely remains unknown. Recently, we have shown that OD exposure of brain microglial cells induces an inflammatory phenotype with the release of mitochondrial DNA (mt-DNA). Therefore, we tested a hypothesis that ODexposure induced secreted mt-DNA signaling drives the inflammation. OD samples were collected from commercial swine operations and a filter sterilized OD extract (ODE) was prepared. Mouse (C57BL/6) microglial cell line was treated with medium or ODE (5%) for 48 hours along with either PBS or mitoapocynin (MA, 10 μM, NOX-2 inhibitor). Microglia treated with control or anti-STING siRNA were exposed to medium or ODE. Next, mouse (C57BL/6) pups were euthanized under an approved protocol, organotypic brain slice cultures (BSCs) were prepared and exposed to medium or ODE with or without MA treatment daily for five days. Culture supernatant, cell pellets and mt-free cytosolic fractions were processed to quantify mt-superoxide, mt-DNA, cytochrome C, TFAM, mitochondrial stress markers and mt-DNA induced signaling via cGAS-STING and TLR9. Data were analyzed using one-way ANOVA and posthoc tests. A p value of ≤ 0.05 was considered significant. ODE exposure increased the mt-superoxide formation andMA treatment decreased the ODE-induced mt-DNA release into cytosol. ODE exposure increased the cytochrome C and TFAM levels. ODE increased MFN1/2 and PINK1 but not DRP1 and MA treatment decreased the MFN2 expression. MA treatment decreased the ODE-exposure induced mt-DNA signaling via cGAS-STING and TLR9. Anti-STING siRNA decreased the ODE-induced increase in IRF3, IFN-β and Iba1 expression. In BSCs, MA-treatment decreased the ODE induced TNF-α, IL-6 and MFN1.Taken together, OD exposure induced mt-DNA signaling could be curtailed through mitochondrial NOX-2 inhibition or STING suppression to reduce neuroinflammation.

Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model

“…TUNEL positive cells were found in the brain after ODE administration, and oral administration of MA(C11) alleviated neurodegeneration by reducing the number of TUNEL positive cells. These results are in agreement with our previous findings from our in vitro and ex vivo models (Massey et al, 2021a ).…”

“…We employed in vitro and ex vivo models (Massey et al, 2021a ) to understand how OD-exposure induces mitochondrial dysfunction. Mitoapocynin (MA) is a mitochondrion targeting cytoplasmic NOX-2 inhibitor with proven effectiveness in reducing toxicant exposure-induced mitochondrial damage (Langley et al, 2017 ).…”

“…Neuroprotective effects of MA(C2) have been demonstrated using in vitro models (Ghosh et al, 2016 ), and the long-acting MA(C11) has been used in in vivo models (Dranka et al, 2014 ; Langley et al, 2017 ). MA treatment of exposed microglia significantly reduced mitochondrial stress responses and prevented secretion of mt-DNA into the cytosol, thereby reducing exposure induced inflammation and mitochondrial damage (Massey et al, 2021a ).…”

Mitochondrial dysfunction in chronic neuroinflammatory diseases (Review)

“…On the one hand, activated microglia contribute to reducing neuroinflammation by phagocytosing and eliminating Aβ, while on the other hand, these microglia release pro-inflammatory cytokines and other inflammatory molecules, thus promoting inflammation ( 86 , 87 ). Notably, emerging research suggests that there is a bidirectional communication between mitochondria and microglia ( 88 , 89 ). Damaged mitochondria release mtDNA fragments into the cytoplasm, which can activate immune responses through Toll-like receptor 9, NLRP3 and stimulator of interferon genes (STING) signaling pathways.…”

Role of Microglia in Sepsis-Associated Encephalopathy Pathogenesis: An Update

“…When mitochondria are damaged, excessive mtDNA is released into the cell, which can further stimulate inflammatory responses in microglia. This mechanism may involve a cyclic GMP-AMP synthase stimulator of interferon gene signaling pathway and cytoplasmic NOX-2 (99). Excessive microglial activation simultaneously causes mitochondrial dysfunction.…”