Organic dust induced mitochondrial dysfunction could be targeted via cGAS-STING or mitochondrial NOX-2 inhibition

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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 OD-exposure 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 post-hoc 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.</jats:p>

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

“…All the experiments were conducted in accordance with an approved protocol from the Institutional Biosafety Committee (IBC, protocol# 19-004) of the Iowa State University. Settled swine barn dust (representing OD) samples were collected from various swine production facilities, placed in sealed bags with a desiccant, and transported on ice to the laboratory and prepared as previously described (Massey et al, 2021b ). The prepared stock (100%) was diluted in cell culture medium to prepare a 1% (v/v) solution to use in our experiments ( Supplementary Table 1 ).…”

“…We performed terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay as per our published protocol (Massey et al, 2021b ). Using formaldehyde perfused brain tissues, we prepared paraffin embedded tissue sections (10 μm).…”