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.

Acute exposure to the organophosphate nerve agent (OPNA) soman (GD) triggers severe and persistent brain injury characterized by seizures, oxidative stress

Introduction: Inhalation of organic dust from concentrated animal production operations induces inflammation in the brain and respiratory tract. We investigated the role of the potassium channel Kv1.3 in models of organic dust (OD)-induced neuroinflammation. Kv1.3 channels play multifaceted roles in microglial immune modulation, cancer, and neurodegenerative diseases, and represent a potential therapeutic target. Methods: We used in vivo (C57BL/6 mice), in vitro (microglial cell line, and primary microglia), and ex vivo (brain slice culture) models of OD-induced neuroinflammation. Sterile OD extract (ODE) was prepared, and mice were exposed to either normal saline or ODE intranasally for 5 weeks (5 days/week) to simulate an occupational exposure scenario. Primary microglia were isolated from neonatal mice for total RNA sequencing (RNA-seq). Results: The ODE-induced expression of Kv1.3 was quantified using in vitro and ex vivo models with and without PAP-1 treatment. Exposure-induced changes in cytokines and markers of reactive species were measured. Using western blot, we quantified phosphorylated p38 MAPK (p-p38 MAPK) and NOX2. We measured microglial Kv1.3 currents using whole-cell patch-clamp. Exposure to ODE increased the expression of Kv1.3 and p-p38 MAPK in mouse microglia without affecting the Kv1.3 currents at the cell surface. Exposure increased the levels of inflammatory cytokines and NOX2. Kv1.3 inhibition with PAP-1 decreased inflammatory markers (TNF-α in BV2 microglia and IL-6 in Brain slice cultures and BV2 microglia), levels of Kv1.3, p-p38 MAPK, NOX2, and nitrites. Conclusion: Our study revealed that pharmacological inhibition of Kv1.3 potassium channels reduces ODE-induced neuroinflammation by decreasing inflammatory and oxidative stress markers.