Organic Dust Induces Mitochondrial and ER Stress Response in Activated Microglia
Authors: Massey Nyzil , Shrestha Denusha , Mahadev‐Bhat Sanjana , Karriker Locke A , Kanthasamy Anumantha G , Charavaryamath Chandrashekhar 
Published: June 2021

Occupational exposure to animal production units and other industries may create risk for respiratory and other diseases due to exposure to organic dust (OD) and gases. OD is rich in microbial products and particulate matter varying in size. OD induced respiratory diseases and underlying inflammation is being studied but the effect of OD exposure on vital organs such as brain is not fully understood. Microglial cells of the brain are chief immune cells that respond to external dangers and drive neuroinflammation. Cellular homeostasis is largely controlled by organelles such as mitochondria and endoplasmic reticulum (ER). Therefore, we tested a hypothesis that mitochondrial and ER dysfunction is central to microglial driven neuroinflammation upon exposure to OD.

Settled organic dust from commercial swine operations was collected, a sterile organic dust extract (ODE) prepared and filter sterilized. Mouse (C57BL6) microglial cell line were treated with ODE or medium followed by ethyl pyruvate (EP, 2.5 μM) or mitoapocyanin (MA, 10 μM). EP suppresses activations of PKCα, protein kinase IV and the phosphorylation of HMGB1 whereas MA is a mitochondrial‐targeted anti‐oxidant.

24‐hours after treatments, microglial gross morphology and ultrastructural changes were analyzed. Mitochondrial respiration was measured (Seahorse assay) and markers for mitochondrial fusion (MFN1 and MFN2), fission (DRP1) and mitophagy (PINK1 and parkin) and ER stress (XBP1, ATF4 and GRP90) were analyzed (Western blot and qRT‐PCR). Released mitochondrial DNA (mtDNA) and cytosolic cytochrome C were measured. Microglia were stained with Rhod‐2AM dye (intracellular calcium) and Mitotraker (selective mitochondrial dye) to delineate calcium uptake by mitochondria. Following ODE exposure, microglia were stained for Mitosox (mitochondrial superoxide) and caspase 1, 3 and 9 were quantified. Data was analyzed using one‐way ANOVA followed by post‐hoc analysis. A p value of < 0.05 was considered significant.

Compared to control, ODE treated cells appeared activated with increase in size with robust branching. Compared to controls, ODE exposure of microglia impaired the mitochondrial respiration whereas both EP and MA improved mitochondrial respiration significantly. ODE exposure induced mitochondrial hypertrophy and swelling with ER fragmentation and appearance of prominent calcium sequestration bodies. ODE exposure increased the expression of MFN2 and DRP1 along with elevated mitochondrial DNA and cytosolic cytochrome C levels, increased mitochondrial calcium influx and mitochondrial superoxide production. ODE exposure increased expression of spliced XBP1 mRNA indicating ER stress along with increase in caspase 3 and 9 levels. However, EP treatment reduced the microglial activation, cytosolic mtDNA release, and decreased the expression of MFN2, DRP1 and spliced XBP1 mRNA.

Collectively, our data indicate that ODE exposure activates microglia and induces mitochondrial and ER dysfunction, and suggest that ROS and HMGB1 may contribute to the stress response.

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Iowa State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.