Nrf2 Activation Protects Against Organic Dust and Hydrogen Sulfide Exposure Induced Epithelial Barrier Loss and K. pneumoniae Invasion.

Agriculture workers report various respiratory symptoms owing to occupational exposure to organic dust (OD) and various gases. Previously, we demonstrated that pre-exposure to hydrogen sulfide (H2S) alters the host response to OD and induces oxidative stress. Nrf2 is a master-regulator of host antioxidant response and exposures to toxicants is known to reduce Nrf2 activity. The OD exposure-induced lung inflammation is known to increase susceptibility to a secondary microbial infection. We tested the hypothesis that repeated exposure to OD or H2S leads to loss of Nrf2, loss of epithelial cell integrity and that activation of Nrf2 rescues this epithelial barrier dysfunction. Primary normal human bronchial epithelial (NHBE) cells or mouse precision cut-lung slices (PCLS) were treated with media, swine confinement facility organic dust extract (ODE) or H2S or ODE+H2S for one or five days. Cells were also pretreated with vehicle control (DMSO) or RTA-408, a Nrf2 activator. Acute exposure to H2S and ODE+H2S altered the cell morphology, decreased the viability as per the MTT assay, and reduced the Nrf2 expression as well as increased the keap1 levels in NHBE cells. Repeated exposure to ODE or H2S or ODE+H2S induced oxidative stress and cytokine production, decreased tight junction protein occludin and cytoskeletal protein ezrin expression, disrupted epithelial integrity and resulted in increased Klebsiella pneumoniae invasion. RTA-408 (pharmacological activator of Nrf2) activated Nrf2 by decreasing keap1 levels and reduced ODE+H2S-induced changes including reversing loss of barrier integrity, inflammatory cytokine production and microbial invasion in PCLS but not in NHBE cell model. We conclude that Nrf2 activation has a partial protective function against ODE and H2S.

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

Increased incidences of neuro-inflammatory diseases in the mid-western United States of America (USA) have been linked to exposure to agriculture contaminants. Organic dust (OD) is a major contaminant in the animal production industry and is central to the respiratory symptoms in the exposed individuals. However, the exposure effects on the brain remain largely unknown. OD exposure is known to induce a pro-inflammatory phenotype in microglial cells. Further, blocking cytoplasmic NOX-2 using mitoapocynin (MA) partially curtail the OD exposure effects. Therefore, using a mouse model, we tested a hypothesis that inhaled OD induces neuroinflammation and sensory-motor deficits. Mice were administered with either saline, fluorescent lipopolysaccharides (LPSs), or OD extract intranasally daily for 5 days a week for 5 weeks. The saline or OD extract-exposed mice received either a vehicle or MA (3 mg/kg) orally for 3 days/week for 5 weeks. We quantified inflammatory changes in the upper respiratory tract and brain, assessed sensory-motor changes using rotarod, open-field, and olfactory test, and quantified neurochemicals in the brain. Inhaled fluorescent LPS (FL-LPS) was detected in the nasal turbinates and olfactory bulbs. OD extract exposure induced atrophy of the olfactory epithelium with reduction in the number of nerve bundles in the nasopharyngeal meatus, loss of cilia in the upper respiratory epithelium with an increase in the number of goblet cells, and increase in the thickness of the nasal epithelium. Interestingly, OD exposure increased the expression of HMGB1, 3- nitrotyrosine (NT), IBA1, glial fibrillary acidic protein (GFAP), hyperphosphorylated Tau (p-Tau), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-positive cells in the brain. Further, OD exposure decreased time to fall (rotarod), total distance traveled (open-field test), and olfactory ability (novel scent test). Oral MA partially rescued olfactory epithelial changes and gross congestion of the brain tissue. MA treatment also decreased the expression of HMGB1, 3-NT, IBA1, GFAP, and p-Tau, and significantly reversed exposure induced sensory-motor deficits. Neurochemical analysis provided an early indication of depressive behavior. Collectively, our results demonstrate that inhalation exposure to OD can cause sustained neuroinflammation and behavior deficits through lung-brain axis and that MA treatment can dampen the OD-induced inflammatory response at the level of lung and brain.

Transcriptomic and ultrastructural evidence indicate that anti-HMGB1 antibodies rescue organic dust-induced mitochondrial dysfunction.

Exposure to organic dust (OD) in agriculture is known to cause respiratory symptoms including loss of lung function. OD exposure activates multiple signaling pathways since it contains a variety of microbial products and particulate matter. Previously, we have shown how OD exposure leads to the secretion of HMGB1 and HMGB1-RAGE signaling, and how this can be a possible therapeutic target to reduce inflammation. Cellular mitochondria are indispensable for homeostasis and are emerging targets to curtail inflammation. Recently, we have also observed that OD exposure induces mitochondrial dysfunction characterized by loss of structural integrity and deficits in bioenergetics. However, the role of HMGB1 in OD-induced mitochondrial dysfunction in human bronchial epithelial (NHBE) cells remains elusive. Therefore, we aimed to study whether decreased levels of intracellular HMGB1 or antibody-mediated neutralization of secreted HMGB1 would rescue mitochondrial dysfunction. Single and repeated ODE exposure showed an elongated mitochondrial network and cristolysis whereas HMGB1 neutralization or the lack thereof promotes mitochondrial biogenesis evidenced by increased mitochondrial fragmentation, increased DRP1 expression, decreased MFN2 expression, and increased PGC1α expression. Repeated 5-day ODE exposure significantly downregulated transcripts encoding mitochondrial respiration and metabolism (ATP synthase, NADUF, and UQCR) as well as glucose uptake. This was reversed by the antibody-mediated neutralization of HMGB1. Our results support our hypothesis that, in NHBE cells, neutralization of ODE-induced HMGB1 secretion rescues OD-induced mitochondrial dysfunction.

Organic dust exposure induces stress response and mitochondrial dysfunction in monocytic cells.

Exposure to airborne organic dust (OD), rich in microbial pathogen-associated molecular patterns (PAMPs), is shown to induce lung inflammation. A common manifestation in lung inflammation is altered mitochondrial structure and bioenergetics that regulate mitochondrial ROS (mROS) and feed a vicious cycle of mitochondrial dysfunction. The role of mitochondrial dysfunction in other airway diseases is well known. However, whether OD exposure induces mitochondrial dysfunction remains elusive. Therefore, we tested a hypothesis that organic dust extract (ODE) exposure induces mitochondrial stress using a human monocytic cell line (THP1). We examined whether co-exposure to ethyl pyruvate (EP) or mitoapocynin (MA) could rescue ODE exposure induced mitochondrial changes. Transmission electron micrographs showed significant differences in cellular and organelle morphology upon ODE exposure. ODE exposure with and without EP co-treatment increased the mtDNA leakage into the cytosol. Next, ODE exposure increased PINK1, Parkin, cytoplasmic cytochrome c levels, and reduced mitochondrial mass and cell viability, indicating mitophagy. MA treatment was partially protective by decreasing Parkin expression, mtDNA and cytochrome c release and increasing cell viability.

Ambient hydrogen sulfide exposure increases the severity of influenza A virus infection in swine.

Hydrogen sulfide (H2S) is common in concentrated pig feed operations from the decomposition of manure. Ambient H2S is a respiratory tract irritant and an environmental stressor for caretakers and pigs. Influenza A virus (IAV), a zoonotic pathogen, has caused prior pandemics. The effects of H2S or IAV alone on the respiratory system have been investigated, but their interaction has not. We hypothesized that exposure to environmentally-relevant H2S concentrations increases the pathogenicity of IAV infection in swine. Thirty-five, three-week old pigs of mixed sex were exposed to breathing air or H2S via inhalation 6 hours daily for 12 days. After 7 days, pigs were inoculated with H3N2 IAV (or a placebo). Results showed that ambient H2S increased the severity of respiratory distress and lung pathology. H2S also suppressed IL-IL-1ß, IL-6 and IL-8 cytokine response in BALF and increased viral loads and nasal shedding.

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

Organic dust (OD) exposure in animal production industries poses serious respiratory and other health risks. OD consisting of microbial products and particulate matter and OD exposure-induced respiratory inflammation are under investigation. However, the effect of OD exposure on brain remains elusive. We show that OD exposure of 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. A mouse microglial cell line was treated with medium or organic dust extract (ODE, 1% v/v) along with either phosphate-buffered saline (PBS) or mitoapocynin (MA, 10 µmol). Microglia treated with control or anti-STING siRNA were exposed to medium or ODE. Mouse organotypic brain slice cultures (BSCs) were exposed to medium or ODE with or without MA. Various samples were processed to quantify mitochondrial reactive oxygen species (mt-ROS), mt-DNA, cytochrome c, TFAM, mitochondrial stress markers and mt-DNA-induced signaling via cGAS-STING and TLR9. Data were analyzed and a p value of ≤ 0.05 was considered significant. MA treatment decreased the ODE-induced mt-DNA release into the cytosol. 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-ß and IBA-1 expression. In BSCs, MA treatment decreased the ODE-induced TNF-α, IL-6 and MFN1. Therefore, OD exposure-induced mt-DNA signaling was curtailed through cytoplasmic NOX-2 inhibition or STING suppression to reduce brain microglial inflammatory response.

Pre-exposure to hydrogen sulfide modulates the innate inflammatory response to organic dust.

Animal production units produce and store many contaminants on-site, including organic dust (OD) and hydrogen sulfide (H2S). Workers in these settings report various respiratory disease symptoms. Both OD and H2S have shown to induce lung inflammation. However, impact of co-exposure to both H2S and OD has not been investigated. Therefore, we tested a hypothesis that pre-exposure to H2S modulates the innate inflammatory response of the lungs to organic dust. In a mouse model of H2S and organic dust extract (ODE) exposure, we assessed lung inflammation quantitatively. We exposed human airway epithelial and monocytic cells to medium or H2S alone or H2S followed by ODE and measured cell viability, oxidative stress, and other markers of inflammation. Exposure to 10 ppm H2S followed by ODE increased the lavage fluid leukocytes. However, exposure to 10 ppm H2S alone resulted in changes in tight junction proteins, an increase in mRNA levels of tlr2 and tlr4 as well as ncf1, ncf4, hif1α, and nrf2. H2S alone or H2S and ODE exposure decreased cell viability and increased reactive nitrogen species production. ODE exposure increased the transcripts of tlr2 and tlr4 in both in vitro and in vivo models, whereas increased nfkbp65 transcripts following exposure to ODE and H2S was seen only in in vitro model. H2S alone and H2S followed by ODE exposure increased the levels of IL-1ß. We conclude that pre-exposure to H2S modulates lung innate inflammatory response to ODE.