Role of HMGB1 and mitochondria in organic dust induced airway inflammation

Due to a sharp increase in global demand for protein of animal origin, animal production systems have transformed into industrial-scale operations known as concentrated animal feeding operations (CAFOs). Due to the high animal density within CAFOs, these facilities generate and accumulate various of contaminants such as airborne dust, gases, and microbes. Organic dust (OD) from such large animal confinement facilities is a complex mixture of microbial-associated components and particulate matter known to elicit chronic respiratory diseases in exposed workers. Examination of clinical samples from exposed workers revealed the prevalence of fevers, airway hyperresponsiveness, and an increase in neutrophils, macrophages, and proinflammatory mediators including TNFα, IL-6, and IL-8 (CXCL8) in bronchoalveolar lavage (BAL) fluid. Studies have also shown the release of damage-associated molecular patterns (DAMPs) and activation of multiple overlapping signaling pathways on OD exposure. In the following dissertation, we investigated the role of high mobility group box 1 (HMGB1), a ubiquitously present transcription factor and DAMP, in OD-mediated airway inflammation. HMGB1 has been shown to mediate the activation of innate immune responses and plays a critical role at the intersection of host inflammatory response to sterile and to infectious threats. The goal of our research was to understand the role and impact of HMGB1 in OD-mediated airway inflammation. We show that OD-mediated HMGB1 release amplifies cytokine release and tissue damage. Using experimental strategies that selectively target HMGB1, we effectively reversed activation of specific immune signaling molecules and cytokine release and significantly attenuated damage in OD exposed in vitro and in vivo models. In addition to the myriad of immune signaling and responses, inflammation contributes to cellular structural and functional changes as well. Recently, mitochondria are emerging as therapeutic targets in addition to their essential role in cellular respiration. Emerging evidence shows that exposure to contaminants damages mitochondrial structure and alters function. We identified that OD exposure would induce ultrastructural changes in mitochondria and transcriptional changes in genes encoding proteins related to mitochondrial structure and function. We further investigated how the pathologic (secreted) and physiologic (nuclear) roles of HMGB1 would influence OD-exposure induced mitochondrial dysfunction, and airway inflammation. By using targeted HMGB1 antagonists we identified that HMGB1 could be a critical regulator of mitochondrial structure and function. We showed that neutralization of HMGB1 rescues OD-induced mitochondrial damages at structural and transcriptomic levels. Overall, our results highlight a critical role HMGB1, and mitochondria play in the progression of OD mediated airway inflammation. Identifying a mechanistic correlation between these two factors will likely help develop effective therapeutic strategies.</p

Role of HMGB1 and mitochondria in organic dust induced airway inflammation

Due to a sharp increase in global demand for protein of animal origin, animal production systems have transformed into industrial-scale operations known as concentrated animal feeding operations (CAFOs). Due to the high animal density within CAFOs, these facilities generate and accumulate various of contaminants such as airborne dust, gases, and microbes. Organic dust (OD) from such large animal confinement facilities is a complex mixture of microbial-associated components and particulate matter known to elicit chronic respiratory diseases in exposed workers. Examination of clinical samples from exposed workers revealed the prevalence of fevers, airway hyperresponsiveness, and an increase in neutrophils, macrophages, and proinflammatory mediators including TNFα, IL-6, and IL-8 (CXCL8) in bronchoalveolar lavage (BAL) fluid. Studies have also shown the release of damage-associated molecular patterns (DAMPs) and activation of multiple overlapping signaling pathways on OD exposure. In the following dissertation, we investigated the role of high mobility group box 1 (HMGB1), a ubiquitously present transcription factor and DAMP, in OD-mediated airway inflammation. HMGB1 has been shown to mediate the activation of innate immune responses and plays a critical role at the intersection of host inflammatory response to sterile and to infectious threats. The goal of our research was to understand the role and impact of HMGB1 in OD-mediated airway inflammation. We show that OD-mediated HMGB1 release amplifies cytokine release and tissue damage. Using experimental strategies that selectively target HMGB1, we effectively reversed activation of specific immune signaling molecules and cytokine release and significantly attenuated damage in OD exposed in vitro and in vivo models. In addition to the myriad of immune signaling and responses, inflammation contributes to cellular structural and functional changes as well. Recently, mitochondria are emerging as therapeutic targets in addition to their essential role in cellular respiration. Emerging evidence shows that exposure to contaminants damages mitochondrial structure and alters function. We identified that OD exposure would induce ultrastructural changes in mitochondria and transcriptional changes in genes encoding proteins related to mitochondrial structure and function. We further investigated how the pathologic (secreted) and physiologic (nuclear) roles of HMGB1 would influence OD-exposure induced mitochondrial dysfunction, and airway inflammation. By using targeted HMGB1 antagonists we identified that HMGB1 could be a critical regulator of mitochondrial structure and function. We showed that neutralization of HMGB1 rescues OD-induced mitochondrial damages at structural and transcriptomic levels. Overall, our results highlight a critical role HMGB1, and mitochondria play in the progression of OD mediated airway inflammation. Identifying a mechanistic correlation between these two factors will likely help develop effective therapeutic strategies

Heterogeneous distribution of mitochondria and succinate dehydrogenase activity in human airway smooth muscle cells

Abstract Succinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal‐based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties

TNFɑ Reduces the Maximum Respiratory Capacity of Mitochondria in hASM Cells

Tumor necrosis factor alpha (TNFɑ) is a proinflammatory cytokine that produces inflammation in airway diseases. TNFɑ increases mitochondrial volume density and O2 consumption rate (OCR) in human airway smooth muscle (hASM) cells; however, when normalized for mitochondrial volume density, the OCR per mitochondrion decreases. Our quantitative histochemical technique measures the maximum velocity of the succinate dehydrogenase reaction (SDHmax) in individual hASM cells. We hypothesized that TNFɑ decreases SDHmax per mitochondrion in individual hASM cells. Following TNFɑ treatment, mitochondrial volume density increases, consistent with reduced SDHmax, suggesting increased ATP demand from TNFɑ is met with increased mitochondrial volume density and SDHmax

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.This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at DOI: 10.1007/s00418-021-01978-x. Copyright 2021 The Author(s). Posted with permission

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 PGC1 alpha 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-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-β 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.This pre-print of the article does not reflect post-acceptance improvements or any corrections. The Version of Record is available online at DOI: 10.1007/s00441-021-03422-x. Copyright 2021 The Author(s). Posted with permission

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.This article is published as Massey, Nyzil, Denusha Shrestha, Sanjana Mahadev Bhat, Piyush Padhi, Chong Wang, Locke A. Karriker, Jodi D. Smith, Anumantha G. Kanthasamy, and Chandrashekhar Charavaryamath. "Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model." Frontiers in Cellular Neuroscience 16 (2022): 817046. DOI: 10.3389/fncel.2022.817046. Copyright 2022 Massey, Shrestha, Bhat, Padhi, Wang, Karriker, Smith, Kanthasamy and Charavaryamath. Attribution 4.0 International (CC BY 4.0). Posted with permission

Nrf2 Activation Protects Against Organic Dust and Hydrogen Sulfide Exposure Induced Epithelial Barrier Loss and 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 (HS) 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 HS 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 HS or ODE+HS for one or five days. Cells were also pretreated with vehicle control (DMSO) or RTA-408, a Nrf2 activator. Acute exposure to HS and ODE+HS 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 HS or ODE+HS induced oxidative stress and cytokine production, decreased tight junction protein occludin and cytoskeletal protein ezrin expression, disrupted epithelial integrity and resulted in increased invasion. RTA-408 (pharmacological activator of Nrf2) activated Nrf2 by decreasing keap1 levels and reduced ODE+HS-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 HS