Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds

Background: Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin (IL)-1β. IL-1β plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders. Objective: The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and IL-1β processing. Method: Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, IL-1β enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays. Results: TETBr and TMTOH induced inflammasome aggregate formation and IL-1β release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or LPS+ATP challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways. Conclusion: Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314


Introduction
Since the mid-1900s, an interest in the role of mitochondria in chemical-induced toxicity has developed primarily based on studies demonstrating alterations in oxidative phosphorylation following exposure to a heterogenous group of substances, including triorganotin and tri-organolead compounds (Aldridge et al. 1977;Powers and Beavis 1991). This interest further developed with the implication of mitochondrial function as a target of environmental toxicants (Caito and Aschner 2015;Delp et al. 2019;Meyer et al. 2013;Shaughnessy et al. 2014;Xia et al. 2018). This, as well as work establishing mitochondrial deficits across various human diseases (Khan et al. 2015;Javadov et al. 2020) and modeled in the mouse (Wallace and Fan 2009) led to consideration of mitochondrial dysfunction as a key characteristic of toxicity and prompted the formulation of mitochondrial-based adverse outcome pathways (Dreier et al. 2019;Goodchild et al. 2019) for which causative links over the biological hierarchical processes are of current interest.
Increasing evidence suggests a role for metabolic remodeling by mitochondria in controlling the maintenance and establishment of innate and adaptive immune responses (Viola et al. 2019). Mitochondrial dysfunction occurs in many disease or injury models and is thought to propagate injurious inflammation through inducing mitochondrial reactive oxygen species (mtROS) and releasing endogenous mitochondrial damage-associated molecular patterns (DAMPs), which activate innate immune receptors (Nakahira et al. 2011;Zhou et al. 2011;Shimada et al. 2012), contributing to sustained inflammation (Dela Cruz and Kang 2018). Cross-communication between mitochondrial dysfunction and inflammation has been implicated owing to shared cellular processes (Johansson et al. 2010;Liu et al. 2018b;Mathur et al. 2018;Tschopp 2011). A central mechanism whereby damaged mitochondria propagate inflammation is the activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor 3 (NLRP3) inflammasome. Inflammasomes are multiprotein complexes that involve the common adaptor, apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) apoptosisassociated speck-like protein containing a CARD (ASC), and an effector, caspase-1. In the cytosol of immune cells, this complex forms in response to pathogen-associated molecular patterns or DAMPs that are released upon tissue injury and serves as a key sensor and effector of inflammation (Franchi et al. 2009;Ghiringhelli et al. 2009;Gross et al. 2011). Activated inflammasomes induce caspase-1-dependent processing of the pro-inflammatory cytokines interleukin ðILÞ-1b and IL-18 (Broz and Dixit 2016;Ghiringhelli et al. 2009), which play critical roles for host defense actions against invading factors and in coordinating the inflammatory response (Dinarello 2009;Sims and Smith 2010). The need to maintain this tightly regulated process is demonstrated in the number of inflammatory disorders or multifactorial diseases. NLRP3 inflammasome activation releases IL-1b for host defense actions that are critical to the overall health status of the organism. However, activation can also be associated with rare heritable inflammasomopathies and related diseases that cause excessive IL-1b activation and with a loss of immune regulation in common pathologies, such as cancer, cardiometabolic disease, neurological disorders, and diabetes (Dinarello 2011;Franchi et al. 2009;Kaneko et al. 2019;Swanson et al. 2019;Walsh et al. 2014). Thus, activation or dysregulation of this process can result in a broad spectrum of health effects underlying various disease processes.
Of the canonical inflammasomes, NLRP3 inflammasome is broadly sensitive to exogenous and endogenous activators. NLRP3 inflammasome activation generally requires prior transcriptional cell priming by an activating ligand. This priming step typically involves a nuclear factor kappa-light-chain-enhancer of activated B cells ðNF-jBÞ-dependent) up-regulation of cellular NLRP3, pro-IL-1b transcription, and de novo protein synthesis upon recognition of pro-inflammatory stimuli and Toll-like Receptor (TLR) activation (Bauernfeind et al. 2009;Franchi et al. 2009). Once primed, NLRP3 activation can be induced by a variety of extracellular, sterile, nonpathogenic triggers (Cassel et al. 2009;Dostert et al. 2008;Hughes and O'Neill 2018;Strowig et al. 2012) that work through activating purinergic receptors or ionic membrane pore alterations (see the schematic representation in Figure 1). These sterile activators include cholesterol and uric acid crystals (Duewell et al. 2010;Martinon et al. 2006), aggregated proteins and lipids (Ralston et al. 2017;Sheedy et al. 2013), silica and asbestos (Dostert et al. 2008), aluminum salt adjuvant (Eisenbarth et al. 2008), and polystyrene nanoparticles (Lunov et al. 2011). In addition to these extracellular signaling factors, mitochondrial dysfunction may elicit an inflammasome response upon the release of mitochondrial DNA (Liu et al. 2018b). A number of molecules can directly or indirectly interact with different components of the NLRP3 inflammasome and impede complex assembly. In addition, negative regulatory molecules targeting ion efflux, mitochondrial function, and ROS signaling can block NLRP3 inflammasome Figure 1. Representative schematic of NLRP3 inflammasome priming and activation (Deets and Vance 2021;Guo et al. 2015;Kelley et al. 2019;Martinon et al. 2002). The initial priming signal (A) is provided by activation of cytokines or pathogen-associated molecular patterns (PAMPs) and signaling through Toll-like receptors (TLRs), tumor necrosis factor receptors (TNFRs), and the IL-1 receptor 1 (IL-1R1), lipopolysaccharide (LPS), resulting in transcriptional up-regulation of canonical and noncanonical NLRP3 inflammasome components. The second, triggering, signal (B) then serves to trigger the response and can be provided by any number of PAMPs or damage-associated molecular patterns (DAMPs). These include pore-forming toxins to allow K + efflux, particulates and crystals (b-amyloid, uric acid, silica) that are taken up into the lysosome, and adenosine triphosphate (ATP) as a purinergic type 2 receptor 7 (P2X7) ligand. These signals can activate multiple signaling events such as K + efflux, Ca 2+ flux, lysosomal disruption, and mitochondrial reactive oxygen species (ROS) production, which can lead to NLRP3 inflammasome activation (dashed arrows). Oligomerization of the components to form the inflammasome activates caspase-1 that then cleaves pro-IL-1b and pro-IL-18. Activation leads to the release of mature IL-1b and IL-18, pore formation, pyroptosis, and/or release of exosomes or ASC specks. Note: ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; IjBa, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; p-IjBa, phosphorylated IjBa; Ijj, inhibitor of nuclear factor-jB kinase; IL, interleukin; LBP, lipopolysaccharide binding protein; MDP, muramyl dipeptide; NLRP3, nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing receptor 3. activation (Zheng et al. 2020). Thus, in addition to functioning as a direct activator, changes in mitochondrial function or induction of ROS can serve as underlying cellular mechanisms for NLRP3 activation induced by multiple, disparate triggering stimuli (Nakahira et al. 2011;Shimada et al. 2012;Tschopp and Schroder 2010;Zhou et al. 2011).
Given the complex interaction between mitochondria and inflammation, we hypothesized that mitochondrial dysfunction induced by environmental toxicants represents a mechanism leading to altered immune cell functioning and that triggering of the NLRP3 inflammasome serves as one step along the biological pathway leading to an adverse health outcome. Chemical-induced alterations in mitochondrial membrane potential (MMP) have been used to identify mitochondrial toxicants by Tox21 screening (Attene-Ramos et al. 2015). We considered whether they may be further classified based upon NLRP3 inflammasome activation and IL-1b production and thus provide a better characterization. To examine this possible association, a class of compounds were selected based upon effects on oxidative phosphorylation and immune cell stimulation/dysfunction and with evidence of in vivo toxicity. Several of the tri-organotins, triphenyltin (TPT), triethyltin (TET), trimethyltin (TMT), and tributyltin (TBT), met the criteria of altered mitochondrial (Aldridge et al. 1977;1981;Davidson et al. 2004;Gennari et al. 2000;Nesci et al. 2011;Powers and Beavis 1991;Snoeij et al. 1987) and human and murine immune cell functions (Benya 1997;Brown et al. 2018;Ferraz da Silva et al. 2018;Gomez et al. 2007;Lawrence et al. 2016;Nunes-Silva et al. 2018;Pestka and Zhou 2006;Van Loveren et al. 1990;Whalen et al. 1999;Wu 2019). For example, altered immune function has been reported for TBT with decreased peripheral lymphocytes (Attahiru et al. 1991;Snoeij et al. 1988;Ueno et al. 2009) and thymus atrophy (Snoeij et al. 1988) in rats, T-cell development in mice (Im et al. 2015), macrophage phagocytic activity, intestinal mucosal immune response, and B-lymphocyte proliferation in cod (Harford et al. 2007), and antibody production to immune challenge in catfish (Regala et al. 2001). TPT and/or TMT disrupted lytic function of human natural killer cells in vitro (Gomez et al. 2007;Holloway et al. 2008) and delayed-type hypersensitivity reactions in rats (Snoeij et al. 1985) and exacerbated a proinflammatory response in lipopolysaccharide (LPS)-primed murine macrophages (Pestka and Zhou 2006). TMT and TET demonstrated an inflammatory component associated with neurotoxicity in rodent models (Harry et al. 2003;Kraft et al. 2016;Long et al. 2019;Röhl et al. 2009;Sandström et al. 2019). The implication of immunodeficiency following TBT exposure and the stimulation of neuroimmune cells with TET and TMT, as well as the documented effects on oxidative phosphorylation, suggested the possibility that, in the absence of a direct effect, an inflammatory response could be elicited by alternative processes, such as inflammasome activation. Given the critical role of macrophage-type cells in immune-related responses, we examined the ability of these selected tri-organotins to serve as secondary triggers for NLRP3 inflammasome activation and IL-1b processing. We further examined cells for mitochondrial dysfunction, ROS production, and microRNA (miRNA) and mRNA gene expression profiles to characterize differences across the two tri-organotin compounds-TET and TMT-to investigate the potential to trigger mature IL-1b release.  (Taciak et al. 2018), were maintained in Dulbeccos's Modified Eagle's Medium (DMEM; L-glutamine and sodium pyruvate; #11995-065 Gibco; ThermoFisher) supplemented with 10% fetal bovine serum (FBS; <0:25 EU=mL; Catalog no. 100-108; Lot #A22GOOJ; Gemini Bio-Products) and 100 U=mL penicillin/streptomycin (#P0781; Sigma-Aldrich). Unless otherwise stated, cells were plated in tissue culture plates (Corning) at 20,000 cells/well (62,500 cells=cm 2 ; 96-well); 300,000 cells/well (150,000 cells=cm 2 ; 24-well); 1,000,000 cells/ well (105,000 cells=cm 2 ; 6-well) and allowed to adhere overnight prior to experimental manipulation. Cells were not allowed to become more than 80% confluent in order to prevent loss of the flattened adherent cell characteristic. Cells were maintained at 37°C, 5% CO 2 =5% O 2 , 90% humidity (Nu-5831 tri-gas incubator; Nuaire). Immediately prior to dosing, media volume was decreased by 50%. Corning black-walled optical plates were used for fluorescent imaging.

Primary Bone Marrow-Derived Macrophages
C57BL/6J male mice (6-to 12-wk-old; IMSR Catalog no. JAX:000664, RRID:IMSR_JAX:000664) were euthanized under CO 2 , the femur excised, bone marrow extruded, and primary bone marrow-derived macrophages (BMDMs) collected (Trouplin et al. 2013). Animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals following an approved animal protocol from the National Institute of Environmental Health Sciences Animal Care and Use Committee. Isolated cells were initially plated 1,000,000 cells/well per 12-well plate in 3 mL DMEM (Gibco #11965-092; 10% FBS, 100 U=mL penicillin/streptomycin) supplemented with 10% L929 DMEM conditioned-media (CM) as a source of macrophage colony stimulating factor to induce hematopoietic cell differentiation into macrophages. Cells were allowed to adhere at 37°C, 5% CO 2 =5% O 2 , 90% humidity (Nu-5831 tri-gas incubator; Nuaire), followed by half DMEM/CM change on days in vitro 3 (DIV3) and full change to DMEM on DIV5 to remove nonmacrophage contaminating cells. On DIV6, a half-media change occurred with tri-organotin addition.

Exposure Levels
Human levels for TBT and TPT have been detected in the nanograms-per milliliter range (TBT ranging in whole blood between <0:01 and 85 and in serum between <0:02 and 0.05; TPT in whole blood between <0:4 and 0.56 and in serum between 18 and 0.67) (Sousa et al. 2014). Occupational exposure to TMT in 216 factory workers (personal air sampling over 8 h detected between 0.008 and 0:028 mg TMT=m 3 ) resulted in the detection of TMT in 61 workers ranging up to 0:264 lg=mL in plasma and 211:8 mg=g creatine in urine (Tang et al. 2013). The limited data available for TET estimated the toxic dose for an adult to be ∼ 70 mg of TET over 8 d (Barnes and Stoner 1959) and in children, ∼ 4:5 mg (Fontan et al. 1955). In vitro assessments of these tri-organotins included primary human cortical neurons or astrocytes after 24-h exposure to 10 −3 M TMTBr or 10 −5 M TETCl, maintaining >80% viability (Cristòfol et al. 2004). In primary hippocampal neurons, normal viability was maintained over 24 h with 1 lM TMT (Hou et al. 2017). In primary rat microglia, median lethal concentration values of 24:8 lM for TMT, 4:3 lM for TET, and 0:7 lM for TBT with 24 h were determined, with 80% viability observed at 2 lM TMT or 1 lM TET (Röhl et al. 2009). In LPS-primed RAW 264.7 cells, 6-h exposure to 1 lM TPT decreased Tnfa but not cell viability (Pestka and Zhou 2006) and Sandström et al. (2019) reported normal cell viability and an increase in Il6 and Inos in BV-2 cells at 1 lM TMTCl for 24 h and limited alterations in three-dimensional brain cell cultures with 0.5 or 1 lM for 10 d.

Cell Viability
Cell viability was determined using CyQuant Direct Cell Proliferation Assay (C35013; Molecular Probes). Cells were incubated with CyQuant reagent (37°C; 60 min) and measurements read at 508 nm excitation=562 nm emission (BioTek Synergy four-plate reader). The live cell number was estimated based upon a standard curve and calculated as the percentage of the control. Cell viability of ∼ 75-85% was used to select dose levels for further study.

IncuCyte Tracking of ASC Oligomerization
ASC speck formation has been effectively used as a readout for inflammasome activation (Stutz et al. 2013). Real-time imaging of NLRP3 inflammasome aggregate assembly was conducted using a RAW 264.7-ASC reporter cell line, as previously described (Bowen et al. 2020). HEK293T/17 cells (ATCC #CRL-11268; RRID: CVCL_1926) were transiently transfected with pMD2G (RRID: Addgene_12259), pUMVC (RRID:Addgene_8449) and retroviral transfer vector pRP-ASC-ESCBLerulean (RRID:Addgene_41840) using Invitrogen Lipofectamine 2000 (#1168019; ThermoFisher) (Barde et al. 2010). To determine titers (transducing units per milliliter), HEK293Ts cells (50,000 cells/well 6-well plate) were incubated at 37°C, 5% CO 2 for 24 h. The media was replaced and 10 lL of nonconcentrated or 0:5 lL of concentrated virus added. The media was replaced at 24 and 72 h after infection. At 5-d postinfection, chromosomal DNA was isolated using DNeasy Blood and Tissue Kit (#69506; Qiagen), diluted 1:40, and 5 lL diluted DNA was used in qPCR. Diluted retro/lentiviral plasmids and human genomic DNA were used to create standard curves for qPCR analysis. The following primers for gag (lentiviruses, forward: 5 0 -GGAGCTAGAACGATTCGCAGTTA; and reverse: 5 0 -GGTT-GTAGCTGTCCCAGTATTTGTC), psi (retroviruses, forward: 5 0 -GCAGCATCGTTCTGTGTTGT; and reverse: 5 0 -GCTCGA-CATCTTTCCAGTGA), and actin (forward: 5 0 -TCCGTGTGG-ATCGGCGGCTCCA; and reverse 5 0 -CTGCTTGCTGATCCA-CATCTG) were used to with SYBR green to perform qPCR using Roche LightCycler 96 Instrument, and LightCycler 96 software was used to extrapolate the copies of gag/psi and actin per sample. In addition, the cells were fixed in 1% formalin and flow cytometry (BD LSRFortessa cell analyzer) was used to determine the percentage of fluorescent cells. Gag/psi copies relative to actin copies/two per cell were calculated and the titer was determined per copies in 100,000 cells times the virus dilution factor. Post-48-h transfectionsupernatant was used for the transduction of RAW 264.7 cells (Stutz et al. 2013). Cell culture conditions were identical to normal RAW 264.7 cells. Cells were exposed to PBS or LPS (33 ng=mL) for 3 h, then to TETBr (10 lM), TMTOH (1:25 lM), or ATP (5 mM). To eliminate extracellular ATP and inhibit activation of purinergic receptors (P2XRs), apyrase (5 U=mL), an enzyme that converts ATP to ADP, was added to each well 1 h prior to organotin. Timelapse images were obtained using an IncuCyte S3 live-cell analysis system (20-manification objective) (Sartorius) by IncuCyte ZOOM 2015A software and formation of ASC aggregates was determined (Stutz et al. 2013). Regions of interest (ROI) images were standardized by total number of cells, and threshold for ASC speck size and intensity, and ASC speck number was determined using Fiji ImageJ (Schindelin et al. 2012).

Nitrite Production
Nitrite accumulation in culture medium was measured as an indirect indicator of nitric oxide synthesis using a GREISS Reagent kit (G2930; Lot #122106; Promega). Briefly, a 50-lL aliquot of 100 lL phenol-free cell media and 50 lL sulfanilamide were held (RT; dark; 10 min), followed by 50 lL N-1-napthylethylenediamine (RT; dark; 10 min). Absorbance at 548 nm was recorded (BioTek Synergy four-plate reader). Background-corrected data was calculated relative to a sodium nitrite standard curve.
Immuno-Spin Trapping 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) reacts with secondary radicals to form nitrone adducts that remain covalently attached as radical reporters and immune-spin trapping allows for stabilization for antibody identification (Ramirez and Mason 2005). DMPO-tagged secondary radicals were determined in whole-cell fractions and mitochondrial fractions of RAW 264.7 cells. Cells were expanded to 80-90% confluency in a T75 flask. Media was changed to serum-free DMEM media containing 40 mM DMPO (D048; Lot #GB635; Donjindo Molecular Technologies) for 30 min prior to exposure to either PBS, TMTOH, or TETBr for 6 h. Cells were detached with 0.25% trypsin, centrifuged (200 × g; 10 min; RT), rinsed, and resuspended in 1 mL PBS. A 200-lL cell lysate aliquot was collected and stored at −80 C for <2 d. Mitochondria was isolated by differential centrifugation (Darley-Usmar et al. 1987). Cells were resuspended in ice-cold mitochondria isolation buffer {10 mM HEPES (pH 7.4), 250 mM sucrose, 10 mM 2-[2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid (pH 7.4), and 5 mg=mL bovine serum albumin (BSA)}. The solution was transferred into a chilled 0:15-mm Dounce tissue homogenizer and after 12 strokes at 600 rpm, cellular debris and unbroken cells were pelleted from the solution by centrifugation at 1,000 × g for 10 min at 4°C. Cellular debris and unbroken cells were pelleted from the solution by centrifugation (500 × g; 10 min; 4°C). The supernatants, containing isolated mitochondria, were transferred into a new tube and centrifuged for 10 min at 10,000 × g and 4°C. The pellets containing isolated mitochondria were washed once with ice-cold isolation buffer without BSA. The final mitochondrial pellet obtained was resuspended in the isolation buffer containing protease inhibitors and kept on ice. Eight hundred-microliter mitochondrial aliquots were centrifuged (200 × g; 10 min; RT), and the pellets were stored at −80 C for <2 d. Protein concentrations were determined by Pierce BCA assay (ThermoFisher). Nitrone adducts were detected by DMPO ELISA (Ramirez and Mason 2005;Taetzsch et al. 2015). Briefly, a 96-well ELISA plate (NUNC MaxiSorp; ThermoFisher Scientific) was coated with the sample (100 lg=well) at 4°C overnight. The plate was blocked with a 1% casein solution (Sigma-Aldridge) and 5% sucrose in PBS for 2 h. Anti-DMPO (1:1,000 in 1% casein/PBS) was applied for 1 h, washed three times with PBS, followed by HRP-antirabbit (1:1,000 in 1% casein/PBS). The wells were washed three times with PBS and 3,3 0 ,5 0 5-tetramethylbenzidine liquid substrate (100 lL; Sigma-Aldridge) was applied for 20 min. The reaction was stopped with 50 lL of 2N sulfuric acid solution, and the plate was read at 450Å on a Tecan plate reader (Tecan US).

Seahorse Flux Analyzer
Cells were plated in a Seahorse XF96 microplate (25,000 cells/ well; 80 lL DMEM/10% FBS/antibiotics; Agilent Technologies) and allowed to adhere for 1 h, then incubated for 20 h under standard conditions. Cells were exposed to PBS, TETBr (1.25 or 10 lM), or TMTOH (1:25 lM) for 6 h. The media was removed and replaced with 180 lL Seahorse XF media supplemented with 4:5 g=L glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (pH 7.4) and maintained under ambient air conditions at 37°C for 1 h. Oxygen consumption rates (OCRs) were measured using an XF96 Extracellular Flux Analyzer (Agilent Technologies). Sensor cartridges delivered a final concentration of 0:9 lM oligomycin, 0:75 lM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 lM rotenone for the mitochondrial stress test using Agilent Wave software (version 2.4.0). Prior to and immediately following the assay, cells were visualized by light microscopy to confirm cell viability and culture uniformity.

miRNA and mRNA Arrays
Based on the comparable ability to serve as a secondary trigger for mature IL-1b release, we examined the response of primary LPSprimed BMDMs to a 6-h exposure to TMTOH (1:25 lM) or TETBr (10 lM) for associated molecular profiles. Total RNA was isolated from three samples (6 × 10 6 cells) using TRIzol (Invitrogen), treated with 2:5 lL DNase I and 10 lL Buffer RDD (#79254, RNase-Free DNase Set; Qiagen) (RT; 10 min), followed by column separation (#74104, RNeasy mini prep; Qiagen). miRNA analysis was conducted using Affymetrix GeneChip (version 4; Affymetrix) following the Affymetrix hybridization protocols. Total RNA (110 ng) was labeled using Affymetrix FlashTag Biotin HSR, according to the manufacturer's protocol. On average, 14:1 lg of labeled sample was hybridized (16 h; 48°C) in a rotating hybridization oven using Affymetrix Eukaryotic Target Hybridization Controls and protocol. On the same samples, mRNA analysis was conducted using Affymetrix Mouse Genome 430 2.0 GeneChip arrays (Affymetrix). Total RNA (50 ng) was amplified (Nugen WT-Ovation Pico RNA Amplification System) and labeled (Nugen Encore Biotin Module protocol). Amplified biotin-cDNAs (5:5 lg) were fragmented and hybridized (16 h; 45°C). Slides were stained with streptavidin/phycoerythrin using a double antibody staining procedure and then washed for antibody amplification (GeneChip Hybridization; Wash and Stain Kit). Arrays were scanned (Affymetrix Scanner 3000) and data were obtained using Transcriptome Analysis Console software (version 4.0). miRNA and RNA Expression Array Data Analysis miRNA data were preprocessing and normalized using Affymetrix Expression Console (build 1.4.1.46). Only mouse miRNAs were retained for downstream analysis. The mouse Genome 430 array data were preprocessing and normalized using the Affymetrix package. The Robust Multichip Analysis (RMA) approach was applied for normalization. To identify differentially expressed miRNAs and mRNAs, the empirical Bayes moderated t-statistics method implemented in the limma package (Ritchie et al. 2015) was performed. The fold-change differences of probe-sets expression were calculated based on normalized log 2 -transformed RMA values. p-Values were adjusted (Benjamini and Hochberg 1995) to control false discovery rate and correct for multiple hypothesis testing. miRNAs with a p ≤ 0:05 and absolute fold-change >1:5 and mRNA probe sets with an adjusted p ≤ 0:05 and absolute foldchange >2 were considered as differentially expressed. Predicted and validated miRNA-binding sites of known genes were determined using miRWalk (http://mirwalk.umm.uni-heidelberg.de) and miRDB (http://mirdb.org) programs. Targeted enrichment analyses were carried out with Qiagen Ingenuity Pathway Analysis software to test for pathway or functional group in significantly differentially expressed genes. Nontargeted enrichment analysis was conducted by gene set enrichment analysis to evaluate whether genes in a gene set were enriched at the top or bottom of the entire expression data as ranked by fold-change. Statistical analyses were performed using R/Bioconductor. Data were deposited in the National Center for Biotechnology Information Gene Expression Ominbus (GSE155128).

Statistical Analysis
Statistical analyses were performed using GraphPad Prism software (version 8.4,GraphPad Software,Inc.). In experiments where ATP or LPS were used as assay positive controls, that data was not included in the analyses. Data from experiments comparing across tri-organotin exposure in non-LPS-primed cells were evaluated using a one-way analysis of variance (ANOVA). Data from experiments examining the response of cells to LPS following triorganotin exposure or in assessing the response to tri-organotins in LPS-primed cells were analyzed by a two-way ANOVA with triorganotin and LPS as factors. ASC speck formation was analyzed by repeated measures ANOVA. Independent group mean comparisons were conducted with Tukey's or Dunnett's multiple comparisons tests. Bartlett's test was used for homogeneity of variance. ATP-linked respiration and maximum respiration were analyzed by a Kruskal-Wallis test followed by a Dunn's test. Significant p-values are listed as adjusted p-values where appropriate. All experiments were run with technical duplicates or triplicates, and we confirmed findings across at least two independent experiments. Group sample sizes are stated in the figure legends.

TNFa Protein Release
To determine whether tri-organotin compounds induced a proinflammatory response, levels of TNFa and IL-1 protein released into the media were determined after 6 h of exposure. Cells exposed to TPTOH or Bis(TBT)Ox showed levels of TNFa similar to that observed with the PBS vehicle. Slightly higher protein levels (12%) were observed in cells exposed to TMTOH (p = 0:002) or TETBr (p = 0:022) as compared with controls ( Figure 3A). LPS significantly elevated TNFa levels across all groups (p < 0:0001); however, in comparison to PBS, elevations in response to LPS exposure were ∼ 50% lower following subsequent exposure to TMTOH (p < 0:0001) or TETBr (p < 0:0002). In LPS-primed cells, TNFa was ∼ 50% higher in cells exposed to TPTOH (p < 0:0001). No difference was observed with Bis(TBT)Ox.

IL-1 Protein Release
NLRP3 inflammasome activation induces the cellular release of mature IL-1b (Martinon et al. 2002). As an initial evaluation, HEK-Blue IL-1R cells were used as an indicator of released bioactive IL-1 (IL-1a and IL-1b) from RAW 264.7 cells. A higher level of total IL-1 was observed with the positive control, within range of the maximum level of the assay. Levels following tri-organotin exposure were not significantly different from PBS exposure ( Figure  3B). IL-1b ELISA was next used to determine the release of total IL-1b into the medium of RAW 264.7 cells. No increases were observed in non-LPS-primed cells exposed to the tri-organotins ( Figure 3C) In LPS-primed cells, however, significant higher levels ( ∼ 2-fold) were observed with TMTOH (p < 0:0001) and TETBr (p = 0:0262), and a 10-times higher level with the NLRP3 activator, ATP (p < 0:0001) ( Figure 3C). No elevations were observed with TPTOH or Bis(TBT)Ox.
In the absence of elevations in total IL-1b with TPTOH or Bis(TBT)Ox in LPS-primed cells, Western blots to discriminate between pro-(37 kD) and mature (17 kD) IL-1b protein were conducted for TMTOH and TETBr only. In RAW 264.7 cells, IL-1b was not observed in either the supernatant or lysate in the absence of LPS-priming ( Figure 3D). With LPS-priming, triggering of NLRP3 inflammasome activation with the positive control, ATP, induced a release of mature IL-1b within 30 min. A secondary exposure to TMTOH or TETBr resulted in the release of low levels of IL-1b ( Figure 3D). Pro-IL-1b was also detected in the lysate of LPS-primed cells exposed to TMTOH or TETBr ( Figure 3D). Quantitation of protein band density across three gels and five independent samples demonstrated a ratio of released pro-( ∼ 45%) or mature-( ∼ 55%) IL-1b to total protein across exposures ( Figure 3E). In primary murine BMDMs ( Figure 3F), mature IL-1b release from LPS-primed cells was detected following exposure to ATP or tri-organotins.

ASC Speck Assembly
ASC speck formation has been effectively used as a readout for inflammasome activation (Stutz et al. 2013). Visualization of ASC speck formation was examined over 24 h by live-cell imaging of cerulean-tagged ASC aggregation in RAW 264.7-ASC cells ( Figure 4A). Quantitation of ASC speck formation was conducted at 6 and 16 h ( Figure 4B). In cells exposed to PBS, LPS, or ATP, a diffuse cytoplasmic fluorescence was observed. In LPS-primed cells, a 30-min exposure to ATP resulted in a dense coalesced staining pattern representative of aggregates, suggestive of NLRP3 inflammasome assembly. Non-LPS-primed cells exposed to TMTOH maintained a diffuse cerulean staining Data represent means ± SDs as percentages of controls (n = 6). (B) Estimates of mitochondrial stress using JC-10 assay. Data represent mean percentages of maximum response to antimycin ± SD (n = 6). (C,D) Cell viability following 6-h exposure to various doses of (C) TMTOH (0-5lM) or (D) TETBr (0-40lM) in nonprimed and LPS-primed cells (33 ng=mL, 3 h). Data were analyzed by one-way ANOVA followed by a Dunnett's test and represent means ± SDs (n = 6). The numerical data corresponding to this figure are shown in Excel Table S5. **** p < 0:0001; *** p < 0:001; ** p < 0:01; * p < 0:05. Note: ANOVA, analysis of variance; LPS, lipopolysaccharide; PBS, phosphate buffered saline; SD, standard deviation.
pattern that did not change significantly over time. This differed for TETBr, where the compound alone was sufficient to induce a minor level of aggregate assembly at 6 h (p = 0:003), which increased by 16 h (p < 0:0001). In LPS-primed cells, ASC speck formation was significantly higher with TMTOH (6 h p = 0:0049; 16 h p < 0:0001) and TETBr (p = 0:006; 16 h p < 0:0001) as compared with LPS-controls. ASC speck assembly was not observed for TPTOH ( Figure S2). Because extracellular ATP can serve as an autocrine/paracrine trigger for NLRP3 activation via activation of P2XR, cells were treated with the ATP-hydrolytic enzyme apyrase. The higher level of ASC speck formation observed with LPS priming was partially inhibited with apyrase, suggesting a contribution from extracellular ATP. For TMTOH, ASC speck formation was effectively inhibited by apyrase at 6 and 16 h. For TETBr, the significantly higher level of ASC speck formation at 16 h in nonprimed and LPS-primed cells was observed with apyrase (p < 0:01), suggesting a direct triggering effect in addition to that of extracellular ATP.

Nitrite Production and Cellular and mtROS Levels in Cells Exposed to Tri-Organotins
At 6 h, nitrite production remained low but was ∼ 20% higher in cells exposed to TETBr, as compared with PBS exposure and non-LPS-primed cells exposed to TMTOH (1:25 lM; TMT) or TETBr (10 lM; TET) for 6 or 16 h or with apyrase coexposure for 16 h. Images were captured using a 20-magnification objective on an IncuCyte S3 live-cell analysis system and IncuCyte ZOOM 2015A software. (B) Quantitation of relative ASC speck aggregation in nonprimed and LPS-primed cells in the absence or presence of apyrase with exposure to TMTOH or TETBr. Box plots represent median, first and third quartiles, and minimum and maximum values (n = 6) analyzed by two-way ANOVA followed by Tukey's multiple comparisons tests. * p < 0:01 compared with LPS-matched control; #p < 0:01 compared with matched group at 6 h. (C, D) Representative (C) contour plots and (D) histograms of flow cytometry for active caspase-1 at 6 h (see Figure S1 for information on gating). (E) Flow cytometric analysis of percentage active caspase-1 + cells and mean fluorescence intensity (MFI). (F) Cell size (forward-scattered light) and percentage cell death (PI + ) with tri-organotin exposure. Data were analyzed by one-way ANOVA and graphs represent individual values, means ± SDs (n = 3). The numerical data corresponding to this figure are shown in Excel Table S7. * p < 0:05, ** p < 0:01. Note: ANOVA, analysis of variance; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; FITC, fluorescein isothiocyanate; FSC, forward scatter channel; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; NLRP3, nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing receptor 3; PBS, phosphate buffered saline; PI, propidium iodide; PIPE-A, propidium iodide detected in the PE-A channel; SD, standard deviation; TET, triethyltin; TETBr, triethyltin bromide; TMT, trimethyltin; TMTOH, trimethyltin hydroxide.

Mitochondrial Bioenergetics
Given that we did not observe elevations in ROS levels by triorganotins, we examined potential alterations in mitochondrial bioenergetics induced by TMTOH or TETBr. In control cells, OCR and extracellular acidification rate (ECAR) profiles-surrogate measures for oxidative phosphorylation and glycolysis, respectively -demonstrated expected response patterns for basal respiration and following FCCP and rotenone stressors ( Figure 5D,E).

LPS-and LPS/ATP-Stimulated Cytokine Release
We evaluated whether the exposure altered the ability of RAW 264.7 cells to respond to LPS ( Figure 6). No differences were observed with tri-organotin exposure alone, compared with controls. LPS significantly elevated Tnfa (p < 0:0001) in all groups compared with vehicle, with no differences observed due to triorganotin exposure. Il1a and Il1b were elevated by LPS in all groups (p < 0:0001); however, the level of induction was significantly attenuated in cells exposed to TMTOH ( ∼ 30%) or TETBr ( ∼ 60%) (p < 0:0001). IL-1 receptor activity, as determined by HEK-Blue IL-1R cells, was higher in cells exposed to PBS or TMTOH (p < 0:001) and subsequently treated with LPS. This was not observed in cells exposed to TETBr. Tlr4 was significantly elevated with TETBr (3-fold; p < 0:0001) and the relative lower levels observed with LPS-priming were similar across groups. Arg2 was elevated with TETBr ( ∼ 2-fold; p < 0:001) and in all groups with LPS-priming (p < 0:0001). Il10 was slightly (2-fold) elevated with TETBr and with LPS priming; however, the elevation in LPSprimed cells was significantly less ( ∼ 50%) in TMTOH and TETBr exposed cells as compared with PBS (p < 0:0001).

Gene Expression Profiles
miRNAs generally target more than one gene in a signaling pathway. Thus, to gain a better understanding of the target genes associated with the miRNAs altered by each tri-organotin, mRNA expression array profiles were examined (Figure 9). A predominant shift was observed with TETBr that was less with TMTOH ( Figure 9A). When we examined targets for miR-151-3p, which was lower with both tri-organotins, 416 targets predicted by the TargetScan program were represented in the mRNA array. Functional analysis was performed on the up-regulated mRNA targets and the results indicated cyclic adenosine monophosphate (cAMP)-mediated signaling and AMP-activated protein kinase (AMPK) signaling to be the top enriched pathways ( Figure 9C, Table S1). Three miRNAs (miR-6909-5p, miR-7044-5p, miR-7686-5p) were found to be expressed at a higher level with both tri-organotins. We then applied a functional analysis of the mRNA expression data, focusing on down-regulated mRNA targets of these miRNAs. Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis signaling, signal transducer and activator of transcription 3 (STAT3) pathway, as well as IL-22, IL-12, and IL-10 signaling, were among the top enriched pathways ( Figure 9D; Excel Table S3).
To better understand these four miRNAs' potential RNA activation function, we performed additional analyses to identify potential miRNAs' binding sites in the 5 0 -untranslated region of mRNAs. From target mRNAs predicted by miRWalk and miRDB programs, we identified 11 potential targets for commonly down-regulated miRNA (miR-151-3P), with 8 positively correlated with miR-151-3P. However, no significantly enriched pathways were identified. Of the 58 potential targets for commonly up-regulated miRNAs (miR-7686-5P, miR-7044-5P, and Cell viability estimates at 21 h of the tri-organotin with LPS were ∼ 95 ± 7% for TMTOH and 90 ± 5% for TETBr, relative to control (n = 4). mRNA levels were normalized to Gapdh. Data were analyzed by a two-way ANOVA for each end point followed by Dunnett's multiple comparisons tests. The numerical data corresponding to this figure are shown in Excel Table S10. * p < 0:01; *** p < 0:0001 compared with vehicle of respective vehicle (non-LPS) or LPS+ATP group. Note: ANOVA, analysis of variance; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; LPS, lipopolysaccharide; PBS, phosphate buffered saline; ROI, region of interest; TETBr, triethyltin bromide; TMTOH, trimethyltin hydroxide. miR-6909-5P), 47 positively correlated with these up-regulated miRNAs. The fibroblast growth factor signaling pathway (FGF12 and FGF7) was found to be significantly enriched (Table S2). When we applied a functional enrichment analysis on the differentially expressed mRNAs, a differential pattern of pathways was observed between TETBr and TMTOH LPS-primed cells (Excel Table S4). The top enriched canonical pathways were related to apoptosis (such as unfolded protein response and p53 signaling) or cell survival (such as hypoxia and other endoplasmic reticulum-stress related pathways), and senescence.

Discussion
Inflammation can be viewed as a complicated series of local immune responses to deal with a threat to the microenvironment. The appropriate regulation of the initial cellular response to tissue damage facilitates recovery, whereas uncontrolled neuroinflammation can induce secondary injury. Although environmental agents can directly or indirectly, as a result of cell injury, induce a pro-inflammatory response, they may also show the ability to modify a normal response of immune cells. The fact that this may occur in the absence of a pro-inflammatory induction raises a concern in addressing how exposures might contribute across a broad spectrum of human health or with various comorbidities. The interactive role of mitochondria with innate immune responses, including NLRP3 inflammasome activation, offers a cellular mechanism by which factors known to alter mitochondrial function may initiate or dysregulate an immune response. In examining a set of tri-organotin compounds that have been previously demonstrated to induce a spectrum of effects on oxidative phosphorylation and to alter aspects of immune cell function, we demonstrated that the two known neurotoxicants, TETBr and TMTOH, showed evidence of inducing IL-1b release in LPSprimed macrophages. Although the levels were low, the relative levels observed were within ranges previously reported for NLRP3 inflammasome activation in macrophages following oxidized low-density lipoprotein (Sheedy et al. 2013) or silica (Dostert et al. 2008). Prior studies have suggested that ROS activation is an essential element for NLRP3 inflammasome activation (Abais et al. 2015); however, we observed no induction of cellular or mtROS yet, alterations in mitochondrial bioenergetics. Further examination of alterations related to IL-1b release in Figure 8. miRNA expression in BMDM exposed to TMTOH or TETBr. (A) Heatmap showing the differentially expressed miRNAs in LPS-primed cells upon 6-h exposure to TMTOH (1:25 lM) or TETBr (10 lM) as compared with LPS-primed cells exposed to vehicle (PBS) (B) Volcano plots of differentially expressed miRNA in LPS-primed cells following the second exposure to TMTOH or TETBr with four common miRNAs labeled (miR-6909-5p, miR-7044-5p, miR-7686-5p, and miR-151-3p). (C) Venn diagram representing distinct and common differentially expressed miRNAs in both LPS-primed TMTOH-or TETBr-exposed cells compared with LPS-primed cells exposed to vehicle (PBS). Three biological replicates for each group were included in the analysis. The numerical data corresponding to (C) are shown in Excel Table S2. Note: BMDM, bone marrow-derived macrophage; LPS, lipopolysaccharide; miRNA, microRNA; PBS, phosphate buffered saline; TET, triethyltin; TETBr, triethyltin bromide; TMT, trimethyltin; TMTOH, trimethyltin hydroxide.
LPS-primed cells identified differential miRNA and mRNA expression profiles with TMTOH or TETBr exposure that may contribute to understanding the cellular response to such insults and how they may manifest as immune cell dysregulation.
The field of immunometabolism emphasizes the importance of mitochondria in immune signaling (Lynch 2020;O'Neill et al. 2016;West 2017), with several studies demonstrating metabolic reprograming of macrophages to meet energy demands when responding to stimuli. In macrophages, a significant decrease in oxidative phosphorylation can reflect damage to mitochondria and diminished cellular function, but it can also indicate elevated glycolytic rate (Ganeshan and Chawla 2014) and underlie the metabolic shift observed with a pro-inflammatory response (Sanman et al. 2016). Although early work suggested that mitochondrial inhibition would be greater with TPT or TBT (Powers and Beavis 1991), we found that TETBr and TMTOH lowered Figure 10. Real-time qPCR of (A) P2rx7, (B) P2rx4, and (C) A20 levels in BMDM exposed to TMTOH or TETBr. mRNA levels were quantified by real-time qPCR (TaqMan) in BMDM following exposure to vehicle (PBS) or LPS for 3 h followed by the addition of vehicle (PBS), TMTOH (1:25 lM; TMT), or TETBr (10 lM; TET) for 6 h. Relative mRNA amounts were calculated using a normalized standard curve and expressed as ratios of target gene to Gapdh. Data were analyzed by a two-way ANOVA for each end point followed by a Tukey's test for multiple comparisons. Data represent mean relative mRNA ± SDs (n = 6). See Figure S4 for BMDM cell morphology. The numerical data corresponding to this figure are shown in Excel Table S11. **** p < 0:0001; ** p < 0:01 compared with non-LPS-primed cells exposed to vehicle (PBS) within each exposure group. #p < 0:01 compared with LPS-primed cells+vehicle. Note: ANOVA, analysis of variance; BMDM, bone marrow-derived macrophage; LPS, lipopolysaccharide; PBS, phosphate buffered saline; qPCR, quantitative polymerase chain reaction; SD, standard deviation; TET, triethyltin; TETBr, triethyltin bromide; TMT, trimethyltin; TMTOH, trimethyltin hydroxide. Figure 9. Transcriptomic analysis of gene expression changes in BMDM cells exposed to TMTOH or TETBr. Three biological replicates were identical to samples analyzed for mRNA expression. (A) Representative heatmap of the differentially expressed mRNAs in LPS-primed cells upon 6-h exposure to TMTOH (1:25 lM) or TETBr (10 lM) as compared with LPS-primed cells exposed to vehicle (PBS). (B) Venn diagram of the distinct and common differentially expressed genes under each exposure condition as compared with LPS-primed cells. (C) Functional analysis of up-regulated mRNA targets for miR-151-3p, which was lower with both tri-organotins exposed cells relative to LPS-primed cells. (D) Functional analysis of down-regulated mRNA targets for miRNAs [miR-6909-5p, miR-7044-5p, miR-7686-5p] that were higher in both LPS-primed TMTOH-and LPS-primed TETBr-exposed cells as compared with LPS-primed cells. The numerical data corresponding to this figure are shown in Table S1 and Excel Table S3). Note: AMPK, adenosine monophosphateactivated protein kinase; BMDM, bone marrow-derived macrophage; cAMP, cyclic adenosine monophosphate; G, guanine nucleotide; IGF1, insulin-like growth factor 1; IL, interleukin; LPS, lipopolysaccharide; miRNA, microRNA; PBS, phosphate buffered saline; RAR, retinoic acid receptor; STAT3, signal transducer and activator of transcription 3; TET, triethyltin; TETBr, triethyltin bromide; TMT, trimethyltin; TMTOH, trimethyltin hydroxide; TSP1, Thrombospondin 1. mitochondrial basal respiration and ECAR and stimulated IL-1b release in LPS-primed macrophages. As related to overall toxicity of the trialkyltin compounds, this would agree with the highest level of toxicity observed with the lower homologs, TMT and TET (Mushak et al. 1982;Snoeij et al. 1987) but would be in contrast to the previous severity ranking for oxidative phosphorylation (Powers and Beavis 1991). Mushak et al. (1982) reported that, regardless of the toxicity across tri-organotins, all showed significant levels in organs; however, detectable levels in the blood were observed only for TMT and TET. Evidence of immunotoxicity suggested a similar mode of action and potency for TBT and TPT (EFSA 2004); however, given the difference in response in the present study, it is speculated that alternative modes of action are recruited for TET and TMT. During mitoenergetic dysfunction, the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival. A loss in mitochondrial ATP production is compensated for by pyruvate metabolism into lactate (Bonora et al. 2012;Liemburg-Apers et al. 2015). However, the lower levels observed for TETBr in ATP-linked respiration was accompanied by a lower, rather than higher, glycolytic rate. The recent work by Tannahill et al. (2013) showing an altered LPS-induced IL-1 response with pharmacological inhibition of glycolysis might provide a basis for this difference. Given that alterations in mitochondria can modify a cell's ability to respond to inflammatory signals, we shifted the exposure paradigm to examine alterations in the response to LPS or to LPS+ATP in exposed cells. Under this paradigm, we observed no effect on Tnf a but did observe a significant inhibition of Il1a and Il1b induction, demonstrating a low-level effect on proper macrophage response to a pro-inflammatory stimulus and suggestive of immunodeficiency.
NLRP3 inflammasome activation has been linked to the recognition of mitochondrial danger signals and the generation of mtROS (Cruz et al. 2007;Nanayakkara et al. 2019) with many identified NLRP3 inflammasome activators shown to also trigger mtROS production (Tschopp and Schroder 2010). We now report that neither tri-organotin elevated cellular nor mtROS, which would exclude mtROS elevation as a critical factor in inflammasome activation. This would be within the support for both mtROS-dependent and ROS-independent activation mechanisms (Jabaut et al. 2013;Muñoz-Planillo et al. 2013;Sanman et al. 2016). In addition to mtROS activation, mitochondrial function as well as nitric oxide have been shown as being important for regulating NLRP3 inflammasome activation and IL-1b release (Hernandez-Cuellar et al. 2012;Mao et al. 2013;Tran and Kitami 2019). However, in the present study, a prominent role for ROS or nitric oxide was not identified. One possible alternative mechanism for the NLRP3 inflammasome aggregate formation may be related to alterations in ion channels or elevated ion permeability due to membrane damage (Di Virgilio et al. 2018;Hafner-Bratkovi c and Pelegrín 2018). Particulate activators can induce NLRP3 inflammasome through a sequence of events involving extracellular release of ATP through hemichannel opening and signaling through P2XRs. Membrane pore formation can trigger K + efflux and extracellular ATP to engage P2RX7 (Franceschini et al. 2015;Kahlenberg and Dubyak 2004;Mariathasan et al. 2006). When released into the extracellular space, ATP functions as a ubiquitous DAMP, acting at the metabotropic G-protein-coupled P2Y-receptors and the ionotropic P2RX7 for NLRP3 inflammasome activation and ATP-dependent K + efflux activation (Di Virgilio et al. 2018;Franceschini et al. 2015;Gombault et al. 2013;Hafner-Bratkovi c and Pelegrín 2018). P2X7 receptors are frequently expressed with another P2X receptor subtype, P2X4, with which they can form heteromeric receptors (Guo et al. 2007). Decreasing levels of P2X4 have been linked as a mechanism to minimize P2X7 receptor-mediated cell death by lowering intracellular Ca 2+ (Kawano et al. 2012) and may account for the differences observed with TETBr. TET and TMT are effective inhibitors of ATP activated by Na + and K + (Na + -K + -ATPase) . Alterations in K + may contribute to the subtle NLRP3 inflammasome assembly observed with TETBr in nonprimed cells and the elevation in P2rx7 seen with priming. In addition, the diminished but not eliminated response with apyrase further suggests a role for K + efflux. Although this data would imply higher levels of extracellular ATP, this interpretation requires caution given that commercially available apyrase preparations reportedly contain K + levels that are sufficient to inhibit NLRP3 activation (Muñoz-Planillo et al. 2013;Madry et al. 2018). In comparison, TMT inhibits K + influx , which may account for the difference in direct induction of ASC speck formation as compared with TETBr. These findings suggest that triorganotins can serve as effective inflammasome triggering agents in LPS-primed cells. Although the levels are low, the relative levels observed were within ranges previously reported for NLRP3 inflammasome activation in macrophages following oxidized low-density lipoprotein (Sheedy et al. 2013) or silica (Dostert et al. 2008).
miRNAs serve as major players in the regulation of NLRP3 inflammasome activation at both the priming and secondary trigger steps (O'Neill et al. 2011;Rebane and Akdis 2013;Zamani et al. 2020). This tight regulation is dependent upon the inhibitory effects of miRNAs on inflammatory processes (Coll and O'Neill 2010) as a negative feedback mechanism to down-modulate inflammatory cytokine production (Ceppi et al. 2009). Several miRNAs have been demonstrated to target mRNA transcripts of genes coding for components of the NLRP3 inflammasome complex (Bauernfeind et al. 2012;Boxberger et al. 2019;Tezcan et al. 2019;Zamani et al. 2020). Although a priming stimulus triggers NLRP3 transcription, protein translation can be interrupted by binding of miRNAs (miR-223, miR-22, miR-30e, and miR-7) to the untranslated region. In contrast, lower expression of miRNAs, such as miR-7 and miR-30e, may lead to a loss of regulatory control of NLRP3 activation . Given that both TETBr and TMTOH were able to serve as secondary triggers for ASC complex assembly and IL-1b release, it was of interest that differences in only four miRNAs were common to both, with one, miR-151-3p, lower and three (miR-6909-5p, miR-7044-5p, and miR-7686-5p) higher. Little is known regarding these specific miRNA species, with the exception of miR-7686-5p and miR-151-3p. miR-7686-5p has been reported to be up-regulated in the testes of mice exposed to a mixture of environmental endocrine disruptors (Buñay et al. 2017). miR-151-3p has recently been shown to contribute to LPS-stimulated up-regulation of STAT3 and IL-6 production (Liu et al. 2018a). Differences observed in LPS-primed cells were supported by the gene expression array identifying the STAT3 signaling pathway. Functional analysis of the targets for shared miRNAs also included signaling pathways for apoptosis, Wnt beta-catenin, the pro-inflammatory cytokine IL-12 and the IL-10 family of anti-inflammatory cytokines (IL-10, IL-22). IL-12 can signal for the activation of STATs (Trinchieri 2003); however, IL-10 serves as a negative regulator of IL-12 (Aste-Amezaga et al. 1998) and can inhibit NLRP3 inflammasome activation by reducing mtROS production (Ip et al. 2017). Thus, the overall profiles suggested an active cellular process to regulate the overall inflammatory response and to facilitate cell survival. This would be consistent with the growing body of evidence suggesting a threshold of NLRP3 activity acting as a safeguard mechanism to prevent inflammasome overactivation (Bortolotti et al. 2018). Additional experiments based upon the differential profiles generated by the triorganotin compounds and the level of inflammasome activation may help to identify pivot points of this threshold and provide a framework for examining associations between various mitochondrial toxicants and immune dysfunction. One pivotal point may be at the (de)ubiquitinating enzyme A20 or TNFa-induced protein 3 (Tnfaip3, A20/tnfaip3), a target gene of let-7, miR-125a, miR-125b, which regulates crucial stages in immune cell homeostasis, such as NF-jB activation and apoptosis, and directly influences NLRP3 inflammasome in macrophages (Duong et al. 2015;Vande Walle et al. 2014).
Overall, the data suggests that these specific tri-organotins, TMTOH and TETBr, which are known as mitochondrial toxicants, can serve as secondary triggers for NLRP3 inflammasome activation; however, the driving underlying mechanism remains in question. Differences between tri-organotins were demonstrated in mitochondrial bioenergetics, inflammasome activation, molecular profiles, and LPS response. The excess stimulation with TETBr suggested that aspects of this response reflected cell survival mechanisms involving an integrated stress response (Pakos-Zebrucka et al. 2016). Thus, the outcome likely depends on severity, number of cells recruited, secondary stimulatory factors such as ion-channel function, and recruitment of cell survival pathways. With NLRP3 inflammasome activation, inflammatory contribution of IL-1b and IL-18 is clear; however, with pyroptosis, the excretion of exosomes (Zhang et al. 2017;Cypryk et al. 2018) and oligomeric NLRP3 inflammasome particles (Baroja-Mazo et al. 2014;Franklin et al. 2014) are factors that act upon adjacent cells to activate the NF-jB signal pathway, alter lysosome integrity, and enhance or prolong the immune response (Venegas et al. 2017). Each of these factors may represent a biological process of concern for downstream adverse effects.
Using a relatively high-throughput live-cell imaging approach adapted from pharmaceutical inflammasome-activation screening approaches (Redondo-Castro et al. 2018), we provide data suggesting that environmental factors with mitotoxic properties may serve as secondary triggers for inflammasome activation at levels that fail to induce a pro-inflammatory response. It is likely that, although a mitochondrial component may be associated with this immune cell response, it is not a defining characteristic. Thus, examination of NLRP3 inflammasome activation may offer an approach for examining downstream events distinguishing between mitotoxicants, or other classes of compounds, suspected of producing immune cell dysfunction. Further examination of chemical classes with mitochondrial activity is required to determine whether this approach can be used to develop a causal link and biological hierarchy leading to adverse health effects given the critical need for appropriate immune cell functioning.