NOCAS_36695LipopolysaccharrideLipopolysaccharide (LPS)
DTXSID4036695PR:000001096Toll-like receptorPR:000001155Toll-like receptor 4FMA:84050CytokineFMA:241981ChemokineFMA:86578InterleukinGO:0002221pattern recognition receptor signaling pathwayGO:0002224toll-like receptor signaling pathwayGO:0034142toll-like receptor 4 signaling pathwayGO:0002534cytokine production involved in inflammatory responseGO:0090195chemokine secretionGO:0006956complement activation1increasedLipopolysaccharride2018-05-29T07:05:552018-05-29T07:05:55Graphene oxide nanoparticles2017-02-15T04:41:082017-02-15T04:41:08Carbon nanotubes2017-08-09T08:01:552017-08-09T08:03:46Insoluble nano-sized particles2018-05-29T07:09:362018-05-29T07:09:36Virus2018-05-29T07:10:012018-05-29T07:10:0110090mouse10116ratWCS_9606human10116ratsAcute phase proteins expression, IncreasedIncreased expression of APPOrgan2017-06-29T02:25:542023-04-21T08:33:04Systemic acute phase responseSystemic APRMolecular2017-06-29T02:27:062023-03-28T09:53:38AtherosclerosisAtherosclerosisIndividual2017-06-29T02:33:212023-03-28T09:45:00Substance interaction with the lung resident cell membrane componentsInteraction with the lung cell membraneMolecular<p><span style="color:#000000"><span style="font-family:Calibri">The human lung consists of approximately 40 different resident cell types that play different roles during homeostasis, injury, repair and disease states (Franks et al., 2008; Luettich et al., 2021). Of these, resident airway epithelial cells, alveolar/interstitial macrophages and dendritic cells are well characterised for their ability to sense the danger upon interaction with harmful substances and relay the message to mount the necessary immune/inflammatory response. The resident macrophages are present in all tissues, and in a steady state, macrophages contribute to epithelial integrity, survey the tissue for invading pathogens or chemicals and maintain an immunosuppressive environment. Their main function is to clear the incoming irritants and microbes. They are named differently based on the tissue type and their specific functions (Kierdorf et al., 2015).</span></span></p>
<p><br />
<strong><span style="color:#000000"><span style="font-family:Calibri"><em>Substance interactions:</em></span></span></strong></p>
<p><br />
<span style="font-family:Calibri"><span style="color:#000000">The chemicals or pathogens interact with cellular membrane to gain access to the organisms’ interior. A predominant interaction mechanism involves the recognition of innate immune response agonists by pattern recognition receptors (PRRs) present on resident cells such as epithelial and alveolar macrophages. PRRs are also present on other immune and parenchymal cells. PRRs can be activated by two classes of ligands. Pathogen associated molecular patterns (PAMPs) are microbial molecules derived from invading pathogens. PAMPs will not be discussed further as pathogens are not the focus for the AOP presented here. The other class of ligands are called danger associated molecular patterns (DAMPs) that include cellular fragments, nucleic acids, small molecules, proteins and even cytokines released from injured or dying cells (Bianchi, 2007). Most fibrogenic stressors discussed in this AOP act via DAMPs-driven PRR activation. High aspect ratio (HAR) materials such as asbestos or carbon nanotubes (CNTs) pierce the cellular membrane of epithelial cells or resident macrophages resulting in cell injury or non-programmed cellular death. Alveolar macrophages trying to engulf HAR fibres that are long and stiff undergo frustrated phagocytosis because of their inability to engulf the piercing fibres and subsequently lead to cell injury (Boyles et al., 2015; Brown et al., 2007; Donaldson K et al., 2010; </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Dörger et al., 2001; </span></span></span><span style="font-family:Calibri"><span style="color:#000000">Mossman and Churg, 1998</span></span><span style="font-family:Calibri"><span style="color:#000000">). The cellular debris from injured or dying cell then serves as ligands </span><span style="color:red">for</span><span style="color:#000000"> PRRs (Nakayama, 2018), leading to cell activation. In case of pro-fibrotic insoluble particles such as silica, coal dust and nanomaterials (NMs), the particle adsorbed opsonins such as, immunoglobulins, complement proteins, or serum proteins act as ligands to the receptors on the macrophage cell surface (Behzadi et al., 2017). The tissue response to these materials resembles that observed following foreign body invasion in lungs. </span></span></p>
<p><span style="color:#000000"><span style="font-family:Calibri">Toll-like receptors (TLRs) are highly conserved PRRs that are associated with fibrogenic stressors (Desai et al., 2018). Inhibition of TLR-4 is protective against bleomycin-induced fibrosis (Li et al., 2015). However, the exact role and mechanisms by which TLRs mediate lung fibrosis are yet to be uncovered and some studies have shown TLRs to be protective against lung fibrosis (Desai et al., 2018). Asbestos and silica crystals are suggested to engage scavenger receptors present on the macrophages. Mice deficient in class A scavenger Macrophage receptor with collagenous structure (MARCO) are shown to induce reduced fibrogenic response following chrysotile asbestos exposure; although, the direct binding of MARCO by asbestos is not investigated in the study (Murthy et al., 2015). In case of soluble substances such as bleomycin, paraquat (Dinis-Oliveira et al., 2008) (N,N'-dimethyl-4, 4′-bipyridinium dichloride) and other soluble fibrogenic chemicals, direct damage of lung epithelial cells and resulting cellular debris or secreted cytokines (DAMPs) serve as triggers for downstream cascading pro-inflammatory events, tissue injury and fibrosis. Engagement of PRRs and consequent cell activation is observed in various organisms including flies and mammals (Denholm and Phan, 1990; Matzinger, 2002).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><em><strong>Detection of DAMPs or homeostasis-altering molecular processes:</strong></em></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Cellular interaction with substances or particles can be measured by assessing the release of DAMPs from stressed, injured or dying cells - indicative of binding of PRRs on the cell surface. Release of DAMPs is reflective of substance interaction with resident cells and their activation, a key step in the process of inflammation.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The release of DAMPs can be measured by the techniques listed in the published literature (Nikota et al., 2017; Rabolli et al., 2014; Suwara et al., 2014).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><em><strong>Targeted Enzyme-linked immunosorbent assays (ELISA) (routinely used and recommended):</strong></em></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>ELISA </u> – permits quantitative measurement of antigens in biological samples. For example, in a cytokine ELISA (sandwich ELISA), an antibody (capture antibody) specific to a cytokine is immobilised on microtitre wells (96-well, 386-well, etc.). Experimental samples or samples containing a known amount of the specific recombinant cytokine are then reacted with the immobilised antibody. Following removal of unbound antibody by thorough washing, plates are reacted with the secondary antibody (detection antibody) that is conjugated to an enzyme such as horseradish peroxidase, which when bound, will form a sandwich with the capture antibody and the cytokine (Amsen and De Visser, 2009). The secondary antibody can be conjugated to biotin, which is then detected by addition of streptavidin linked to horseradish peroxidase. A chromogenic substrate can also be added, which is the most commonly used method. Chromogenic substrate is chemically converted by the enzyme coupled to the detection antibody, resulting in colour change. The amount of colour detected is directly proportional to the amount of cytokine in the sample that is bound to the capture antibody. The results are read using a spectrophotometer and compared to the levels of cytokine in control samples where cytokine is not expected to be secreted or to the samples containing known recombinant cytokine levels.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Interleukin (IL)-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> and -1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> is activated or secreted into the cytosol following stimulus (Di Paolo and Shayakhmetov, 2016). Targeted ELISA can be used to quantify IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α </span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> or IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> that is released in the culture supernatant of the cells exposed to toxicants, in bronchoalveolar lavage fluid and serum of exposed animals. The assay is also applicable to human serum, cerebrospinal fluid, and peritoneal fluids.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Similarly, other alarmins can also be quantified by ELISA. Western blot is another method that can be used to quantify the release of various alarmins using specific antibodies. Real-time reverse transcription-polymerase chain reaction (qRT-PCR) or ELISA assays can also be used to quantify expression of genes or proteins that are regulated by the receptor binding – e.g. downstream of TLR binding.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Frustrated phagocytosis and cellular uptake of NMs:</em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro, interaction of NMs with the cellular membrane is investigated by assessing their uptake by lysosomes (Chen et al., 2013; Nel et al., 2009; Varela et al., 2012). Immunohistochemistry methods targeting lysosome specific proteins are regularly employed for this purpose. In co-localisation experiments, lysosomal marker Lysosomal-associated membrane protein 1 (LAMP1) antibody is used to detect particle co-localisation with lysosomes. A combination of Cytoviva hyperspectral microscope and immunolocalisation (Decan et al., 2016) or confocal microscopy to visualise co-localisation of fluorescence labelled nanoparticles with lysosomal markers have been used. </span></span></p>
<p style="text-align:justify"><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="background-color:white"><span style="color:black">Frustrated phagocytosis is assessed using <span style="color:black">microscopic techniques </span>such as, time-lapse microscopy, backscatter electron microscopy and others (Donaldson et al., 2010; Murphy et al., 2012; Padmore et al., 2017; Pascolo et al., 2013; </span><span style="color:#212529">Schinwald</span><span style="color:black"> et al., 2012).</span> In addition, MIE 1668 of AOP303 notes other indirect methods for measuring frustrated phagocytosis. </span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:red">Cellular co-culture models of the pulmonary epithelium:</span></em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Complex co-culture systems, such as those containing epithelial cells and immune cells, better model the environment of the lung epithelium and can be used to study the interaction of potentially pro-fibrotic fibres and particles with resident lung cells. This type of model has been used, alongside electron microscopy, to study lung cell interactions with CNTs following 24 Hr <em>in vitro</em> exposure (Clift <em>et al.,</em> 2014). More recently, the EpiAlveolar model, which contains primary human alveolar epithelial cells, endothelial cells, as well as fibroblasts was assessed for its ability to predict fibrosis induced by CNTs (Barasova <em>et al.,</em> 2020). Using laser scanning, fluorescence, and enhanced darkfield microscopy, CNT interaction with the resident cells of the model was shown, and this interaction induced the formation of holes in the epithelial model (Barasova <em>et al.,</em> 2020). While new co-culture models are a better recapitulation of the native lung environment as compared to traditional mono-cultures, the increased complexity necessitates enhanced expertise in tissue culture techniques, and can make them less practical as compared to submerged mono culture methods. </span></span></span></p>
<p><strong><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><em><span style="color:red">Ex vivo model of the lung – Precision cut lung slices (PCLS):</span></em></span></span></strong></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Even closer to the <em>in vivo</em> condition than co-culture models, PCLS techniques capture the native lung architecture, cell-cell communication and cellularity of the lung. </span></span></span><span style="color:red; font-family:"Arial",sans-serif; font-size:11pt"><span style="font-size:16px">Advancement in culturing and cryopreservation techniques has increased accessibility and use of PCLS for longer term studies (Bai <em>et al</em>., 2016, Neuhaus <em>et al</em>., 2017).</span> </span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">These slices can be cultured <em>ex vivo</em> for up to a week with minimal reduction in viability, and the technique has recently been assessed for its applicability to assess nanomaterial induced fibrosis <em>ex vivo</em> (Rahman <em>et al.,</em> 2020). Using multi-walled carbon nanotubes and darkfield microscopy, interaction between the nanofibers and the lung epithelium could be determined. The main downside of this technique is the animal requirement, which precludes their use in a first-pass screening context for the MIE. </span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Human, mouse, rat.</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Although the expression of DAMPs following exposure to pro-fibrotic substances is not assessed across species, it is known that alarmins are released after trauma or injury, and their release is important for initiating the inflammatory response in all species including humans. The immediate acute inflammatory response involving DAMP signalling is also observed in human idiopathic pulmonary fibrosis (IPF); however, anti-inflammatory drugs have proven ineffective for treating IPF. Danger signalling axis including uric acid, adenosine triphosphate and IL-33/ST2 has been proven to promote lung fibrosis in animals. </span></span></p>
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<p><span style="color:red"><span style="font-family:Calibri">35. </span></span>Rahman L, Williams A, Gelda K, Nikota J, Wu D, Vogel U, Halappanavar S. 21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision-Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial-Induced Lung Fibrosis. Small. 2020 Sep;16(36):e2000272. doi: 10.1002/smll.202000272.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">36. </span></span>Schinwald A, Donaldson K. Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo. Part Fibre Toxicol. 2012 Aug 28;9:34. doi: 10.1186/1743-8977-9-34.<span style="color:#000000"><span style="font-family:Calibri"> </span></span></p>
<p><span style="color:#000000"><span style="font-family:Calibri">37. </span></span>Suwara MI, Green NJ, Borthwick LA, Mann J, Mayer-Barber KD, Barron L, Corris PA, Farrow SN, Wynn TA, Fisher AJ, Mann DA. IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts. Mucosal Immunol. 2014 May;7(3):684-93. doi: 10.1038/mi.2013.87.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">38. </span></span>Varela JA, Bexiga MG, Åberg C, Simpson JC, Dawson KA. Quantifying size-dependent interactions between fluorescently labeled polystyrene nanoparticles and mammalian cells. J Nanobiotechnology. 2012 Sep 24;10:39. doi: 10.1186/1477-3155-10-39. </p>
2018-01-02T12:27:142023-04-25T10:51:29Increased, secretion of proinflammatory mediatorsIncreased proinflammatory mediatorsCellular<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including <a href="https://bioregistry.io/genecards:IL2">IL-2</a>, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL7">IL-7</a>, <a href="https://bioregistry.io/genecards:IL9">IL-9</a>, <a href="https://bioregistry.io/genecards:IL15">IL-15</a>, <a href="https://bioregistry.io/genecards:IL21">IL-21</a>, <a href="https://bioregistry.io/genecards:IL3">IL-3</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a> and Granulocyte-macrophage colony stimulating factor (<a href="https://bioregistry.io/genecards:CSF2">GM-CSF</a>) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (<a href="https://bioregistry.io/genecards:IL1a">IL-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="color:blue"><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> </span></span></u></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL1b">IL-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL1ra">IL-1r</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL18">IL-18</a>, <a href="https://bioregistry.io/genecards:IL36a">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36b">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36g">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36Ra">IL-36R</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL37">IL-37</a>), <a href="https://bioregistry.io/genecards:IL6">IL-6 </a>family, Tumor necrosis factor (<a href="https://bioregistry.io/genecards:TNF">TNF</a>) family, <a href="https://bioregistry.io/genecards:IL17">IL-17</a>, and Interferon gamma (<a href="https://bioregistry.io/genecards:IFNg">IFN</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">) (Turner et al., 2014). While <a href="https://bioregistry.io/genecards:IL4">IL-4</a> and <a href="https://bioregistry.io/genecards:IL5">IL-5</a> are considered T helper (Th) cell type 2 response, <a href="https://bioregistry.io/genecards:IFNg">IFN</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> is suggested to be Th1 type response.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, <a href="https://bioregistry.io/genecards:IL1">IL-1</a> family cytokines, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a>, <a href="https://bioregistry.io/genecards:IL6">IL-6</a>, <a href="https://bioregistry.io/genecards:TNFa">TNF</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, IFN-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:red">Literature evidence for its perturbation:</span></em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials</span>. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">B], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">B luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte Chemoattractant Protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>Real-time reverse transcription-polymerase chain reaction (qRT-PCR)</u> – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan T et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>Enzyme-linked immunosorbent assays (ELISA)</u> – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red"><u>Cell models</u> - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used <em>in vitro</em> due to the large literature base, and ease of use, but do not adequately mimic the <em>in vivo</em> condition (Sundarakrishnan <em>et al.,</em> 2018, Sharma <em>et al.,</em> 2016). Recently, the EpiAlveolar <em>in vitro</em> lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNT, and researchers noted increases in the pro-inflammatory molecules TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">, IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">, and the pro-fibrotic TGF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red"> using ELISA (Barasova <em>et al.,</em> 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the <em>in vivo</em> pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments. </span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Human, mouse, rat</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.</span></span></p>
CL:0000255eukaryotic cellHighMaleHighFemaleHighAdultsHighHighHigh<p><span style="color:#000000"><span style="font-family:Calibri">1. </span></span>Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. </p>
<p><span style="color:red"><span style="font-family:Calibri">2. </span></span>Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">3. </span></span>Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">4. </span></span>Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">5. </span></span>Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">6. </span></span>Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">7. </span></span>Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">8. </span></span>Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">9. </span></span>Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">10. </span></span>Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">11. </span></span>Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">12. </span></span>Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P<sub>2</sub>) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">13. </span></span>Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.</p>
<p><span style="color:red"><span style="font-family:Calibri">14. </span></span>Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7. </p>
<p><span style="color:red"><span style="font-family:Calibri">15. </span></span>Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7. </p>
<p><span style="color:red"><span style="font-family:Calibri">16. </span></span>Sundarakrishnan A, Chen Y, Black LD, Aldridge BB, Kaplan DL. Engineered cell and tissue models of pulmonary fibrosis. Adv Drug Deliv Rev. 2018 Apr;129:78-94. doi: 10.1016/j.addr.2017.12.013.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">17. </span></span>Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">18. </span></span>Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099.</p>
2018-01-02T13:12:112023-04-21T09:51:325ae3e3f5-22eb-4fa1-9f33-6e6809eebee0ff079b83-3346-4367-8ccd-567f413dcf1f<p style="margin-left:7px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Innate immune response is the first line of defence in any organism against invading infectious pathogens and toxic substances. It involves tissue triggered startle response to cellular stress and is described by a complex set of interactions between the toxic stimuli, soluble macromolecules and cells (reviewed in Nathan, 2002). The process culminates in a functional change defined as inflammation, purpose of which is to resolve infection and promote healing. In lungs, the interaction of toxic substances with resident cells results in cellular stress, death or necrosis (Pouwels et al., 2016) leading to release of intracellular components such as alarmins (Damage associated molecular patterns [DAMPs], Interleukin (IL)-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">, High mobility group box 1 [HMGB1]). Released alarmins (danger sensors) bind cell surface receptors such as Interleukin 1 Receptor 1 (IL-1R1), Toll Like Receptors (TLRs) or others leading to activation of innate immune response signalling.</span></span></span></p>
<p style="margin-left:7px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">For example, binding of IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> to IL-1R1 can release Nuclear factor kappa B (NF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">B) resulting in its translocation to nucleus and transactivation of pro-inflammatory genes including cytokines, growth factors and acute phase genes. The signalling also stimulates secretion of a variety of pro-inflammatory mediators. Overexpression of IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> in cells induces increased secretion of pro-inflammatory mediators. Products of necrotic cells are shown to stimulate the immune system in an IL-1R1-dependent manner (Chen et al., 2007).</span></span></span></p>
<p style="margin-left:7px; margin-right:12px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">The secreted alarmins activate resident cells pre-stationed in the tissues such as mast cells or macrophages leading to propagation of the already initiated immune response by releasing more eicosanoids, cytokines, chemokines and other pro-inflammatory mediators. Thus, secreted mediators signal the recruitment of neutrophils, which are the first cell types to be recruited in acute inflammatory conditions. Neutrophil influx in sterile inflammation is driven mainly by IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> (Rider P, 2011). IL-1 mediated signalling regulates neutrophil influx in silica-induced acute lung inflammation (Hornung et al., 2008). IL-1 signalling also mediates neutrophil influx in other tissues and organs including liver and peritoneum. Other types of cells including macrophages, eosinophils, lymphocytes are also recruited in a signal-specific manner. Recruitment of leukocytes induces critical cytokines associated with the T helper type 2 immune response, including Tumor necrosis factor alpha (TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">), IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">, and IL-13.</span></span></span></p>
<p style="margin-left:7px; margin-right:12px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">The biological plausibility of this relationship is high. There is a mechanistic relationship between the MIE (Event 1495) and KE1 (Event 1496) which has been evidenced in a number of both in vitro and in vivo model systems in response to stressors such as, asbestos, silica, bleomycin, carbon nanotubes, and metal oxide nanoparticles (NPs) (Behzadi <em>et al.,</em> 2017; Denholm & Phan 1990; Dostert et al., 2008; Mossman & Churg 1998). </span></span></span></p>
<p style="margin-left:7px; margin-right:12px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Increased expression of IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> or IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> following lung exposure to multi-walled carbon nanotubes (MWCNTs), bleomycin, micro silica particles, silica crystals, and polyhexamethyleneguanidine phosphate has been shown to be associated with neutrophil influx in rodents (Gasse et al., 2007; Girtsman et al., 2014; Hornung et al., 2008; Nikota et al., 2017; Rabolli et al., 2014; Suwara et al., 2014). Inhibition of IL-1 function by knocking out the expression of IL-1R1 using IL-1R1 knockout mice or via treatment with IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> or IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> neutralising antibodies results in complete abrogation of lung neutrophilic influx following exposure to MWCNTs (Nikota et al, 2017), cigarette smoke (CS) (Halappanavar et al., 2013), silica crystals (Rabolli et al., 2014) and bleomycin (Gasse et al., 2007). IL1-R1, Myeloid differentiation primary response protein (Myd88) or the IL-33/St2 signaling are involved in pulmonary fibrosis induced by bleomycin (Gasse et al., 2007; Xu et al., 2016).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Empirical support for this KER is moderate. There are limited <em>in vitro</em> studies, which show a temporal and dose-dependent relationship between these two events, using the upregulation of specific surface receptors as a proxy for direct membrane interaction (Chan et al., 2018; Denholm & Phan, 1990; Roy et al., 2014). There are also studies that provide general support for the idea that an interaction with the lung resident cell membrane components leads to increased, secretion of pro-inflammatory and pro-fibrotic mediators (<a href="https://aopwiki.org/system/dragonfly/production/2021/11/26/9tlt67fjhl_MIE_KE1_Table_1.pdf"><u>Table 1</u></a>).</span></span></span></p>
<p style="text-align:justify"><strong><span style="font-family:"Arial",sans-serif">Dose-Response Evidence:</span></strong></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">There are a few studies which provide evidence for a dose-response relationship in this KER. An <em>in vitro</em> study demonstrated a concentration-response relationship, in which silica exposure induced increases in pro-inflammatory cytokines through scavenger receptors in cultured bone marrow-derived murine mast cells. Cells were exposed to 6.25, 12.5, 25 or 50 </span><span style="font-family:Symbol">m</span><span style="font-family:"Arial",sans-serif">g/cm<sup>2</sup> silica dioxide (SiO<sub>2</sub>) for 24 h. Macrophage scavenger receptor (MSR2) expression increased over time at 50 </span><span style="font-family:Symbol">m</span><span style="font-family:"Arial",sans-serif">g/cm<sup>2</sup> and in a concentration dependent relationship. Moreover, Tumor necrosis factor alpha (TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">)</span><span style="font-family:"Arial",sans-serif">, IL-13 and Monocyte chemoattractant protein-1 (MCP-1) increased in a concentration-dependent manner (Brown et al., 2007). This provides indications that at higher concentrations of the stressor, the interaction with the lung resident cell membrane components (Event 1495) leads to an increased secretion of pro-inflammatory mediators (Event 1496). </span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-family:"Arial",sans-serif">Temporal Evidence:</span></strong></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><em><span style="font-family:"Arial",sans-serif">In vitro</span></em><span style="font-family:"Arial",sans-serif"> and <em>in vivo</em> studies have demonstrated temporal concordance of the KEs. </span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">TLR4 signal pathway was evaluated in differentiated macrophages exposed to silica at 2.5 </span><span style="font-family:Symbol">m</span><span style="font-family:"Arial",sans-serif">g/cm<sup>2</sup>. After 16 and 24 h, the mRNA expression level of TLR4 increased. Moreover, the protein expression level of TLR-4 and related MyD88/Toll-interleukin-1 receptor domain containing adaptor protein (TIRAP) pathway increased at 24 h. Release of IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">, IL-6, IL-10, and TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> was induced by silica exposure at 24 h. Pre-treatment with resatorvid (TAK-242), an inhibitor of TLR4 signaling, suppressed the release of the cytokines (Chan et al., 2018).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Macrophages exposed to zinc oxide NPs at 2.5 </span><span style="font-family:Symbol">m</span><span style="font-family:"Arial",sans-serif">g/mL for 24 h increased the expression level of TLR6 and MyD88, TNF receptor-associated factor (TRAF), and IL-1 receptor-associated kinase (IRAK). At 24 h, they also observed an increase in the mRNA and protein levels of the pro-inflammatory cytokines IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">, IL-6, and TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">. These results demonstrated that ZnO NPs induced pro-inflammatory mediators by TLR stimulation and Mitogen-activated protein kinases (MAPKs) activation (Roy et al., 2014).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif">The pro-inflammatory IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"> induced granulocyte migration and can be produced as a result of cellular detection of pathogen associated molecular patterns (PAMPs). Mice exposed to 2.5 mg/mouse of silica by instillation showed an increase of mRNA expression of pro-IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"> in bronchoalveolar lavage fluid (BALF) at 6, 12, and 24 h post-exposure in a time-dependent manner. At early time points (1 h, 3 h, 6 h), there was an increase in the release of an alarmin (IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Arial,sans-serif">) which indicates that the alarmin was released due to cell damage leading to cytokine production and an inflammatory reaction. Moreover, at 24 h, the levels of mature IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"> and neutrophil accumulation in BALF increased. Neutralization or deletion of IL-1</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"> reduced the observed responses (Rabolli et al., 2014).</span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Epithelial damage can lead to the release of alarmins. In this stead, conditioned media from primary human bronchial epithelial cells (PBECs) exposed to thapsigargin was able to induce a pro-inflammatory response in primary human lung fibroblasts. PBECs were exposed to thapsigargin (a tumor promoter in mammalian cells) 20 </span><span style="font-family:Symbol">m</span><span style="font-family:"Arial",sans-serif">M for 2 h. After that, the cell culture medium was replaced, and cells were incubated for 24 h. At this time, the medium was recovered and used to culture lung fibroblast for 5 h. This conditioned media from epithelial cells damage contains the alarmin IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">, which induced increased gene expression of IL-6, IL-8, MCP-1, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) in fibroblasts. These responses were reduced with anti-IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> treatment (Suwara et al., 2014).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Heijink et al. 2015 conducted a similar strategy to identify the relationship between DAMPs and pro-inflammatory mediator release after exposure to CS. Neutrophils treated with CS bubbled for 1 min, released high levels of HMGB1 as a consequence of necrotic cell death. The cell-free supernatant, which contains HMGB1, was used to culture human bronchial epithelial cells, and after 24 h it promoted the production of the C-X-C motif chemokine ligand (CXCL)8 or IL-8 by lung epithelial cells. Pharmacological inhibitors, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC) and Receptor for advanced glycation endproducts (RAGE) antagonist peptide (RAP), reduced the effect of CXCL8 release.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">HMGB1 and Heat shock protein 70 (HSP-70) can be released by damaged hepatocytes. In a study, mice were treated with acetaminophen 350 mg/Kg for 3 and 6 h. At these time points, the liver perfusate was obtained and an increase in HSP-70 and HMGB1 protein levels was observed. RAW 264.7 cells (a macrophage cell line) treated with the liver perfusate exhibited increased mRNA expression levels of MCP-1 and IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif"> (Martin-Murphy et al. 2010).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Female mice were intratracheally administered with bleomycin at 5 mg/kg to represent idiopathic pulmonary fibrosis. IL-33, a molecule that can act as a DAMP, increased in lungs after 3 and 7 days of treatment. In serum, at 7-, 14- and 28-days post-exposure, IL-4 and IL-13 increased. It was concluded that IL-33/ST2 signaling pathway is involved in pulmonary fibrosis by bleomycin (Xu et al., 2016).</span></span></span></p>
<p style="margin-left:7px; margin-right:8px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">Attenuation or complete abrogation of KE1 <span style="color:red">(Event 1496) </span>and KE2 <span style="color:red">(Event 1497) </span>following inflammogenic stimuli is observed in rodents lacking functional IL-1R1 or other cell surface receptors that engage innate immune response upon stimulation. However, following exposure to MWCNTs, it has been shown that absence of IL-1R1 signalling is compensated for eventually and neutrophil influx is observed at a later post-exposure time point (Nikota et al., 2017). In another study, acute neutrophilic inflammation induced by MWCNTs was suppressed at 24 hr in mice deficient in IL-1R1 signalling; however, these mice showed exacerbated neutrophilic influx and fibrotic response at 28 days post-exposure (Girtsman et al., 2014). The early defence mechanisms involving DAMPs is fundamental for survival, which may necessitate activation of compensatory signaling pathways. As a result, inhibition of a single biological pathway mediated by an individual cell surface receptor may not be sufficient to completely abrogate the lung inflammatory response. Forced suppression of pro-inflammatory and immune responses early after exposure to substances that cannot be effectively cleared from lungs, may enhance the injury and initiate other pathways leading to exacerbated response.</span></span></span></p>
<p style="margin-left:7px; margin-right:7px; text-align:justify"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Arial",sans-serif">A majority of the in vivo studies are conducted with only one dose and thus, it is difficult to derive quantitative dose-response relationships based on the existing data. However, it is clear from the studies referenced above that greater concentrations or doses of pro-fibrotic substances results in higher release of alarmins, and consequently, higher pro- inflammatory signalling. The above studies also demonstrate strong temporal relationships between the individual KEs.</span></span></span></p>
<p style="text-align:justify"> </p>
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2018-01-05T13:18:172023-04-25T13:30:01ff079b83-3346-4367-8ccd-567f413dcf1ff6fe448c-8956-4117-84c7-63afd8cbd5bf2020-03-01T10:32:072020-03-01T10:32:07f6fe448c-8956-4117-84c7-63afd8cbd5bf21e943f0-9311-4cd0-bcfd-ea10dcf7bbfb2017-06-29T02:37:312017-06-29T02:37:3121e943f0-9311-4cd0-bcfd-ea10dcf7bbfb09471412-4cd7-496f-a79c-bb231095e6bf2023-03-28T09:58:352023-03-28T09:58:35Substance interaction with lung resident cell membrane components leading to atherosclerosisInteraction with lung cells leading to atherosclerosis<p>Claudia Torero Gutierrez<sup>1</sup>, Sarah Søs Poulsen<sup>1</sup>, Jorid Birkelund Sørli<sup>1</sup>, Håkan Wallin<sup>2</sup>, Sabina Halappanavar<sup>3</sup>, Carole L. Yauk<sup>4</sup>, Ulla Vogel<sup>1,*</sup></p>
<p><sup>1</sup>The National Research Centre for the Working Environment, Denmark</p>
<p><sup>2</sup>Statens Arbeidsmiljøinstitutt, Norway</p>
<p><sup>3</sup>Health Canada, Canada</p>
<p><sup>4</sup>University of Ottawa, Canada</p>
<p>*Corresponding author: Ulla Vogel (ubv@nfa.dk)</p>
Under development: Not open for comment. Do not citeUnder DevelopmentIncluded in OECD Work Plan1.55<p>Cardiovascular disease (CVD) is the leading cause of death worldwide, being responsible for 31% of all deaths in 2012 (WHO: http://www.who.int). The term CVD covers all diseases of the cardiovascular system, including atherosclerosis, which is manifested as increased plaque deposition or build-up in the arteries. Atherosclerosis is normally asymptotic disease and is initiated by a biological, chemical or physical insult to the artery walls. This leads to the expression of cell adhesion molecules (selectins, VCAM-1 and ICAM-1) on the endothelial lining of the arteries, which facilitates the activation, recruitment, and migration of monocytes through the endothelial monolayer [1;2]. Inside the intima layer, the monocytes differentiate into macrophages and internalize fatty deposits (mainly oxidized low-density lipoprotein). This results in them transforming into foam cells, which is a major component of the atherosclerotic fatty streaks. The fatty streaks reduce the elasticity of the artery walls and the foam cells promote a pro-inflammatory environment by secretion of cytokines and ROS. In addition, foam cells also induce the recruitment of smooth muscle cells to the intima. Added together, these changes lead to the formation of plaques on the artery walls. A fibrous cap of collagen and vascular smooth muscle cells protects the necrotic core and stabilizes the plaque [3;4]. However, blood clots can be formed if the plaque ruptures. These may travel with the bloodstream and obstruct the blood flow of smaller vessels, eg. the coronary arteries, which ultimately can lead to myocardial infarction.</p>
<p><br />
Inhalation of particulate matter, chemicals and pathogens have been related to increased pulmonary inflammation. Whereas a normal immune reaction is crucial for effective elimination of incoming threats, chronic and unresolved inflammation has been linked to both adverse pulmonary and adverse systemic effects in humans. In concordance with this, various retrospective and prospective epidemiological studies have linked pulmonary exposure to respirable air particulates with increased the risk of developing CVD [5-8]. Inhalation of particles has been proposed to affect the cardiovascular system in several different ways, including through disruption of vasomotor function and through acceleration of plaque progression in atherosclerosis [9;10]. We recently showed that a sustained pulmonary inflammatory response occurs concurrently with a persistent acute phase response (APR) in the lungs and in the plasma after exposure to particulate matter in mice [11-13]. Both responses were dose-dependent [14] and the most differentially expressed genes were the serum amyloid A (Saa) isoforms, with <em>Saa3</em> showing the greatest fold changes [11;13-15]. The SAAs are characterized as APR proteins. Similar to the APR protein C-reactive protein (CRP), elevated plasma levels of SAA protein are a risk factor for CVD in human [16-19]. However, in contrast to CRP, increased plasma protein levels of SAA is still related to CVD after Mendelian randomization, suggesting a causal relationship [20;21]. Indeed, studies in rodents have shown that increased levels of SAA increase plaque progression in ApoE−/− mice [22;23].</p>
<p>As stated earlier, there are many different ways by which pro-fibrotic stressors can interact with the components of cell membrane and often involve multiple interactions at the same time. Few studies investigate the exact interaction between the stressor and the cellular membrane components. Asbestos and silica crystals engage scavenger receptors present on the macrophages (Murthy et al., 2015). Bleomycin binds high affinity bleomycin binding sites present on rat alveolar macrophage surfaces, leading to macrophage activation (Denholm and Phan, 1990). However, the consequences of such interactions such as, the release of PRR agonists DAMPs (alarmins) from dying or injured cells, increased gene or protein synthesis downstream of receptor binding or in the case of NMs, their cellular uptake, are measured routinely as indicative of occurrence of such interactions (Nel et al., 2009; Cheng et al., 2013). Because of the phys-chem properties such as surface charge, NMs and asbestos like materials can bind to cellular macromolecules and cell surface/membrane components, which in turn, facilitate their uptake and intracellular sequestration by the cells (NIOSH, 2011a; Pascolo et al., 2013). Several DAMPs that can be effectively measured in biological samples and cultured cells include High Mobility Group Binding 1 (HMGB1) protein, Heat Shock proteins (HSPs), uric acid, annexins, and S100 proteins (Bianchi, 2007). Of all DAMPs, interleukin (IL)-1α is the most commonly measured alarmin. IL-1α is the principal pro-inflammatory moiety and is a designated ‘alarmin’ in the cell that alerts the host to injury or damage (Di Paolo and Shayakhmetov, 2016). It is shown that administration of necrotic cells to mice results in neutrophilic inflammation that was entirely mediated by IL-1α released from the dying or necrosed cells and consequent activation of IL-1 Receptor 1 (IL-1R1) signalling (<a href="#auth-1"><strong>Suwar</strong></a><a href="#a1"><strong>a</strong></a> et al., 2014). IL-1α is released following exposure to MWCNTs (Nikota et al., 2017) and silica (Rabolli et al., 2014). Although IL1-b is not a designated alarmin, its secretion following exposure to stressors is routinely assessed and is linked to initiation of cell or tissue injury.</p>
<p>Other high aspect ratio fibres such as asbestos and CNTs induce frustrated phagocytosis and acute cell injury (Boyles et al., 2015; Dörger et al., 2001; Brown et al., 2007; Kim et al., 2010; Poland et al., 2008), leading to DAMP release (Nikota et al, 2017), inflammation and immune responses.</p>
adjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedNot SpecifiedUnspecificNot SpecifiedAdultNot SpecifiedNot SpecifiedNot SpecifiedNot SpecifiedNot SpecifiedNot SpecifiedNot Specified<p> 1. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006; 6(7):508-519.</p>
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<p> 3. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917):868-874.</p>
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<p>11. Bourdon JA, Halappanavar S, Saber AT, Jacobsen NR, Williams A, Wallin H et al. Hepatic and pulmonary toxicogenomic profiles in mice intratracheally instilled with carbon black nanoparticles reveal pulmonary inflammation, acute phase response, and alterations in lipid homeostasis. Toxicol Sci. 2012; 127(2):474-484.</p>
<p>12. Poulsen SS, Saber AT, Mortensen A, Szarek J, Wu D, Williams A et al. Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease. Toxicol Appl Pharmacol. 2015; 283(3):210-222.</p>
<p>13. Poulsen SS, Saber AT, Williams A, Andersen O, Kobler C, Atluri R et al. MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol. 2015; 284(1):16-32.</p>
<p>14. Saber AT, Jacobsen NR, Jackson P, Poulsen SS, Kyjovska ZO, Halappanavar S et al. Particle-induced pulmonary acute phase response may be the causal link between particle inhalation and cardiovascular disease. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2014; 6(6):517-531.</p>
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