key: cord-0714705-hqrppqfe authors: You, Dorothy J.; Bonner, James C. title: Susceptibility Factors in Chronic Lung Inflammatory Responses to Engineered Nanomaterials date: 2020-10-03 journal: Int J Mol Sci DOI: 10.3390/ijms21197310 sha: 2fe00888a0e72c56ba49217ffff5e805c25db8b3 doc_id: 714705 cord_uid: hqrppqfe Engineered nanomaterials (ENMs) are products of the emerging nanotechnology industry and many different types of ENMs have been shown to cause chronic inflammation in the lungs of rodents after inhalation exposure, suggesting a risk to human health. Due to the increasing demand and use of ENMs in a variety of products, a careful evaluation of the risks to human health is urgently needed. An assessment of the immunotoxicity of ENMs should consider susceptibility factors including sex, pre-existing diseases, deficiency of specific genes encoding proteins involved in the innate or adaptive immune response, and co-exposures to other chemicals. This review will address evidence from experimental animal models that highlights some important issues of susceptibility to chronic lung inflammation and systemic immune dysfunction after pulmonary exposure to ENMs. Inflammation is the consequence of an innate immune response of the host to stimuli such as pathogens (e.g., bacteria, fungi, viruses), allergens, or toxic chemicals and pollutants [1] [2] [3] . The steps in the inflammatory response can be defined as (1) induction, (2) peak, and (3) resolution [1, 2, [4] [5] [6] [7] . The induction phase involves innate immune cells (e.g., macrophages) detecting the pathogen or other foreign agent and orchestrating the recruitment of other innate immune cells (e.g., neutrophils, eosinophils) to the site of infection and/or injury [1, 2, [5] [6] [7] . The peak of inflammation involves recruited innate immune cells (e.g., macrophages, monocytes, neutrophils) engulfing pathogens or inhaled agents, triggering key defensive mechanisms, notably the generation of reactive oxygen species (i.e., respiratory burst) from macrophages and the release of neutrophil extracellular traps (NETs) from recruited neutrophils [1] [2] [3] [4] [5] [6] [7] . Recruited innate immune cells also secrete a plethora of cytokines (e.g., IL-6, IL-1β, TNF-α) and chemokines (e.g., CXC and CC motif chemokines) that have numerous functions (e.g., cell proliferation, cell migration, cell death, alteration of epithelial or endothelial barrier permeability) and also serve as extracellular signals that bridge the innate and adaptive immune systems. The resolution of inflammation involves clearance of foreign agents, removal of recruited host immune cells, and repair of damaged tissue [8, 9] . During an acute inflammatory response, the body's innate immune system defends against the foreign agent in order to remove the invading threat and to heal the damaged tissue through the recruitment of various immune cells such as neutrophils, monocytes, macrophages, lymphocytes, and plasma cells [1] [2] [3] [4] [5] [6] [7] [8] [9] . The induction of acute inflammation is a rapid process that occurs within hours and resolves within days, where the immune process is more localized to the specific injury site [9, 10] . This acute inflammatory response is critical to restore the body back to a state of homeostasis after injury [5] [6] [7] [8] [9] [10] . However, when acute inflammation fails to resolve or multiple exposures dysregulate the immune system, this may lead to unresolved chronic inflammation [4] [5] [6] [7] [8] [9] [10] . [76, 77] . There is some limited evidence that occupational ENM exposure (i.e., MWCNTs) is associated with increased pro-fibrotic mediators in serum and induced sputum of workers [74] . Therefore, repeated pulmonary exposures to ENMs may possess a new risk for chronic unresolved lung inflammation in humans. There is a large gap of knowledge in terms of which susceptibility factors are most important in determining the severity of ENM-induced chronic respiratory diseases in humans, such as asthma, fibrosis, and cancer. Therefore, in this review, we will summarize some of the susceptibility factors that appear to be important in determining the severity of lung inflammatory responses and systemic immunotoxicity in experimental animals exposed to ENMs. These susceptibility factors include deficiency or dysregulation of specific genes, sex, pre-existing diseases, and co-exposures to other chemicals, allergens, or microbial agents that may increase the severity of chronic unresolved inflammatory responses of the lung to ENMs (Figure 1 ). In addition, we will identify some gaps in our understanding of susceptibility to ENM-induced lung inflammation that should help to better predict the risks to human health. The signal transducers and activators of transcription (STATs) are a family of seven transcription factors that remain inactivated in the cytoplasm until activation by extracellular signaling proteins like cytokines or growth factors binding to their cognate receptors on the cell surface [78] . Among them, STAT1 is known as a regulator of apoptosis, growth arrest, and development and maintenance of T helper 1 (TH1) cells [78, 79] . Therefore, the actions of STAT1 are critical to oppose the development of T helper 2 (TH2) cells. It has been previously shown that Stat1 Knockout (KO) mice are susceptible to mortality caused by viral and bacterial infection, primarily due to the fact that the STAT1 pathway is a major signaling pathway through which interferons (IFNs) mediate antiviral activity [80] . Stat1 KO mice are also susceptible to bleomycin-induced lung fibrosis with heightened proliferative responses to growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) [81] . As summarized in Table 1 , Stat1 KO mice exhibit increased fibrosis induced by rigid Susceptibility factors in the chronic lung inflammatory response to engineered nanomaterials (ENMs). The signal transducers and activators of transcription (STATs) are a family of seven transcription factors that remain inactivated in the cytoplasm until activation by extracellular signaling proteins like cytokines or growth factors binding to their cognate receptors on the cell surface [78] . Among them, STAT1 is known as a regulator of apoptosis, growth arrest, and development and maintenance of T helper 1 (T H 1) cells [78, 79] . Therefore, the actions of STAT1 are critical to oppose the development of T helper 2 (T H 2) cells. It has been previously shown that Stat1 Knockout (KO) mice are susceptible to mortality caused by viral and bacterial infection, primarily due to the fact that the STAT1 pathway is a major signaling pathway through which interferons (IFNs) mediate antiviral activity [80] . Stat1 KO mice are also susceptible to bleomycin-induced lung fibrosis with heightened proliferative responses to growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) [81] . As summarized in Table 1 , Stat1 KO mice exhibit increased fibrosis induced by rigid MWCNTs with higher levels of TGF-β1 in the bronchoalveolar lavage fluid (BALF) and increased Smad2/3 phosphorylation in lung tissue [82] . Furthermore, STAT1 plays a critical role to protect against allergen-induced airway remodeling and exacerbation by tangled MWCNTs [83] . From a clinical perspective, patients with idiopathic pulmonary fibrosis have lower levels of STAT1 protein than normal individuals [84] . Therefore, STAT1 deficiency likely plays an important role in lung inflammation that results in chronic lung diseases and can be a risk factor to increase susceptibility toward ENMs. T-box transcription factor TBX21 (T-bet) is a transcription factor that plays a critical role in the development and differentiation of T H 1 cells in the lung [85] . T-bet regulates IFN-γ production and inhibits T H 2 cell development [85] . It has been reported that mice with targeted deletion of T-bet have decreased production of IFN-γ and increased production of T H 2 cytokines, such as IL-4 and IL-13, leading to the spontaneous development of allergic airway remodeling (eosinophilic infiltration, airway mucous cell metaplasia, and subepithelial fibrosis) similar to pathological changes observed in the lungs of asthma patients [86] . As summarized in Table 1 , homozygous T-bet KO mice exhibit enhanced mucous cell metaplasia three weeks after exposure to nickel nanoparticles (NiNPs) compared to wild type mice [87] . The enhanced mucous cell metaplasia in T-bet KO mice induced by NiNPs was accompanied by an increase in MUC5AC and MUC5B mRNAs in the lung [87] . Furthermore, numbers of inflammatory cells, including eosinophils and lymphocytes, persisted in the BALF of T-bet KO mice for at least three weeks after the final NiNP exposure [87] . Moreover, T-bet KO mice developed more interstitial lung fibrosis after NiNP exposure compared to wild type mice [87] . MWCNT exposure via oropharyngeal aspiration also increased mucous cell metaplasia in T-bet KO compared to the wildtype mice, but to a lesser extent compared to NiNPs [87] . Therefore, T-bet appears to play an important role in the polarization of T cells towards a T H 1 phenotype and suppression of allergic T H 2 lung inflammation. Since T-bet KO mice are susceptible to chronic allergic lung inflammation caused by NiNPs and MWCNTs, this suggests that individuals with T-bet deficiency would be more susceptible to certain types of ENMs. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor that controls the expression of antioxidant proteins [88] . Nrf2 is activated to increase the production of antioxidants such as drug-metabolizing enzymes including glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 upon exposure to oxidative stress in cells [88] . Activation of Nrf2 has been implicated as a protective factor that counteracts the pathogenesis of lung disease in mice and humans upon inhaled oxidants including ozone, cigarette smoke, and air pollution particles [89] . It has been reported that a lack of Nrf2 results in a severe outcome inpatients with respiratory infections, chronic obstructive pulmonary disease (COPD), asthma, idiopathic pulmonary fibrosis, and lung cancer [89] . As summarized in Table 1 , exposure of Nrf2 KO mice to MWCNTs by oropharyngeal aspiration showed a higher level of inflammation and fibrosis with increased inflammatory cell infiltrates in the lungs 7 days after the initial exposure compared to the wild type mice [90] . Moreover, increased ROS generation, oxidative damage, and lung inflammation in Nrf2 KO mice exposed to MWCNTs suggests that Nrf2 is critical towards suppressing the lung inflammatory response [90] . Another study, summarized in Table 1 , showed that Nrf2 KO mice exposed to silica nanoparticles via intranasal instillation displayed increased generation of reactive oxygen species (ROS) and decreased total antioxidant capacity compared to wild type mice [91] . This study also suggested that Nrf2 protects against the oxidative stress induced by silica nanoparticles [91] . Therefore, the role of Nrf2 in controlling antioxidant protein expression may be critical to resolve inflammatory responses through suppression of ROS generated by ENMs. The source of these ROS could be directly generated from ENMs via surface chemistry, or indirectly by inflammatory cells (e.g., macrophages) undergoing a respiratory burst in response to ENM exposure. P53 is a transcription factor and tumor suppressor that is pivotal in the progression of lung cancer and also appears to play a role in pulmonary fibrosis [92] . For example, idiopathic pulmonary fibrosis patients have a higher incidence of mutated p53 genes and overexpression of mutated p53 [93, 94] . Human lung epithelial (BEAS-2B) cells exposed to SWCNTs for 12-24 weeks showed increased resistance to apoptosis and a decrease in p53 activation in vitro [95] . Furthermore, mesothelioma formation in the pleural lining of the abdominal cavity in p53 heterozygous mice (p53 +/-) has been reported following intraperitoneal injection of MWCNTs [96] . However, that study lacked a comparison to wild type p53 +/+ mice. As summarized in Table 1 , an increased incidence of larger granulomas, lymphoid aggregates, and epithelial cell hyperplasia was observed in the lungs of p53 +/mice, compared to wild type mice, eleven months after oropharyngeal exposure to rigid (Mitsui-7) MWCNTs [97] . Therefore, p53 could play a role in suppressing chronic lung inflammation and granuloma formation upon pulmonary exposures to different ENMs, including SWCNTs and MWCNTs. Deficiency of p53 in experimental animals and humans is a susceptibility factor in pulmonary diseases, including cancer and pulmonary fibrosis. 2.1.5. BMAL1 Brain and muscle ARNT-like protein (BMAL1) is a transcription factor that controls circadian rhythm and regulates ROS generation [98] . Bmal1 KO mice showed higher platelet aggregation and adhesion, indicating a higher risk for cardiovascular disease compared to wild type mice [99] . Moreover, most of the Bmal1 KO mice die at an age between 26 and 52 weeks, mainly due to excessive ROS production and a chronic oxidative stress state in various tissues, resembling an early aging phenotype [100] . As summarized in Table 1 , both wild type and Bmal1 KO mice have been evaluated for pro-inflammatory responses after exposure to zinc oxide nanoparticles (ZnONPs) or MWCNTs delivered to the lungs via oropharyngeal aspiration over 5 weeks [98] . MWCNT exposure caused an increase in inflammatory responses, cell counts in BALF, oxidative stress, and procoagulant effects in the serum of Bmal1 KO mice compared to wild type mice [98] . On the other hand, ZnONP exposure showed a decrease in inflammatory and oxidative responses but increased the procoagulant effect in Bmal1 KO mice compared to the wild type mice [98] . Although MWCNTs and ZnONPs produced opposite inflammatory responses in Bmal1 KO mice, the dysregulation of coagulation induced by both of these ENMs in Bmal1 KO mice indicates that BMAL1 plays an important role in preventing adverse cardiovascular effects of ENMs [98] . However, different mechanisms appear to be involved in the chronic inflammatory responses to MWCNTs and ZnONPs. NADPH oxidases are enzymes that are responsible for producing superoxide radicals (O 2 •-) [101] . Neutrophils kill invading pathogens by generating reactive oxygen species (ROS) such as O 2 •- [101] . ROS have recently been shown to be a critical factor in controlling the resolution of inflammation by regulating neutrophils and macrophages by sending "eat-me" signals [101] . NADPH oxidase-deficient mice or gp91 phox−/− that specifically lacks the gp91 phox subunit of the enzyme have been used to evaluate lung inflammation after exposure to SWCNTs [102] . This study, summarized in Table 2 , showed that gp91 phox−/− KO mice had augmented lung inflammation with higher numbers of neutrophils, apoptotic cells, pro-inflammatory cytokines (TNF-α, CCL2, and IL-6), and reduced TGF-β1 at 1, 7, and 28 days after pharyngeal exposure to SWCNTs [102] . The results also suggest that the impaired resolution of the inflammatory response may develop into a chronic unresolved inflammatory state, due to prolonged increase in neutrophils and pro-inflammatory cytokines, in NADPH oxidase deficient mice [102] . Thus, NADPH oxidase could play a protective role in the resolution of ENM-induced lung inflammation by releasing ROS to regulate neutrophils and macrophages. Cyclooxygenase-2 (COX-2), also known as prostaglandin-endoperoxide synthase-2 (PTGS2), is an enzyme that has been implicated in asthma and fibrosis [103] . Along with T H 2 cytokines, including IL-13 and IL-5, COX-2 has been implicated in asthma pathogenesis [104, 105] . However, unlike IL-13 and IL-5, which are increased in individuals with asthma, COX-2 deficiency has been linked to increased severity of asthma progression [103] [104] [105] [106] [107] . Particularly, individuals with asthma have a reduced level of mRNA encoding COX-2 in airway epithelial cells [106] . Furthermore, a study using airway epithelial cells collected from endobronchial brushings from healthy individuals, when treated with IL-13 to mimic the asthmatic microenvironment, had reduced COX-2 mRNA expression levels [107] . Lower levels of COX-2 in airway epithelial cells from the healthy individuals treated with IL-13 resulted in lower prostaglandin E 2 (PGE 2 ) [107] . Therefore, these data indicate that COX-2 plays a protective role in the asthmatic lung and loss of COX-2 and PGE 2 results in susceptibility to allergic airway inflammation. As summarized in Table 2 , Ptgs2 KO mice sensitized to ovalbumin allergen through repeated intranasal aspiration followed by a single oropharyngeal aspiration of MWCNTs were found to be more susceptible to eosinophilic lung inflammation, airway mucous cell metaplasia, and airway fibrosis compared to wild type mice [103] . Compared to wild type mice, the Ptgs2 KO mice also had significantly higher Th2 cytokines including IL-13, Th1 cytokines such as CXCL10, and the Th17 cytokine IL-17A [103] . Overall, this study showed that Ptgs2 KO mice were susceptible to the exacerbation of allergen-induced airway disease by MWCNTs [103] . Deficiency in COX-2 could therefore be a possible mechanism involved in chronic lung inflammation induced by certain types of ENMs such as MWCNTs. Thus, COX-2 appears to be an enzyme that is needed for the resolution of chronic airway inflammation and loss of COX-2 could lead to susceptibility to ENM-induced lung inflammation. Tissue inhibitor of metalloproteinase 1 (TIMP1) is a glycoprotein that acts as an extracellular signaling molecule to control cell growth, apoptosis, differentiation, angiogenesis, and oncogenesis [108] . TIMP1 also plays a role to control extracellular matrix by mediating the activity of matrix metalloproteinases (MMPs) [108] . Furthermore, it has been reported that high expression of TIMP1 is associated with liver and pulmonary fibrosis in animal models [109] [110] [111] . It has been demonstrated that MWCNT-induced lung fibrosis in mice corresponded with highly upregulated TIMP1 at both the mRNA and protein level in lung tissue [112] . Moreover, Timp1 KO mice exposed to MWCNTs as summarized in Table 2 had reduced lung fibrosis, suppressed myofibroblast differentiation, and lower activation of extracellular signal-regulated kinase (ERK) signaling, indicating that TIMP1 plays a pro-fibrotic role in the progression of MWCNT-induced lung fibrosis through activation of the intracellular ERK pathway [112] . Thus, TIMP1 may aggravate MWCNT-induced lung inflammation and may contribute to the susceptibility to chronic lung disease. Myeloperoxidase (MPO) is an abundant enzyme in inflammatory cells especially produced by neutrophils and plays a vital defense role in the innate immune system [113] . Intratracheal instillation of MWCNTs and SWCNTs in rats has been shown to consistently increase the concentration of MPO in BALF [114] . Increased MPO was also associated with an increased number of total cells and neutrophils in the BALF, suggesting that it could be a potential biomarker for pulmonary toxicity induced by ENMs [114] . However, MPO has been reported to effectively mediate the oxidative biodegradation of CNTs (SWCNTs or MWCNTs), leading to resolution of inflammation [113] . To support this, MPO KO mice exposed to SWCNTs (summarized in Table 2 ) showed less efficient clearance of the nanomaterials [115] . This same study showed that MPO KO mice had a slightly weaker acute neutrophilic inflammatory response at day 1 after exposure to SWCNTs but significantly greater lung fibrosis at day 28. Thus, these findings suggest that increased MPO from pulmonary injury induced by ENMs could be playing a vital role in both initiating and resolving inflammation. As such, MPO has been suggested to be a double-edged sword where it can both be responsible for the initiation of inflammation and the resolution of inflammation caused by pulmonary exposure to ENMs. Apolipoprotein E (ApoE) is a protein that is responsible for fat metabolism in the body [116] . It has been reported that ApoE KO mice have increased vascular dysfunction to particulates like diesel exhaust and fullerene C60 (C60) nanoparticles [117, 118] . As summarized in Table 2 , intratracheal instillation of different ENMs, including carbon black nanoparticles (CBNPs), gold nanoparticles (AuNPs), C60 nanoparticles, and SWCNTs, increased DNA damage in BALF inflammatory cells, increased neutrophils in BALF, and higher BALF protein levels in ApoE KO mice compared to wild type mice [119] . ApoE KO mice with repeated exposure to MWCNTs via intratracheal instillation were also more susceptible to oxidative DNA damage in lung tissue compared to the wildtype mice [120] . Suzuki et al. also found that pharyngeal aspiration to single or double-walled CNTs caused endothelial progenitor cell (EPC) dysfunction and reduced migration function of EPCs in ApoE KO mice [121] . Dysregulation of EPCs in ApoE KO mice could contribute to the development of atherosclerosis [121] . Another study using ApoE KO mice treated with MWCNTs via intratracheal instillation showed a positive association between pulmonary inflammation and oxidative stress, along with the expression of genes involved in vascular activation [122] . Increased vascular activation from MWCNTs also could play a role in exacerbating the progression of atherosclerosis [122] . Overall, these studies suggest that a lack of ApoE is a susceptibility factor in ENM-induced lung inflammation and cardiovascular disease. Aryl hydrocarbon receptor (AhR) is a transcription factor that was originally shown to bind and become activated by environmental toxicants such as dioxins and polycyclic aromatic hydrocarbons [123] . Once activated, AhR transactivates phase I and phase II metabolizing enzymes such as cytochrome P450 1A1 [123] . Recent studies have shown that AhR is not only involved in metabolizing toxicants but may also be involved in the pulmonary immune and pro-inflammatory responses [124] . For example, a study using AhR KO mice exposed to ZnONPs via oropharyngeal aspiration (Table 3) showed that these mice had reduced cell numbers, total protein, LDH activity, and pro-inflammatory cytokine production in BALF compared to the wild type mice [125] . No increase in CYP1A1, a downstream target of AhR, was detected in AhR KO mice [125] . In addition, AhR KO mice did not display an increase in kynurenine (KYN), which is an endogenous AhR agonist [125] . Therefore, this work suggests that AhR is involved in mediating ZnONP-induced pulmonary inflammation. Furthermore, impaired/imbalanced AhR expression could be a potential factor in determining susceptibility to ENM-induced chronic lung inflammation. CCR5 is a chemokine receptor that is known to mediate the T H 1 response and binds several chemokines including RANTES/CCL5, MIP-1α, and MIP-1β [126, 127] . A study by Park and colleagues, summarized in Table 3 , compared the inflammatory response from Ccr5 KO and wild type mice after exposure to SWCNTs via intratracheal instillation [128] . A single intratracheal instillation of SWCNTs significantly reduced the number of neutrophils in Ccr5 KO mice, yet KO mice had more frequent histopathological lesions compared to the wildtype mice [128] . The lung inflammatory response of Ccr5 KO mice was dominated by B cells and CD8+ T cells, while the wild type mice were mostly dominated by T cells and CD4+ T cells in the lungs [128] . Moreover, SWCNTs also increased IL-6, IL-13, and IL-17 in BALF of Ccr5 KO mice compared to the wildtype mice [128] . These data suggested that the delay in the resolution of inflammation could be due to impaired cell migration to the inflammation site, which was supported by reduced numbers of neutrophils [128] . The authors of this work suggested that a delay in the resolution of inflammation resulted from shifting from a Th1 type response to a Th2 type response in the Ccr5 KO model [128] . Overall, these findings suggest that Ccr5 KO mice appear to be more susceptible to SWCNT-induced chronic lung inflammation due to the delay in the resolution of inflammation [128] . Therefore, CCR5 may play an important role in the resolution of inflammation induced by ENMs. IL-1 is a family of cytokines that play a critical role in innate immunity [129] . There are 11 members in the family, and IL-1β, IL-1α, and IL-33 are the main IL-1 family members [129, 130] . IL-1 family members provide non-specific immune responses toward foreign pathogens [129] . However, IL-1 also can mediate an adaptive immune response [129] . Therefore, IL-1 family members are pivotal cytokines that are involved in both acute and chronic inflammation [129] . Among the IL-1 family, the role of IL-1β has been studied the most extensively in ENM-induced lung inflammation. IL-1β secretion from inflammatory cells is regulated by inflammasomes, intracellular scaffolds that cleave pro-IL-1β to mature IL-1β when they detect extracellular signals [129] . The inflammasome is a critical complex to orchestrate IL-1 function [129] . For example, IL-1β is first produced as a precursor form, termed pro-IL-1β, that is lacking in biological activity [129] . In order to increase IL-1β production and secretion, the multiprotein complex inflammasome is assembled and serves as a docking scaffold for caspase-1 to cleave the inactive pro-IL-1β to active IL-1β. It has been shown that MWCNTs and other nanoparticles can activate the inflammasome assembly through lysosomal disruption [129, 131] . For example, mice exposed with MWCNTs through oropharyngeal aspiration had increased IL-1β secretion in BALF [132] . Different studies have shown that IL-1α and IL-1β play roles in ENM-induced lung inflammation [131, 132] . However, the role of IL-1β remains controversial, since some work has shown that reduced inflammasome activation results in greater pulmonary fibrosis induced by ENMs. For example, mice sensitized to house dust mites and challenged with MWCNTs showed reduced IL-1β in BALF but had more severe airway fibrosis after 21 days with increased pro-fibrogenic cytokines, including PDGF-A and PDGF-B mRNAs, compared to HDM or MWCNT treatment alone [132] . Another study, summarized in Table 3 , showed that a 24 h acute exposure to MWCNTs, containing either low or high nickel content, through oropharyngeal aspiration in IL-1R KO mice had reduced acute inflammation and airway resistance but increased IL-6 protein production compared to the wild type mice [133] . However, this study also showed that the exposure to MWCNTs induced a significantly higher number of pulmonary granulomas and significant inflammation in IL-1R KO after 28 days compared to wild type mice [133] . The results of this study also showed that total inflammatory cells were reduced at both 24 h and 28 days post exposure in IL-1R KO mice [133] . It was suggested that the resolution of inflammation was inefficient in IL-1R KO mice, resulting in increased granuloma and inflammation after 28 days [133] . Similarly, another study also showed that acute inflammation induced by intratracheal instillation of MWCNTs was suppressed 24 h after the exposure in IL-1R KO mice, but fibrotic lesions still developed in KO mice after 28 days [134] . Other work using IL-1R KO mice exposed to rod-like MWCNTs by oropharyngeal aspiration (see Table 3 ) showed significantly reduced neutrophils in the BALF and lower levels of mRNA encoding CXCL5 (a neutrophil chemokine) at 4 h compared to the wildtype mice [135] . After the 28-day exposure to rod-like MWCNTs, neutrophils were still reduced in the BALF and TNF-α mRNA expression levels were suppressed compared to the wild type mice [135] . However, no changes were observed in T H 2 related signals including mRNAs encoding IL-13 and TGF-β1, compared to the wild type mice [135] . Overall, the available evidence suggests that impaired IL-1 signaling caused by MWCNTs dysregulates the immune system, resulting in reduced acute inflammation or inefficient resolution of inflammation and resulting in pulmonary fibrosis and granuloma formation. Osteopontin (OPN; secreted phosphoprotein 1 or SPP1) is a cytokine that is involved in various physiological and pathological processes, including inflammation, fibrosis, and bone remodeling [136] . Elevated OPN is expressed in tissues undergoing an intense wound healing process or during fibrogenesis [136] . It has been shown that both acute and chronic exposures to MWCNTs can induce higher OPN production levels [136] . A study using Opn KO mice showed that inhalation exposure to MWCNTs via pharyngeal aspiration (see Table 3 ) induced inflammation that was dependent on OPN [136] . The study also showed that Opn KO mice had reduced fibrotic formation and myofibroblast accumulation in the lungs compared to the wild type mice [136] . They suggested that OPN production and secretion in turn could activate TGF-β1 to promote fibrosis in the lungs [136] . Rats exposed to different doses of TiO 2 particles via inhalation showed a significant increase in lung OPN mRNA and OPN protein in BALF in a dose-response manner [137] . Therefore, OPN appears to play a critical role in tissue remodeling. Individuals with excessive production of OPN might be more susceptible to unresolved chronic lung inflammation due to exposure to ENMs, resulting in excessive tissue remodeling and fibrosis in the lungs. Table 3 . Summarized list of receptors and cytokines/chemokines that may be involved in the susceptibility to ENM-induced chronic lung inflammation. Single exposure Reduced acute inflammation and airway resistance but increased IL-6 protein production within 1 day. [133] Induced significantly higher number of pulmonary granulomas formation and significant inflammation post 28 days. Increased IL-6, IL-13, and IL-17 in BALF. More frequent histopathological lesions were detected. Interleukin 6 (IL-6) is major pro-inflammatory cytokine and vital regulator for both the innate and adaptive immune system [138] . Studies have shown that IL-6 is increased systemically upon inhalation exposure to ENMs in mice and humans [139] [140] [141] . Specifically, pulmonary instillation of MWCNTs induced levels of systemic IL-6 in the heart, and MWCNTs coated with Zn also induced IL-6 mRNA levels in the heart and liver [141, 142] . IL-6 can mediate both pro-inflammatory and anti-inflammatory functions [138] . An acute increase in IL-6 facilitates neutrophil recruitment to sites of tissue injury in order to induce acute inflammation [143] . However, IL-6 also activates STAT3 signaling which facilitates the reduction in neutrophils in acute inflammation [144] . Sustained IL-6 production also induces prolonged STAT3 activation and neutrophil recruitment to promote unresolved chronic inflammation [145] . Prolonged STAT3 activation has been implicated in many chronic inflammatory diseases including asthma, fibrosis, cancer, and hepatitis [145] . Therefore, adequate regulation of IL-6 is required for the initiation and resolution of inflammation. For example, a study showed that IL-6 can prevent the initiation but at the same time, promote the progression of lung cancer [146] . We recently reported that IL-6 is highly induced in the lungs of mice following acute exposure to NiNPs by oropharyngeal aspiration [147] . Il6 KO mice have not yet been evaluated after inhalation exposure to ENMs. However, it is likely that IL-6 plays a major role in determining susceptibility to chronic inflammation. Sex can be a key determinant of susceptibility to ENM-induced chronic lung inflammation [148] [149] [150] . In this review, we will discuss this topic in the context of biological sex differences, including chromosome, sex organs, and endogenous hormonal profiles between males and females as defined by the Office of Research on Women's Health at the National Institute of Health [151] . Sex is recognized as a critical factor in determining the susceptibility of individuals to respiratory disease [148, [152] [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] [163] . Furthermore, according to epidemiology studies, there are significant differences in susceptibility between acute and chronic respiratory disorders [148, 152, [156] [157] [158] [159] 164] . Epidemiology studies show that men are more susceptible to acute lung inflammation from viral or bacterial infections and they have worse a prognosis compared to females [158, 164, 165] . As seen with the novel coronavirus SARS-CoV-2, epidemiology studies have found that a greater number of men are infected compared to women [166] . This trend was also seen in the MERS-CoV and SARS-CoV-1 viruses [166] . In contrast to men, women are more susceptible to developing chronic lung inflammation such as asthma [164, 167] . Thus, clear susceptibility differences exist between men and women with regards to respiratory diseases, suggesting differences in pulmonary immune responses. To date, there is a lack of data depicting sex as a key determinant of susceptibility to ENM-induced chronic lung inflammation. However, there is some evidence showing susceptibility of either male or female rodents to ENM-induced lung inflammation. For example, Ray et al. found that a single dose of MWCNTs delivered via oropharyngeal aspiration caused a greater acute and chronic inflammatory response in female mice compared to male mice [148] . Kasai et al. also found that female rats were more susceptible to lung inflammation induced by rigid-MWCNTs via whole-body inhalation compared to male rats [168] . On the other hand, Ray and colleagues also showed that male mice were more susceptible to lung inflammation induced by repeated chronic exposure to crystalline silica (cSiO 2 ) compared to female mice [148] . Others have shown that cSiO 2, delivered to the lungs of mice by intratracheal instillation, caused less severe lung inflammation in female mice than male mice [169, 170] . Furthermore, it was demonstrated in the same study that male mice treated with estradiol through subcutaneous injection also had reduced cSiO2-induced lung inflammation compared to male mice that received no exogenous estradiol [169, 170] . In addition, lung fibrosis was more severe in male mice compared to female mice or male mice with supplementary estradiol [169, 170] . Our lab has also recently reported that male mice were susceptible to lung inflammation with both acute and sub-chronic exposure to NiNPs with or without LPS [147] . Epidemiological and animal studies suggest that sex is a critical factor in determining the susceptibility and resolution of lung inflammation. Moreover, the available literature using rodents suggests that susceptibility to acute and chronic lung disease induced by ENMs is determined in part by sex. Furthermore, epidemiology data related to ENMs are lacking due to the fact that nanotechnology is an emerging industry and human exposures are relatively new. Thus, more extensive studies should be conducted to carefully analyze the risk of ENMs to unresolved chronic lung inflammation and how the risk may differ between sexes. Pre-existing lung diseases, including asthma, chronic bronchitis, COPD, or inflammation induced by microbial infection (e.g., bacterial, fungal, or viral), would likely render individuals susceptible to inhaled ENMs. As mentioned earlier, asthma affects approximately 300 million people worldwide and 26 million in the United States [21, 22] . Individuals with asthma or other pre-existing lung disorders may be at a higher risk of developing more severe pulmonary disease, i.e., exacerbation, with exposure to ENMs. Our group previously reviewed the toxicology of ENMs in asthma in more detail [171] . Ovalbumin (OVA) is a commonly used allergen to induce an asthmatic phenotype in rodents, including AHR and T H 2-mediated airway inflammation [172] . It has been shown that silica nanoparticles (SNPs) caused respiratory toxicity and exacerbated OVA-induced inflammation with higher T H 2 cytokine levels, including IL-13 [173] . Titanium dioxide nanoparticles (TiO 2 NPs), carbon black nanoparticles (CBNPs), MWCNTs, or zinc oxide nanoparticles (ZnONPs) have been reported to exacerbate allergic inflammatory responses in mice with increases in both T H 1 and T H 2 cytokines [132, [174] [175] [176] . Collectively, these studies suggest that individuals suffering from asthma or other pre-existing respiratory disorders maybe at a higher risk for developing severe chronic lung inflammation upon exposure to ENMs. Inhalation exposure to particulate matter can lead to more severe cardiovascular conditions in individuals with pre-existing cardiovascular disorders [177] . In addition, researchers have established a relationship between occupational exposure to dust and ischemic heart diseases [178] . While there is a lack of information on cardiovascular disease and ENMs in humans, studies using rodents suggest that pulmonary exposure to ENMs could exacerbate pre-existing cardiovascular conditions in humans [179] [180] [181] . Additionally, several specific mediators discussed below play important roles in determining susceptibility to cardiovascular disease after ENM exposure. Thrombospondin (TSP-1) is a protein involved in wound healing and regulating blood pressure [182] [183] [184] [185] . TSP-1 binding to the cell surface receptor CD47 in vascular smooth muscles has been reported to inhibit endothelial nitric oxide synthase (eNOS) [182, 186] . In addition, acetylcholine (ACh)-stimulated activation of eNOS leads to a decrease in blood pressure, but TSP-1 has been reported to block the Ach-stimulated decrease in blood pressure [186] . TSP-1 also has another receptor, CD36, that may also play a role in suppressing eNOS activity [183] . Thus, the impaired signaling axis of TSP-1/CD47/CD36 has been implicated in different cardiovascular diseases, including cardiac hypertrophy, impaired angiogenesis, and pulmonary hypertension [184, 185, [187] [188] [189] . Studies with Tsp1 KO mice and CD47 KO mice suggest that TSP-1 is critical to regulate endothelial function when exposed to ENMs [184, 185] . Two studies by Mandler et al. showed that Tsp1 KO mice exposed to MWCNTs through oropharyngeal aspiration resulted in microvascular dysregulation that could play a critical role in developing cardiovascular diseases [184, 185] . This finding suggested that impairment of TSP-1 is critical to cardiovascular function and leads to greater susceptibility to severe cardiovascular disorders upon exposure to MWCNTs. A study using mice treated with a high dose of small, tangled MWCNTs or large, thick MWCNTs by intratracheal instillation showed a significantly higher level of serum amyloid A 3 (SAA3), haptoglobin, total cholesterol, and low-density lipoprotein in plasma [190] . The accumulation of SAA3 lipoprotein is induced by atherosclerotic plaque formation and is associated with chronic vascular inflammation [191, 192] . Another study using different ENMs including MWCNTs, SWCNTs, and CB nanoparticles also showed that exposure via intratracheal instillation increased transcript level of SAA3 in the lungs in a time-and dose-dependent manner [192] . An increase in plasma proteins, along with an increased transcript level of SAA3, in mice after pulmonary exposure to MWCNTs, suggests that the risk of developing or exacerbating cardiovascular diseases could be high [190, 192] . The low-density lipoprotein (LDL) receptor (LDLR) binds both LDL and haptoglobin [193] . A recent study used Ldlr KO mice to examine systemic inflammation caused by indium dioxide (In 2 O 3 ) nanoparticles. After a single pharyngeal aspiration dose of In 2 O 3 nanoparticles, Ldlr KO mice developed significantly severe atherosclerotic lesions compared to wild type mice [193] . The Ldlr KO mice also showed that the aorta had a much higher transcript level of IL-6 and MCP-1 (CCL2), suggesting ongoing inflammation in the heart [193] . Ldlr KO mice also showed an increase in total cholesterol and low-density lipoprotein in the plasma compared to wild type mice after treatment [193] . Therefore, this study emphasized the protective role of the LDLR in limiting cardiovascular disease after pulmonary exposure to an ENM. Moreover, these findings suggest that the pulmonary exposure to In 2 O 3 nanoparticles can induce systemic inflammation that may exacerbate the progression of pre-existing cardiovascular diseases. As mentioned earlier, ApoE is a protein responsible for fat metabolism and ApoE KO mice represent a model for atherosclerosis in humans [116, 194] . Thus, deficiency in ApoE not only results in increased susceptibility to chronic lung inflammation, but also could lead to increased susceptibility to cardiovascular diseases. A study showed that intratracheal instillation of TiO 2 NPs significantly increased total cholesterol, nitric oxide, and eNOS in ApoE KO mice compared to wild type mice [195] . Another study also showed long term exposure to nickel nanoparticles (NiNPs) viawhole-body inhalation system exacerbated atherosclerosis in ApoE KO mice [196] . Furthermore, the study also showed that different vascular effects, including significant mitochondrial damage in the aorta and severe progression of atherosclerosis, were seen after pulmonary exposure via intratracheal instillation to carbon black nanoparticles (CBNPs) in ApoE KO mice [196] . Collectively, these mouse studies suggest that ApoE is likely important for suppressing cardiovascular inflammation in humans after pulmonary exposure to ENMs and that reduced ApoE would render individuals susceptible to the adverse effects of ENMs on the cardiovascular system. Although few studies have investigated the systemic effects of inhalation exposure to ENMs, some work has focused on the liver to determine metabolic changes after inhalation exposure to ENMs. A recent study showed that intratracheal instillation of MWCNTs in female mice could affect liver lipid metabolism of their offspring [197] . The data showed that the weight of offspring was slightly reduced, and histopathological changes were observed in the liver tissue [197] . Intratracheal instillation of nickel oxide NPs to the lungs of rats caused pathological changes in the liver, including cellular edema and inflammatory cell infiltration, increased total nitric oxide synthase (NOS), and increased in liver-related enzymes including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) [198] . Another study showed that sub-chronic inhalation of lead oxide nanoparticles in mice via whole-body inhalation resulted in the translocation of nanoparticles to systemic organs, including the kidney, liver, and spleen [199] . They found that lead oxide nanoparticles caused hepatic necrosis, remodeling, and degeneration of hepatocytes [199] . Furthermore, other studies have also demonstrated that inhalation exposure to silver nanoparticles, iron oxide nanoparticles, and MWCNTs results in translocation to the liver [200, 201] . Repeated intratracheal instillation of TiO 2 NPs to the lungs of rats also showed significant hepatocyte necrosis and fibrosis [202] . Furthermore, the increased presence of inflammatory cells in the liver and formation of liver fibrosis was seen in the liver through pulmonary exposure via intratracheal instillation to CBNPs in ApoE KO mice [202] . Therefore, these studies suggest that inhalation exposure to different ENMs has the potential to elicit systemic inflammation in the liver and could exacerbate pre-existing liver conditions such as hepatic steatosis, hepatitis, and cirrhosis. However, to our knowledge, there are no published reports on the effects of ENMs in mouse models of pre-existing liver disease. The spleen has been shown to be an important target of systemic immune responses to inhaled ENMs, specifically to MWCNTs [203] [204] [205] . Whole-body inhalation of MWCNTs in mice has been shown to cause systemic splenic immunosuppression [204] . Immune function was measured on spleen-derived cells and showed suppressed T cell-dependent antigen responses and reduced proliferative ability of T cells following mitogen stimulation after 14 days of MWCNTs inhalation exposure [204] . In addition, an increase in IL-10 and NAD(P)H oxidoreductase 1 mRNA level was detected only in the spleens and not in the lungs [204] . Following this study, the same group investigated the mechanism for immune suppression caused by MWCNT inhalation exposure and found that secreted transforming growth factor-beta (TGF-β) from the lungs had an effect on suppressing the immune function of splenocytes [203] . They demonstrated that activation of this signal in the lung from inhalation exposure to MWCNTs activated the cyclooxygenase pathways in the spleen, resulting in suppressed immune function and T cell dysfunction [203] . Another study showed that inhalation exposure to MWCNTs in rats caused immune dysfunction in the spleen [205] . After 13 weeks of exposure, rats developed systemic inflammation with increased production of inflammatory cytokines from splenic macrophages [205] . They also found a decrease in IL-2 mRNA expression in T-lymphocytes [205] . Thus, inhalation exposure to MWCNTs can affect the normal immune response generated by the spleen and could result in immunosuppression from T cell dysfunction. The blood-brain barrier (BBB) consists of capillary tight junctions that selectively allow movement of specific molecules, ions, and cells between the blood and central nervous system [206] . Under healthy conditions, the BBB has capillary tight junctions that prevent particles entering the central nervous system (CNS) [206] . However, during cerebrovascular inflammation caused by inhalation of pollutants or toxicants, the tight junction of the BBB becomes destabilized with increased permeability, thereby enabling unwanted molecules or particles like ENMs to enter the CNS [207, 208] . Furthermore, some studies have found that inhalation exposure to nanoparticles or ultrafine particles can reach the brain in mammals [209] [210] [211] [212] . While such exposures that result in ENM translocation across the BBB could have deleterious effects on the CNS, some studies have shown potential benefits of using ENMs in nanomedicine to treat brain diseases [213] [214] [215] . Thus, the toxicity of ENMs in the brain still remains unclear. While some ENMs may not have a toxic effect on the brain, there is evidence that some ENMs elicit neuroinflammation. For example, mice exposed to MWCNTs via oropharyngeal aspiration induced BBB disruption after 4 h, which led to the recruitment of phagocytic microglia causing neuroinflammatory responses [216] . Another study also showed aerosolized exposure of MWCNTs to rats increased mitochondrial ROS formation in different parts of the brain [217] . Intranasal instillation of aluminum NPs in rats induced ERK and p38 MAPK activation in the brain, suggesting penetration through the BBB [218] . Lastly, two weeks after an intranasal instillation of AgNPs in mice, RNAseq analysis showed that 73 genes were affected in the cerebrum and 144 genes were changed in the cerebellum [219] . Therefore, these studies suggest that pulmonary exposure to different ENMs results in translocation across the BBB, causing neuroinflammation. However, the use of ENMs that are able to penetrate the BBB could also be beneficial to treat neurological disorders. More rigorous studies should be conducted in order to analyze the risk related to ENM exposure in the brain. Co-exposures to a variety of toxic agents (e.g., metals, chemicals, microbial-derived products) could render individuals susceptible to the pro-inflammatory, pro-fibrotic, or carcinogenic effects of ENMs. For example, lipopolysaccharide (LPS) derived from Gram-negative bacteria can induce acute lung injury through neutrophilic inflammation [220] [221] [222] . LPS is ubiquitous in the environment and therefore, co-exposure to LPS and ENMs in occupational settings is likely [223] [224] [225] [226] . Studies have shown that ENMs, including MWCNTs and NiNPs, can exacerbate LPS-induced lung inflammation in mice or rats [147, 220] . A single co-exposure to LPS and NiNPs in mice via oropharyngeal aspiration caused significantly higher acute neutrophilic lung inflammation and greater IL-6 secretion in the BALF compared to LPS or NiNPs alone [147] . Greater acute lung inflammation induced by the co-exposure corresponded to heightened phosphorylation of STAT3 in lung tissue. Repeated co-exposure of mice to LPS and NiNPs in a sub-chronic exposure study produced greater monocytic lung inflammation and greater CCL2 in BALF compared to LPS or NiNPs alone [147] . Greater sub-chronic lung inflammation induced by LPS and NiNP co-exposure was associated with reduced STAT1 in lung tissue. MWCNT exposure has been reported to exacerbate PDGF signaling and pulmonary fibrosis in rats that were pre-exposed to LPS and had pre-existing neutrophilic lung inflammation [220] . Moreover, CBNPs also enhanced LPS-induced lung inflammation by increasing IL-1β and macrophage chemoattractant protein (MIP-1) [227] . Smaller CBNPs elicited a more profound effect by exacerbating severe LPS-induced lung inflammation [227] . Moreover, circulating fibrinogen levels were much higher in the serum of mice co-exposed to LPS and CBNPs, compared to LPS or CBNP exposure alone [227] . Proteolytic allergens from the house dust mite (HDM) Dermatophagoides pteronyssinus are common indoor allergens [176, 228, 229] . Early life exposure to HDM allergens, including Der p1 and Der p2, has been linked to the development of asthma in humans [230] [231] [232] . Pre-exposure of mice to HDM extract by repeated intranasal aspiration exacerbated lung inflammation and airway fibrosis caused by a single dose of MWCNTs delivered by oropharyngeal aspiration [132] . HDM-induced serum IgE levels were amplified by MWCNTs in this study [132] . In contrast, pre-exposure of mice to MWCNTs by 30 days of inhalation prior to repeated intranasal aspiration of HDM extract also exacerbated lung inflammatory lesions, but reduced HDM-induced serum IgE levels [176] . Therefore, while these two studies showed that either pre or post exposure to MWCNTs exacerbates HDM-induced chronic lung inflammation, the mechanisms involved are likely different. Overall, co-exposure to ENMs and ubiquitous allergens or LPS can exacerbate lung inflammation in rodents, suggesting that such co-exposures would increase lung disease severity in humans. Some challenges remain that must be overcome to improve risk assessment of ENMs. As the lungs are constantly exposed to a variety of inhaled toxicants and microbial agents, single ENM exposures studied in the lab using rodent models or in vitro cell culture models might not reflect a real-world exposure scenario. However, studies on co-exposures to ENMs and other inhaled toxicants or microbial agents are lacking. Therefore, more studies should be performed, using a tiered approach involving both in vitro and in vivo models to address how co-exposure to toxicants or microbial pathogens affects the immune response to inhaled ENMs. Another challenge is the knowledge gap between predicting the immunotoxicity of ENMs in rodents or humans in vivo using cell culture models in vitro. This is due, at least in part, to the complexity of the innate and acquired immune system that involves numerous cell types (e.g., macrophages, neutrophils, T cells, B cells) and subpopulations of each of these cell types. Some of these limitations could be overcome by co-culture models or 'organ-on-a-chip' technologies, coupled with in silico approaches. Furthermore, to our knowledge, no studies using mice have been performed to differentiate the immune response generated by inhalation exposure to ENMs between different age groups. As age and sex can be critical susceptibility factors in chronic lung inflammation, more studies should be done to carefully assess the toxicity of ENMs, comparing male and female mice of varying ages. This is also a challenge, as research on the immunotoxicity of ENMs is increasingly moving towards in vitro and in silico approaches, based on the growing number of ENMs to be evaluated and ethical concerns of using animals in research. Nevertheless, complex issues of sex and age are difficult to recapitulate using only in vitro or in silico approaches. Herein, we have summarized different susceptibility factors, including sex, pre-existing disease state, deficiency of specific genes, susceptible organ systems, and co-exposures to other agents that might influence the pulmonary and/or systemic immune response generated by exposures to different types of ENMs. The available literature discussed in this review article illustrates that multiple factors play critical roles in determining host susceptibility to the adverse effects of ENMs on the immune system. Future research should continue to emphasize susceptibility factors and susceptible populations in risk assessments to avoid underestimating adverse outcomes in humans caused by occupational or environmental exposure to ENMs. Pathogen recognition and inflammatory signaling in innate immune defenses Inflammatory responses and inflammation-associated diseases in organs Lung inflammation caused by inhaled toxicants: A review Resolution of in flammation: State of the art, definitions and terms Resolution of chronic inflammatory disease: Universal and tissue-specific concepts -7) promotes resolution of neutrophilic inflammation in a model of antigen-induced arthritis in mice ROCK inhibition drives resolution of acute inflammation by enhancing neutrophil apoptosis Resolution of pulmonary inflammation induced by carbon nanotubes and fullerenes in mice: Role of macrophage polarization Resolution of acute inflammation and the role of Resolvins in immunity, thrombosis, and vascular biology Acute inflammation and metabolism Key mechanisms governing resolution of lung inflammation Resolution of acute inflammation in the lung Bioactive lipids and chronic inflammation: Managing the fire within Chronic inflammation and asthma Innate control of adaptive immunity: Beyond the three-signal paradigm Inflammation and cancer: Tissue regeneration gone awry The cytokine network in chronic obstructive pulmonary disease Liver inflammation and fibrosis Susceptibility to chronic inflammation: An update Mechanisms of airway hyper-responsiveness in asthma: The past, present and yet to come The Global Asthma Report Most Recent National Asthma Data Estimating production data for five engineered nanomaterials as a basis for exposure assessment Mesenchymal cell survival in airway and interstitial pulmonary fibrosis Toxicity of engineered nanomaterials: A physicochemical perspective Safety assessment for nanotechnology and nanomedicine: Concepts of nanotoxicology Engineered nanomaterials (ENMs) and their role at the nexus of Food A Multilaboratory toxicological assessment of a panel of 10 engineered nanomaterials to human health-ENPRA project-The highlights, limitations, and current and future challenges Implications of engineered nanomaterials in drinking water sources A systematic review of reported exposure to engineered nanomaterials Nanoparticles as a potential cause of pleural and interstitial lung disease Risks, release and concentrations of engineered nanomaterial in the environment Engineered nanomaterials for antimicrobial applications: A review Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world Toxicological assessment of nanoparticle interactions with the pulmonary system Synergistic effect of functionalized nickel nanoparticles and Quercetin on inhibition of the SMMC-7721 cells proliferation Biopharmaceutics and therapeutic potential of engineered nanomaterials Novel therapeutics and diagnostics strategies based on engineered nanobiomaterials Engineered nanomaterials as potential candidates for HIV treatment: Between opportunities and challenges PFOS induced lipid metabolism disturbances in BALB/c mice through inhibition of low density lipoproteins excretion Research and Markets Carbon Nanotubes (CNT) Market by Type, Method, Application-Global Forecast to 2023 Carbon nanotubes and its applications-A review A review of functionalized carbon nanotubes and graphene for heavy metal adsorption from water: Preparation, application, and mechanism Toxicity of carbon nanotubes: A review Carbon nanotubes: Evaluation of toxicity at biointerfaces The regulating role of carbon nanotubes and Graphene in lithium-ion and lithium-sulfur batteries Medical applications for the flow of carbon-nanotubes suspended nanofluids in the presence of convective condition using Laplace transform Modeling the interaction between anti-cancer drug penicillamine and pristine and functionalized carbon nanotubes for medical applications: Density functional theory investigation and a molecular dynamics simulation Immunological effects among workers who handle engineered nanoparticles Inhaled nanomaterials and the respiratory microbiome: Clinical, immunological and toxicological perspectives Multi-walled carbon nanotubes: A cytotoxicity study in relation to functionalization, dose and dispersion Differential crosstalk between global DNA methylation and metabolomics associated with cell type specific stress response by pristine and functionalized MWCNT Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs Physicochemical predictors of Multi-Walled Carbon Nanotube-induced pulmonary histopathology and toxicity one year after pulmonary deposition of 11 different Multi-Walled Carbon Nanotubes in mice Respiratory tract toxicity of Titanium dioxide nanoparticles and multi-walled carbon nanotubes on mice after intranasal exposure The impact of nanomaterial characteristics on inhalation toxicity Dysregulation of macrophage activation profiles by engineered nanoparticles Ongoing inflammation enhances the toxicity of engineered nanomaterials: Application of an in vitro co-culture model of the healthy and inflamed intestine Interaction of nanoparticles with immunocompetent cells: Nanosafety considerations Health implications of engineered nanomaterials Exposure to multiwalled carbon nanotubes and allergen promotes earlyand late-phase increases in airway resistance in mice Profibrotic activity of Multiwalled carbon nanotubes upon prolonged exposures in different human lung cell types Multi-walled carbon nanotube-induced genotoxic, inflammatory and pro-fibrotic responses in mice: Investigating the mechanisms of pulmonary carcinogenesis The significance of nanoparticles in particle-induced pulmonary fibrosis Inhaled Multiwalled carbon nanotubes potentiate airway fibrosis in murine allergic asthma Carbon nanotubes as delivery systems for respiratory disease: Do the dangers outweigh the potential benefits? STAT1 attenuates murine allergen-induced airway remodeling and exacerbation by carbon nanotubes Advances in nanotechnology and asthma Titanium and gold nanoparticles in asthma: The bad and the ugly Lung exposure to nanoparticles modulates an asthmatic response in a mouse model Fibrosis biomarkers in workers exposed to MWCNTs Pathologic and molecular profiling of rapid-onset fibrosis and inflammation induced by multi-walled carbon nanotubes Multiwalled carbon nanotubes induce a Fibrogenic response by stimulating reactive oxygen species production, activating NF-κB signaling, and promoting fibroblast-to-Myofibroblast transformation Cytotoxicity screening and cytokine profiling of nineteen nanomaterials enables hazard ranking and grouping based on inflammogenic potential A road map for those who don't know JAK-STAT Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells Inborn errors of human STAT1: Allelic heterogeneity governs the diversity of immunological and infectious phenotypes Susceptibility of signal transducer and activator of transcription-1-deficient mice to pulmonary Fibrogenesis STAT1-dependent and -independent pulmonary allergic and fibrogenic responses in mice after exposure to tangled versus rod-like multi-walled carbon nanotubes Role of signal transducer and activator of transcription 1 in murine allergen-induced airway remodeling and exacerbation by carbon nanotubes Microarray profiling reveals suppressed interferon stimulated gene program in fibroblasts from scleroderma-associated interstitial lung disease T-bet: A bridge between innate and adaptive immunity Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet Nickel nanoparticles cause exaggerated lung and airway remodeling in mice lacking the T-box transcription factor, TBX21 (T-bet) Role of nrf2 in oxidative stress and toxicity Role of Nrf2 and its activators in respiratory diseases Suppression of basal and carbon nanotube-induced oxidative stress, inflammation and fibrosis in mouse lungs by Nrf2 Nrf2 protects against oxidative stress induced by SiO2 nanoparticles p53 mutations in human cancers Expression and alteration of ras and p53 proteins in patients with lung carcinoma accompanied by idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis: A disease with similarities and links to cancer biology Carbon nanotubes induce malignant transformation and Tumorigenesis of human lung epithelial cells Induction of mesothelioma in p53+/-mouse by intraperitoneal application of multi-wall carbon nanotube Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice Deficiency in core circadian protein Bmal1 is associated with a prothrombotic and vascular phenotype Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock An overview from structure to innate immunity-associated pathologies Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes Role of Cyclooxygenase-2 in exacerbation of allergen-induced airway remodeling by multiwalled carbon nanotubes Upregulation of IL-13 concentration in vivo by the IL13 variant associated with bronchial asthma Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma Deficiency of cyclooxygenases transcripts in cultured primary bronchial epithelial cells of aspirin-sensitive asthmatics Selective downregulation of prostaglandin E 2-related pathways by the TH2 cytokine IL-13 Tissue inhibitor of Metalloproteinase-1 promotes NIH3T3 fibroblast proliferation by activating p-Akt and cell cycle progression Common and distinct mechanisms of induced pulmonary fibrosis by particulate and soluble chemical fibrogenic agents Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis Timp-1 is a key factor of fibrogenic response to bleomycin in mouse lung TIMP1 promotes multi-walled carbon nanotube-induced lung fibrosis by stimulating fibroblast activation and proliferation Peroxidase-mediated biodegradation of carbon nanotubes in vitro and in vivo Usefulness of myeloperoxidase as a biomarker for the ranking of pulmonary toxicity of nanomaterials Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice Involvement of Apolipoprotein E in excess fat accumulation and insulin resistance Diesel exhaust particles induce endothelial dysfunction in apoE-/-mice Modest vasomotor dysfunction induced by low doses of C60 fullerenes in apolipoprotein E knockout mice with different degree of atherosclerosis Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE-/-mice Cardiovascular health effects of oral and pulmonary exposure to multi-walled carbon nanotubes in ApoE-deficient mice Single-and double-walled carbon nanotubes enhance atherosclerogenesis by promoting monocyte adhesion to endothelial cells and endothelial progenitor cell dysfunction Vascular effects of multiwalled carbon nanotubes in dyslipidemic apoe-/-mice and cultured endothelial cells The aryl hydrocarbon receptor: An environmental sensor integrating immune responses in health and disease Role of the aryl hydrocarbon receptor (AhR) in lung inflammation Involvement of the cytokine-IDO1-AhR loop in zinc oxide nanoparticle-induced acute pulmonary inflammation Th1-and Th2-related chemokine and chemokine receptor expression on the ocular surface in endotoxin-induced uveitis Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver Chemokines in dengue fever CCR5 plays an important role in resolving an inflammatory response to single-walled carbon nanotubes Overview of the IL-1 family in innate inflammation and acquired immunity Disease-associated functions of IL-33: The new kid in the IL-1 family Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1α and IL-1β An allergic lung microenvironment suppresses carbon nanotube-induced Inflammasome activation via STAT6-dependent inhibition of caspase-1 IL-1R signalling is critical for regulation of multi-walled carbon nanotubes-induced acute lung inflammation in C57Bl/6 mice Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: Insights from an adverse outcome pathway framework A single aspiration of rod-like carbon nanotubes induces asbestos-like pulmonary inflammation mediated in part by the IL-1 receptor Osteopontin enhances multi-walled carbon nanotube-triggered lung fibrosis by promoting TGF-β1 activation and myofibroblast differentiation Osteopontin expression in particle-induced lung disease IL-6: A regulator of the transition from neutrophil to monocyte recruitment during inflammation Vanadzins, I. The effect of different workplace nanoparticles on the immune systems of employees Effects of naked gold nanoparticles on Proinflammatory cytokines mRNA expression in rat liver and kidney Pulmonary instillation of MWCNT increases lung permeability, decreases gp130 expression in the lungs, and initiates cardiovascular IL-6 transsignaling Atomic layer deposition coating of carbon nanotubes with zinc oxide causes acute phase immune responses in human monocytes in vitro and in mice after pulmonary exposure Advanced role of neutrophils in common respiratory diseases IL-6 regulates neutrophil trafficking during acute inflammation via STAT3 STAT3 activation in response to IL-6 is prolonged by the binding of IL-6 receptor to EGF receptor Interleukin-6 prevents the initiation but enhances the progression of lung cancer Sex differences in the acute and subchronic lung inflammatory responses of mice to nickel nanoparticles Sex differences in the inflammatory immune response to multi-walled carbon nanotubes and crystalline silica The role of sex in particle-induced inflammation and injury Effect of cell sex on uptake of nanoparticles: The overlooked factor at the Nanobio interface NIH Office of Research on Women's Health (ORWH) Lung cancer in patients with idiopathic pulmonary fibrosis: Clinical characteristics and impact on survival Hormones, sex, and asthma Sex differences in chronic obstructive pulmonary disease mechanisms Sex-specific differences in mitochondria biogenesis, morphology, respiratory function, and ROS homeostasis in young mouse heart and brain Male sex hormones exacerbate lung function impairment after Bleomycin-induced pulmonary fibrosis Sex differences in immune variables and respiratory infection incidence in an athletic population Sex-based differences in susceptibility to respiratory and systemic pneumococcal disease in mice Sex differences in the incidence and severity of respiratory tract infections Sex differences in immune responses Sex differences in respiratory function Respiratory regulation by steroids in newborn rats: A sex-specific balance between allopregnanolone and progesterone receptors Sex bias in asthma prevalence and pathogenesis Sex and inflammation in respiratory diseases: A clinical viewpoint Age and testosterone mediate influenza pathogenesis in male mice Epidemiological and clinical characteristics of 99 cases of 2019-Novel Coronavirus (2019-nCoV) pneumonia in Wuhan, China: A descriptive study Working Group of GA2LEN WP 2.5 Gender. Impact of gender on asthma in childhood and adolescence: A GA2LEN review Lung carcinogenicity of inhaled multi-walled carbon nanotube in rats Gender influences the response to experimental silica-induced lung fibrosis in mice Secreted Phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice The toxicology of engineered nanomaterials in asthma TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice Toxic and adjuvant effects of silica nanoparticles on ovalbumin-induced allergic airway inflammation in mice Effect of TiO2Nanoparticles on Inflammasome-Mediated Airway Inflammation and Responsiveness Zinc oxide nanoparticles induce eosinophilic airway inflammation in mice Inhalation exposure to multi-walled carbon nanotubes alters the pulmonary allergic response of mice to house dust mite allergen Air pollution exposure and cardiovascular disease Occupational exposure to dust: Inflammation and ischaemic heart disease Impact of pulmonary exposure to gold core silver nanoparticles of different size and capping agents on cardiovascular injury Pulmonary cerium dioxide nanoparticles exposure differentially impairs coronary and mesenteric arteriolar reactivity Heterogeneous vascular bed responses to pulmonary titanium dioxide nanoparticle exposure. Front. Cardiovasc Thrombospondin-1 signaling through CD47 inhibits self-renewal by regulating c-Myc and other stem cell transcription factors Inhibition of VEGFR2 activation and its downstream signaling to ERK1/2 and calcium by thrombospondin-1 (TSP1): In silico investigation Thrombospondin-1 mediates multi-walled carbon nanotube induced impairment of arteriolar dilation Microvascular dysfunction following multiwalled carbon nanotube exposure is mediated by thrombospondin-1 receptor CD47 Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial-dependent vasorelaxation TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction Diabetes impairs angiogenesis and induces endothelial cell senescence by up-regulating thrombospondin-CD47-dependent signaling Role of thrombospondin-1 and thrombospondin-2 in cardiovascular diseases (Review) Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease Serum amyloid A directly accelerates the progression of atherosclerosis in Apolipoprotein E-deficient mice Particle-Induced Pulmonary Acute Phase Response Correlates with Neutrophil Influx Linking Inhaled Particles and Cardiovascular Risk Aggravation of atherosclerosis by pulmonary exposure to indium oxide nanoparticles Plaque-associated endothelial dysfunction in apolipoprotein E-deficient mice on a regular diet. Effect of human apolipoprotein AI Cardiovascular effects of pulmonary exposure to titanium dioxide nanoparticles in ApoE knockout mice Long-term inhalation exposure to nickel nanoparticles exacerbated atherosclerosis in a susceptible mouse model Effects of exposure of adult mice to multi-walled carbon nanotubes on the liver lipid metabolism of their offspring Role of oxidative stress in liver toxicity induced by nickel oxide nanoparticles in rats Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs Toxic effects of the Fe2O3 nanoparticles on the liver and lung tissue Subchronic inhalation toxicity of silver nanoparticles Liver histopathological alteration after repeated intra-tracheal instillation of titanium dioxide in male rats Mechanisms for how inhaled multiwalled carbon nanotubes suppress systemic immune function in mice Pulmonary and systemic immune response to inhaled multiwalled carbon nanotubes The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks Structure and function of the blood-brain barrier Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice The outdoor air pollution and brain health workshop Translocation of inhaled ultrafine manganese oxide particles to the central nervous system Inhalation of uranium nanoparticles: Respiratory tract deposition and translocation to secondary target organs in rats Neurotoxicity of silica nanoparticles: Brain localization and dopaminergic neurons damage pathways The neurotoxicity induced by engineered nanomaterials Brain tumor-targeted therapy by systemic delivery of siRNA with Transferrin receptor-mediated core-shell nanoparticles Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma Correction: Tailored theranostic apolipoprotein E3 porphyrin-lipid nanoparticles target glioblastoma Serum-borne bioactivity caused by pulmonary multiwalled carbon nanotubes induces neuroinflammation via blood-brain barrier impairment Toxicity of multi-wall carbon nanotubes inhalation on the brain of rats Aluminum nanoparticles induce ERK and p38MAPK activation in rat brain Genomics-based screening of differentially expressed genes in the brains of mice exposed to silver nanoparticles via inhalation Bacterial lipopolysaccharide enhances PDGF signaling and pulmonary fibrosis in rats exposed to carbon nanotubes TLR4 and infectious disease diversity Lipopolysaccharide (LPS) exposure differently affects allergic asthma exacerbations and its amelioration by intranasal curcumin in mice The uninvited guest The inflammatory response in humans after inhalation of bacterial endotoxin: A review Lipopolysaccharide (LPS) inhalation in healthy subjects increases neutrophils, lymphocytes and fibronectin levels in bronchoalveolar lavage fluid Induction of the lung Myofibroblast PDGF receptor system by urban ambient particles from Mexico City Effects of airway exposure to nanoparticles on lung inflammation induced by bacterial endotoxin in mice EAACI Guidelines on Allergen Immunotherapy: House dust mite-driven allergic asthma. Allergy Eur Proteome and allergenome of the European house dust mite Dermatophagoides pteronyssinus The relative risks of sensitivity to grass pollen, house dust mite and cat dander in the development of childhood asthma Sensitization to common allergens and its association with allergic disorders at age 4 years: A whole population birth cohort study Home environmental interventions for house dust mite This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license We thank Alexia Taylor-Just for reading the manuscript and providing helpful edits. The authors declare no conflict of interest.