Increased proinflammatory mediators leads to Recruitment of inflammatory cells

The biological plausibility of this KER is high. There are very well established functional relationships between the secreted signalling molecules and the chemotactic effects on pro-inflammatory cells (Harris, 1954; Petri & Sanz 2018).

Increased proinflammatory mediators means more proinflammatory cytokines, chemokines, vasoactive amines, and lipid mediators (Villenueve et al, https://doi.org/10.1093/toxsci/kfy047). Increased Signaling from these Cytokines and Chemokines promote leukocyte recruitment to areas of infection, including monocytes and neutrophil (Leick et al, doi: 10.1007/s00441-014-1809-9). The leukocytes will differentiate into mature proinflammatory cells, in response to mediators they encounter in the local tissue microenvironment (Villenueve et al, https://doi.org/10.1093/toxsci/kfy047). With higher levels of leukocytes from increased proinflammatory mediators, it causes an increase in proinflammatory cells (Libby, https://doi.org/10.1093/cvr/cvv188).

The empirical support for this KER is moderate. There are many studies which show temporal and dose-dependent recruitment of immune cells following increases in pro-inflammatory mediators. However, these mediators exhibit pleiotropy, and knockdown or knockout of a single pathway or mediator can result in compensation and recruitment of immune cells at a later time, as is seen in Nikota et al.,. 2017. (Chen et al., 2016; Nikota et al., 2017; Schremmer et al., 2014) (Additional studies available in Table 1.).

Dose-Response Evidence:

Many studies provide dose-response evidence of this KER. For example, in vitro and in vivo studies testing stressors at different doses/concentrations have demonstrated a dose-response relationship; at the higher dose of the stressor, the pro-inflammatory mediators increased, leading to an increase of pro-inflammatory cell recruitment. 

Ma, et al. (2016) studied inflammatory responses in male BALB/c mice exposed to multi-walled carbon nanotubes (MWCNT) administered intravenously at different doses (0.5-4 mg/kg) for 2 days. A dose-dependant relationship was found between the levels of the inflammatory mediators IL-6 and TNF-a and the MWCNT dose. At the highest dose, 4 mg/Kg, white blood cells, lymphocytes, and neutrophils levels increased.

Porter et al. (2020) have demonstrated that MWCNT caused dose-dependent and time-dependent pulmonary inflammation in male C57BL/6J mice. Animals received a single dose of 2.5, 10, or 40 mg/mouse. At 40 mg/mouse, IL-1b and IL-18 increased at one day post-exposure. Moreover, polymorphonuclear leukocyte increased on day 1, and after 7 days the number of inflammatory cells was higher.

Zinc oxide nanoparticles (NPs) can induce metal fume fever and acute inflammation. Female C57BL/6J mice were intratracheally instilled once at 11, 33, and 100 mg/kg with coated ZnO NPs. Inflammatory responses were evaluated after 1, 3, and 28 days of exposure. An increase in serum amyloid A3 mRNA in lung tissue was observed at 33 and 100 mg/kg. Neutrophils accumulated in BAL fluid after 28 days of exposure in a dose-dependant manner (Hadrup et al., 2019).

Polyhexamethyleneguanidine phosphate (PHMG-P) is used as a disinfectant. PHMG-P at 0.3, 0.9, and 1.5 mg/kg was instilled into the lungs of mice. At 7- and 14-days post-exposure an increase in the levels of pro-inflammatory markers (IL-1b, IL-6 and CXCL1) and an increase in mRNA levels of MCP1, MMP2, and MMP12 was seen. Moreover, on day 7, neutrophils were recruited to the inflamed site. These changes were observed in a dose-response manner (Song et al., 2014).

Bourdon et al. 2012 evaluated the toxicity of carbon black nanoparticles (CBNPs) in mouse lung and liver. C57BL/6 mice were exposed to Printex 90 CBNPs with 0.018, 0.054, or 0.162 mg, and after 1, 3, and 28 days of the single instillation, BAL fluid was analyzed. Polymorphonuclear cell counts in BAL increased in a dose-dependant manner with the strongest recruitment 1- and 3-days post-exposure and remained elevated at day 28. CBNP also increased the expression of Saa3 mRNA levels in lung tissue on days 1, 3, and 28 in a dose-dependant manner. Although this response decreased over time, the expression of Saa3 mRNA increased at all time points, which indicates a persistent acute phase response.

A study evaluated the mechanisms of toxicity after exposure to PM2.5 in a tri-culture system: A549 cells and THP-1 differentiated macrophages in the apical chamber; meanwhile, EA.hy926 endothelial cells were cultured in the basolateral chamber. The system was exposed to PM2.5 at three different concentrations 20, 60, and 180 mg/ml for 24 h. An increase in the pro-inflammatory mediators IL-6, IL-8, and TNF-a was observed, as well an increase in mRNA expression of MMP9, ICAM-1, and CAV-1. These genes are involved in the movement and recruitment of leukocytes in sites of inflammation. Changes were observed in a concentration-dependant manner (Wang et al., 2019).

In another study female C57BL/6 mice were exposed to 18, 54, or 162 mg of MWCNT/mouse via single intratracheal instillation. An increased gene expression of Cxcl1, IL-6, Mt2, Saa1, and Saa2 was observed in a dose-dependent manner at 24 h post-exposure. Moreover, an increase in the recruitment of pro-inflammatory cells was observed in a dose-dependent manner (Poulsen et al., 2013).

Temporal Evidence:

There is significant evidence of the temporal relationship between the two KES.  In vitro and in vivo studies have demonstrated that pro-inflammatory mediators (Event 1496) increased prior to the recruitment of pro-inflammatory cells (Event 1497).

Female C57BL/6J mice were exposed to carbon nanoparticles at 20 mg/mouse via intratracheal instillation. An increase in the levels of cytokines CXCL1, CXCL2, and CXCL5 at 3 h post-exposure was observed, with peaks after 12 and 18 h post-exposure. These pro-inflammatory mediators preceded neutrophil recruitment (12 and 24 h post-exposure) (Chen et al., 2016). Alveolar macrophages (AM) were isolated from lungs 3 to 12 h after CNP instillation, but they did not show a pro-inflammatory response. The authors suggest that AM are not involved in the initiation of the inflammatory response. Meanwhile, ATII cells induced the highest CXCL levels and acute neutrophilic inflammation.

Nickel oxide NPs intratracheally instilled at one single dose 200 cm²/rat into female Wistar rats induced an increase of pro-inflammatory cytokines in BALF, at 24 and 74 h for CINC-3 and eotaxin, respectively. At 24 h and 48 h, neutrophils were observed, and after 72 h, the levels of neutrophils, eosinophils, and macrophages increased (Lee et al., 2016).

Porter et al. (2002) have shown pulmonary inflammation in rats exposed to crystalline silica aerosol at a concentration of 15 mg/m³ (6h/day, 5 days/week) for 116 days.  Lung disease was linked to TNF-a and IL-10 production in a timely response (10-116 days). The number of polymorphonuclear cells in the BALF increased progressively from day 41 - 116.

One study has demonstrated a dose-response and temporal relationship for these two KEs (Patowary et al., 2020). Female Wistar rats were exposed to oleoresin capsicum (OC) sprays at 2, 6, and 10%, and after 1, 3, and 24 h post-exposure, blood cell and BALF cytokines were evaluated. The pro-inflammatory cytokine TNF-a increased in a dose-dependant manner, and polymorphonuclear cells increased in a time-dependant manner.

Schremmer et al. (2014) have reported the time course of chemotaxis in vitro in response to the challenge of biopersistent particles and their relation to inflammatory mediators.  NR8383 rat alveolar macrophages were challenged with different types of particles for 1, 4, and 16 h. The cell supernatants obtained from different time points were used to evaluate the chemotaxis of unexposed NR8383 macrophages. They found that nanosized silica at 16 mg/cm² induced an elevated transcription of CCL4, CXCL1, CXCL3, and TNF-a in a time-dependant manner. The pro-inflammatory cytokines present in the supernatants induced chemotaxis of unexposed macrophages at 4 and 16 h post-exposure.

Husain et al. (2015) found increased expression of genes related to chemotactic recruitment of pro-inflammatory cells at 3 h and 1 day after exposure to 162 mg/mouse carbon black nanoparticles in female C57BL/6 mice. They observed an increase in the gene expression of pro-inflammatory mediators at day 1 (Cxcl2, Ccl2), day 3 (IL-17, IL-33), day 14 (Cd2), and day 42 (Cxcl) post-exposure. The KE2 (Event 1497) increased over time with the maximum levels of neutrophils, macrophages, eosinophils, and lymphocytes at 4- and 5-days post-exposure. This response suggests chronic inflammation occurs because of an incomplete resolution of acute inflammation.

Rahman et al. 2017 evaluated whether different TiO₂ NPs induce lung inflammation. C57BL/6 mice were exposed to 18, 54, 162, or 486 mg/mouse of TiO₂ NPs via single intratracheal instillation. At 1-day post-exposure, gene expression analysis showed more changes in genes associated with inflammation and fibrosis. Moreover, after 1- and 28-days post-exposure, an increase in cell counts in BALF was observed in a dose-dependant manner.

Ho et al., 2013 evaluated the inflammatory response in mice exposed to coated quantum dots (QD705-PEG, QD705-COOH) at 12 or 60 mg/mouse. At 2-, 17- and 90-days post-exposure, an increase in the level of TNF-a, IL-1b, IL-6, CXCL1, CCL2, CCL1, CCL17, and CXCL13 mRNA levels in lungs was observed and the amount of polymorphonuclear cells in BALF increased in a dose-dependent manner at day 7 post-exposure. The inflammatory response increased on days 2 and 17, but on day 90 decreased. QD705-COOH induced granulomas persistently presented from 2 to 90 days.

Morimoto et al. 2010 examined the different kinds of cytokines related to lung inflammation by nickel oxide exposure. Rats were intratracheally exposed to 0.33 mg/Kg and 0.66 mg/kg nickel oxide NPs and were sacrificed at day 3, after 1 week, 1, 3, and 6 months post exposure. Infiltration of alveolar macrophages in lung tissue and BALF was observed from day 3 to 3 months post exposure, with higher levels after 1 and 3 months. Before the recruitment of inflammatory cells, an increase in the level of pro-inflammatory cells as MCP-1 and IL-1b in BALF was observed. Nickel oxide nanoparticles induced a persistent inflammatory effect.

Kamata et al. (2011) studied the impact of carbon black nanoparticles on susceptible subjects with predisposing lung disease and the effects of nanoparticles on inflammation and fibrotic changes. To achieve this goal, female C57BL/6J mice were intratracheally administered with bleomycin 20 mg/mouse and carbon black nanoparticles 10 mg/mouse. Evaluations were performed post-exposure at different time points. An increase of IL-6 and CCL2 in BALF was observed at days 2 and 7. After 7- and 14-days, a recruitment of pro-inflammatory cells was observed. Oxidant injury (evaluated as nitrotyrosine expression) was observed after 7 days and 14 days. The levels of TGF-b₁ increased over time with the highest level at day 14. Finally, they observed an increase in lung collagen deposition, and a decrease in lung compliance at day 21.

Attenuation or complete abrogation of KE1 (Event 1496) and KE2 (Event 1497) 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 MWCNT was suppressed at 24 hr in mice deficient in IL1R1 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 signalling 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.

Most of the studies evaluate one dose at different time points or one-time point at different concentrations. Moreover, some studies have demonstrated that a stressor can lead to the recruitment of pro-inflammatory cells, but the presence of pro-inflammatory mediators was not determined (Westphal et al., 2015).

Recruitment of pro-inflammatory cells is a key event that is complicated to replicate in vitro conditions as cell migration is induced by cooperative chemotactic mediators (Gouwy et al., 2015) which are produced and released from different cells. Therefore, more kinetics studies in co-culture techniques are needed to fill this gap.

Activated pro-inflammatory cells secrete pro-inflammatory mediators, and those mediators' goal is to cause signalling and response, which can lead to chronic inflammation (https://aopwiki.org/events/1497). Chronic inflammation means proinflammatory mediators increase and increased recruitment of inflammatory cells acts in a positive feedback loop, which continues a pro-inflammatory environment.