key: cord-0941604-0qjfozm7
authors: Shahbaz, Muhammad Ali; Martikainen, Maria-Viola; Rönkkö, Teemu J.; Komppula, Mika; Jalava, Pasi I.; Roponen, Marjut
title: Urban air PM modifies differently immune defense responses against bacterial and viral infections in vitro
date: 2020-09-25
journal: Environ Res
DOI: 10.1016/j.envres.2020.110244
sha: 4cea69a28a9f0f7ffae57c8e33d870234f466514
doc_id: 941604
cord_uid: 0qjfozm7

Epidemiological evidence has shown the association between exposure to ambient fine particulate matter (PM) and increased susceptibility to bacterial and viral respiratory infections. However, to date, the underlying mechanisms of immunomodulatory effects of PM remain unclear. Our objective was to explore how exposure to relatively low doses of urban air PM alters innate responses to bacterial and viral stimuli in vitro. We used secondary alveolar epithelial cell line along with monocyte-derived macrophages to replicate innate lung barrier in vitro. Co-cultured cells were first exposed for 24 hours to PM(2.5-1) (particle aerodynamic diameter between 1 and 2.5μm) and subsequently for an additional 24 hours to lipopolysaccharide (TLR4), polyinosinic-polycytidylic acid (TLR3), and synthetic single-stranded RNA oligoribonucleotides (TLR7/8) to mimic bacterial or viral stimulation. Toxicological endpoints included pro-inflammatory cytokines (IL-8, IL-6, and TNF-α), cellular metabolic activity, and cell cycle phase distribution. We show that cells exposed to PM(2.5-1) produced higher levels of pro-inflammatory cytokines following stimulation with bacterial TLR4 ligand than cells exposed to PM(2.5-1) or bacterial ligand alone. On the contrary, PM(2.5-1) exposure reduced pro-inflammatory responses to viral ligands TLR3 and TLR7/8. Cell cycle analysis indicated that viral ligands induced cell cycle arrest at the G2-M phase. In PM-primed co-cultures, however, they failed to induce the G2-M phase arrest. Contrarily, bacterial stimulation caused a slight increase in cells in the sub-G1 phase but in PM(2.5-1) primed co-cultures the effect of bacterial stimulation was masked by PM(2.5-1.) These findings indicate that PM(2.5-1) may alter responses of immune defense differently against bacterial and viral infections. Further studies are required to explain the mechanism of immune modulation caused by PM in altering the susceptibility to respiratory infections.

Ambient particulate matter, Toll-like receptor, Lipopolysaccharides, Poly: IC, ORN R-0006, Respiratory infections.

Exposure to ambient air pollution causes up to 4.2 million annual premature deaths worldwide (Cohen, et al. 2017) . One major component of air pollution is particulate matter (PM). PM is a mixture of solid and liquid particles (organic and inorganic-derived particles) dispersed in ambient air. Its size, shape, and composition vary depending on the source of origin. Generally, PM originates from natural (e.g. airborne dust, volcanic activity, and pollen) or anthropogenic sources (e.g. industry and traffic primarily by different combustion processes). It has been classified according to aerodynamic diameter into coarse (≤10 μm), Fine (≤2.5 μm), and ultra-fine (≤0.1 μm) PM (US EPA 2016). The size of the particles directly links with the potential of PM for causing detrimental health outcomes. PM with an aerodynamic diameter less than 2.5 µm (PM 2.5 ) are among the most studied air pollutants of health concern since smaller particles are more likely to penetrate deeper in the lungs and encounter lung surface (Xing. et al. 2016 ). PM 2.5 is not a self-contained pollutant; it contains a heterogeneous combination of solid and liquid particles, which not only includes chemicals but also biological fractions (Kelly and Fussell 2012) . Epidemiological evidence suggests that there is an increasing risk of developing bacterial and viral respiratory infection with exposure to ambient air pollution, in particular, fine particulate matter (Ciencewicki and Jaspers 2007; Croft, et al. 2019; Horne, et al. 2018; Zhang, et al. 2019 ). Furthermore, the recent coronavirus epidemic emphasizes the need for a detailed understanding of the link between air pollution and respiratory infection. Nationwide U.S cross-sectional study has highlighted the association between a small increase in long term exposure to PM 2.5 and an increased risk of mortality due to COVID-19 infection (Wu, et al. 2020 ). In experimental models, fine PM exposure has increased the risk of pneumonia due to the deleterious effect on alveolar macrophages and alveolar epithelium (Migliaccio, et al. 2013; Mushtaq, et al. 2011 ). As reviewed in Wei and Tang (2018) few studies have focused on the immunemodulatory effect of fine PM and its effects on the immune response to respiratory infections. Therefore, it is crucial to explore links between fine PM exposure and alteration in the immune response to bacterial and viral respiratory infections since large populations J o u r n a l P r e -p r o o f are exposed to air pollutants and respiratory infections may spread more rapidly in the densely populated areas.

Alveolar epithelium and alveolar macrophages serve as pillars in the innate immune system of the respiratory tract. Inhaled pathogens must evade the innate immune system to establish infections (Bhattacharya and Westphalen 2016) . In vitro co-culture of secondary epithelial cells (A549 cells) and THP1 monocytes, differentiated into macrophage-like cells have been used in immunological and toxicological studies on the respiratory health to better understand the effect of cell-cell interaction and to better mimic the first line of defense i.e., alveolar epithelium and macrophages (Dehai, et al. 2014; Holownia, et al. 2015 ).

Family of Toll-like receptors (TLRs) expressed by epithelial and dedicated immune cells are an important component in pathogen recognition and innate immune response (Medzhitov 2001) . Activation of TLRs on airway epithelial cells has been shown to induce the production of several cytokines, chemokines, and antimicrobial peptides (Guillot, et al. 2005; Hertz, et al. 2003; Sha, et al. 2004 ). The importance of TLRs for the host defense in the lung has been demonstrated by the increased susceptibility of TLR knockout mice towards viral or bacterial infections (Takeuchi, et al. 2000; Wetzler 2003 ). This is because each member of TLR family recognizes specific pathogen-associated molecular patterns (PAMPs), e.g., TLR3 is activated by virus-derived double-stranded RNA (Alexopoulou, et al. 2001 ) and TLR4 by bacterial lipopolysaccharides (Beutler 2002) , whereas TLR7 and TLR8 recognize single-stranded viral RNA (Diebold, et al. 2004; Heil, et al. 2004 

For the co-culture experiments, A549 cells were seeded in 12-well plates at a seeding density of 120,000 cells per well in FBS supplemented media and allowed them to attach for 4 hours. Once the A549 cells were attached to the bottom of the well, media in the wells was aspirated and activated macrophage-like THP-1 cells were seeded at a seeding density of 24,000 cells per well on top of attached A549 cells. The co-cultured cells were incubated at 37°C and 5% CO 2 for 40 hours before exposing the cells to the first exposure. The seeded co-culture yields approximately 400,000-600,000 cells per well after the end of the exposure. One hour before the exposure the co-culture medium was replaced with fresh medium supplemented with 5% FBS, 2mM L-Glut, and 100 U/ml (pen/strep).

The study was designed as a two-step submerged exposure. Co-cultured cells were exposed to three concentrations of PM 2.5-1 (25µg/ml, 50µg/ml, and 100µg/ml corresponding to 6.6, 13.2, and 26.3 µg/cm 2 ) in duplicate wells for 24 hours at 37°C and 5% CO 2 .

The PM 2.5-1 -exposed cells were then co-exposed for next 24 hours to fixed doses of three lysis buffer was added to each well. Cells were then lysed for 2 hours at 37°C and 5% CO 2 followed by 30 minutes at room temperature on a plate shaker to release formazan from the cells. After incubation, absorbance was measured at 570 nm using a Synergy H1, Microplate reader (BioTek, USA). The absorbance values of the exposed cells were then normalized against the untreated controls and the percentage of cellular metabolic activity was calculated by the following formula: ((absorbance exposed/absorbance control) *100 %). Figure 1 . IL-6 pro-inflammatory cytokine production was assessed after two-step submerged exposure. Co-cultured cells were first exposed to three doses (25, 50, PM stimulation increased the production of IL-6 in a dose-dependent manner in the cocultured cells. Of the studied viral ligands, TLR7/8 induced the highest production of IL-6

whereas TLR3 induced a marginal increase in IL-6 production compared to unexposed control cells. TLR4 also significantly increased the IL-6 production relative to the unexposed control. In co-exposure to PM and TLR7/8, the IL-6 release of the cells increased significantly at the lowest PM dose when compared to responses of the cells after PM exposure only.

However, PM and TLR7/8 co-exposure resulted in much lower IL-6 concentrations than for TLR 7/8 ligand alone. With the highest PM dose, IL-6 levels were not only lower than after exposure to TLR-7/8 ligand alone but they were also decreased when compared to IL-6 release after the cells were exposed to same PM mass without the ligand. Co-exposure to PM and TLR3 induced lower cytokine secretion than PM alone. In contrast, co-exposure to PM and TLR4 caused IL-6 production that was higher than that of cells exposed to either PM or TLR4 alone ( Figure 1 ). PM increased the production of TNF-α dose-dependently. TLR7/8 induced the highest production of TNF-α, whereas TLR3 was not able to induce TNF-α production in our cell model. TLR4 induced slight but non-significant increase in TNF-α production. Ιn PM and TLR7/8 co-exposures, production of TNF-α was approximately the same for all PM doses and comparable to the response after exposure to highest PM dose alone, but significantly lower than after exposure to TLR7/8 alone. Interestingly, when the cell model was coexposed to PM and TLR3, cytokine secretion was lower than in stimulation with PM alone, but the difference was not statistically significant. In contrast to other two ligands, cells primed PM responded to TLR4 exposure by significantly enhanced TNF-α production ( Figure   2 ). In co-exposure to PM and TLR7/8, the production of IL-8 increased slightly for the lowest two doses when compared to respective PM dose alone. However, no significant indication of additive effects of PM and TLR7/8 was observed at any of the dose levels. Similar to TLR7/8, co-exposure to PM and TLR3 induced roughly the same level of cytokine secretion than PM. In contrast, co-exposure to PM and TLR4 caused IL-8 production that was higher than that of cells exposed to PM or TLR4 alone ( Figure 3 ). Figure 7 . Cell cycle analysis was performed using PI staining after two-step submerged exposure described in Figure 1. A. Sub-G1 phase, Fig B. G1-G0 phase, and Fig C. G2-M phase. We characterized the cytokine response of A549-THP1 co-culture to viral TLR ligands (TLR3, TLR7/8) and bacterial TLR ligand (TLR4). Results showed that both viral ligands caused an increase in IL-6 and IL-8 levels compared to control. However, response to TLR7/8 ligand was very high compared TLR3 ligand response. Our results are partially consistent with the study on primary airway epithelial cells, showing that exposure to TLR3 ligand increased IL-6, IL-8, and TNF-α production of the cells (Lever, et al. 2015) . Interestingly, in our study TLR3 ligand did not induce TNF-α production. TNF-α serves an important physiological role of activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Κβ) and

influx of neutrophils to the site of inflammation (Schütze, et al. 1995) . A study on the A549-THP1 co-culture has concluded that TNF-α was secreted by activated macrophages and A549-THP1 co-culture when exposed to PM but not by PM-exposed A549 cells (Kasurinen, et al. 2018 ). Furthermore, TNF-α has been detected in neither control A549 cells nor the cells exposed to cigarette smoke (Holownia, et al. 2016 with the results of the study that has been conducted on human lung mucoepidermoid carcinoma (H292) and THP-1 cells. LPS stimulation induced significant pro-inflammatory cytokines in both cell lines (Liu, et al. 2018 ).

Co-cultures primed with PM 2.5-1 on subsequent exposure with viral ligand TLR7/8 showed no additive effect on the response of pro-inflammatory cytokines as the response remained roughly the same as for PM 2.5-1 alone. This indicates that in PM 2.5-1 primed co-cultures the pro-inflammatory response was altered against TLR7/8 ligand. Another viral ligand TLR3 behaved differently as it alone induced very low levels of IL-8 and IL-6 but not induce the production of TNF-α in co-culture. In addition, when PM primed co-cultures were exposed to TLR3 ligand, the production of IL-8 and IL-6 was not further increased. This indicates that in addition to low inflammatory potential of TLR3 ligand, it may also suppress responses induced by PM.

Several studies have indicated that urban particulate matter decreases the ability of the macrophage to phagocytize and weakens the capacity of alveolar macrophages as well as alveolar epithelium to mount an effective immune response against viral stimuli (Migliaccio, et al. 2013; Xu, et al. 2008; Zhou and Kobzik 2007) . Results from an in vivo study on mice also that ultrafine carbon particle exposure suppresses the early immune response in the lung. However, at day 7 inflammation and viral exacerbation increased drastically (Lambert, et al. 2003) . Therefore, in our study, it could be assumed that prior exposure to PM 2. In this study, any of the stimulations did not induce significant changes to ROS stress.

Moreover, no drastic changes in viability were seen. Activation of pro-inflammatory Lastly, we also studied the cell cycle phase distribution of exposed cells. Exposure to PM 2. conditions for viral replication (Dove, et al. 2006 ). Therefore, we assumed that the increase in the number of cells in the G2-M phase is associated with viral ligand-induced cell cycle arrest to progeny viral production. However, viral ligands failed to induce modifications in the G2-M phase in PM 2.5-1 primed co-cultures. From our results, it can be speculated that prior exposure to PM may alter the viral virulence. On the other hand, bacterial ligand TLR4

increased the number of cells in the Sub-G1 phase, with no effect on G1-G0 and G2-M phases. In correlation to the results from thiol assay, which accounts for the early apoptotic cells; pretreatment of cells with PM 2.5-1 slightly increased the number of cells in the Sub-G1

phase, especially at higher doses of PM 2.5-1. The response was, however, not statistically significant when compared to PM 2.5-1 alone but it shows that co-exposure with TLR4 ligand has some effect on the number of cells in the Sub-G1 phase. Therefore, our results indicate that PM 2.5-1 priming modifies immune responses against bacterial and viral stimulation in the studied cell model.

PM 2.5-1 exposure altered the pro-inflammatory cytokine response to both bacterial and viral stimuli in the alveolar lung cell model. PM 2.5-1 increased the sensitivity of the A549-THP1 coculture to produce pro-inflammatory cytokines, which potentially leads to hyperinflammatory response against bacterial infection. Instead, virus-mediated proinflammatory effects were suppressed if the co-culture model of the alveolar barrier was primed with PM 2.5-1. These findings provide insight into the underlying immunomodulatory effects of fine particulate matter, which potentially leads to susceptibility to respiratory infections.

The work was supported by Päivikki and Sakari Sohlberg Foundation, Juho Vainio Foundation, and the Academy of Finland grants (319245, 294081, 287982). 

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The authors wish to thank Ms Hanne Vainikainen, MSc Tuukka Ihantola and MSc Henri Hakkarainen for their technical support.