Background
Exposure to ambient air pollution is an adverse health risk to respiratory health, particularly in the young, elderly and those with co-morbidities such as heart disease [
1,
2]. In the young, epidemiological and toxicological research studies consistently demonstrate air pollution as a major risk factor in the onset of asthma [
3,
4]. This is well illustrated by rising rates of asthma observed in developing countries such as China where expanding industrialization correlates with raised airborne pollution [
1]. While in the elderly, long term exposure to particulate matter (PM) has been implicated in developing COPD [
1,
2,
5,
6]. Not surprisingly, hospital admission rates for breathing difficulties have been shown to rise during times of raised ambient air pollution concentrations [
3]. Despite the risks of air pollution exposure being well accepted, the precise mechanisms leading to the onset of these chronic airway diseases are poorly understood [
6‐
9].
Ambient air pollution, comprised in part by AMP, is a complex mixture of organic compounds, different sized particles and chemicals [
10]. The US EPA refers to the inhalable solid phase of ambient air pollution as PM categorized as; coarse (≤ 10 μm), fine (≤ 2.5 μm) and ultrafine (≤ 0.1 μm) [
11]. Accumulation mode particles (AMP) straddle the ultrafine particulate (UFP) and fine categories making up the inhalable vapour cloud of PM [
12]. AMP’s are largely sourced from engine combustion. Due to their small size these are subject to wind and other climatic conditions which enable dispersal and exposure far from their source of origin [
13]. As AMP are small enough to penetrate alveolar spaces and capillary walls, exposure to this particulate size fraction has been shown to result in respiratory disease and exacerbation, with exposure also linked to cardiovascular disease [
12,
13].
To date, the majority of air pollution toxicology studies have explored the role of whole ambient mixtures and individual chemical components on respiratory health [
7‐
9]. Due to the number of stimuli within ambient air, identifying the causes and/or interactions responsible for the onset of disease is difficult, as these can trigger a variety of host defence mechanisms when inhaled [
14‐
16]. Oxidative particulates and/or reactive oxygen species generated by particulate phagocytosis have been shown to drive proinflammatory pathways which can cause long-term lung damage and airway disease [
17‐
19]. There is also evidence to suggest Toll like receptor (TLR)-2 and TLR-4 activation in these PM driven inflammatory processes as part of an inflammasome driven response [
20‐
23].
The TLR family are well described pattern recognition receptors that detect characteristic microbial motifs to signal the presence of invading microbial organisms [
24]. TLR function forms part of the innate immune system and induce pro-inflammatory cytokine release. These signals alert and activate surrounding tissues and the adaptive immune system [
24]. Bacteria are detected by TLR-2 and TLR-4 which recognise components of Gram positive and Gram negative bacterial cell walls known as lipotoeic acid (LTA) and lipopolysaccharide (LPS, also known as endotoxin) respectively [
24]. Recognition of either LTA and LPS by TLR’s induces a cascading inflammatory response which can be severe as in the case of sepsis when bacteria are found in blood [
25].
Both LTA and LPS form a significant immuno-stimulatory component of ambient air. This has been shown by reduced inflammatory responses in cell cultures treated with ambient PM preparations mixed with polymixin B, a compound binding the Lipid A moiety of LPS [
26]. While exposure to LPS has been shown to exacerbate asthma there is conflicting evidence to suggest it also modulates allergic airway responses [
27,
28]. The role of TLR-2 and TLR-4 in responses to ambient PM has been further elucidated in alveolar macrophages, a key phagocyte in the lung [
29]. However, the overall effects of LPS and PM (including AMP) deposited in the lower airways and the impact of this on lung function and immune modulation has not been fully investigated.
In this study we aimed to delineate the individual and combined effects of LPS and AMP on airway inflammation and potential contribution to airway disease. Due to the well documented inflammatory effects of LPS, we hypothesized that airway inflammation induced by exposure to AMP would be augmented when AMP was co-administrated with LPS. Using a mouse model, the inhaled effects of nebulised AMP, LPS and AMP administered together with LPS on lung function, inflammatory cell infiltrate and cytokine responses in bronchoalveolar lavage and lung parenchymal tissue. To determine the role of TLR-4 in AMP and LPS induced airway inflammation, results were compared between wildtype mice and mice not expressing TLR-4. As PM size fractions contain a mixture of compounds which includes attached LPS [
10,
30], an inert fluorescent bead model was used in order to clearly assess the impact of AMP delivered with a known amount of LPS attached.
Discussion
The results of this study clearly demonstrate that the inflammatory effects of inhaled particulate matter are heavily influenced by the presence of LPS. Airway resistance and sensitivity were shown to correlate inflammatory cytokine responses to inhaled LPS and AMP-LPS measured in bronchoavleolar lavage. While these responses were more pronounced when signalled by TLR-4, inflammation was also observed in TLR-4 knock-out mice indicating other LPS recognition pathways. A larger TNF-α response observed in TLR-4 knockout mice treated with AMP-LPS compared to LPS alone, suggest alternate recognition or a divergent signalling pathway for this treatment combination. As there were no changes observed in IL-10 or IL-13 expression measured in lung parenchymal tissue, this suggests the inhaled preparations used in this study did not have an effect on airway tissue remodelling. Interestingly, elevated macrophage numbers observed in mice treated with inhaled latex beads alone, used as the model for AMP in this study, was not mimicked by increased inflammatory cytokine levels or augmented airway responses when compared to control non-treated mice.
Raised ambient PM levels are shown to be directly correlated to asthma admissions in health care centres, with long term exposure linked to the onset of lung cancer and COPD [
2]. In this study, we did not find any significant change in lung function resulting from AMP exposure. However, augmented airway resistance and airway sensitivity responses to methacholine were observed in wildtype mice exposed to LPS and AMP-LPS. As LPS is found ubiquitously in the environment our data suggests LPS attached to inhalable AMP induces changes in lung function rather than AMP alone. As AMP exposure is linked to the onset of chronic diseases such as COPD, asthma and even cardiovascular disease a longer study period may be more suitable. This would allow tracking of slow onset of symptoms which underlie these diseases in response to ongoing long-term exposure to inhaled AMP.
Neutrophilic inflammation present in wildtype mice treated with LPS and AMP-LPS compared to TLR4−/− mice indicates this response was driven by the presence of TLR-4 driven by the presence of LPS. In the absence of TLR-4, cellular inflammation to LPS and AMP-LPS was dominated by the presence of macrophages. AMP alone also induced increased macrophage infiltration compared to control mice, however this was observed irrespective of TLR-4 expression. Elevated macrophage numbers suggests either strengthened recruitment to the lung to clear inhaled particles, or impaired clearance, a hallmark of alveolar macrophages overloaded with phagocytosed particles [
43‐
48]. On the other hand, larger neutrophil numbers in response to particle inhalation have been shown to correlate the onset of cancer tumors, an observation which dissipates when particle deposition shifts from the alveolar space to lung interstitium [
49]. Airway deposition of AMP particles was not characterised in this study; however, these observations clearly demonstrate a greater number of macrophages with unchanged neutrophil numbers compared to non-treated control mice. Therefore, these findings suggest an interstitial lung deposition of AMP with induced inflammatory responses independent of TLR-4 expression for the first time. Interestingly, eosinophil numbers were significantly higher in wildtype mice treated with AMP-LPS compared to LPS. Indeed distinct TLR-4 driven cellular compartments have been shown to activate neutrophilic and eosinophilic responses in response to different allergens [
50], which may explain the larger eosinophil responses observed in wildtype mice treated with AMP-LPS compared to LPS alone. As eosinophilia is closely associated with the onset of asthma and allergy [
50,
51], further investigation of this finding may elucidate the cellular mechanisms underlying allergic airway disease caused by exposure to particulates.
Of those treated wildtype mice, LPS or AMP-LPS induced the largest inflammatory cytokine responses measured in BAL. Augmented responses to LPS and AMP-LPS were also observed in TLR4−/− mice, illustrating proinflammatory signalling mechanisms other than TLR-4 activated by LPS. Furthermore, TNF-α levels were significantly greater in BAL of AMP-LPS treated TLR4−/− mice compared to LPS treated mice, suggesting this combination was signalled by yet another mechanism. As we found evidence for LPS being attached to AMP, elevated TNF- α levels may have been induced by alternate receptors for LPS (such as scavenger receptors), or endocytosed resulting in recognition by intracellular pattern recognition receptors for LPS; including nucleotide-binding oligomerization domain (NOD) receptors contained in cellular inflammasomes [
51]. Indeed, Shi et al. have shown binding of LPS by caspase 11 is critical for activation of this intracellular process [
51]. Augmented TNF-α responses have been shown in the presence of eosinophilia [
52,
53]. Thereby the combination of elevated non-TLR4 driven TNF-α and TLR-4 driven eosinophilia observed in AMP-LPS treated mice suggest AMP-LPS is a stronger stimulus for allergic inflammation in the airways than LPS alone. Despite a larger number of macrophages observed in AMP treated mice, these did not display inflammatory cytokine responses that were significantly different to those measured in non-treated control mice.
Interestingly, IL-10 or IL-13 measured in the lung parenchyma remained unchanged in response to all inhaled treatments for wildtype and TLR4−/− mice. This is surprising given the long-standing relationships between AMP exposure and airway disease development characterised by airway remodelling co-ordinated by these cytokines [
54,
55]. Within the context of this study, this novel finding suggests TLR-4 driven inflammatory responses activated by LPS recognition appear to be predominantly secreted (BAL). However, as long term-low grade inflammation activity can go undetected in subepithelial tissues for long periods [
55], a larger study period may elucidate mechanisms pertinent to slow onset airway disease attributable to AMP exposure such as COPD and related cardiovascular disease [
56]. Importantly, IL-13 responses are associated with allergen associated airway disease such as asthma [
57]. Elevated TNF-α responses measured in BAL to LPS suggests modulation of this response by type-2 inflammatory cytokines such as IL-4 or IL-5. Although closely affiliated with IL-4, IL-13 responses observed in lung parenchyma did not correlate LPS induced TNF-α responses in BAL [
58]. Therefore, findings from a longer study period which include analysis of IL-4 or IL-5 may be valuable to our overall understanding of immune-modulated airway disease in response to allergic stimuli carried in inhaled air such as LPS, which has remained elusive to date [
9].
Conclusions
In conclusion, we have shown the presence of LPS in AMP preparations has an influential impact on induced airway and inflammatory BAL responses in the lung which are augmented by the presence of TLR-4. Importantly, dominant macrophage responses observed in BAL from AMP treated mice over all other treatments, suggest interstitial lung deposition, triggered regardless of TLR-4 expression for the first time. Despite this, inflammatory cytokine responses were not observed in the lung parenchymal tissues in response to any treatment, suggesting a longer study period may be needed to observe pro-fibrotic changes that underlie airway disease caused by long-term AMP inhalation. Interestingly, when AMP was attached to LPS larger TNF-α responses independent of TLR-4 expression were observed in BAL suggesting activation of allergic responses by non-TLR4 pathways. If augmented by TLR-4 driven eosinophilia as observed in AMP-LPS treated mice, these findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways over LPS alone. Taken together, these results demonstrate divergent response pathways in the lung to AMP and LPS, with larger allergy affects observed in AMP-LPS which have not been shown before. Therefore, these findings contribute novel information to the field investigating the onset of allergic and non-allergic airway disease, such as asthma and COPD, as a result of PM exposure and warrants further investigation.