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Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses… Fonceca, Angela M; Zosky, Graeme R; Bozanich, Elizabeth M; Sutanto, Erika N; Kicic, Anthony; McNamara, Paul S; Knight, Darryl A; Sly, Peter D; Turner, Debra J; Stick, Stephen M Jan 22, 2018

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RESEARCH Open AccessAccumulation mode particles and LPSexposure induce TLR-4 dependent andindependent inflammatory responses in thelungAngela M. Fonceca1*, Graeme R. Zosky2, Elizabeth M. Bozanich2, Erika N. Sutanto2,3, Anthony Kicic1,2,3,4,Paul S. McNamara5, Darryl A. Knight6,7,8, Peter D. Sly9, Debra J. Turner2 and Stephen M. Stick1,2,3,4AbstractBackground: Accumulation mode particles (AMP) are formed from engine combustion and make up the inhalablevapour cloud of ambient particulate matter pollution. Their small size facilitates dispersal and subsequent exposurefar from their original source, as well as the ability to penetrate alveolar spaces and capillary walls of the lung wheninhaled. A significant immuno-stimulatory component of AMP is lipopolysaccharide (LPS), a product of Gram negativebacteria breakdown. As LPS is implicated in the onset and exacerbation of asthma, the presence or absence of LPS inambient particulate matter (PM) may explain the onset of asthmatic exacerbations to PM exposure.This study aimed to delineate the effects of LPS and AMP on airway inflammation, and potential contribution to airwaysdisease by measuring airway inflammatory responses induced via activation of the LPS cellular receptor, Toll-like receptor4 (TLR-4).Methods: The effects of nebulized AMP, LPS and AMP administered with LPS on lung function, cellular inflammatoryinfiltrate and cytokine responses were compared between wildtype mice and mice not expressing TLR-4.Results: The presence of LPS administered with AMP appeared to drive elevated airway resistance and sensitivity viaTLR-4. Augmented TLR4 driven eosinophilia and greater TNF-α responses observed in AMP-LPS treated mice independentof TLR-4 expression, suggests activation of allergic responses by TLR4 and non-TLR4 pathways larger than those inducedby LPS administered alone. Treatment with AMP induced macrophage recruitment independent of TLR-4 expression.Conclusions: These findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways thenLPS alone.Keywords: Asthma, TLR-4, PM, LPS, AMP, COPDBackgroundExposure to ambient air pollution is an adverse healthrisk to respiratory health, particularly in the young, eld-erly and those with co-morbidities such as heart disease[1, 2]. In the young, epidemiological and toxicologicalresearch studies consistently demonstrate air pollutionas a major risk factor in the onset of asthma [3, 4]. Thisis well illustrated by rising rates of asthma observed indeveloping countries such as China where expandingindustrialization correlates with raised airborne pollution[1]. While in the elderly, long term exposure to particu-late matter (PM) has been implicated in developingCOPD [1, 2, 5, 6]. Not surprisingly, hospital admissionrates for breathing difficulties have been shown to riseduring times of raised ambient air pollution concentra-tions [3]. Despite the risks of air pollution exposure be-ing well accepted, the precise mechanisms leading to theonset of these chronic airway diseases are poorly under-stood [6–9].* Correspondence: angela.fonceca@uwa.edu.au1School of Paediatrics and Child Health, University of Western Australia,Nedlands, WA, AustraliaFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Fonceca et al. Respiratory Research  (2018) 19:15 DOI 10.1186/s12931-017-0701-zAmbient air pollution, comprised in part by AMP, is acomplex mixture of organic compounds, different sizedparticles and chemicals [10]. The US EPA refers to theinhalable solid phase of ambient air pollution as PM cate-gorized 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 categoriesmaking up the inhalable vapour cloud of PM [12]. AMP’sare largely sourced from engine combustion. Due to theirsmall size these are subject to wind and other climaticconditions which enable dispersal and exposure far fromtheir source of origin [13]. As AMP are small enough topenetrate alveolar spaces and capillary walls, exposure tothis particulate size fraction has been shown to result inrespiratory disease and exacerbation, with exposure alsolinked to cardiovascular disease [12, 13].To date, the majority of air pollution toxicology stud-ies have explored the role of whole ambient mixturesand individual chemical components on respiratoryhealth [7–9]. Due to the number of stimuli within ambi-ent air, identifying the causes and/or interactions re-sponsible for the onset of disease is difficult, as thesecan trigger a variety of host defence mechanisms wheninhaled [14–16]. Oxidative particulates and/or reactiveoxygen species generated by particulate phagocytosishave been shown to drive proinflammatory pathwayswhich can cause long-term lung damage and airway dis-ease [17–19]. There is also evidence to suggest Toll likereceptor (TLR)-2 and TLR-4 activation in these PMdriven inflammatory processes as part of an inflamma-some driven response [20–23].The TLR family are well described pattern recognitionreceptors that detect characteristic microbial motifs tosignal the presence of invading microbial organisms [24].TLR function forms part of the innate immune systemand induce pro-inflammatory cytokine release. Thesesignals alert and activate surrounding tissues and theadaptive immune system [24]. Bacteria are detected byTLR-2 and TLR-4 which recognise components of Grampositive and Gram negative bacterial cell walls known aslipotoeic acid (LTA) and lipopolysaccharide (LPS, alsoknown as endotoxin) respectively [24]. Recognition of ei-ther LTA and LPS by TLR’s induces a cascading inflam-matory response which can be severe as in the case ofsepsis when bacteria are found in blood [25].Both LTA and LPS form a significant immuno-stimulatory component of ambient air. This has beenshown by reduced inflammatory responses in cell cul-tures treated with ambient PM preparations mixed withpolymixin B, a compound binding the Lipid A moiety ofLPS [26]. While exposure to LPS has been shown to ex-acerbate asthma there is conflicting evidence to suggestit also modulates allergic airway responses [27, 28]. Therole of TLR-2 and TLR-4 in responses to ambient PMhas been further elucidated in alveolar macrophages, akey phagocyte in the lung [29]. However, the overall ef-fects of LPS and PM (including AMP) deposited in thelower airways and the impact of this on lung functionand immune modulation has not been fully investigated.In this study we aimed to delineate the individual andcombined effects of LPS and AMP on airway inflamma-tion and potential contribution to airway disease. Due tothe well documented inflammatory effects of LPS, we hy-pothesized that airway inflammation induced by exposureto AMP would be augmented when AMP was co-administrated with LPS. Using a mouse model, the inhaledeffects of nebulised AMP, LPS and AMP administered to-gether with LPS on lung function, inflammatory cell infil-trate and cytokine responses in bronchoalveolar lavageand lung parenchymal tissue. To determine the role ofTLR-4 in AMP and LPS induced airway inflammation, re-sults were compared between wildtype mice and mice notexpressing TLR-4. As PM size fractions contain a mixtureof compounds which includes attached LPS [10, 30], aninert fluorescent bead model was used in order to clearlyassess the impact of AMP delivered with a known amountof LPS attached.MethodsAnimalsWe used commercially available fluorescent polystyrenebeads as a model for inert AMP (Fluoresbrite™ polychro-matic red microspheres, Polysciences Inc., Pennsylvania,USA; herein referred to as AMP) exposure as has beenused previously [31]. In this study, mice with a mutatednon-functional TLR-4 expression (C3H/HeJ, referred toas TLR4−/−) and mice of the same strain expressingTLR-4 (C3H/HeN, referred to as wildtype, WT) wereused in order to assess changes in respiratory mechanicsand lung inflammation in response to nebulized treat-ments of AMP, LPS and a mixed AMP-LPS preparation.TLR4−/− mice have been previously characterized withdysfunctional TLR-4 expression due to a spontaneousproline to histidine point mutation in the TLR-4 signal-ing sequence [32]. This mouse model is commonly usedas a negative control for TLR-4 expression in studies in-vestigating TLR-4 responses [33, 34]. Mice were used at7–9 weeks of age (Animal Resource Centre, Murdoch,Western Australia) and housed in a controlled environ-ment with a 12 h light to dark cycle with unrestrictedaccess to food and water. All experiments presentedwere approved by the Telethon Kids Institute’s AnimalEthics Committee (approval reference #128) and carriedout in accordance with the recommendations of theAustralian code for the care and use of animals forscientific purposes 8th edition (2013).Ten mice of each strain were grouped to receive thefollowing nebulized treatments: LPS alone (50 μg/ml,Fonceca et al. Respiratory Research  (2018) 19:15 Page 2 of 10Salmonella typhimurium, Sigma-Aldrich, St. Louis, Mis-souri, USA); 0.5 μm polystyrene beads alone (50 μg/ml,equating to approximately 7.26 × 1010 particles/ml ofAMP; both AMP and LPS (AMP-LPS, 50 μg/mL); ordouble-distilled water (control). Double distilled waterand AMP nebulization preparations contained < 0.015EU/ml (detection limit) using the Limulus amebocytelysate (LAL) assay (Sigma-Aldrich, Missouri, USA).Double distilled water induced less airway resistance tomethacholine at doses above 3 mg/ml during challengecompared to endotoxin free 0.9% saline, confirmingsuitability of this as a control for these studies (seeAdditional file 1: Figure S1) [35]. To best represent shortterm exposure inducing an inflammatory response, micewere exposed to their allocated treatment at constantflow of 3 ml/min for 30 min at the same time for sixconsecutive days. Nebulized aerosols were delivered toanimals via an UltraNeb™ nebulizer (DeVilbiss, Somerset,Pennsylvania, USA), as described previously [36]. Ac-cording to DeVilbiss, nebulized droplet size distributiongenerated ranges from 0.5-3 μm [37]. As a solution of0.5 μm polychromatic spheres were used, the overallnebulization range was deemed to fit the accumulationmode particle size range (0.1–2.5 μm) for the purposesof this study.Lung function measurementsLung function was assessed using a modification of thelow frequency, forced oscillation technique (LFOT).Mice were initially anaesthetized with an intraperitonealinjection of a solution containing xylazine (2 mg/ml,Troy Laboratories, NSW, Australia) and ketamine(40 mg/ml, Troy Laboratories, NSW, Australia) at a doseof 0.01 mg/g. Mice were then tracheotomized and a10 mm section of polyethylene tubing (1.27 mm OD,0.86 mm ID) inserted into the trachea. Mice were venti-lated at 450 breaths/min with a tidal volume of 8 ml/kgand a positive end expiratory pressure (PEEP) of 2 cmH2O using a computer-controlled ventilator (flexiVent,SCIREQ Inc., Montreal, Canada). This system was usedfor ventilation and measurement of respiratory mechan-ics as previously described [38, 39].Before commencing lung function measurements,mouse lung volume history was standardized using 5deep inflations to total lung capacity. Respiratory imped-ance (Zrs) was measured using an oscillatory signal con-taining 19 frequencies ranging from 0.25 to 19.625 Hzduring pauses in ventilation. Zrs was partitioned intocomponents representing the mechanical properties ofthe airways and lung tissue parenchyma using a fourparameter model with constant phase tissue impedance[38, 39]. Partitioning of Zrs in this way allows calculationof parameters representing airway resistance, tissuedamping and tissue elastance [40, 41].Methacholine challengeFollowing measurement of baseline Zrs, mice wereexposed to a saline aerosol for 90s (Ultraneb™ 99, Devil-biss, Somerset, Pennsylvania, USA). Five measurementsof Zrs were then obtained, averaged and used as thecontrol measurements for MCh challenge. The aerosolprocedure was repeated with half log incremental dosesof MCh from 0.1 to 30 mg/ml. Measurements of Zrswere recorded every minute for 5 min after each MChaerosol and the maximum response calculated. Fromthese, data dose response curves for airway resistance(Raw) were constructed. Sensitivity to MCh was deter-mined by calculating the MCh dose required to producea 200% increase in Raw in response to the saline chal-lenge at 30 mg/ml using interpolation [41]. Maximumresponses in Raw and airway sensitivity were used tocompare lung function responses between groups.Inflammatory cell countsFive additional animals per group were anaesthetisedand tracheotomised for bronchoalveolar lavage (BAL)used for inflammatory cell infiltrate and inflammatorycytokines analysis as previously described [40]. Briefly,BAL fluid was collected by slowly infusing and with-drawing a 1 ml aliquot of 0.9% saline from the lungthree times. The resulting fluid was centrifuged at2000 rpm for 4 min. Supernatant was collected andstored at −80 °C for later analysis. The cell pellet was re-suspended in saline and a portion stained with trypanblue to determine viability and total cell count (TCC).The remaining portion was centrifuged onto slides andstained with Leishman’s (Sigma-Aldrich, St Louis, Mis-souri, USA) to obtain differential cell counts.Inflammatory cytokine responsesBronchoalveolar lavage (BAL)Analysis of BAL for the presence of secreted pro-inflammatory cytokines known to be secreted in responseto LPS and PM [42]. Interleukin (IL)-6, Interferon (IFN)-γ,and Tumor necrosis factor (TNF)-α was completed usinga cytokine bead array assay (BD Biosciences California,USA) as per manufacturers’ instructions, with a detectionrange of 20-5000 pg/ml for all cytokines within the array.These measurements were completed in BAL from fivemice of each mouse strain for each treatment using anoptimized sample dilution factor.Lung parenchymaSoluble protein was extracted from a single mouse lunglobe to gain a measure of subepithelial pro-inflammatoryresponses indicative of airway remodelling and developingchronic airway inflammation. For this reason expressionof immune-regulatory, IL-10 and pro-fibrotic cytokine,IL-13, were examined using ELISA (R&D Systems,Fonceca et al. Respiratory Research  (2018) 19:15 Page 3 of 10Abdington, UK; detection ranges: IL-10, 31.2-2000 pg/mland IL-13, 62.5-4000 pg/ml) using optimised sample dilu-tion factors. Specifically, IL-10 was chosen as it is secretedin response to LPS and is thought to protect the lungagainst lung injury by reducing the production of proin-flammatory cytokines, chemokines and transcription fac-tors implicated in airway remodelling. Whereas IL-13 isknown to be involved in subepithelial fibrosis related tothe onset of asthma and COPD. Data was calculated andnormalized to 100 μg of total soluble protein as measuredusing the Pierce BCA assay (Thermo-Pierce, Rockford,USA) for comparative purposes.Statistical analysisSPSS Levene’s test was used to test for equal varianceacross all the groups of data compared. Following verifi-cation, an independent t-test was then used to determinestatistically significant differences between (a) controlsand treatment groups and (b) single treatments betweenWT and TLR4−/− mice. Due to differences in baselinelung function (see results Table 1), responses to MChwere expressed as a percentage of baseline with graphsare shown as mean ± SEM. Due to the range of data,cytokine responses are presented as box and whisperplots depicting interquartile range and 2.5 and 97.5percentiles with medians. A minimum of 4 biologicalreplicates and p values < 0.05 considered significantlydifferent were used for all data sets.ResultsLung functionBaseline lung function responses were recorded for eachanimal prior to saline and methacholine (MCh) challenge(Table 1). Baseline airway resistance (Raw) values were el-evated in all TLR4−/− mice compared to wildtype (WT)mice of the same treatment (PM p = 0.04, LPS p = 0.03,PM-LPS p = 0.035), including control mice (p = 0.035); in-dicating this mouse strain had more sensitive airwaysoverall. However, baseline Raw did not vary considerablybetween mice of the same strain treated with AMP, LPSor AMP-LPS nebulisations (Table 1).At 30 mg/ml MCh, Raw was significantly augmentedin WT mice treated with nebulized LPS or AMP-LPScompared to control mice (p = 0.04 and 0.03respectively, Fig. 1a). Sensitivity to MCh as determinedby interpolation, was significantly less in WT micetreated with LPS (p = 0.008) and AMP-LPS (p = 0.017)compared to controls. This was not observed in LPS andAMP-LPS treated TLR4−/− mice, indicating more sensi-tive airways in response to LPS and AMP-LPS treat-ments in the presence of TLR-4 (Fig. 1b). Responses forall other doses of MCh used for challenge can be foundin Additional file 2: Figure S2.Cellular responses measured in bronchoalveolar lavageBronchoalveolar lavage (BAL) inflammatory total cellcounts were larger in WT mice treated with AMP, LPS (p< 0.001) and AMP-LPS (p < 0.001) compared to WT con-trol mice. Total cell counts in WT mice treated with LPS(p < 0.001) and AMP-LPS (p < 0.001) were greater thansimilarly treated TLR4−/− mice (Fig. 2a). Neutrophilswere the predominant cell type in LPS and AMP-LPStreated WT mice compared to control and AMP treatedmice (p < 0.001) (Fig. 2b). In contrast, neutrophils werebarely detectable in TLR4 −/− mice irrespective of expos-ure. Macrophages were the dominant cell type in micetreated with AMP irrespective of strain (p = 0.01 for bothstrains) and in TLR4−/− mice treated with LPS (p = 0.007)and AMP-LPS (p = 0.04) compared to respective controls(Fig. 2c). Eosinophil numbers were greater in WT micetreated with LPS (p = 0.006) and AMP-LPS (p < 0.001); forwhich numbers were larger in AMP-LPS treated mice (p= 0.024) compared to those treated with LPS. Lymphocyteand epithelial cell numbers were not significantly differentbetween controls and any of the treatments given foreither strain. Other cytokine responses measured usingthe commercial kit can be found in Additional file 3: Fig-ure S3.Inflammatory cytokine responsesBronchoalveolar lavageSignificantly increased levels of IFN-γ, IL-6 and TNF-αwere observed in wildtype and TLR4−/− mice treatedwith LPS (Wildtype: IFN-γ p = 0.02; IL-6 p = 0.002;TNF-α p = 0.001, TLR4−/−: IFN-γ p < 0.001; IL-6 p <0.001; TNF-α p < 0.001) and AMP- LPS (Wildtype: IFN-γ p < 0.001, IL-6 p < 0.001, TNF-α p = 0.001, TLR4−/−:IFN-γ p < 0.001; IL-6 p < 0.001; TNF-α p < 0.001), withTable 1 Baseline lung function for mice studies completed in control and treated wildtype (WT) and TLR4 (TLR4 −/−) mutant mice.Baseline lung function measurements taken before methacholine challenge were not significantly different between treatments inWT and TLR4−/− mice. Greater Raw values were measured for all TLR4−/− mice compared to WT mice of the same treatment,indicating that overall, this mouse strain had more sensitive airwaysTreatment Control AMP LPS AMP-LPSMouse strain WT TLR4−/− WT TLR4−/− WT TLR4−/− WT TLR4−/−Raw (hPa.s.ml−1) 0.35 0.45* 0.34 0.46* 0.34 0.40* 0.34 0.45*(0.01) (0.02) (0.01) (0.02) (0.01) (0.03) (0.01) (0.02)*p < 0.05 between WT and TLR4−/−,()indicates SDFonceca et al. Respiratory Research  (2018) 19:15 Page 4 of 10greater amount of cytokine in wildtype mice for thesetreatments (LPS p = 0.028 and APM-LPS p < 0.001)(Fig. 3). Only AMP-LPS treated TLR4−/− mice had sig-nificantly more TNF-α compared to LPS treated TLR4−/− mice (p = 0.032). The amounts of these cytokineswere not significantly different in AMP treated micecompared to control mice for both strains.Lung parenchymaSoluble protein was extracted from one whole mouselung lobe and analysed by ELISA for IL-10 and IL-13expression showed no differences in these cytokines forany treatment in WT or TLR4−/− mice compared tocontrols (p > 0.05 for all; Fig. 4). Similarly, there wereno differences observed between WT and TLR4−/−mice for any of the individual inhaled treatmentsadministered (p > 0.05).DiscussionThe results of this study clearly demonstrate that the in-flammatory effects of inhaled particulate matter are heavilyinfluenced by the presence of LPS. Airway resistance andsensitivity were shown to correlate inflammatory cytokineresponses to inhaled LPS and AMP-LPS measured inbronchoavleolar lavage. While these responses were morepronounced when signalled by TLR-4, inflammation wasalso observed in TLR-4 knock-out mice indicating otherLPS recognition pathways. A larger TNF-α response ob-served in TLR-4 knockout mice treated with AMP-LPSFig. 1 Airway resistance (a) and sensitivity to methacholine at30 mg/ml (b) for wildtype (WT) and mice not expressing TLR4 (TLR4−/−) for each treatment. Airway resistance (Raw) values for thelargest methacholine challenge (MCh, 30 mg/ml) are presented as apercentage of the initial saline challenge given to each animal givenprior to commencing MCh challenges (a). Raw was augmented inWT mice treated with nebulized LPS and AMP-LPS only. Airwaysensitivity was calculated by interpolating the amount of MChneeded to cause a doubling of baseline responses (b). WT micetreated with LPS and AMP-LPS required significantly less MCh thanthat needed for control mice, indicating more sensitive airways dueto LPS and AMP-LPS treatments in the presence to TLR-4 (*p < 0.05compared to controls). No significant differences were observed inairway resistance or sensitivity between treatments of LPS or AMP-LPSwithin each mice strain (p > 0.05)Fig. 2 Bronchoalveolar lavage (BAL) total (a) and differential cellcounts (b, c) for wildtype (WT) and mice not expressing TLR4 (TLR4−/−) for each treatment. Elevated total cell counts in BAL were observedin WT mice treated with AMP, LPS and AMP-LPS compared to WTcontrol mice. Cell counts in WT mice treated with LPS and AMP-LPSwere also greater than similarly treated TLR4−/− mice. Neutrophilswere the predominant cell type in LPS and AMP-LPS treated WT mice.Macrophages dominated counts in AMP treated WT and TLR4−/−mice, as well as LPS and AMP-LPS treated TLR4−/− mice compared torespective controls. Greater eosinophil numbers were observed in WTmice treated with LPS and AMP-LPS; for which numbers were greaterin AMP-LPS treated mice. Lymphocyte and epithelial cell numberswere not significantly different between controls and any of the treat-ments given for either strain (*p < 0.05 between treatment and control;# = p < 0.05 between strains for the same treatment; α = p < 0.05between treatment compared to all other treatments for thesame strain)Fonceca et al. Respiratory Research  (2018) 19:15 Page 5 of 10compared to LPS alone, suggest alternate recognition or adivergent signalling pathway for this treatment combin-ation. As there were no changes observed in IL-10 or IL-13 expression measured in lung parenchymal tissue, thissuggests the inhaled preparations used in this study didnot have an effect on airway tissue remodelling. Interest-ingly, elevated macrophage numbers observed in micetreated with inhaled latex beads alone, used as the modelfor AMP in this study, was not mimicked by increased in-flammatory cytokine levels or augmented airway responseswhen compared to control non-treated mice.Raised ambient PM levels are shown to be directlycorrelated to asthma admissions in health care centres,with long term exposure linked to the onset of lungcancer and COPD [2]. In this study, we did not find anysignificant change in lung function resulting from AMPexposure. However, augmented airway resistance andairway sensitivity responses to methacholine were ob-served in wildtype mice exposed to LPS and AMP-LPS.As LPS is found ubiquitously in the environment ourdata suggests LPS attached to inhalable AMP induceschanges in lung function rather than AMP alone. AsAMP exposure is linked to the onset of chronic diseasessuch as COPD, asthma and even cardiovascular diseasea longer study period may be more suitable. This wouldallow tracking of slow onset of symptoms which underliethese diseases in response to ongoing long-term expos-ure to inhaled AMP.Neutrophilic inflammation present in wildtype micetreated with LPS and AMP-LPS compared to TLR4−/−mice indicates this response was driven by the presenceof TLR-4 driven by the presence of LPS. In the absenceof TLR-4, cellular inflammation to LPS and AMP-LPSwas dominated by the presence of macrophages. AMPalone also induced increased macrophage infiltrationcompared to control mice, however this was observed ir-respective of TLR-4 expression. Elevated macrophagenumbers suggests either strengthened recruitment to thelung to clear inhaled particles, or impaired clearance, ahallmark of alveolar macrophages overloaded withphagocytosed particles [43–48]. On the other hand, lar-ger neutrophil numbers in response to particle inhal-ation have been shown to correlate the onset of cancertumors, an observation which dissipates when particledeposition shifts from the alveolar space to lung intersti-tium [49]. Airway deposition of AMP particles was notcharacterised in this study; however, these observationsclearly demonstrate a greater number of macrophageswith unchanged neutrophil numbers compared to non-treated control mice. Therefore, these findings suggestan interstitial lung deposition of AMP with induced in-flammatory responses independent of TLR-4 expressionfor the first time. Interestingly, eosinophil numbers weresignificantly higher in wildtype mice treated with AMP-Fig. 3 Cytokine responses measured in bronchoalveolar lavagecollected from treated wildtype (WT) (□) and mice not expressingTLR4 (TLR4−/−) (■). Significantly elevated IFN-γ, IL-6 and TNF-α wasobserved in WT and TLR4−/− mice treated with LPS and AMP-LPS,with these results being greater in WT mice. Only AMP-LPS treatedTLR4−/− mice had significantly more TNF-α compared to LPS treatedTLR4−/− mice. The amount of these cytokines was not significantlydifferent in AMP treated mice compared to control mice for bothstrains (* indicates p < 0.05 compared to controls, α = p < 0.05 singletreatment compared to all other treatments for the same strain)Fonceca et al. Respiratory Research  (2018) 19:15 Page 6 of 10LPS compared to LPS. Indeed distinct TLR-4 drivencellular compartments have been shown to activateneutrophilic and eosinophilic responses in response todifferent allergens [50], which may explain the larger eo-sinophil responses observed in wildtype mice treatedwith AMP-LPS compared to LPS alone. As eosinophiliais closely associated with the onset of asthma and allergy[50, 51], further investigation of this finding may eluci-date the cellular mechanisms underlying allergic airwaydisease caused by exposure to particulates.Of those treated wildtype mice, LPS or AMP-LPSinduced the largest inflammatory cytokine responsesmeasured in BAL. Augmented responses to LPS andAMP-LPS were also observed in TLR4−/− mice, illustrat-ing proinflammatory signalling mechanisms other thanTLR-4 activated by LPS. Furthermore, TNF-α levels weresignificantly greater in BAL of AMP-LPS treated TLR4−/−mice compared to LPS treated mice, suggesting thiscombination was signalled by yet another mechanism. Aswe found evidence for LPS being attached to AMP, ele-vated TNF- α levels may have been induced by alternatereceptors for LPS (such as scavenger receptors), or endocy-tosed resulting in recognition by intracellular pattern rec-ognition receptors for LPS; including nucleotide-bindingoligomerization domain (NOD) receptors contained in cel-lular inflammasomes [51]. Indeed, Shi et al. have shownbinding of LPS by caspase 11 is critical for activation of thisintracellular process [51]. Augmented TNF-α responseshave been shown in the presence of eosinophilia [52, 53].Thereby the combination of elevated non-TLR4 drivenTNF-α and TLR-4 driven eosinophilia observed in AMP-LPS treated mice suggest AMP-LPS is a stronger stimulusfor allergic inflammation in the airways than LPS alone.Despite a larger number of macrophages observed in AMPtreated mice, these did not display inflammatory cytokineresponses that were significantly different to thosemeasured in non-treated control mice.Interestingly, IL-10 or IL-13 measured in the lung par-enchyma remained unchanged in response to all inhaledtreatments for wildtype and TLR4−/− mice. This is sur-prising given the long-standing relationships betweenAMP exposure and airway disease development charac-terised by airway remodelling co-ordinated by these cy-tokines [54, 55]. Within the context of this study, thisnovel finding suggests TLR-4 driven inflammatoryresponses activated by LPS recognition appear to be pre-dominantly secreted (BAL). However, as long term-lowgrade inflammation activity can go undetected in sube-pithelial tissues for long periods [55], a larger studyperiod may elucidate mechanisms pertinent to slowonset airway disease attributable to AMP exposure suchas COPD and related cardiovascular disease [56]. Im-portantly, IL-13 responses are associated with allergenassociated airway disease such as asthma [57]. ElevatedTNF-α responses measured in BAL to LPS suggestsmodulation of this response by type-2 inflammatory cy-tokines such as IL-4 or IL-5. Although closely affiliatedwith IL-4, IL-13 responses observed in lung parenchymadid not correlate LPS induced TNF-α responses in BAL[58]. Therefore, findings from a longer study periodwhich include analysis of IL-4 or IL-5 may be valuable toour overall understanding of immune-modulated airwaydisease in response to allergic stimuli carried in inhaledair such as LPS, which has remained elusive to date [9].ConclusionsIn conclusion, we have shown the presence of LPS inAMP preparations has an influential impact on inducedairway and inflammatory BAL responses in the lungwhich are augmented by the presence of TLR-4. Import-antly, dominant macrophage responses observed in BALfrom AMP treated mice over all other treatments, sug-gest interstitial lung deposition, triggered regardless ofTLR-4 expression for the first time. Despite this,Fig. 4 Cytokines measured in lung parenchymal tissue from wildtype (WT) (□) and mice not expressing TLR4 (TLR4−/−) (■) treated with AMP, LPSand AMP-LPS. There were no observed differences in IL-10 and IL-13 protein expression measured from whole mouse lung lobe analysed byELISA for between mouse strains for any treatment or for any treatment compared to control non-treated mice (p > 0.05). Results were normalisedto 100 μg of total soluble protein for comparative purposes (n =minimum of 4 for each group)Fonceca et al. Respiratory Research  (2018) 19:15 Page 7 of 10inflammatory cytokine responses were not observed inthe lung parenchymal tissues in response to any treat-ment, suggesting a longer study period may be neededto observe pro-fibrotic changes that underlie airway dis-ease caused by long-term AMP inhalation. Interestingly,when AMP was attached to LPS larger TNF-α responsesindependent of TLR-4 expression were observed in BALsuggesting activation of allergic responses by non-TLR4pathways. If augmented by TLR-4 driven eosinophilia asobserved in AMP-LPS treated mice, these findingssuggest AMP-LPS as a stronger stimulus for allergicinflammation in the airways over LPS alone. Taken to-gether, these results demonstrate divergent responsepathways in the lung to AMP and LPS, with largerallergy affects observed in AMP-LPS which have notbeen shown before. Therefore, these findings contributenovel information to the field investigating the onset ofallergic and non-allergic airway disease, such as asthmaand COPD, as a result of PM exposure and warrantsfurther investigation.Additional filesAdditional file 1: Figure S1. Airway resistance in TLR4−/− micetreated with double distilled water (ddH2O) and saline. Saline responseswere significantly greater for methacholine challenges larger than 3 mg/ml (* p < 0.05). (TIFF 356 kb)Additional file 2: Figure S2. Airway resistance in wildtype (WT) andTLR4−/− mice for all treatment groups across for all methacholinechallenges used. Raw was significantly greater in WT mice treated withLPS and AMP-LPS compared to control mice at 30 mg/ml MCh(*p < 0.05). (TIFF 1196 kb)Additional file 3: Figure S3. Additional cytokines measured inbronchoalveolar lavage (BAL) and lung parenchyma. MCP-1 was measuredin BAL using cytokine bead array assay (20-5000 pg/ml detection range)and IL-8 in lung parenchyma using ELISA (15.6-1000 pg/ml detection range)using optimised sample dilution factors. No significant difference withtreatment was observed for these cytokines. (TIFF 558 kb)AcknowledgementsNone to declareFundingThis work was supported by grants from the National Health and MedicalResearch Council of Australia and Princess Margaret Hospital for ChildrenFoundation. Stephen M. Stick is a NHMRC Practitioner Fellow.Availability of data and materialsThe datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request.Authors’ contributionsAMF, GZ, EMB and ENS performed the experiments and completed theanalysis. AMF, AK, PSM, DAK, DJT and SMS conducted the data interpretationand manuscript preparation. AMF, PDS, DJT and SMS conceived the idea anddesigned the experiment. All authors read and approved the finalmanuscript.Ethics approval and consent to participateMouse study approved by Telethon Kids Institute Animal Ethics Committee,approval reference number 128.Consent for publicationNot applicable, authors agree to pay journal processing fee should themanuscript be accepted for publication.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1School of Paediatrics and Child Health, University of Western Australia,Nedlands, WA, Australia. 2Telethon Kids Institute, Subiaco, WA, Australia.3Department of Respiratory Medicine Princess Margaret Hospital for ChildrenPerth, Subiaco, WA, Australia. 4Centre for Cell Therapy and RegenerativeMedicine, School of Medicine and Pharmacology, The University of WesternAustralia, Nedlands, WA 6009, Australia. 5Department of Women’s andChildren’s Health, Institute of Translational Medicine, University of Liverpool,Liverpool, UK. 6School of Biomedical Sciences and Pharmacy, University ofNewcastle, Callaghan, NSW, Australia. 7Priority Research Centre for Asthmaand Respiratory Disease, Hunter Medical Research Institute, Newcastle, NSW,Australia. 8Department of Anesthesiology, Pharmacology and Therapeutics,University of British Columbia, Vancouver, Canada. 9Queensland Children’sMedical Research Institute, University of Queensland, Royal Children’sHospital, Herston, QLD, Australia.Received: 1 September 2017 Accepted: 13 December 2017References1. 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