These cancers are characterized by instability at short tandem repeats (STRs) and in telomeric DNA. We have investigated telomere length in saliva DNA from LS and control families, using single telomere analysis at XpYp and 12q and by qPCR to measure total telomeric DNA. Single telomere analysis showed a trend for shorter XpYp telomeres in +/−MSH2 carriers compared to +/MLH1− carriers or controls, but this was masked in the comparative analysis of total telomeric DNA. Comparison of age-adjusted telomere length within families showed that neither +/−MSH2 or +/−MLH1 children had consistently shorter or longer telomeres than their MMR+/− parent, indicating the absence of an inter-generational effect on telomere length. Unexpectedly however, wildtype children in families with MSH2 mutations, had significantly longer XpYp telomeres than their MMR+/− parent. Altogether our data suggest that MMR insufficiency, particularly in +/−MSH2 carriers, increases telomere instability and somatic cell turnover during the lifetime of LS mutation carriers but has minimal consequences for telomere length in the germline.Lynch syndrome (LS) is an inherited predisposition to early onset of various cancers, caused by mutation in a DNA mismatch repair (MMR) gene. In heterozygous MMR MLH1 or MSH2 mutations show a higher level of instability at short tandem repeat (STR) loci , with a bias towards deletions, than seen in their non-carrier relatives. This shows that heterozygous MMR+/− cells have a limited capacity for MMR arising from haplo-insufficiency [Telomeres are the essential capping structures of chromosomes with well-established roles in ageing and cancer. Short telomeres activate a DNA damage response and cells with one or a few dysfunctional telomeres enter a senescent state. Cancer cells usually acquire mutations that allow them to bypass this replication barrier but must eventually activate a telomere maintenance mechanism ,2,3. Inhficiency ,6,7.n often interspersed with sequence-variant repeats at the proximal end of the telomere [n strand and the requirement to unwind the t-loop capping structure. TRF1, an essential component of shelterin, is required for the efficient replication of telomeric DNA as it facilitates G-quadruplexes removal and prevents the manifestation of fragile telomeres [MSH2 gene, resulting in frequent gains and losses of telomeric repeats [Telomeric DNA comprises tandem arrays of (TTAGGG)telomere . Thus tetelomere . There aelomeres . We have repeats ,12. Telo repeats . It is tMSH2 mutations, while data from other studies indicating that such observations may be explained by other factors [MSH2+/− and MLH1+/− carrier status affects telomere length within LS families. We did not find evidence of an inter-generational effect on telomere length between MMR+/− parents and children. In contrast, single telomere length analysis (STELA) showed that wild type (WT) children in LS families tended to have some longer telomeres than their MMR+/− parent and siblings, particularly when MSH2 was mutated. Overall the data support the hypothesis that MMR insufficiency, particularly when MSH2 is mutated, increases telomere instability and subsequent cell turnover in somatic tissues but has little consequence for telomere length in the germline where telomerase is active.It has been proposed that LS families show genetic anticipation, with the first cancer appearing at an earlier age in successive generations. This hypothesis is controversial  with som factors ,16. The  factors . Genetic factors , and oth factors . However factors . In thisn = 10), comprised 37 individuals with a mean age 45.3 ± 18.4 (range 18.2 to 82.6 years) and were recruited with approval from the University of Leicester’s Research Ethics Committee (Ref: njr-61d3). Lynch syndrome families (n = 24) comprising 91 individuals with a mean age 47.0 ± 15.8 (range 19.2 to 73.1 years) were recruited in the Birmingham Women and Children’s Hospital (BWH) and the Leicester Royal Infirmary (LRI) under the ethical approval for the ‘Molecular Pathology of Human Genetic Disease (HumGenDis)’ study . Saliva samples were collected from two generations in each family with the older generation being designated as ‘parents’ and the younger generation as ‘children’. The one exception was the BWHF105 family, with one saliva sample also collect form a third generation. In this family, the individual BWHF105 G4P9 is the child in one comparison and the parent in another.Saliva samples were collected from adult individuals (18 years or older) in control and LS families. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki. Control families . The samples were coded and telomere length analyses conducted blind. STELA was used to amplify XpYp and 12q telomeres as described previously . The anaLinear regression and other statistical analyses were conducted using Graph-Pad Prism, version 7.0. Age-adjusted telomere lengths (ΔTel) were calculated as described previously  and chilHigh molecular weight DNA was extracted from the saliva samples collected from the control and LS families described in Median telomere lengths were measured in saliva DNA from control and LS families using STELA at XpYp and 12q  and tota+/−) also showed a significant decline with age. In contrast, the graphs of median XpYp and 12q telomere lengths versus age in the LS cohort showed lower r2 and r values and the slopes were not significantly different from zero  reduced the number of child-parent comparisons that could be made, but it confirmed that WT and not MSH2+/− or MLH1+/− children tend to have longer XpYp telomeres than their MSH2+/− or MLH1+/− parent  . The dif− parent a. A simi− parent b.n DNA and STELA to measure median lengths at the XpYp and 12q telomeres in saliva DNA samples from control and LS families [The aim of this study was to determine whether haplo-insufficiency for DNA MMR impacts on telomere length in heterozygous carriers of MMR gene mutations. The various methods for telomere length measurement each have advantages and disadvantages . Therefofamilies ,21.+/− carriers did not show a strong negative correlation between telomere length and age. This may suggest that there is a wider distribution of telomere lengths at any age among MMR+/− carriers, which could be a consequence of MMR+/− driven telomere instability. In addition, the cross-sectional and Y-intercept analyses showed that adult +/−MSH2 carriers tended to have shorter XpYp telomeres, than controls and MLH1+/− carriers. MSH2 is involved, as a heterodimer with MSH6 or MSH3, in the detection and binding to mismatches or insertion/deletion loops in DNA but also in the role that the MMR machinery plays in the cell cycle response to DNA damage. The data presented raise the possibility that MSH2 deficiency has a greater effect on telomere dynamics than MLH1 deficiency but the consequences may vary between telomeres. The question then arises as to why some telomeres might be more adversely affected by MSH2 deficiency than others. One possibility is variation in the composition and density of telomere variant repeats (TVRs) in the proximal regions of human telomeres, as this is known to vary considerably between chromosome ends, alleles and populations [The cross-sectional analyses of XpYp and 12q telomere lengths in the healthy controls showed significant shortening with age, in line with other studies ,24. In culations ,12,25,26ulations ,12,26, aulations . MSH2 is mutated but these differences were masked in the bulk analysis of all telomeres (using T/S-qPCR, n abundance from all telomeres, but may under-represent very short or very long telomeres [The subtle differences we identified between individual telomeres indicate that telomere length dynamics differ between controls and MMR mutation carriers, particularly if elomeres . In this+/− children would show shorter age-adjusted telomere length than their MMR+/− parents. In fact, comparison of parent-child age-adjusted telomere lengths showed that MMR+/− children did not have significantly shorter or longer telomeres than either parent. Consequently, we did not find evidence of an inter-generational effect on telomere length that might be attributed to shorter telomere length in gametes. In contrast, WT children had significantly longer age-adjusted XpYp telomeres than their MMR+/− parents, with a stronger effect in MSH2 families, and a similar non-significant trend at 12q. Moreover, MSH2+/− carriers tend to start adulthood with shorter telomeres than WT individuals. Altogether the data presented here show there is a complex relationship between telomere instability and turnover of somatic cells during the lifetime of MMR+/− carriers, in particular MSH2+/− individuals.For MMR haplo-insufficiency to contribute to genetic anticipation in LS families we proposed that MMR


The Household Air Pollution Intervention Network (HAPIN) trial aims to assess health benefits of a liquefied petroleum gas (LPG) cookfuel and stove intervention among women and children across four low- and middle-income countries (LMICs). We measured exposure contrasts for women, achievable under alternative conditions of biomass or LPG cookfuel use, at potential HAPIN field sites in India, to aid in site selection for the main trial.We recruited participants from potential field sites within Villupuram and Nagapattinam districts in Tamil Nadu, India, that were identified during a feasibility assessment. We performed.N = 79) using either biomass or LPG as their primary cookfuel and (ii) before-and-after measurements on pregnant women (N = 41), once at baseline while using biomass fuel and twice – at 1 and 2 months – after installation of an LPG stove and free fuel intervention. We involved participants to co-design clothing and instrument stands for personal and area sampling. We measured 24 or 48-h personal exposures and kitchen and ambient concentrations of fine particulate matter (PM2.5) using gravimetric samplers.(i) cross-sectional measurements on women  kitchen PM2.5 concentrations in biomass and LPG using homes were 134 μg/m3 [IQR:71–258] and 27 μg/m3 [IQR:20–47], while corresponding personal exposures were 75 μg/m3 [IQR:55–104] and 36 μg/m3 [IQR:26–46], respectively. In before-and-after analysis, median 48-h personal exposures for pregnant women were 72 μg/m3 [IQR:49–127] at baseline and 25 μg/m3 [IQR:18–35] after the LPG intervention, with a sustained reduction of 93% in mean kitchen PM2.5 concentrations and 78% in mean personal PM2.5 exposures over the 2 month intervention period. Median ambient concentrations were 23 μg/m3 [IQR:19–27). Participant feedback was critical in designing clothing and instrument stands that ensured high compliance.An LPG stove and fuel intervention in the candidate HAPIN trial field sites in India was deemed suitable for achieving health-relevant exposure reductions. Ambient concentrations indicated limited contributions from other sources. Study results provide critical inputs for the HAPIN trial site selection in India, while also contributing new information on HAP exposures in relation to LPG interventions and among pregnant women in LMICs.NCT02944682; Prospectively registered on October 17, 2016.ClinicalTrials.Gov. The online version contains supplementary material available at 10.1186/s12889-020-09865-1. Household air pollution (HAP) from the use of solid fuels  for cooking and heating is a leading risk factor for population health . The heaHAP exposure and its potential adverse health effects have been examined in India over the last several decades . Recent Globally, several field-based randomized controlled trials (RCTs) have been conducted to evaluate the effect of clean cooking interventions on multiple health outcomes  Trial has been designed to overcome many of the evidence gaps for health benefits from HAP interventions, while also attempting to address the challenges encountered in previous HAP RCTs. It aims to assess the health benefits  of a LPG cook-stove and fuel intervention prospectively in four Intervention Research Centers (IRCs): Guatemala, India, Peru and Rwanda .in preparation), we undertook an assessment of expected exposure contrasts associated with the planned intervention, potentially avoiding costly investments on sites that could fail either due to lack of intervention feasibility or lack of exposure reduction.One of the key inputs for successful conduct of HAP RCTs is the availability of detailed site-specific information gathered prior to the main trial through formative research. After an initial scoping phase to identify potential sites based on feasibility . Trial-wide exploration of potential exposure contrasts for all sites is reported in Liao and Kirby (under review).Specifics of site selection in India, including feasibility-based assessments for how sites were chosen, are being reported elsewhere . Briefly, these include low rates of LPG penetration, low anticipated uptake of LPG among primary biomass cook-fuel users over the 5 years of the trial, and low anticipated air pollution cross-contamination from neighbouring households and other ambient sources . This exercise identified five blocks (an administrative revenue unit defined for implementation of welfare schemes) in the districts of Villupuram and Nagapattinam in the southern state of Tamil Nadu, India, as potential candidate sites .We designed an exposure monitoring strategy at candidate study blocks in VP and NP districts to establish the potential exposure contrasts that could be expected with an LPG intervention. The protocol in India broadly followed common pilot phase protocols designed for implementation across HAPIN countries with some differences across sites necessitated by local access to air pollution monitors, housing types, economic and cultural conditions  using gravimetric samplers (described below). We also elicited participant feedback to design acceptable clothing for wearing the monitors.The exposure monitoring strategy was two-pronged with (i) cross-sectional measurements on women, under routine conditions of either primary biomass or LPG cook-fuel use and (ii) before-after measurements on pregnant women over a 3 month period: once at baseline while using biomass fuel, and at 1 and 2 months after installing an LPG stove and providing free fuel. We measured 24 or 48 h personal exposures and kitchen, near-household (outdoor), and ambient concentrations of fine particulate matter (PMN = 79) aged (18–64) with a BMI < 40 kg/m2 from a convenience sample of households selected from each of the 5 identified blocks across the two districts, with representation from households that used biomass (N = 48) or LPG(N = 31) as the primary cookfuel. Women were approached directly by study staff to ascertain eligibility and were administered informed consent. For these measurements, we recruited women over two successive time periods. In the first period we recruited women aged 18–35 years, followed by women aged 35–64 years in the second period. The age groups were chosen to mirror eligibility for enrollment as pregnant women or older adult women in the main trial.For cross-sectional measurements, we recruited women (N = 41) aged 18–35 years who used biomass exclusively as a cooking fuel, with a gestational age of 9 to < 20 weeks  by screening those seeking routine antenatal care at local primary health centers in each of the five study blocks. Eligibility was based on: (i) ultrasound assessments at registered ultrasound centers in the study area to determine gestational age; (ii) reported exclusive biomass use and the absence of an LPG connection at baseline, so as to pilot the process of securing an LPG connection as required in the main trial; and (iii) reported availability of the participant over the 3 month study period at the same residence (without an intention to move). Smokers and women with pre-existing health conditions (including previously diagnosed hypertension) were excluded for cross-sectional as well as before-after measurements. Also, since these measurements were performed sequentially, there was no instance of more than one eligible woman in the same household.For before-and-after measurements, we recruited pregnant women (The inclusion criteria across the two arms were designed such that we could: (i) test the feasibility of being able to perform exposure measurements on pregnant and other older women, similar to those expected to be enrolled in the main trial; and (ii) evaluate exposure contrasts under conditions of routine LPG use versus HAPIN efficacy trial conditions (i.e. providing a free LPG stove and fuel).An LPG intervention was provided to pregnant women enrolled for before-and-after measurements. For each household with an eligible pregnant woman, the study team prepared the required documentation for securing a licensed LPG connection (access to LPG in India requires refills to be supplied via authorized distributors of public sector oil marketing companies). Upon receipt of the connection license, which includes an LPG regulator, the study team supplied a two-burner stainless steel LPG stove and a hose connecting the stove to the regulator, which is attached to an LPG cylinder Figure S. InstallAll households received a simple behavioural reinforcement message  developeField staff visited households every week to monitor adherence and confirm exclusive LPG use. This included recording any visible signs of biomass stove use at the time of visit (such as presence of cinder/ash on the stove or a warm stove); asking participants if they had to use the biomass stove for any specific purpose (over the preceding week); eliciting feedback on the ease of routine cooking with LPG, including from available family members; and recording primary and/or secondary biomass stove dismantling/disablement. Queries regarding LPG stove repairs were also addressed during the weekly visits.2.5 concentrations in the kitchen and wherever possible, in the near-home outdoor space, with simultaneous personal exposure measurements on women. Cross-sectional assessments included 24 or 48-h measures and were performed over two separate 8 week periods to include young women (aged 18–35) and older women (aged 35–64) respectively. Before-after measurements were performed on pregnant women (aged 18–35) at baseline, and again at 1 and 2 months after the LPG intervention, and included only 48-h measures.We conducted 24- or 48-h measurements of PM2.5 measurements and filter weighing protocols are furnished in Supplementary Information (n = 41) or ECM samplers (n = 21)). We also changed filters over two consecutive 24-h periods on the SKC/Casella samplers (n = 15) to compare 24 vs. 48 h averages in the same household. The ECM samplers also collected real-time nephelometric data that are not included in analyses presented in this paper. 48-h ambient measurements were performed using the MiniVol™  at the VP and NP sites by placing the sampler (one in each site) on the rooftop of a willing community resident’s home.Details of PMormation . Briefly2.5 mass depositions were blank adjusted. The median value of field blanks was 2 μg for 15 mm filters, 2 μg for 37 mm filters, and 3 μg for 47 mm filters.We collected 21 field blank filters  to account for changes in filter mass due to handling and transportation. Final PMn = 44, 7.7%) by ±20% or (b) all filters with damage, such as holes or tears . Our final dataset included 521 gravimetric PM2.5 samples from the following environments: 216  from the kitchen area, 95 from the near outdoor environment and 210  personal exposures.We excluded a total of 53 measurements with (a) sampling times that deviated from the expected sample duration  to assess cooking practices, identify other potential sources of air pollution, and record self-reported compliance in wearing the monitoring equipment. Customised vests (referred to here as “wearables”) and equipment housings (such as stands) were tested in other pre-existing SRIHER field sites prior to deployment at newly identified HAPIN sites. We also elicited detailed qualitative participant feedback on the acceptability of wearables, devices, and device housings (such as stands) with respect to comfort of fabric choice and vest designs, weight of alternative monitoring devices, and inconvenience caused by equipment housings/stands.2 or history of high blood pressure). In the before-and-after arm, 81 were screened and 41 were enrolled, with 3 women unwilling to receive an ultrasound, 11 unwilling to participate, and 26 found ineligible (LPG usage at baseline (17); gestational age < 9 weeks or > 20 weeks (9)).A total of 120 households were enrolled from 17 villages across the two sites between April 2017 and December 2017. In the cross-sectional arm, 96 women were screened and 79 were enrolled, with 6 unwilling to participate and 11 found ineligible  reporting use of more than one stove. Most women (88–96%) enrolled were also the primary cooks in the family. Table SWe made multiple modifications prior to arriving at wearables and equipment housings that were accepted widely by participants. Additional details of the modifications, samples of vests and area sampling equipment housings, feedback from participants as well as compliance information, are provided in Table SAmong pregnant women who were enrolled for before-and-after measurements, 100% adherence  was achieved throughout the 3 month intervention period. On the average, households received 2 cylinder refills in both the VP site and the NP site over the observation period. No stove or regulator repairs were requested by households. At the NP site, 10 households dismantled/removed their primary biomass stove out of their own volition. Qualitative feedback indicated overwhelming satisfaction with receiving the stove and free LPG refills .r = 0.92 for 24 vs 48 h measures in the same households; r = 0.94 between SKC/Casella vs UPAS co-located measurements and r = 0.87 between SKC/Casella vs ECM co-located measurements). Scatter plots for observed correlations are shown in Supplementary Information  measurements were matched to respective personal sampling durations. We pooled 24 or 48-h averages recorded in the cross-sectional arm and averages measured across all instruments in both cross-sectional and before-after arms based on observed correlations  concentrations were 31 μg/mThe number of measurements performed using a particular instrument and sampling durations varied by site precluding a direct comparison of measurement results stratified by instrument, sampling duration, and site. Additional information regarding this is furnished in supplementary information  Figure b, S3c. DTrials of environmental health interventions are typically complex and are predicated on an expectation that interventions will reduce exposures and subsequently improve health. For studies of HAP, understanding existing sources of exposure and the potential for their reduction via a stove intervention is key to the success of the program. Often, these factors have not been well documented in previous literature. We provide here evidence of the ability of such an intervention to reduce exposures in rural Tamil Nadu, India. The exposure results reported here quantify the reduction in air pollution exposure that may be expected via an LPG stove intervention in Villuparum and Nagapattinam, Tamil Nadu, and strengthen the case for their selection for the HAPIN trial in India. As no prior HAP research has been conducted in these areas, the findings also provide site-specific baseline information relevant to global HAP reduction efforts and provide valuable information on LPG-related exposures in India.2.5 measurements in the districts of Villupuram and Nagapattinam. Further, we performed measurements under alternative scenarios of biomass and LPG use that could be expected in the sites: in our cross-sectional arm, our measurements represent routine fuel use in the study site; in our before-and-after arm, we mimic the HAPIN efficacy trial and performed measurements under ideal anticipated main trial conditions where households were provided with a licensed LPG connection, a high quality stove, free fuel, and behavioural reinforcements.We report results from 521  24/48 h PMIn addition to information on the exposure contrast (described below), this exercise confirmed many other exposure measurement-related feasibilities for the main trial. The selected study sites are located 250–450 km from SRIHER (the parent institution for study investigators in India) located in Chennai, India. We established temporary field offices to house staff and equipment, arranged for transportation logistics (for both staff and samples), coordinated exposure measurements alongside biomarker assessments, enabled maximal participant compliance through customization of clothing which held personal samplers, and augmented available analytical capacities in the SRIHER laboratory for filter weighing. Further, we made elaborate comparisons of device specific parameters related to charging and calibration as well as 24 vs. 48 h sampling durations that can be used alongside information from other HAPIN sites to refine main trial protocols. Finally, the HAPIN central core team built a large workforce of trained personnel at the India IRC for executing the complex monitoring protocols in preparation for the main trial.2.5 when compared to use of biomass among women and pregnant women. Only a handful of studies that include information on HAP exposures among pregnant women [The use of LPG stoves and fuel by households at the proposed study sites in India was associated with significantly lower kitchen concentrations and personal exposures to PMnt women , 39–42 ont women  are curr2.5 exposures for pregnant women over a 2 month follow up period (after an LPG stove/free fuel intervention) with post–intervention values consistently below the World Health Organization annual average Interim-1 PM2.5 Target (WHO-ITG1) of 35 μg/m3.We observed sustained reductions (90 to 93%) in kitchen concentrations and (73 to 78%) in personal PM3 in India [3 in a recent RCT in Nigeria [3 and 13 μg/m3), but also observed low levels amongst biomass users at baseline (46.0 μg/m3 and 27 μg/m3) and attribute this to short durations spent near the cook stove during the dry season .Recent studies involving clean fuel interventions among pregnant women report levels that are well above the WHO-ITG1 levels. Unmeasured traditional stove use and high levels of background ambient concentrations have been cited as possible reasons for post intervention kitchen concentrations around 76 μg/min India  and pers Nigeria . A study Nigeria , observe2.5 have seldom been described as part of pilot studies for HAP RCTs. The feasibility study for the RCT in Malawi was conducted using the “Chitetozo” improved biomass cook-stove. Personal CO was monitored on just 4 participants over two separate 24-h periods with no significant differences between baseline and follow up [2.5 concentrations from 1414 μg/m3 to 130 μg/m3, during the cooking period [Exposure contrasts for PMg period . Perhapsg period , 19, butSeveral recent RCTs have incorporated stove interventions using clean fuels (such as LPG and ethanol), but likewise few have presented exposure results. Among trials recently concluded in Ghana, Malawi, Nepal, Peru and Nigeria , 26–28, The exposure contrast results from the pilot phase of the HAPIN trial in India provide comprehensive and substantive evidence of the suitability of the candidate sites and proof of principle that large sustained reductions in personal exposure are feasible.2.5 exposures and birthweight in a recent cohort study conducted by the same investigators in Tamil Nadu [The exposure reductions achieved with the introduction of LPG among biomass users and the prevalent differences between existing LPG and biomass users suggest that use of LPG is likely to result in health benefits based on known exposure-response relationships for acute lower respiratory infections in infants, birth-weight, and blood pressure in HAP settings . Furthermil Nadu , we estimil Nadu ); as welmil Nadu . The obshttps://mausam.imd.gov.in/).The investigators optimized available resources to generate the most relevant pieces of information for site selection for the HAPIN trial within a short time period of 9 months. However, this posed some limitations. We could not add a control arm for the before-after measurement group. This may have resulted in some residual confounding. Seasonal differences were minimal at the study sites over the period of the intervention, making it unlikely that associated household practices (such as boiling bath water and using the biomass cook-stove for heating) explain observed differences. The seasonal variations at the sites are also otherwise minimal with temperatures remaining in the hot temperate range throughout the year ; and (iv) occasional use of the secondary biomass stove in LPG using households during the cross-sectional phase of measurements (n = 2). While these effects are most likely non-differential between LPG and biomass users  or between baseline and follow-up (before-after arm), recording these observations will allow us to address these more carefully in the analyses for the main trial.The field staff made several observations that may have had a bearing on the observed exposure contrast Table Sc includiWe conducted a limited number of ambient measurements at the sites using real-time instrumentation. The measured concentrations remained low and stable throughout the monitored period. While initial site selection  indicated an absence other major local sources, including brick kilns or hazardous industries, contributions from sources such as garbage or agricultural burning could be important and may not have been fully captured by the sparse density of ambient measurements. Further, contributions from second-hand smoke (from smokers in the household), smoke from neighbours, use of kerosene lamps as a secondary lighting source, and dust could attenuate differences in personal exposures , 49. FulFinally, we could not measure child exposures in the HAPIN pilot phase in India, as has been done at the HAPIN Guatemala site  on accouThe HAPIN RCT is a very complex environmental health trial aimed at demonstrating the health impacts of an LPG stove and fuel intervention among four diverse biomass using communities in the countries of India, Guatemala, Rwanda and Peru. Results from the HAPIN trial can be very valuable for policy makers in the respective countries to draw implications of an LPG intervention for exposure reductions and health benefits for pregnant women, young children and older women among rural populations. Evidence from the study has the potential to considerably augment the global thrust on expanding clean household energy solutions.Given the potential burden to participants, field staff, and others in the community, it was critical to establish feasibility among multiple axes at potential field sites in India and the other HAPIN IRCs. We find it critical that site selection processes be described comprehensively to afford insights into baseline conditions. Further, given the relative paucity of results from pilot studies for HAP RCTs in published literature, the methods and results add important information for the design of future research studies. Finally, this study adds valuable kitchen concentration and exposure data for LPG, which is generally lacking from the wider literature.Recently published study protocols of the HAPIN main trial draw heavily on the results from the pilot phase across all countries –33. The Additional file 1.


The effect of various amounts of carboxylated nitrile butadiene rubber (XNBR) functionalized halloysite nanotubes (XHNTs) on the cure characteristics, mechanical and swelling behavior of XNBR/epoxy compounds was experimentally and theoretically investigated. The morphology of the prepared XNBR/epoxy/XHNTs nanocomposites was imaged using scanning electron microscopy (SEM). The effects of various XNBR-grafted nanotubes on the damping factor of nanocomposites were evaluated by dynamic mechanical thermal analysis (DMTA). The cure behavior characterization indicated a fall in the scorch time, but a rise in the cure rate with higher loading of XHNTs into the XNBR/epoxy nanocomposites. SEM micrographs of tensile fracture surfaces were indicative of a rougher fracture surface with a uniform dispersion state of nanotubes into the polymer matrix in the XNBR/epoxy/XHNTs nanocomposites. The stress–strain behavior studies of XNBR/epoxy/XHNTs nanocomposites showed a higher tensile strength up to 40% with 7 wt % XHNTs loading. The theoretical predictions of uniaxial tensile behavior of nanocomposites using Bergström–Boyce model revealed that some of the material parameters were considerably changed with the XHNTs loading. Furthermore, the used theoretical model precisely predicted the nonlinear large strain hyperelastic behavior of nanocomposites. Rubber composites and nanocomposites have received continued attention in recent years ,2,3. A wg), cure behavior and mechanical properties. Sahoo et al. [The carboxylic functional group creates a potential for XNBR to mix with various fillers and polymers with sufficient interfacial interactions in the mixing and curing processes such as epoxy polymers. Epoxy resins are widely used in various applications such as thermosetting composites , thermalo et al.  studied Halloysite nanotubes (HNTs) are naturally occurs nano cylinders which recently used in various polymer nanocomposites for their high thermal and mechanical properties ,17,18. WOur findings suggested a detailed study of the cure behavior and mechanical characteristics of the XNBR/epoxy nanocomposites containing various concentrations of XNBR grafted halloysite nanotubes (XHNTs). The main objective of this research focused on the theoretical and experimental evaluations of the stiffness and stress–strain behavior of the XNBR/epoxy/XHNTs nanocomposites through using appropriate large strain hyperelastic theoretical models. The predictions on the stiffness analysis and uniaxial stress–strain behavior of nanocomposites were evaluated in comparison with the experimental results of tensile experiments of XNBR/epoxy/XHNTs containing various nanotube loadings.The mechanical behavior of large strain rubber like materials such as XNBR/epoxy/XHNTs nanocomposites could be predicted through using Bergström–Boyce model which was discussed in details in our previous work . The unis in Equation (2) which is a dimensionless material parameter.In contrast, the Cauchy stress tensor for time dependent viscoelastic behavior could be represented as :(2)TB=sμThere are some of the material parameters in the Bergström–Boyce model which could be affected by incorporation of XHNTs into the XNBR/epoxy matrix. These material parameters could be separately calculated for each nanocomposite by matching the experimental data of tensile tests with the theoretical model through using an optimization method.Carboxylated nitrile butadiene rubber (XNBR) was Krynac X160, supplied by Lanxess Elastomers  which contains 32.5% by weight of acrylonitrile and 1% by weight of carboxylic acid group. The diglycidyl ether of bisphenol A (DGEBA) type epoxy resin, KER828, with epoxy group content of 5260–5420 mmol/kg, was obtained from Kumho P&B Chemicals, Seoul, South Korea. XNBR grafted halloysite nanotubes (XHNTs) synthesized in accordance with our previous works through using halloysite nanotubes (HNTs), ultrafine grade, was procured from Imerys Tableware Asia Limited . Other ingredients such as zinc oxide and acetic acid were laboratory reagent grades which prepared from Merck Co.  and used as received.The XNBR/epoxy/XHNTs nanocomposites with various formulations in accordance with The morphology of XNBR/epoxy/XHNTs nanocomposites was determined by Vega II XMU scanning electron microscope (SEM) from Tescan Brno s.r.o., Brno, Czech Republic. The SEM analysis was carried out on the cryogenically fractured surfaces after coating with gold powders by sputtering technique.The cure behavior of prepared samples was studied by using a Monsanto Rheometer R-100 testing instrument operated at 175 °C with 3° arc at a period of 30 min in accordance with ASTM D2084.The uniaxial stress–strain behavior of XNBR/epoxy nanocomposites containing various XHNTs loading was monitored according to ASTM D412 through using Universal tensile testing machine, Instron 6025 model  operated at room temperature at an extension speed of 500 mm/min with an initial gauge length of 25 mm.The phase structure of the prepared nanocomposites was evaluated from dynamic mechanical thermal analysis (DMTA) using Triton Technology Tritec 2000DMA . The storage modulus and damping factor of prepared samples were recognized in tension mode at a constant heating rate of 3 °C/min and a frequency of 1 Hz in a strain of 0.02 mm from −100 °C to 100 °C.3), 3/mol) and Swelling of rubber nanocomposites was extensively studied ,27 Swellequation :(5)υ=wpdequation :(6)χ=0.4Another approach to determine the cross-link density of a cured rubber system is the following equation which uses a hypothesis that the cross-link density has a direct relation with the Young’s modulus of the rubber :(7)υe=E3Furthermore, the cross-link density parameter of a cured rubber could be calculated using the modulus at the rubbery plateau region in the plot of storage modulus versus temperature :(8)υst=EThe cure behavior of the XNBR/epoxy nanocomposites containing various XHNTs loading represented in The SEM photomicrographs of tensile fracture surfaced of XNBR/epoxy and XNBR/epoxy nanocomposite containing various loading of XHNTs are compared in The uniaxial stress-stain behavior of various XNBR/epoxy/XHNTs nanocomposites are depicted in μ, with higher XHNTs concentrations which may be due to the induction of the chain mobility restrictions by nanotubes because of some interactions between the XHNTs and polymer matrix [κ, is high for all samples due to the used incompressibility hypothesis in the modeling of stress–strain behavior [s, decreased with incorporation of nanotubes into the XNBR/epoxy matrix. Another parameter is the strain exponential factor, C, which increases with higher loadings of XHNTs. It should be noticed that when this parameter tends toward −1, the mechanical behavior of material become more hyperelastic [cutτ, indicated the higher values with XHNTs concentrations.The uniaxial large strain behavior of various XNBR/epoxy/XHNTs was evaluated through using Bergström–Boyce model by the Nelder–Mead simplex optimization procedure  in MCalir matrix . As indibehavior . Furtherbehavior . The resrelastic . Consequr chains . TherefoA comparison between the experimental data of uniaxial stress–strain behavior of prepared nanocomposites and predicted results was carried out as shown in The results of DMTA measurements containing storage modulus and damping factor (tanδ) are demonstrated in The results of calculated swelling ratio and cross-link density for various XNBR/epoxy/XHNTs nanocomposites as a function of XHNTs loading were demonstrated in The cross-link density calculated in accordance with the stress–strain curves and storage modulus were investigated in The XNBR/epoxy nanocomposites containing various concentrations of XNBR grafted halloysite nanotubes (XHNTs) were prepared through using a two roll mills. The results of cure rheometer show that the introduction of XHNTs into the XNBR/epoxy matrix cause a rise in the maximum torque while decreases the scorch and optimum cure times. The morphology investigations of nanocomposites revealed a rougher fracture surface of nanocomposites with a unique dispersion of nanotubes into the rubber matrix. Tensile strength and modulus at 300% elongations increased with higher nanotubes loadings while there are some decreases in the values of elongation at break. The theoretical evaluations for uniaxial stress–strain behavior of XNBR/epoxy/XHNTs nanocomposites cleared that some of material parameters of used Bergström–Boyce model significantly varied with the XHNTs concentrations. Nevertheless, the model could precisely predict the large strain hyperelastic behavior of XNBR/epoxy/XHNTs nanocomposites. However, there is some deviations from experimental values of stress–strain data at the regions of ultimate elongations. Furthermore, the results of theoretical investigations show a more deviation from experimental values with higher loadings of XNBR grafted nanotubes. The results of dynamic mechanical analysis demonstrated a higher values of storage modulus with higher loadings of XHNTs, while there is some diminution of damping factor. Swelling studies of nanocomposites suggested that the nanotubes act as obstacle to reduce the diffusion of the used solvent into the bulk of rubber matrix. The results preferred to make a rubber based nanocomposite with higher mechanical behavior which could be prepared through using general mixing equipment.


The present study summarizes the demographics, subspecialty training, and academic productivity of contemporary leaders in Canadian Otolaryngology – Head & Neck Surgery (OHNS) training programs across Canada.h-index and number of publications, obtained from Scopus. Characteristics of CPs and PDs were compared using statistical analysis.Demographic data regarding chairpersons (CPs) and program directors (PDs) were obtained from publicly-available faculty listings and online sources, and included employment institution, residency training, fellowship training status, gender, and years of post-graduate experience. Research productivity was measured using the p = 0.001, Fisher’s exact test; φ = 2.63, p = 0.001). On average, individuals with a graduate (Master’s) degree had a significantly higher H-index  and greater number of publications . Compared to PDs, CPs had a significantly higher average h-index score  and accrued more years of post-graduate experience . There were no differences in the proportions of CPs and PDs with graduate degrees. There appeared to be a decline in research productivity beginning 3 years after academic appointment.Cross-sectional data was obtained from a total of 27 CPs and PDs from 13 accredited OHNS training programs across Canada active on July 1, 2019. All academic leaders completed at least 1 year of fellowship training. Head and neck oncology represented 77% of CPs and 59% of academic leaders overall, while pediatric otolaryngology represented 43% of PDs. Females represented 11% of academic leaders. There was a significant association between location of residency training and employment, with 56% (15/27) of physicians working where they had trained  all leaders completed fellowship training; 2) head and neck surgical oncology was the most common fellowship training subspecialty; 3) leaders were likely to be employed at the institution where they trained; 4) a Master’s degree may be associated with increased research productivity; 5) there is a potential risk of decreased productivity after appointment to a leadership position; and 6) women are underrepresented in academic leadership roles. In 2015, Eloy and colleagues [A similar overview has not been conducted in the Canadian setting. Examination of the characteristics and productivity of Canadian academic leaders will provide an understanding of the pathways for promotion in a Canadian context. It may also reveal possible issues with diverse representation within academic leadership. Therefore, the aim of the present study is to summarize the demographics, subspecialty training, and academic productivity of contemporary leaders in OHNS training programs across Canada. A secondary objective is to evaluate the impact of academic appointment on research productivity.https://www.entcanada.org/cso/canadian-departments-otolaryngology/) and Royal College of Physicians and Surgeons of Canada  websites and validated using departmental websites.Demographic data on departmental CPs and PDs from 13 accredited Canadian OHNS programs were obtained from publicly-available faculty listings after July 1, 2019. Current leaders were identified from the Canadian Society of Otolaryngology – Head & Neck Surgery  with general OHNS subspecialty training reported on faculty listings were categorized according to their focus of clinical practice  after consensus agreement among all authors. Research impact and productivity were measured using the dividual –6. Indivh-index ≥ 1. Data was aggregated using Microsoft Excel 2018 , and statistical analyses were performed using SPSS version 24.0 . A p-value less than 0.05 was considered statistically significant.Statistical analysis was used to explore the strength of correlation between training and employment location using a Cramér’s V. Characteristics were compared between chairs and program directors using a t-test for continuous variables and a Chi-Square or Fisher’s exact test for categorical variables. To achieve the secondary objective of evaluating the impact of academic appointment on research productivity, the average number of publications over a standardized measure of time before and after academic appointment was plotted and compared using a t-test. Inclusion criteria for this analysis included appointment prior to January 2018  and Data was gathered from all 13 accredited OHNS training programs, with information available from 27 academic leaders, summarized in Table All academic leaders were fellowship trained. Among CPs, head and neck surgical oncology (77%), facial plastic surgery 15%), and laryngology (15%) were the most common areas of fellowship training .Females represented 3 of 14 21%) PDs, none of the CPs, and altogether only 11% of the academic leaders overall  and higher mean h-index scores . The remainder of the CPs were associate professors 23%). Overall, there was a significant difference in the composition of academic ranks . Individuals with a graduate degree had a significantly higher h-index  and average number of publications  compared to those without a graduate degree.In total, 9 academic leaders (33%) earned a graduate (Master’s) degree, including 5 CPs (38%) and 4 PDs (29%). There was no difference in the proportion of CPs and PDs with graduate degrees (p = 0.64). However, we noted subjective trends of increasing research productivity leading up to appointment and peaking 3 years after appointment, followed by a notable decline in productivity  academic leaders meeting our inclusion criteria. We found that there were no significant differences before and after academic appointment  has estimated that females represent 23% of practicing OHNS surgeons in Canada . Despite8 has estDespite the underrepresentation of females in otolaryngology and academic leadership, there is evidence that the gender proportions are becoming more balanced. The 23% female proportion of Canadian OHNS surgeons shown in 2018 CMA report represents a significant increase from the previous figure of less than 10% in the early 2000’s . Represeh-index and academic productivity were gathered from Scopus similar to the methods of Eloy et al. [The cross-sectional study design provided a snapshot of the current academic leadership profile and did not examine trends over time or describe the characteristics of other otolaryngologists across Canada. Data on y et al. ; these dy et al. . MoreoveThis study provides a cross-sectional overview of academic leaders in Canadian OHNS programs, and demonstrated the following key findings: 1) fellowship training was universal; 2) head and neck surgical oncology is the most common fellowship training subspecialty; 3) leaders were more likely to be employed at the institution where they trained; 4) a graduate (Master’s) degree may be associated with increased research productivity; 5) there is a potential risk of decreased academic productivity after appointment to a leadership position; and 6) women are currently underrepresented in academic leadership roles.


Background: Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen and have a highly specialised but plastic phenotype that is governed by signals in the local microenvironment. AMs are thought to maintain immunological homeostasis in the steady state, but have also been implicated in the pathogenesis of allergic airway disease (AAD). In this study, we aimed to better understand these potentially contrasting AM functions using transcriptomic analysis.Methods: Bulk RNA sequencing was performed on AMs (CD11c+ Siglec F+ CD64+ CD45+ SSChi) flow cytometry sorted from C57BL/6 mice during experimental AAD driven by repeated house dust mite inhalation (AMsHDM), compared to control AMs from non-allergic mice. Differentially expressed genes were further analysed by hierarchical clustering and biological pathway analysis.Results: AMsHDM showed increased expression of genes associated with antigen presentation, inflammatory cell recruitment and tissue repair, including several chemokine and matrix metalloproteinase genes. This was accompanied by increased expression of mitochondrial electron transport chain subunit genes and the retinoic acid biosynthetic enzyme geneRaldh2. Conversely, AMsHDM displayed decreased expression of a number of cell cycle genes, genes related to cytoskeletal functions and a subset of genes implicated in antimicrobial innate immunity, such asTlr5,Il18 andTnf. Differential gene expression in AMsHDM was consistent with upstream effects of the cytokines IL-4 and IFN-γ, both of which were present at increased concentrations in lung tissue after HDM treatment.Conclusions: These data highlight diverse gene expression changes in the total AM population in a clinically relevant mouse model of AAD, collectively suggestive of contributions to inflammation and tissue repair/remodelling, but with decreases in certain steady state cellular and immunological functions. In addition, deficiencies in immune regulatory mechanisms have been associated with the incidence and severity of asthma and experimental allergic airway disease (AAD) phenotypes2. Characterising the cellular and molecular determinants that control the balance between pro- and anti-inflammatory mechanisms in AAD is therefore central to understanding the immunological basis of allergic asthma.Immunity must be tightly regulated in the respiratory tract, to avoid overexuberant immune responses in delicate lung tissue without compromising immunity to airborne pathogens. Allergic asthma is a disease of chronic pulmonary inflammation and airway remodelling that involves pro-inflammatory functions of diverse leukocytes of the innate and adaptive immune response, typically driven by type 2 immunity3 and are essential for pulmonary surfactant metabolism5. They differentiate from foetal precursors in the first week of life and can self-renew during adulthood independently of circulating monocytes7, although replacement from bone marrow-derived monocytes is observed with advancing age9. AMs have a distinctive transcriptional and cell surface marker profile that is imprinted by the airway microenvironment, including high surface expression of the integrin CD11c and sialic acid-binding lectin (Siglec) F10. AMs are relatively poor activators of adaptive immunity compared to other macrophage subsets11 and their depletionin vivo results in enhanced pulmonary immune responses13, leading to the view that AMs act to maintain pulmonary immune homeostasis in health14. AMs also show a muted response to the type 2 cytokine IL-4 when delivered to naïve mice, compared to interstitial macrophages (IMs) that exist outside of the airspaces of the lung15, consistent with a relative hyporesponsiveness of AMs to a number of activating stimuli in the steady state. However, AMs are also crucial drivers of early inflammatory responses to pathogens, including respiratory syncytial virus17, and are important for innate protection from respiratory bacterial infections18.Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen21 and delays resolution of elevated Th2 cell numbers following cessation of allergen exposure21, consistent with an anti-inflammatory, pro-resolving role of AMs in the context of aeroallergen exposure. Moreover, adoptive transfer of antigen-pulsed AMs to naïve mice limited subsequent allergic sensitisation to the same antigen, concomitant with increased generation of FoxP3+ Tregs22. However, adoptive transfer of AMs lacking the classical macrophage activation transcription factor IRF5 into naïve mice resulted in an increased type 2 immune response upon subsequent allergen inhalation, indicating that AMs can promote inflammation, rather than tolerance, when dysregulated23. Accordingly, AMs expressed high levels of the major Th1 cell attracting chemokines CXCL9 and CXCL10 in a mouse model of Th1hi AAD24. AMs during AAD are therefore likely to differ markedly in phenotype and function from those at steady state, likely reflecting the inherent plasticity of macrophage phenotypes, particularly when faced with a complex and dynamic set of polarising signals such as during inflammatory responses25. Indeed, expression of molecules associated with allergic inflammation and airway remodelling has been observed in AMs during asthma and experimental AAD29.Both pro- and anti-inflammatory functions have also been suggested for AMs during AAD, based in part on mechanistic experiments in animal models. Depletion of AMs by delivery of cytotoxic clodronate-loaded liposome to the airways of mice augments pulmonary inflammatory responses to allergensin vivo roles and the signals controlling these processes. Herein, we describe results of bulk RNA sequencing (RNA-seq) on AMs sorted from mice during a well-established model of AAD driven by inhalation of the clinically relevant aeroallergen house dust mite (HDM), against those from allergen-naive control mice. AMs from HDM-treated mice, as compared to controls, showed decreased expression of genes related to certain steady state behaviours , but marked increases in genes associated with inflammatory cell recruitment, extracellular matrix (ECM) remodelling, oxidative metabolism and generation of the immunoregulatory molecule, retinoic acid. Gene expression signatures of AMs during HDM-driven AAD were suggestive of regulation by multiple upstream cytokine signals, including IFN-γ and IL-4. This work provides insights into potential phenotypic changes to AMs during AAD using an unprecedented RNA-seq approach, which will aid understanding of their protective and pathogenic functionsin vivo. The complex and potentially conflicting pro- and anti-inflammatory functions of AMs during AAD warrant further studies of the molecular features of AMs in the allergic lung, to better understand their likelyAdditional information on key materials is presented inad libitum access to food and water and were maintained under a 12 hour light/dark cycle. Mice were housed under specific pathogen free conditions in cages of 5. Experiments were initiated with mice at 7–9 weeks of age and weighing approximately 20 g. At the time of experiment initiation, mice had not undergone any prior treatments and had been housed for 1 week in the Imperial College London facility to acclimatise. All work with live animals was performed according the Animals (Scientific Procedures) Act 1986 and local guidelines at Imperial College London.A total of 10 mice were used in this study. Female C57BL/6J mice were purchased from Charles River Laboratories and housed at Imperial College London in individually ventilated cages with wood shaving bedding throughout experiments. Mice had32 and our own extensive experience with the HDM mouse model. However, we were unable to obtain sufficient AM RNA from one mouse in the HDM group, resulting in final group sizes of N=5 for PBS treatment and N=4 for HDM treatment. The experimental unit for all analysis was 1 individual mouse. A total of 25 μg of HDM extract (Greer Laboratories) in 25 μl sterile PBS  or 25 μl PBS control, were delivered intranasally to mice under brief isoflurane anaesthesia (3.5% isoflurane with oxygen as the carrier gas) in an inhalation chamber 5 days per week with a 2 day break, for 3 weeks in total, as described previously33. The anaesthetic protocol was selected to be minimally invasive and cause deep nasal breathing to promote allergen inhalation. Allergen challenges were performed during light hours, between 9 am and 12.30 pm. PBS treatments were performed first, immediately followed by HDM treatments. Mice were euthanised and analysed 24 hours after the final intranasal challenge. PBS-treated mice were euthanised and dissected immediately before HDM-treated mice, with all dissections conducted within an approximately 2 hour period.Individual cages of mice were assigned to either HDM or PBS control groups prior to inspection or unique identification by the researcher performing procedures, to minimise the potential for subjective bias. A target group size of 5 was determined based on a previous study in sorted macrophages during type 2 immune responsesg for 5 minutes at 4°C to obtain cell pellets. Total BAL cell counts were determined by staining cells with 0.02 % crystal violet (Sigma-Aldrich) in 700 mM acetic acid in PBS and counting manually using haemocytometer slides.Mice were euthanised by intraperitoneal overdose with 150 μl of 200 mg/ml pentobarbital (approximately 1500 mg/kg), followed by terminal exsanguination via cardiac puncture to avoid damage to the lungs and trachea. Mice were tracheotomised with 19 G cannulas and lungs lavaged a total of 7 times with separate 1 ml volumes of 10 mM EDTA in PBS , leaving the fourth volume of EDTA in the lungs for 5 minutes to promote detachment of cells adherent to the pulmonary epithelium. Lavage volumes were pooled and centrifuged at 300Post-lavage lungs were removed and individual right lung lobes dissected and flash frozen in liquid nitrogen. Right middle lung lobes were homogenised for protein analysis using a FastPrep-24 bead homogeniser and Lysing Matrix D  at a concentration of 50 mg tissue/ml of Hank’s buffered salt saline  without calcium or magnesium, supplemented with 1 cOmplete Protease Inhibitor cocktail tablet (Roche) per 50 ml solution.- CD45+ FSChi SSChi Siglec F+ CD11c+ CD64+ on a BD FACSAria using a 100-μm nozzle and purity sort settings. Purity checks showed 97.0% and 90.4% for PBS and HDM groups, respectively.BAL cell pellets were washed once in flow cytometry grade PBS (Thermo Fisher), before suspending in sort buffer  containing fluorescently-conjugated antibodies to cell surface markers as detailed in- CD45+ Siglec F+ CD64) in BAL were determined by analysing data from 105 events acquired during sorts, analysed using FlowJo software (TreeStar) and multiplied by total crystal violet cell counts.Numbers of AMs and eosinophils  and reactions stopped using an equal volume of 0.18 M sulphuric acid. Results were determined by measuring absorbance at 450 nm using a Tecan Sunrise plate reader and Magellan software and unknown values interpolated into standard curves in Prism v7 using the second order polynomial equation.Cells were pelleted and lysed in RLT buffer supplemented with 1% β-mercaptoethanol within 30 minutes of sorting, passing lysates through QIAShredder columns (QIAGEN) as per manufacturer’s instructions. RNA was extracted from lysates using the RNeasy Micro Plus kit (QIAGEN) according to manufacturer’s instructions. RNA quantity and integrity was determined using the 2200 Tapestation and RNA Screentape reagents (Agilent). All RNA integrity (RIN) values were ≥9.5.A total of 200 ng of total RNA from each sample was converted to mRNA sequencing libraries using the Illumina TruSeq Stranded mRNA HT kit, following the standard HT protocol and using 15 PCR amplification cycles. Library cDNA concentrations were determined using QuantiFluor double-stranded DNA system and GloMax plate reader (Promega) and fragment sizes determined using the Agilent 2100 Bioanalyzer, allowing library concentrations to be normalised to 10 nM. Libraries were pooled and run within single lanes of the Illumina HiSeq 4000, obtaining 75 base pair (bp) paired end reads.StrandNGS software package , using the developer’s recommended settings. Reads required minimum match length of 25 bp, sequence identity of ≥90% and a maximum of 5% gaps in overlap to map to a genomic location and only the best matching genomic location for each read was considered. Reads were filtered if they mapped to five or more different genomic locations, if they aligned to mouse ribosomal RNA or UniVec sequences or if both paired reads did not align to the same genomic location. In total, >92% of all reads in each sample were successfully aligned using these criteria. Alternative open access aligner tools also based on the Burrows-Wheeler transform includeBurrows-Wheeler Aligner andBowtie 2. Open access tools such asclean_reads can be used to filter Univec sequences from FASTQ files prior to alignment and ribosomal RNA contamination can be detected and filtered in post-alignment BAM files usingRSeQC.Demultiplexed sequence files were subjected to FASTQC analysis (Babraham Institute), removing reads with mean phred scores of ≤20. Filtered reads were aligned to the UCSC Mm10 reference genome and transcriptome using the COBWeb aligner (based on the Burrows-Wheeler transform) within the31 within StrandNGS.DESeq is an open access Bioconductor package that can be run outside of StrandNGS. A lower normalised expression limit of 8, required in ≥75% of samples in either experimental group, was imposed to filter low abundance and potential non-AM contaminant transcripts. Principal component analysis (PCA) was performed on all filtered genes usingClustVis34, using single value decomposition to calculate the two major principal components and visualise data after unit variance scaling of normalised expression values. Differential expression analysis was performed on filtered genes using a moderated unpairedt-test within StrandNGS, using the Benjamini-Hochberg method to adjust thep value for the false discovery rate (FDR). Comparable differential expression analysis can be performed on a matrix of genes and normalised expression values using thelimma Bioconductor package. Unless otherwise stated, differentially expressed genes refer to genes with a fold change ≥2 and an FDR-adjustedp-value <0.01 in AMs from HDM-treated mice compared to AMs from PBS controls.Aligned reads were assigned to genes and transcripts according to the Refseq (2013) annotations within StrandNGS, with each read contributing a score of up to 1.0 to an exon, based on the proportion of the read length mapping to the exon. Transcripts were quantified from aligned reads using the DESeq methodClustering of unscaled differentially-expressed genes was performed using Ward’s minimum variance method within StrandNGS, clustering on Euclidean distance between data points. A list of all differentially-expressed genes and their expression clusters is included as extended data for this article. Clustering of smaller selected gene sets was performed using ClustVis after unit variance scaling expression values for each gene, again clustering using Ward’s method and based on Euclidean distance.Ingenuity Pathway Analysis  and core analyses performed, restricting the considered interactions to those observed experimentally inMus musculus. Open access alternative tools for pathway analysis includeReactome andPANTHER. Canonical pathways were determined to be highly enriched within gene lists if they had a positive or neutral/not applicablez-score for activation and a Fisher’s exactp-value of <0.01 and up to 15 of the top enriched pathways reported for each gene set . Representative genes from highly enriched pathways in gene lists were manually selected on the basis of a combination of i) greatest fold difference between experimental groups, ii) involvement in >1 of the highly enriched pathways and iii) a well-established role in the pathway/biological process of interest. Where relevant, individual canonical pathways were visualised using the Path Designer tool and fold change values overlaid as colours.Selected gene lists and their associated normalised expression and fold change values were uploaded toz-score. Genes with differential expression patterns consistent with activation of upstream cytokines of interest were visualised using the Path Designer tool, adding known regulatory interactions at the level of expression or transcription between genes and the upstream cytokine of interest to generate a network and overlaying fold change values as colours.Upstream regulator analysis was performed on total differentially expressed genes and results filtered to limit to cytokines and growth factors with a positive activationin vitro-stimulated macrophages were obtained from the publicly available GEO records associated with published microarray data35 and used to generate .grp gene set files, which were tested simultaneously for enrichment in a .gct file of normalised expression values of all genes filtered on the lower expression threshold using the standard gene set enrichment analysis protocol36. FDR-correctedq-values and normalised enrichment scores are presented. Gene lists used for GSEA are included in the Open Science Framework repository entry supporting this work (seeExtended data).Lists of differentially expressed genes inp-value of <0.05 was considered statistically significant using the notation: *,p < 0.05; **,p < 0.01, ***,p < 0.001 and ns, not significant.Cell counts and lung cytokine concentrations were compared between PBS and HDM groups using two-tailed, unpaired, Student’s t-tests in GraphPad Prism v7. A33. Accordingly, bronchoalveolar lavage (BAL) from HDM-treated mice contained increased numbers of eosinophils compared to PBS-treated non-allergic controls, without a concomitant increase in AM numbers (+ Siglec F+ CD64+ CD45+ SSChi AMs were flow cytometry sorted from BAL (Methods). SeeUnderlying data for flow cytometry output values37.To examine gene expression changes in AMs during AAD driven by a clinically-relevant aeroallergen, mice were repeatedly exposed to inhaled HDM extract for 3 weeks , a modelnumbers . CD11c+rom BAL  of HDM- HDM) and from PBS-treated mice (hereafter ‘control AMs’) to differ substantially in gene expression, with marked separation between the groups in PC1  were observed in AMsHDM compared to control AMs, with roughly equal numbers of up- and down-regulated genes  andItgax (CD11c) among upregulated genes and the apoptotic cell uptake receptorAxl among downregulated genes . Highly enriched pathways were observed in all gene clusters except for C10  and C11 .To better understand the observed changes in gene expression in AMsAtr,Rad51 andRad52, and cell cycle genes, such asPlk1 and genes encoding several cyclins and cyclin-dependent kinases, in AMsHDM was apparent from these clusters  contained genes highly enriched for pathways related to cell cycle progression and DNA replication and repair, including ‘Mitotic Roles of Polo-Like Kinase’ and ‘Role of BRCA1 in DNA Damage Response’ . Specifilusters . The ‘ArHDM  in AMsHDM along with these cell signalling genes resulted in significant over-representation of the ‘fMLP Signaling in Neutrophils’ pathway in C5, suggesting that AM responsiveness to formyl peptide receptor ligands may be reduced during AAD.The remaining downregulated gene clusters, C2 and C5, showed relatively high expression in control AMs that was reduced in AMsHDM , suggestndVasp . C5 was alling’ . DownregCollectively, these data suggested that steady state processes in AMs, such as cell cycle progression, cytoskeletal functions and innate sensing of certain pro-inflammatory stimuli, may be disrupted during allergic airway inflammation.HDM downregulate a subset of genes involved in innate immunityAMsHDM that were not obvious from the cluster-based analysis. To assess this, pathway analysis was repeated on all downregulated genes together. The results of this analysis were highly dominated by the previously noted decreases in cell cycle and DNA repair-related pathways, which topped the list of most enriched pathways  genes, includingH2-Aa,H2-Ab1 andH2-Eb1 , which catalyses the second step of the tricarboxylic acid cycle  genes, such asAMsHDM . Indeed,M , which generates the immunoregulatory molecule retinoic acid, which has been implicated in tolerogenic mechanisms of AMs in the context of allergic inflammation22.Cluster C9 was unique among DEG clusters in being enriched for several pathways of small molecule catabolism, including ‘Histamine Degradation’ and various ethanol degradation pathways . These erol AMs . NotableThus, in the context of substantial allergic inflammation, gene expression of a key immunoregulatory enzyme, RALDH2, is increased in AMs, which could potentially indicate a regulatory role for AM-derived retinoic acid during active allergic inflammation.HDM compared to controls, with little to no detectable signal in control AMs , monocytes  and eosinophils  and tissue inhibitor of metalloproteinase (TIMP) gene expression changes were examined in the whole dataset. Of the chemokine genes detected in AMs, the majority showed increased expression in AMs Ccl24;. Not allontrols . Thus, AMmp8 displayed significantly greater expression in AMsHDM than control AMs  and M(IL-4) signatures  were significantly enriched in AMsHDM compared to control AMs  and alternative (IL-4) activation responses of macrophages. Lists of upregulated genes afterrol AMs , providiHDM show altered regulation of diverse gene sets, suggestive of regulation by multiple cytokine signals in the allergic lung.Thus, AMs23, suggestive of heterogeneity and/or functional plasticity of AMs in the allergic lung. A number of studies have described altered expression of disease-associated genes and proteins in asthmatics and murine AAD models by targeted methods such as flow cytometry, quantitative PCR and immunohistochemistry29. However, no studies to date have examined AM gene expression globally in a clinically relevant mouse model of airway allergen inhalation. Our transcriptomic analysis highlighted several distinct gene expression signatures in the total AM population present following repeated HDM challenge that differed from that in control AMs, overall suggestive of increased inflammatory effector function but decreases in certain steady state AM processes.AMs appear to have a complex role in the immunology of AAD. Both protective and pathogenic roles have been attributed to AMs in the context of AAD1. It is therefore notable that the genes with the most strikingly increased expression in AMsHDM were enriched for chemokines associated with allergic inflammation, MMPs associated with ECM reorganisation47 and other genes involved in tissue repair and remodelling.Chemotaxis of inflammatory cells to the lung and remodelling of the stromal cells and ECM of the airway wall are key pathobiological features of asthma, which involve the coordinated action of several cell populationsHDM, including chemoattractants of both Th2 cells (Ccl1748 andCcl849) and eosinophils (Ccl2450), suggestive of a role for AMs in bringing key effector cells into the lung in response to HDM exposure. AMsHDM also exhibited increased expression of several major agonists of CCR2 and CCR1 51. Since both CCR2 and CCR1 mediate monocyte recruitment into the lung52, these results indicate that the distinct AMs present in established AAD may act to further shape the mononuclear phagocyte compartment of the allergic lung by promoting accumulation of monocyte-derived cells, which are key local drivers of allergic inflammation53. Indeed, AMs have previously been identified as the major producers of CCL6 during IL-13-driven type 2 lung inflammation, production of which was required for maximal airway inflammation and remodelling54. MMP activity has also been proposed to facilitate migration of inflammatory cells into the airspaces55. It can therefore be speculated that AM-derived chemokines and MMPs cooperate to locally promote inflammation during established AAD. We were interested to observe thatMmp8, unlike other MMP genes, was decreased in AMsHDM as compared to control AMs. This suggests that complex changes might occur to the ECM degradation capabilities of AMs during HDM-driven AAD that warrant further study.A diverse array of chemokine genes were upregulated in AMsCollectively, these inflammation and tissue repair/remodelling gene expression signatures strongly suggest an active contribution of AMs to the pathogenesis of AAD following repeated HDM exposure and a shift away from their relatively immunologically quiescent steady state phenotype.HDM displayed increased expression of a large number of genes related to antigen processing and presentation, consistent with the ability of cytokines elevated in the HDM-allergic airway such as IFN-γ and IL-4 to upregulate MHC-II on macrophages56. The likely consequences of increased antigen processing/presentation genes by AMsHDM are not clear, since although AMs readily internalise HDM upon inhalation, they are sessile cells and do not carry antigen to draining lymph nodes57. Increased expression of MHC-II and related genes may instead reflect potential for local interactions between inflammatory AMsHDM and CD4 T cells. Indeed, we have observed co-localisation of CD4 T cells and CD11c+ mononuclear phagocytes, the majority of which are Siglec F+ AMs, during established HDM-driven AAD41.AMsHDM were enriched for genes related to cytoskeletal functions and related signalling pathways. Downregulation of genes related to such cytoskeleton and intracellular signalling pathways have also been observed in AMs following LPS inhalation39, suggesting that these gene expression changes may reflect common features of AM responses to distinct pro-inflammatory stimuli. Since actin polymerisation signalling is important for macrophage phagocytosis58, decreased expression of genes involved in actin cytoskeleton signalling could be indicative of decreased phagocytic capacity in AMsHDM, as has been observed in human asthmatics59. Indeed, AMsHDM also showed a significant decrease in mRNA expression for the apoptotic cell uptake receptor AXL, suggestive of a possible decrease in the capacity for efferocytosis using this receptor. However,Axl expression in these cells remained relatively high so it is unclear whether this decrease would have functional impact. DecreasedAXL mRNA has also been observed in human asthmatic AMs and is associated with impaired efferocytosis of apoptotic cells in these patients38, while AXL knockout mice display impaired resolution of lung inflammation following influenza infection60, consistent with an important function of this receptor in maintaining and restoring immune homeostasis in the airway. The likely functional consequences of a decrease in expression ofAxl and phagocytosis-related cytoskeletal genes, but increased antigen presentation and processing genes in AMsHDM are not clear from these data alone. Functional assays of the capacity of AMsHDM to phagocytose different cell types, such as apoptotic leukocytes and pathogenic bacteria, will shed more light on these results.Despite a clear increase in antigen processing and presentation genes, two downregulated gene clusters in AMsHDM was accompanied by increased expression of genes related to oxidative metabolism, particularly those encoding mitochondrial ETC subunits, suggesting that capacity for ATP generation in AMs by oxidative phosphorylation is increased during HDM-driven AAD. Similar upregulation of electron transport chain genes has previously been reported during the acute response of AMs, but not IMs, to LPSin vivo39, suggestive of a common feature of activated AMs in several inflammatory settings. Moreover, increased electron transport chain gene expression has also been demonstrated in peritoneal macrophages following IL-4Rα-dependent activation during nematode infection32. Notably, IL-4Rα-dependent peritoneal macrophages also displayed increased expression ofCcl8 andCcl2432 during anti-nematode immunity and polarisation of GM-CSF-differentiated human monocyte-derived macrophages with IL-4 increased expression of both oxidative metabolism genes and the chemokineCCL2261. Collectively, these findings suggest that an association exists between oxidative metabolic gene expression and chemokine production by macrophages activated in type 2 immune settings, particularly since increased oxidative phosphorylation is a central feature of macrophages activated alternatively by type 2 cytokines such as IL-462. Such a relationship between macrophage activation and metabolism is unlikely to be limited to oxidative phosphorylation, since inhibition of glycolysis has been shown to reduce the IL-4-induced gene expression in murine AMs that was enhanced uponex vivo culture15. Although gene expression is not a perfect surrogate for cellular metabolic phenotype these data prompt more detailed analysis of AMHDM metabolism to reveal whether these cells indeed break their reported steady state metabolic quiescence15 and whether metabolic reprogramming regulates their production of inflammatory mediators in the allergic airway.Increased pro-inflammatory gene expression in AMsHDM were pro-inflammatory. AMsHDM had reduced expression of the antimicrobial inflammatory cytokine genesTnf andIl18, as well asTlr2, which was previously reported in AMsHDM at the level of cell surface expression and associated with impaired antibacterial immunity40. AMsHDM also showed reduced expression of the positive regulator of TLR signalling,Trem1, as observed previously for neutrophils in a similar HDM model40, along with a substantial decrease inTlr5, which is essential for certain antibacterial responses in AMs63. Notably, TLR5 has also been shown to recognise flagellin present in HDM extracts to promote AAD, suggesting that AMHDM sensing of HDM could be altered in our AAD model, although the specific role of AMs in TLR5-dependent sensing of flagellin in HDM extracts is not known64. Our transcriptional data therefore support the notion that certain antimicrobial functions in AMs are impaired during allergic airway inflammation40.Not all gene expression differences in AMsAldh1a2, encoding the retinoic acid biosynthetic enzyme RALDH2, in AMsHDM. Since AM-derived retinoic acid has been proposed to contribute to their tolerogenic capacity22, increased RALDH2 expression by AMsHDM could present a mechanism of protective immune regulation by these cells even during established allergic inflammation. An active immune regulatory role of AMs during AAD and not just prior to allergic sensitisation would go some way to explain why AM depletion enhances HDM-driven AAD even when cells are depleted when allergic inflammation is already established21. However, it is important to note that retinoic acid function is context-dependent and can be pro-inflammatory in certain settings65.We also observed increased expression ofHDM were consistent with IL-4Rα-dependent, alternatively-activated macrophages, as expected given the type 2 immune environment of the HDM-allergic lung. However, gene expression signatures in AMsHDM were also suggestive of upstream influence from other cytokines, notably the classical macrophage activator IFN-γ. This mixed transcriptional response supports the idea that the classical/alternative macrophage activation paradigm fails to capture the phenotype of tissue macrophagesin vivo, where they are exposed to several cytokine signals simultaneously66, and that a spectrum of activation states exists based on combinatorial effects of different signals67. The increased expression of both IL-4 and IFN-γ-induced genes in AMsHDM is interesting with these ideas in mind, since IFN-γ and IL-4 are considered to be opposing and antagonistic activators of macrophages68. However, a subset of genes induced by these cytokines are relatively refractory to cross-inhibition by the opposing cytokine, indicating that mixed IFN-γ/IL-4 activation states are possible in macrophages68. One interpretation of the mixed gene expression profile observed in bulk AMsHDM is therefore that these cells are a relatively homogeneous population that integrates signals from several cytokines present in the allergic airway, including type 2 cytokines and IFN-γ, to achieve an intermediate activation state. Alternatively, several AM subsets may exist during HDM-driven AAD, some with an IL-4-dominated transcriptional profile and others with greater expression of classical macrophage activation genes, which could conceivably contribute to distinct protective and/or pathogenic AM functions in the allergic lung. Single cell transcriptomic and immunophenotypic analysis of AMsHDM will be valuable in untangling this.Several of the gene expression changes in AMsHDM population may reflect cells of different ontogeny, since AMs derived from circulating inflammatory monocytes have been characterised both after primary influenza infection and bleomycin-induced lung injury in mouse models and shown to be transcriptionally and functionally distinct from prenatally-derived long term-resident AMs70. It is therefore possible that the bulk AMHDM population observed in our model comprises AMs resident throughout the treatment period and one or more additional subpopulations derived from inflammatory monocytes. Decreased expression of a large number of cell cycle genes in AMsHDM relative to control AMs could result from incorporation of monocyte-derived macrophages into the total AM population, since resident AMs undergo steady state proliferation in the absence of inflammation7 and express high levels of cell cycle genes relative to IMs and pulmonary monocytes39, so expression of cell cycle genes could conceivably be diluted within the bulk AMHDM population by monocyte-derived inflammatory AMs that have exited the cell cycle. Alternatively, cell cycle gene expression in resident AMs could be decreased during HDM-driven AAD. A recent study showed cell cycle gene expression to be decreased in murine AMs present after self-limiting pneumococcal infection71, suggesting that this may be a common feature of inflammation-experienced AMs.Heterogeneity within the bulk AMHDM reflects plasticity of resident AMs or local differentiation and instruction of infiltrating monocytes by signals in the allergic airway environment. If multiple AMHDM subpopulations of different ontogeny exist, it will be important to determine the protective and/or pathogenic functions of each subset in AAD, given that monocyte-derived AMs, rather than tissue-resident AMs, have been identified as drivers of bleomycin-induced pulmonary fibrosis69 and mediators of prolonged innate protection againstStreptococcus pneumoniae infection following resolution of influenza infection70. Furthermore, it will be important to determine how long distinct AMHDM transcriptional signatures and phenotypes persist following cessation of allergen exposure, given the importance of AMs in resolution of HDM-driven AAD21.Further studies specifically labelling and characterising monocyte-derived versus resident AMs in the HDM AAD model will be necessary to determine the extent to which gene expression observed in AMs72, several differences exist between human and murine lungs and it will therefore be important to examine these and other molecular signatures in AMs from human asthmatics. Nonetheless, our AMHDM gene expression provide useful insights into potential widespread changes in AM behaviour in the allergic lung, including reductions in steady state functions and increases in those related to AAD pathogenesis, likely governed by a combination of cytokine signals. We believe that these findings will prompt and support further study of AMs in experimental AAD and human asthma. Collectively, our data identify substantial gene expression changes in AMs during experimental AAD compared to those present in naïve mice in the steady state. Although the HDM inhalation mouse model recapitulates many of the hallmark features of allergic asthmaGSE148590;https://identifiers.org/geo:GSE148590.NCBI Gene Expression Omnibus: Transcriptomic analysis of airway macrophages from a murine experimental allergic airway disease model. Accession numberThis accession contains the raw and processed data files from the RNA-seq study. A list of differentially expressed genes and their associated expression clusters is provided as extended data.https://doi.org/10.17605/OSF.IO/8Q3S737.Open Science Framework: Supporting material for 'Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease'.This project contains the following underlying data:ELISA_absorbance_values. .Data are available under the terms of the This paper delivers an original and in-depth profile of gene expression in airway macrophages (AW), using bulk RNA sequencing, in the lungs of mice in the steady state and during an allergic airway disease (AAD) model. The authors are leaders in this arena and the study is underpinned by a robust HDM AAD model and sequencing analysis. The attention to detail in the comprehensive Methods section is notable.Tlr5) and metabolism (Aldh1a2).  The paper is well written and highly informative. It raises new insight on multiple aspect of functions of AM in allergic lung tissue, including cell heterogenicity, innate immunity  include the specific cell surface profile ("CD11c+ Siglec F+ CD64+ CD45+ SSChi") of AM. Fig 1. The addition of BAL IFN-g and IL-4 expressing CD4 cells in BAL would illustrate the Th1 and Th2 cellular dynamics of this specific HDM AAD model. The inclusion is not essential; but would extend discussions on Figure 7, including panel 7D, on AM in the prevailing lung cytokine environs. Figures 2B and 2D, 3B, 4B, 5B, 6B. "Expression of representative genes for clusters and pathway annotations shown in A". These are not clear and would benefit from presentation in a different graphic format. Tlr5 is evidently different between PBS v HDM, but are the others genes? Data are mean (n= 4 or n=5) showing statistical differences or just inferred relative expression? For example, Fig 3B, Minor comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunology. Mouse models of inflammatory diseases, including pulmonary inflammation.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We are grateful for this thoughtful and thorough review of our manuscript. We have revised the manuscript in response to the feedback provided as detailed below.In the abstract (Methods) include the specific cell surface profile ("CD11c+ Siglec F+ CD64+ CD45+ SSChi") of AM. We have now included this in the Methods section of the Abstract to more clearly describe the studyFig 1. The addition of BAL IFN-g and IL-4 expressing CD4 cells in BAL would illustrate the Th1 and Th2 cellular dynamics of this specific HDM AAD model. The inclusion is not essential; but would extend discussions on Figure 7, including panel 7D, on AM in the prevailing lung cytokine environs. Thank you for this suggestion. It is indeed likely that CD4 T cells are providing the cytokines that instruct gene expression in AMs. Unfortunately, we did not acquire these data at the time of analysis of our HDM model. We agree that this is non-essential for the manuscript as it is, but understanding the sources of cytokines that drive different gene expression profiles will be an important component of future studies of AMs in AAD.Tlr5 is evidently different between PBS v HDM, but are the others genes?Figures 2B and 2D, 3B, 4B, 5B, 6B. "Expression of representative genes for clusters and pathway annotations shown in A". These are not clear and would benefit from presentation in a different graphic format. Data are mean (n= 4 or n=5) showing statistical differences or just inferred relative expression? For example, Fig 3B,We agree that the presentation of these data in the original manuscript was misleading, as the log2-transformed normalised expression values were shown, resulting in very small visible distances between points and small to invisible error bars. This was also highlighted as a point for improvement by Reviewer 1. To better reflect the variance within groups and the differences between groups, we have now replaced these dot plots with row-centred heatmaps.We thank the reviewer again for their constructive feedback. et al. performed bulk RNA-seq on sorted AMs from the BAL of HDM or PBS challenged mice. In their study “Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease”, Branchett The data presented by the authors demonstrates a shift in the gene expression signature of AMs upon HDM challenge, with a decrease in steady state associated genes and an increase in genes related to inflammatory cell recruitment and ECM remodeling.  The authors further validated upstream key regulators of AMs gene expression during AAD (IFN-g and IL-4) which opens possibilities of future experiments to functionally characterize AMs in AAD. Altogether, the article is very clear and well written. The conclusions drawn by the authors are adequate and correspond to the data shown. Overall, we think this study is interesting and relevant and deserves to be indexed in Wellcome Open Research. While the study is descriptive in nature, it still provides substantial information and is a good resource for further investigation of the complex role of AMs in allergic asthma.Comments: No major comments.Figure 2B, 2D, 3B, 4B, 5B and 6B: The plots showing the expression of representative genes for the different clusters are unusually depicted. This could be optimized, for example by using a heatmap. Expression values of expression in AMs” could be changed.Figure 3B: wording of figure legend (underlined) “Figure 4C: The pathway diagram could be improved by changing the font size/style to make it clearer and more readable, as the way it is now, a lot of the components of the diagram are overlapping making it very hard to decipher. Figure 7 B and C: the font size could be increased to make the networks clearer. Minor comments:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunology, macrophage biology, lung inflammation.We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We are grateful for this thorough review of our manuscript. We have implemented the suggestions as far as possible and feel that this has improved the presentation of the data.In response to the specific comments:Figure 2B, 2D, 3B, 4B, 5B and 6B: The plots showing the expression of representative genes for the different clusters are unusually depicted. This could be optimized, for example by using a heatmapAll dot plots showing representative gene changes have been replaced with row-centred heat maps to better highlight the magnitude of differential expression between groups.Expression values of expression in AMs” could be changed.Figure 3B: wording of figure legend (underlined) “We apologise for overlooking this small typing error, which has now been corrected.Figure 4C: The pathway diagram could be improved by changing the font size/style to make it clearer and more readable, as the way it is now, a lot of the components of the diagram are overlapping making it very hard to decipher. We are unfortunately unable to make this particular figure element any larger and changes to font sizes would result in further overlap between labels. The figure is provided in high resolution to allow zooming while viewing online. Since the main intention of this panel was to highlight that transcriptional changes were observed across the whole electron transport chain, we hope that it is still informative as it is. The expression values of the individual genes are readily available in the Extended Data table of clustered differentially expressed genes supporting this manuscript.Figure 7 B and C: the font size could be increased to make the networks clearer.We have increased the sizes of the two network panels by 20% to improve readability.We thank the reviewers again for their constructive feedback.