Identication of Persons Who Are Responsive To Wood Smoke Particle-Induced Airway Inammation With Assessment of The Effect of GSTM1, Asthma Status And Sex On This Response.

: 27 Background : We are currently screening human volunteers to determine their sputum 28 polymorphonuclear neutrophil (PMN) response 6 and 24 hours following initiation of exposure to 29 wood smoke particles (WSP). Inflammatory responders (>10% increase in %PMN) are identified 30 for their subsequent participation in mitigation studies against WSP-induced airways 31 inflammation. In this report we compared responder status (N=52) at both 6 and 24hr time 32 points to refine/expand its classification, assessed the impact of the GSTM1 genotype, asthma 33 status and sex on responder status, and explored whether sputum soluble phase markers of 34 inflammation correlate with PMN responsiveness to WSP. 35 Results : In the entire cohort, we found a significant, but very small, decrease in FVC and 36 systolic blood pressure immediately following WSP exposure and sputum %PMNs were 37 significantly increased at 24 hours post exposure, the latter finding was also significantly 38 correlated with sputum IL-1b, IL-6, IL-8, and PMN/mg; a similar response was not found at the 6 39 hour %PMN response. Blood endpoints in the entire cohort showed a significant increase in 40 %PMN and PMN/mg at 6 but not 24 hours. Six-hour responders tended to be 24-hour 41 responders and vice versa, but 24-hour responders also had significantly increased IL-1b, IL-6, 42 IL-8 at 24 hours post WSP exposure. The GSTM1 null genotype significantly (p<0.05) enhanced 43 the %PMN response at 6 hours in the entire cohort, by 24% in the 24-hour responders and not 44 at all in the 6 hours responders. Asthma status enhanced the 24 hour %PMN response in the 45 entire cohort and in the 6- and 24-hour responders. Sex had no effect on %PMN response. Conclusions : The 24 hour time point was more informative than the 6 hour time point in optimally defining airway inflammatory responsiveness to WSP exposure. GSTM1 and asthma 48 status are significant effect modifiers of this response.

inflammation. In this report we compared responder status (N=52) at both 6 and 24hr time 32 points to refine/expand its classification, assessed the impact of the GSTM1 genotype, asthma 33 status and sex on responder status, and explored whether sputum soluble phase markers of 34 inflammation correlate with PMN responsiveness to WSP.

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Results: In the entire cohort, we found a significant, but very small, decrease in FVC and 36 systolic blood pressure immediately following WSP exposure and sputum %PMNs were 37 significantly increased at 24 hours post exposure, the latter finding was also significantly 38 correlated with sputum IL-1b, IL-6, IL-8, and PMN/mg; a similar response was not found at the 6 39 hour %PMN response. Blood endpoints in the entire cohort showed a significant increase in 40 %PMN and PMN/mg at 6 but not 24 hours. Six-hour responders tended to be 24-hour 41 responders and vice versa, but 24-hour responders also had significantly increased IL-1b, IL-6,

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Wood smoke particles (WSP) produced by combustion of biomass contribute to both 61 household and ambient air PM 2.5 air pollution, and are associated with exacerbations of asthma, 62 pneumonia, chronic obstructive pulmonary disease and cardiovascular morbidity 1 . Household 63 WSP reach levels >1000 ug/m 3 . WSP from wildfires accounts for up to 29% of ambient fine size 64 (2.5-micron diameter or less) particulate matter (PM) 2.5 levels in the US airshed and often 65 abruptly produce ambient air PM 2.5 levels >250 ug/m 3 , with firefighters often exposed to levels 66 >750ug/m 3 . 2

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We are currently undertaking proof of concept clinical trials of interventions to mitigate 68 the effect of WSP on airway inflammation using a 2-hour controlled exposure to 500ug/m 3 WSP 69 with exercise sufficient to evoke a minute ventilation of 20 L/min/m 2 body surface area. We are 70 optimizing these studies by only recruiting volunteers with a >10% increase in the 71 polymorphonuclear neutrophil (PMN) content of the sputum differential count following an open-72 label screening challenge to WSP. This procedure eliminates non-informative volunteers and is 73 similar to that described by Holz and colleagues 3 4 who validated an ozone challenge protocol 74 employed to screen proposed anti-inflammatory agents.

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In Holz et al's ozone challenge protocol, only volunteers shown to be responsive to 76 ozone as defined by a >10% increase in PMN content of the sputum differential count following 77 a screening challenge to ozone were recruited into the intervention study. Using this double-78 blinded, placebo-controlled optimized protocol, they found that both inhaled fluticasone and oral 79 prednisone reduced ozone-induced neutrophilic inflammation. Our group led a second study been employed to screen several agents for their action on acute ozone-induced inflammation.

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Additionally, we have used this definition of responsiveness to assess the effect of the GSTM1 83 genotype to low level ozone, 6 and to assess the differences in gene expression between 84 people who are responsive (responders) or non-responsive (non-responders) to ozone 7 .

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This report documents response of 52 volunteers who have undergone our screening 86 WSP protocol. It expands upon our recent preliminary report of 27 volunteers who had 87 completed this screening protocol, where we observed that 67% of healthy volunteers were 88 responders, and that the GSTM1 null genotype appeared to increase inflammatory response to 89 WSP as observed 24 hours after challenge 8 . This report expands on these results with a larger 90 sample size which now includes persons with asthma and defines responder status at 2 post 91 exposure time points, namely at 6 hours and 24 hours post challenge. In this report we compare 92 responders and non-responders at both time points, assess the correlation between the 6 and 93 24 hour neutrophil response and determine the consistency of responsiveness between 94 %PMNs and soluble phase markers of airway inflammation. In addition, we report on the effect 95 of WSP exposure on lung function (forced vital capacity (FVC), forced expiratory volume at 1 96 second (FEV1), respiratory rate) and cardiovascular outcomes (heart rate, systolic and diastolic 97 blood pressure), and evaluated these outcomes in persons defined as PMN responders at both 98 6 and 24 hours post WSP challenge. Finally, we assessed whether the GSTM1 null genotype, 99 asthma status and sex are associated with early and late time point responder status to WSP.

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As the goal of this screening protocol is to identify persons who are responsive to the 118 inflammatory effect of WSP for subsequent entry into an intervention study, we chose to 119 compare post WSP exposure responses to a pre-exposure baseline, rather than undertake an 120 additional clean air control challenge. In support of this approach, we pooled our own historical 121 data from several air control chamber challenges previously undertaken that involved 2-3 hours 122 of exercise like that employed for this WSP challenge. At pre air challenge baseline, the %PMN 123 in sputum was 31.7+3.0 % (mean, SEM, n=66) vs. 29.4+2.9 % (n=68) at 6 hours post air 124 challenge and 37.0+8.0% (n=10) at 24 hours post air challenge (p=0.7). The pre-WSP baseline 125 value was 33.2+3.2 %PMNs (n=50) and was not statistically different (p=0.72) from the 126 baseline, 6h or 24h historic time point values. It has also been reported that FVC and FEV1 127 following an air control session are unchanged or increase slightly. These observations support Endpoints for the Entire Cohort:

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As of April of 2020 (the date at which the protocol was paused due to the COVID-19 pandemic), 135 52 volunteers had completed this WSP screening protocol.

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We also assessed the correlation between the change from baseline of %PMNs at 6 and 151 24 hours with corresponding change from baseline (expressed as %change) of the PMN/mg 152 sputum and sputum levels of IL-1beta, IL-6, IL-8 and TNFalpha using Spearman's Rank

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Correlation as presented in Table 2 below. Only one significant correlation was found at 6 hours 154 (PMN/mg), but 3 cytokine variables and absolute neutrophils were found to be significantly 155 associated at 24 hours post WSP exposure (IL-1beta, IL-6, IL-8, PMN/mg).

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We also measured blood endpoints from volunteers at 6 and 24 hours post WSP 170 exposure (Table 3 above

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We next expanded our regression model to examine if there are any differences in the ratio 216 between the status of GSTM1, Asthmatics, and responder status (with beta1 in Table 4 217 indicates the slope for those variables). We also fit the regression model to allow for other 218 covariates of interest that might influence the response variable, as described fully in Methods.

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Using this technique, we showed the effect of GSTM1, Asthma and Sex on sputum 220 %PMNs as seen in Table 4 below. We found that the GSTM1 null genotype significantly 221 (p<0.05) enhanced only the %PMN variable and none of the other inflammatory variables as 222 follows: in the entire cohort (Table 4a) at 6 hours post WSP exposure; by 24% in the 24-hour 223 responders (Table 4b); and not at all in the 6-hour responders (Table 4c). Likewise, asthma 224 status enhanced just the %PMN variable and did so at 24 hours in each of the cohorts (entire 225 cohort; 24-hour responders; 6-hour responders), with sex having no effect on this measure.

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These factors had no effect on any of the other inflammatory outcomes (data not shown).

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Within the overall cohort, we found that sputum %PMN were significantly elevated at 24 257 hours after WSP exposure. Circulating PMN cell count was also increased at 6 and 24 hours, 258 but no other sputum markers of inflammation, namely soluble phase mediators and PMN/mg,

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were increased after WSP exposure. When we stratified persons on the basis of neutrophilic 260 airway responsiveness to WSP, we found that 64% were responders at 6 hours and 68% were 261 responders at 24 hours. We also found that the FVC and systolic blood pressure were 262 decreased in the overall cohort and in 6 and 24-hour responder cohorts.

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As the goal of the screening protocol is to optimally identify inflammatory responders for 264 intervention studies, we compared results of volunteers defined as responsive using the 6-hour 265 sputum to those defined on the basis of the 24-hour sputum. While the % PMNs were observed 266 to be significantly increased in the 6 hour and 24-hour responder cohorts, we also observed that 267 all of the sputum cytokines except for TNFalpha were increased at 24 hours and not at 6 hours.

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When we examined the correlation between all the sputum inflammatory markers (cytokines 269 and PMN/mg) with change in %PMNs at 6 and 24 hours, we found that while the %PMNs and

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Details of the WSP exposure protocol are described previously. 10 In brief, baseline induced 320 sputum samples were obtained prior to the WSP chamber visit and at 6 and 24 hours following 321 WSP exposure. The WSP chamber used wood smoke generated by heating red oak wood on 322 an electric heating element. Subjects were exposed to 500 μg/m 3 WSP over a 2-hour period

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To assess the effect of GSTM1, Asthma and sex on sputum inflammatory measures, we       Figure 1 Spirometric and Blood Pressure response to WSP in the overall cohort Spirometric and blood pressure endpoints at baseline and immediately after WSP challenge. Fifty subjects provided at least one sputum sample, with 47 (35 healthy volunteers and 12 with asthma) providing matched baseline and 6-hour timepoint sputum samples In ammatory Response to Woodsmoke Particles of the Entire Cohort Sputum % PMN and soluble phase in ammatory mediators 6 and 24 hours post WSP challenge.

Figure 3
Spirometric and blood pressure endpoints at baseline and immediately after WSP challenge of responsive volunteers as de ned by %PMNs at 6 hours. Spirometric and blood pressure endpoints at baseline and immediately after WSP challenge in responders and non-responders In ammatory Response to Woodsmoke Particles of responsive volunteers as de ned by %PMNs at 6 hours. The airway in ammatory response (cells and soluble phase mediators) to woodsmoke particles of 6-hour responders (N=30) and non-responders (N=17) Figure 5 Spirometric and blood pressure endpoints at baseline and immediately after WSP challenge of responsive volunteers as de ned by %PMNs at 24 hours Spirometric and blood pressure endpoints at baseline and immediately after WSP challenge of volunteers Figure 6 In ammatory Response to Woodsmoke Particles of responsive volunteers as de ned by %PMNs at 24 hours The airway in ammatory response (cells and soluble phase mediators) to woodsmoke particles of 24-hour responders (N=28) and non-responders (N=13).