key: cord-0911763-ianzik7v
authors: Roy, Ritwika; Jan, Rohi; Joshi, Uttara; Bhor, Renuka; Pai, Kalpana; Satsangi, P. Gursumeeran
title: Characterization, pro-inflammatory response and cytotoxic profile of bioaerosols from urban and rural residential settings in Pune, India
date: 2020-04-29
journal: Environ Pollut
DOI: 10.1016/j.envpol.2020.114698
sha: 5f96de4f9e7998091df8ab3bbf58fa2e170d35da
doc_id: 911763
cord_uid: ianzik7v

Abstract Microbiota associated with airborne particulate matter (PM) is an important indicator of indoor pollution as they can be pathogenic and cause serious health threats to the exposed occupants. Present study aimed to investigate the level of culturable microbes associated with PM and their toxicological characterization in urban and rural houses of Pune city. Highest concentration of bacterial aerosols observed to be associated with PM10 size fraction in urban site (2136 ± 285 CFU/m3) whereas maximum fungal concentration has been measured in rural houses (1521 ± 302 CFU/m3). Predominantly found bacterial species were Bacillus sp., S. aureus, and Pseudomonas aeruginosa and fungal species were Aspergillus sp., Cladosporium sp., and Penicillium sp. in both urban and rural residential premises. Concentration of endotoxin measured using the kinetic Limulus Amebocyte Lysate assay exhibited that the level of endotoxin in both urban and rural sites are associated with household characteristics and the activities performed in indoor as well as outdoor. Cell free DTT assay confirmed the ability of these airborne microbes to induce the production of reactive oxygen species (ROS) varying along with the types of microorganisms. On exposure of A549 cells to airborne microbes, a significant decrease in cell viability was observed in terms of both necrosis and apoptosis pathway. Elevated production of nitric oxide (NO) and proinflammatory cytokines in epithelial cells and macrophages clearly suggest the inflammatory nature of these airborne microbes. Results derived from the present study demonstrated that the indoor air of urban and rural houses of Pune is contaminated in terms of microbial load. Therefore, attention should be paid to control the factors favoring the microbial growth in order to safeguard the health of exposed inhabitants.

Endotoxin, a cell wall component of gram negative bacteria is a well know pyrogen causing symptomatic 132 effects in the exposed inhabitants. In present study, quantification of endotoxin was assessed as per 133 manufactures instruction in terms of end point chromogenic Limulus Amebocyte Lysate (LAL) assay 134 using endotoxin assay kit (Pyrochrome: Cape Cod Inc.) Briefly, purified extract of Escherichia coli 135 (0113:H10) was used as a control standard endotoxin. PM (100 µl) extract was mixed with LAL reagent 136 (100 µl) followed by incubation at 37ºC for 60 minutes. Then substrate solution was added to the 137 incubated fraction and vortexed for 10 minutes to ensure complete mixing. Presence of endotoxins was 138 confirmed by developing yellow colour and mixture was measured spectrophotometrically (PerkinElmer 139

EnSpire 2300) at 405-410 nm. Concentration of endotoxin was calculated from the standard curve 140 Oxidative potential (OP) of PM in cell free/abiotic system has been considered as one of the most 144 promising screening method of their biological reactivity (Orevik, 2019) . OP describes the inherent 145 ability of particles and their components to induce ROS generation which is an important underlying 146 mechanism for particle toxicity. Therefore, in present study, oxidative potential (OP) of PM 10 and PM 2.5 147 samples along with predominantly found microbial species in urban and rural indoor environments were 148 measured by cell free assay i.e., DTT assay and detail methodology for this assay is provided in our 149 earlier publication (Roy et al., 2019) . 150

Evaluation of cytotoxic potential of PM and its associated micro-biota play a vital role in screening of 151 toxicological properties due to their association with genomic instability leading to the cell death. 152 Therefore, in order to detect the cytotoxic effect, cell viability of indoor PM samples and abundantly 153 identified bacterial and fungal species were studied by using MTT assay. About 80% of total collected 154 samples from both urban (three houses; U1, U2 and U3) and rural sites (two houses; R1 and R2) were 155 used for cell culture exposure and data reported for cytotoxic effect is an average values of total samples 156 analyzed at each site. Preparation of A549 cells (lung epithelial cell line) were detailed out in 157 supplementary file. A549 cells were exposed to collected PM samples and a constant dose (10^6 158 microbes/ml) of microorganisms for 24 hr and assayed in three technical and biological replicates. Cells 159 with media acted as a positive control. Briefly, cells were incubated with PM samples and microbial 160 species for 24 hr and after that 20 µl of MTT solution was added and further incubated the mixture for 4 161 hr followed by centrifugation. Media was removed after 24 hr of exposure and subsequently 100 µl of 162 DMSO was added and finally absorbance was measured at 570 nm by spectrophotometer well plate 163 reader (PerkinElmer EnSpire 2300). Pathways involved in the cell death (in tems of appoptosis and 164 necrosis) on exposure to adequate microbial pathogens can be monitored by widely used 165 phosphatidylserine staining detection method i.e., Annexin PI. Appoptosis is a carefully regulated process 166 of programmed cell death whereas necrosis occurs through differences in plasma membrane integrity and 167 permeability. In present study, cell death on expossure to airborne microbiota was employed by using 168 the Alexa Fluor 488 annexin 5/Dead Cell apoptosis kit with Alexa Fluor 488 annexin v and PI following 169 manufacturer instruction (briefly outlined in supplementary file). The data generated by flow cytometry 170 (Attune NxT Flow cytometer, Thermo Fisher Scientific) are plotted in two-dimensional dot plots in which 171 PI is represented versus Annexin V. These plots can be divided in four regions corresponding to live cells 172 (i.e. annexin V-/PI-), early apoptotic (i.e. annexin V+/PI-), late apoptotic (i.e. annexin V+/PI+) and 173 necrotic (i.e. annexin V-/PI+.) 174

Apart from oxidative potential and cytotoxic effect, one of the prime toxicity governning factor of 175 airborne microbes is inflammatory potential. Pulmonary cells, such as alveolar epithelial cells and 176 macrophages are first to respond to inhaled airborne microbes, therefore, in present study in order to asses 177 the imflammatory activity, both epithelial cells and macrophages (Mouse RAW264.7 macrophage ) were 178 exposed to these airborne microbial speccies. Inflammatory potential of prevalent gram positive, gram 179 negative bacterial and fungal species associated with PM was measured in terms of analysis of immune 180 response of different cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, TNF-α) in lung epithelial cells 181 by the use of the MACSPTEV Cytokine 12 kit according to the manufacturer instruction. In addition, the 182 expression of cytokines such as IL-6 and TNF-α were also measured on expoure to these microbes to 183 macrophages using cytometric bead array (CBA assay, BD Biosciences) as per the manufacturer's 184 protocol. Apart from these cytokines, production of highly reactive signalling molecule i.e., nitric oxide 185 (NO) is also considered as an important marker of immunological activation of the cells. Hence, 186 concentration of stable NO 2 was estimated by Griess reaction detailed out in supplementary file. 187

In order to minimise the risk of error, a number of qulity control parameters were practiced during the 189 study period from collection of samples, microbial analysis to cell culture conditions and toxicological 190 assays. Flow rate calculations were checked every sampling day to assure that the fluctuations in flow rate 191 of the sampler are within the range. Post sampling, collected filter samples were removed from the 192 samplers immediately and transferred to the sample cassettes cleaned with 70% isopropyl alcohol. All the 193 glassware, reagents and plastic wears used for the microbial and toxicity assays were sterilized and these 194 assays were carried out in laminar hood with aseptic conditions. Field and filter blanks were analyzed 195 once per month for each sampling site; in total, 24 blanks were analyzed. Both field and filter blanks were 196 subjected to similar handling, extraction and treatment (culturing, endotoxin measurement, DTT and 197 MTT assay) like sample filters in order to check for contamination. Mass concentration of field and filter 198 blanks were found to be negligible and no contamination was observed. Proper growth of A549 Cells was The variation in microbial load in urban and rural households of Pune might be due to the different 244 household and personal daily activities and as well as meteorological factors such as temperature and 245 relative humidity (RH). Indoor temperature of all rural and urban housing types was reasonably uniform 246

with an average value of 24.8˚C and 26.7 ˚C respectively. However, mean value of RH in rural houses 247 (57.7%) was found to be higher than the urban (53.5%) households. In order to check the influence of 248 temperature and relative humidity (RH) on the survival of airborne microbes, correlation between CFU 249 count of bacterial and fungal aerosols with temperature and RH at both sites were calculated in terms of 250

Spearman's coefficient (Table S6) . Statistically significant (p<0.05) positive correlation has been found 251 between the concentration of microbes in both sites and temperature and RH. Relative humidity is 252 favorable for microbial growth as microbes can absorb this water from their living substrates for their 253 metabolism; therefore increased air humidity helps in growth and release of airborne microbes 254 addition, bacterial load in present study was found to be higher than Chennai, Vishakhapatnam, Delhi, 268

Ethiopia and Iran, and lower than Nigeria, whereas fungal concentration exhibited higher values than 269

Chennai, Vishakhapatnam, Ethiopia, Nigeria and Iran and lower than the values reported in Delhi and 270

Egypt. Comparative study clearly confirmed the high degree of microbial pollution in urban and rural 271 houses of Pune. 272

Significant seasonal variation (p < 0.05) in concentration of PM bound bacteria and fungi at urban and 274 rural sites was observed and tabulated in Table S3b . In present study seasons are classified as winter 275 

Morphology of bacterial colonies found at the urban and rural sampling sites depicted different shapes 289 viz., like round (cocci) and rod (bacillus) and fungal fractions exhibited the presence of spores and 290 hypehae structure (Fig. S5 a and S5 b) . Further, confirmation of these isolates were done in terms of gene 291 sequencing leading to the identification of 5 bacterial and 7 fungal species predominantly found in indoor 292 air. During the study period, common identified bacterias in both rural and urban sites were S. aureus, 293

Bacillus sp., Micrococcus sp., Pseudomonas aeruginosa and E.coli. Among them, two species belong to 294 gram positive Cocci (S. aureus, Micrococcus sp.), Bacillus sp. is spore forming gram positive rod and 295 remaining two Pseudomonas aeruginosa and E.coli. belong to gram negative bacilli. The relative 296 percentage abundance of these microbes associated with PM 10 and PM 2.5 at urban and rural site is depicted 297

in Fig. 1 . At urban site, Pseudomonas aeruginosa was found to be pre-dominant with 59.2% and 51.3% 298 in PM 10 and PM 2.5 samples respectively. Whereas, at rural site S. aureus and Bacillus sp. were dominant 299 in PM 10 and PM 2.5 with percentage abundance of 62.5% and 70% respectively. Profusion of Pseudomonas 300 aeruginosa in urban environment can be a major concern because of its association with respiratory tract 301 infections whereas similarly the dominance of S. aureus in rural houses also may pose health threat to the 302 exposed occupants due to their capability of causing disease through invasion and toxin production such 

Endotoxin, an important biomarker can be linked to various respiratory outcomes and considered as a 320 potent modifier of acute and chronic toxicity. Mean bacterial endotoxin concentration associated with 321 PM 10 and PM 2.5 measured at urban site was 0.98±0.34 EU/m 3 and 0.77±0.22 EU/m 3 respectively while at 322 rural site, the level was recorded as 0.90±0.24 EU/m 3 for PM 10 samples and 0.69±0.24 EU/m 3 for PM 2.5 323 (Fig. 2a) . Overall endotoxin level was little higher in urban houses as compared to the rural one. 324

Endotoxin originates primarily from gram -ve bacteria and in present study concentration of gram -ve 325 bacteria has been found to be maximum in urban houses which supports the comparatively higher level of 326 measured endotoxin at urban site. In present study, urban houses were comprised of pets which have been 327 identified as potential sources of airborne endotoxin (Salonen et al., 2016) . In addition to this, carpet 328 flooring in urban houses and habits of the occupants like tobacco smoking (Fig. S1 ) are also considered as maximum oxidative potential has been exhibited by gram negative bacteria i.e., Pseudomonas aeruginosa 371 which is probably attributed to the oxidative stress induced by Pyocyanin, the most virulence factor 372 produced by this species (Hall et al., 2016) . Oxidative potential of these airborne microbes present in 373 urban and rural households reveals the involvement of these microbial cells in induction of ROS 374 generation and their imperative association with serious inflammation related health risk. 375

Indoor PM samples and prevalently found microbes associated with PM exhibited considerable cytotoxic 377 response towards A549 cells in both urban and rural households. In both size fraction of PM (75µg/ml), 378 statistically significant decrease (p<0.01) in cell viability was observed with maximum cell death in PM 10 379 samples collected from rural houses. Cell viability was decreased to 47.8% and 51.6% for PM 10 and PM 2.5 380 samples respectively in urban site whereas in rural site cell viability decreased to 42.4% and 47.1% for 381 PM 10 and PM 2.5 respectively (Fig. 3a) . The viability from the blank samples from each lot was also 382 calculated and no difference was found as compared to control. Cytotoxic response of PM samples can be 383 characterized by the prominent morphological changes as compared to the untreated cell (control). 384

Microscopic images of A549 cell death on exposure to both PM 10 and PM 2.5 samples from urban and rural 385 site is depicted in Fig. 3a Therefore, in order to address the precise role of microbes associated with PM and bacterial endotoxin for 391 eliciting the cellular toxicity, Pearson's correlation was calculated in present study (Table S6 ). Significant 392 negative correlation has been found between A549 cell viability and microbial (r= -0.74 for urban 393 bacteria, r = -0.69 for rural bacteria; r = -0.77 for urban fungi, r = -0.84 for rural fungi) and endotoxin 394 concentration (r = -0.73 for urban, r = -0.69 for rural) clearly demonstrating the influence of these 395 airborne microbiata and their components in causing the cell death. Further, interaction between 396 individual microbial species abundantly found in indoor air and A549 cells were also evaluated. On 397 exposure of A549 to predominant airborne microbes, significant decrease in cell viability with respect to 398 control was observed. Fungal species induced higher cytotoxic response as compared to the bacterial cells 399 (Fig. 3b) and treated cells appeared to be non-viable with considerable detachment and other 400 morphological changes (Fig. 3b) . All together, maximum cell death was observed (65%) by Aspergillus 401 sp. probably due to their ability to produce secondary metabolites which can be responsible for causing 402 cytotoxic effects (Aiko and Mehta, 2015) and Mucor was found to be least cytotoxic in nature. Gram 

On exposure to microbial pathogens, the immune system responds to irritations through an innate cascade, 428 known as inflammation which is initiated by the release of extracellular mediators. Production of nitric oxide 429 (NO) and proinflammatory cytokines including tumor necrosis factor alpha (TNFα) and various interleukins 430 (IL-6, IL-4, IL-2) are considered as the most potent biomarkers of immunological activation of the cells in the 431 respiratory tract (Huttunen et al., 2012) . In present study, mostly all identified microbes in indoor air trigger 432 the production of NO with respect to control suggesting their ability to cause the adverse respiratory health 433 effects (Fig. 5) . Aspergillus sp. elicited maximum NO production in A549 cells after 24 h exposure with an 

• Total culturable microbes associated with PM were detected in indoor environments.

• Predominantly found microbial isolates are S. aureus, P. aeruginosa and Aspergillus sp.

• LAL assay exhibited eminent level of endotoxin in urban and rural houses.

• Annexin study confirmed cell death pathway induced by prevalent airborne microbes.

• Dominantly found microbes exhibited elevated production of proinflammatory mediators.

IL-17A, IL-22, IL-6, and IL-21 Serum Levels in Plaque-Type Psoriasis in Brazilian Patients

Oxidative Potential Versus Biological Effects: A Review on the Relevance of Cell-606

Assays as Predictors of Toxicity from Airborne Particulate Matter

Predictors and 608 respiratory depositions of airborne endotoxin in homes using biomass fuels and LPG gas for cooking

Fast monitoring of indoor bioaerosol con-centrations with 611 ATP bioluminescence assay using an electrostatic rod-type sampler

Longitudinal study of 613 dust and airborne endotoxin in the home

A Role for Environmental Engineering and Science 615 in Preventing Bioaerosol-Related Disease

Assessment of PM and 617 bioaerosols at diverse indoor environments in a southern tropical Indian region

Terrestrial 620

Macrofungal Diversity from the Tropical Dry Evergreen Biome of Southern India and Its Potential Role 621 in Aerobiology

Sources of airborne microorganisms in the built environment

Microbiome 3,78

Changes in pro-inflammatory cytokines in association with exposure to moisture-damaged building 626 microbes

Review of Quantitative Standards and Guidelines for Fungi 628 in Indoor Air

Assessment of endotoxin levels in the home and current asthma and wheeze in school-age children

Bioaerosol exposure from composting facilities 633 and health outcomes in workers and in the community: A systematic review update

Spatial and temporal 638 variation in endotoxin and PM 10 concentrations in ambient air in a livestock dense area

Association between 641 endotoxin levels in dust from indoor swine housing environments and the immune responses of pigs

Aromatic Hydrocarbons: Toxicity and Health Risk Assessment of Exposed Inhabitants

Exposure to Traffic-647 related Particles and Endotoxin during Infancy Is Associated with Wheezing at Age 3 Years

Endotoxin 650 levels and contribution factors of endotoxins in resident, school, and office environments: A review

The unexpected role of 653 bioaerosols in the Oxidative Potential of

PrtT-regulated 656 proteins secreted by Aspergillus fumigatus activate MAPK signaling in exposed A549 lung cells leading 657 to necrotic cell death

Panton-Valentine leukocidin osteomyelitis in 659 children: a growing threat

Characteristics of biological 661 particulate matters at urban and rural sites in the North China Plain

Aerosol health 663 effects from molecular to global scales

Seasonal Variation of Airborne Viable Bacterial Pollution in 665

Positive and Gram-Negative Bacteria Induce Different Patterns of Cytokine Production in Human 668

Mononuclear Cells Irrespective of Taxonomic Relatedness

Dust, endotoxin, fungi, 670 and bacteria exposure as determined by work task, season, and type of plant in a flower greenhouse

Size-fractionated PM 10 monitoring in relation to the 676 contribution of endotoxins in different polluted areas

A toxicology suite 678 adapted for comparing parallel toxicity responses of model human lung cells to diesel exhaust particles 679 and their extracts

Cytokines and chemokines: At the crossroads 681 of cell signalling and inflammatory disease

Gla-rich 683 protein function as an anti-inflammatory agent in monocytes/macrophages: Implications for calcification-684 related chronic inflammatory diseases

Subcellular and cellular locations of nitric oxide synthase isoforms as 686 determinants of health and disease

Urban PM2.5 oxidative potential: 688 Importance of chemical species and comparison of two spectrophotometric cell-free assays

Assessment of airborne bacteria and fungi in an indoor and outdoor 691 environment

Jumping on the bed and 693 associated increases of PM 10

Characteristics of ambient bioaerosols 696 during haze episodes in China: A review

Airborne endotoxin concentrations in indoor and outdoor 698 particulate matter and their predictors in urban city

aureus. In addition to NO, cytokines are considered as the key modulators participating in acute and chronic 438 inflammation (Turner et al., 2014) . TNF-α, and IL-6 are believed to be the main mediators of host response 439 towards infectious organisms and higher level of these modulators are reported in response to viral infections, 440 and allergic rhinitis (Skovbjerg et al., 2010; Purokivi et al., 2002) . In present study, predominantly found gram 441 positive (S. aureus), gram negative bacterial (Pseudomonas aeruginosa) and fungal (Aspergillus sp.) species 442 associated with PM were subjected to the production of inflammatory cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, 443 IL-10, IL-12, TNF-α) in alveolar epithelial cells and the results revealed the significant production of all these 444 cytokines as compared to the corresponding unexposed controls (Fig.3.5 ). Considerable production of 445 cytokines by these airborne microbes probably is associated with their higher oxidative potential (Viegas et al., 446 2017) . Pseudomonas aeruginosa induced maximum production of IL-5, IL-6 and IL-10 and maximum 447 production of other cytokines (TNF-α, IL-12, IL-2, IL-9) has been triggered by Aspergillus sp. In addition, 448 prominent production of IL-6 and TNF-α as compared to control has been also observed on interaction of 449 chosen microbial with lung macrophages (Fig. 6 ). Significant increase in the expression of representative 450 inflammatory cytokines (IL-6 and TNF-α) clearly indicates acute and short inflammatory response of these 451 airborne microbes of PM. The levels of TNF-α, secreted into the culture medium were highest in GPB treated 452 macrophages whereas, expression of IL-6 was found to be maximum for GNB and Aspergillus sp. treated 453 macrophages. Similar pattern of elevated cytokine (IL-6 and TNF-α) production was also previously reported 454 

In summary, results derived from the present study provide an insight into the toxicological characterization of 462 PM and its microbial components collected from urban and rural households of Pune. Results showed that the 463 microbial load in both sampling sites were highly dependent on household characteristics, activities performed 464 by the occupants and as well as the outdoor influences. Mostly identified microbes in indoor air are pathogenic 465 in nature and their elevated levels in urban and rural houses are a real concern for the respiratory health of 466 exposed residents. Significant oxidative potential, inflammatory activity and cytotoxic behavior of prevalently