Urban Particles increased Streptococcus pneumonaie colonization to human middle ear epithelia cells

Background: Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism underline is not well understood. In this study, we evaluated the effect of urban particles (UP) on S. pneumoniae in-vitro biofilm formation, colonization on Human middle ear epithelium cells (HMEECs) and in mouse nasal cavity and transition to middle ear and lungs. Methods: S. pneumoniae in vitro biofilms and planktonic growth was evaluated in metal ion free medium in presence of UP, and biofilms were quantified by CV-microplate assay, cfu counts and resazurin staining. Biofilm structures were analyzed using scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs was evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected by EZcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization were evaluated using microarray. In vivo colonization of pneumococci in presence of UP and transition to middle ear and lungs were evaluated using intranasal mice colonization model. Results: UP exposure significantly (*p< 0.05) increased pneumococcal in vitro biofilms and planktonic growth. In presence of UP pneumococci formed organized biofilms with matrix, while in absence of UP bacteria was unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilms formation and quorum sensing were up-regulated in pneumococci biofilms grown in presence of UP. The HMEECs viability was significantly (p<0.05) decreased and bacteria colonization was significantly (p<0.05) elevated in co-treatment (UP+S. pneumoniae) in compare to single treatment. Similarly, increased apoptosis and ROS produce were detected in HMEECs treated with UP+ pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, immune response were up-regulated in co-treatment, those genes were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. In vivo study showed increased pneumococcal colonization to nasal cavity in presence of UP and higher transition of bacteria to middle ear and lungs in presence of UP. The HMEECs were maintain and routinely culture in airway epithelial cell growth medium (PromoCell GmbH, Sickingenstr Heidelberg Germany) supplemented with bovine pituitary extract (0.004 mL/mL), epidermal growth factor (10 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/mL), epinephrine (0.5 µg/mL), triiodo-L-thyronine (6.7 ng/mL), transferrin (10 µg/mL), retinoic Acid (0.1 ng/mL) and 1% fetal bovine serum and 5% CO2. 1× 104 HMEECs were seeded in 96-well plate and incubated at 37°C with 5% CO2 for 24 h. Following incubation cells were washed with PBS and medium was replaced with UP (200 µg/mL) containing medium (antibiotic free medium) and further incubated for 8 h. The HMEECs pre-exposed to UP were infected with S. pneumoniae (MOI 10) for 15 h. To educate the molecular mechanism underline for the elevated toxicity of UP + S. pneumoniae, gene expressions analysis was performed using microarray. Global gene expression of HMEECs treated with UP (200 µg/ml) or S. pneumoniae (MOI 10) or co-treatment (UP + S. pneumoniae) were analyzed using Affymetrix microarray chip. Total RNA of HMEECs was extracted using Trizol reagent (Invitrogen, USA), and RNA quality was assessed by Agilent 2100 bioanalyser (Agilent Technologies, USA), and quantity was determined by ND-1000 spectrophotometer (NanoDrop Technologies, USA). The gene expressions of HMEECs were detected by Affymetrix GeneChip® Human Gene 2.0 ST oligonucleotide arrays by manufacturer’s procedure (http://www.affymetrix.com). The change of gene expressions by 2-fold in treated samples with respect to control sample (untreated) was considered significant. The gene ontology (GO) and KEEG pathway analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/), string and Medline databases (http://www.ncbi.nlm.nih.gov/).