Janice Leung

Background: Chronic Obstructive Pulmonary Disease (COPD) is among the leading causes of death worldwide and people living with human immunodeficiency virus (HIV, PLWH) are at a higher risk of developing COPD. However, methods to predict adverse events in COPD are limited and lack precision, especially for PLWH. Previously, patterns of COPD microbial dysbiosis and methylation disruptions have been observed in PLWH with COPD. In this study, we hypothesize that increased risk of mortality is associated with microbiome dysbiosis and differential methylation patterns in the airway epithelium of PLWH with COPD.Methods: Airway epithelial samples from PLWH with (n=32) and without COPD (n=23) and non-HIV infected individuals with (n=35) and without COPD (n=57) were profiled using 16S rRNA sequencing and the Illumina Infinium MethylationEPIC BeadChip. Vital status was captured in the three-year period following bronchoscopy. Microbiome composition and diversity, CpG sites, and methylation age were compared between samples from deceased and survived individuals to identify mortality predictors. Using elastic net regression models, classification models for all-cause mortality were generated using detected features and optimized using the area under the receiver operating characteristics curve (AUC-ROC).Results: We observed a significant increase in alpha diversity in deceased COPD individuals compared to survived participants (p=0.0228), while no difference was observed in PLWH deceased and survived groups (p=0.302). Several pathogens were found to be associated with mortality, including Streptococcus and Veillonella. Top differentially methylated CpG sites associated with death corresponded to cancer-regulating genes, cancer-related pathways and longevity-related pathways. The genus-level microbiome biomarker panel showed promising performance in predicting mortality, especially in PLWH (AUC = 0.875), while the methylation biomarker panel had a weaker AUC performance score of 0.705.Conclusion: Microbial and methylation disturbances in the airway epithelium are associated with mortality in COPD and HIV, with microbiome features demonstrating better predictive performance compared to methylation.

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RATIONALE: People living with HIV (PLWH) appear to have increased proneness to chronic obstructive pulmonary disease (COPD) independent of their cigarette smoke exposure. Previous studies have shown that HIV infection is associated with changes in the airway microbiome and host response, however, the exact mechanism of disease progression is still unknown. We hypothesize that airway epithelial dysbiosis in PLWH increases the susceptibility to COPD in this group.METHODS: Airway epithelial cell brushings were obtained from 18 COPD+HIV+,16 COPD-HIV+, 22 COPD+HIV- and 20 COPD-HIV- subjects. Microbiome, methylation, and transcriptome profiles were measured using 16s amplicon sequencing (Illumina Miseq®), Illumina Infinium Methylation EPIC chip®, and RNA sequencing (NovaSeq6000®), respectively. Microbiome analysis was performed using QIIME 2™, and transcriptome and methylation analyses were performed using R language. The three datasets were integrated using Data Integration Analysis for Biomarker discovery using Latent cOmponents (DIABLO) implemented in the mixOmics R package. Fifty repeats of 10-fold cross-validations and a correlation threshold of 0.7 were set to determine key interactions between bacterial ASVs, CpG methylation sites, and gene transcripts amongst the subjects based on their COPD, HIV, and combined COPD and HIV statuses. RESULTS: The microbiome analysis identified that the groups most associated with disease (COPD+, HIV+, and COPD+HIV+ groups) had reduced alpha diversity (Shannon Diversity Index p=0.0013, p=0023, and p=0.0002, respectively), and significantly disrupted microbial communities (Bray Curtis PERMANOVA p=0.001, p=0.007 and p=0.001, respectively) compared to their relatively ”healthy” counterparts. This was accompanied by changes in the host transcriptome and epigenome, our analysis of which identified top genes and CpG sites that were differentially regulated in patients with COPD and/or HIV. Integration of the three -omes identified features that were correlated with one another at a threshold>0.70. On combining the COPD and HIV statuses of subjects, the multiomic integration identified correlations between the bacterial ASV Bacteroidetes Prevotella and transcriptomic features FUZ, FASTKD3, and ACVR1B, and epigenetic features CpG-FUZ and CpG-PHLDB3. It may be that these features together influence host pathways regulating mucociliary clearance, respiration and energy, cell cycle, and immunity.

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RATIONALE: Despite the reduction in HIV-related mortality since the introduction of antiretroviral therapy, people living with HIV (PLWH) face a higher risk of developing age-associated comorbidities such as chronic obstructive pulmonary disease (COPD). Studies have found that HIV and COPD are independently associated with increased susceptibility and worse manifestations from respiratory viral infections. We hypothesize that COPD in PLWH is marked by increased inflammation in the airway epithelium which may potentiate injury during respiratory viral infections. METHODS: Primary human bronchial epithelial cells obtained from control, COPD, HIV, and HIV/COPD donors were cultured as monolayers and air-liquid interfaces (ALIs). RNA and protein lysates from these cultures were used to quantify HIV receptor mRNA and protein expression via qRT-PCR and western blot, respectively. ALI cultures were infected with respiratory syncytial virus (RSV) for 90 minutes and transepithelial electrical resistance (TEER) values were taken at time points: 0h, 24h, 48h and 72h. ALI cultures underwent immunohistochemistry staining for E-Cadherin quantification and mucus production quantification through Periodic Acid Schiff and MUC5AC staining. RESULTS: Western blot analysis of monolayer lysates demonstrated increased expression of CXCR4 in PLWH with and without COPD and increased expression of CCR5 in PLWH with COPD compared to control individuals. There was a trend towards higher CD4 expression in PLWH with and without COPD compared to control individuals. However, these differences were not found in either protein or mRNA HIV receptor expression in ALIs. TEER data demonstrated a decrease in epithelial resistance in RSV-infected ALIs derived from COPD and PLWH with COPD compared to PLWH without COPD and control individuals. There were no significant differences seen in E-cadherin expression and mucus production in RSV-infected ALIs. CONCLUSION: With an increased expression of HIV canonical receptors in the basal epithelial layer, particularly CCR5 and CXCR4, in PLWH with COPD, a greater interaction between virus and the airway epithelium may be possible, potentially causing damage to the airway epithelium. Furthermore, RSV infection induces greater epithelial permeability in patients with COPD regardless of HIV status. This could indicate that post-viral small airway injury may be driven more by COPD status rather than HIV infection alone.

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