Airway Microbiome and Th17-mediated Inflammation in COPD Among HIV-infected Individuals in a Rural Ugandan Cohort

Rationale: COPD is increasing in prevalence among people living with HIV/AIDS (PLWHA) as widespread use of ART has increased longevity in this population. In rural Ugandan ART clinics, we report COPD prevalence of 6.22%. Currently, it's not fully known what drives chronic lung inflammation in PLWHA population despite being virologically suppressed on ART. There is need to explore factors driving chronic airway inflammation among PLWHA. Airway microbiome has been implicated in the pathogenesis of COPD. Preliminary analysis from our study revealed that, specific microbes were significantly enriched in PLWHA with COPD with more lung bacteria impacted by HIV than COPD. These findings suggest that HIV-associated changes in unique airway microbial genera may be driving COPD among PLWHA in our cohort. Currently, we don't know how such genera drive chronic airway inflammation.

Study objectives: In this study, we will: (1) establish a relationship between airway microbiome and Th17/Treg cellular phenotypes among HIV-infected individuals with COPD; (2) investigate bacterial-mediated Th17 upregulation of pro-inflammatory and pro-fibrotic genes among HIV individuals with COPD and (3) explore the role of bacterial outer membrane vesicles (OMVs) in mediating microbiome driven Th17 immune responses among HIV individuals.

Methods: We will conduct a 2-year case-controlled study, leveraging on the established lung microbiome cohort in rural Nakaseke district of Uganda. We will recruit 80 HIV-infected individuals ≥35 years attending the ART clinic at Nakaseke General Hospital screened for COPD as well as 80 HIV-negative controls ≥35 years attending the pulmonary clinic at Nakaseke General Hospital screened for COPD. In both cases and controls, we will consider 40 stable COPD participants and 40 participants with no COPD. Recruited participants will undergo sputum induction protocol at our newly established negative pressure sputum induction facility at Nakaseke General Hospital following established standard operating procedures. Using induced sputum samples, we will (i) perform 16S sequencing and metagenomics analysis to determine airway bacterial communities, (ii) RNA sequencing and analysis to determine gene expression profiles, mass flow cytometry and analysis to profile immune cells in induced sputum of study participants as well as (iv) ELISA tests to compare OMV levels between participants.

Introduction The improvement in access to antiretroviral therapy (ART) among people living with HIV/AIDs (PLWHA) has resulted in a decrease in HIV-associated morbidity and mortality\[1\]. This is particularly true in low- and middle-income countries (LMICs), which bear the largest burden of HIV\[2\]. The reduction in mortality has substantially increased life expectancy, with estimates among PLWHA now approaching that of the general population\[3\]. Consequently, there has been increased attention among survivors to the emerging burden of non-communicable diseases (NCD), such as chronic obstructive pulmonary disease (COPD)\[4\]. Sub-Saharan Africa, which has the highest density of PLWHA, has experienced dramatic increases in COPD related-morbidity and mortality especially in individuals with history of pulmonary tuberculosis (PTB)\[5\]. In rural Ugandan ART clinics, we reported COPD prevalence of 6.22% with history of PTB being the strongest predictor of COPD risk and reduced lung function\[5\]. Currently, it's not fully known what drives chronic lung inflammation in PLWHA population well-controlled on ART.

In search for answers to the above question, we have been exploring the role of altered airway microbiome in orchestrating chronic airway inflammation in PLWHA. Our research question has been supported by a growing body of knowledge from human and animal studies that alterations in the bacterial communities inhabiting the airway trigger immune responses and chronic airway inflammation\[6-9\]. In a recent study involving 50 HIV+COPD+, 50 HIV+COPD-, 50 HIV-COPD- and 50 HIV-COPD- participants in rural Uganda, we investigated the association between altered airway microbiome and HIV-associated COPD (NCT040702). Preliminary analysis from 16S sequencing on induced sputum samples revealed that, in spite of minor differences in bacterial diversity among PLHWA with COPD compared to controls, commensal microbes belonging to the genera Atopobium, Megasphaera, Actinomyces, Selenomonas, Stomatobaculum and Oribacterium were significantly enriched in PLWHA with COPD while microbes belonging to the genera Prevotella, Porphyromonas, Pseudopropionibacterium, Odoribacter and Filifactor were depleted. Our results clearly show that there are more lung bacteria impacted by HIV than COPD, suggesting that HIV-associated changes in unique airway microbial genera may be driving COPD among PLWHA in our cohort. Currently, what remains unknown is how such genera drive chronic airway inflammation.

Studies from mouse models report Prevotella, Bacteroides and Veillonella to play a significant role in orchestrating lung inflammation through Toll like receptor (TLR) 2/4 signaling on alveolar macrophages, epithelial cells and dendritic cells which prime cells of the adaptive immunity\[8\]. The reported outcome has been aberrant Th17 and reduced Treg immune responses using germ-free and specific pathogen-free mice models \[8\]. Similarly, recent evidence in human studies show that Prevotella and Veillonella genera are associated with a distinct metabolic profile, enhanced pro-inflammatory cytokine expression characterized by elevated Th17 cellular response\[9\]. Microbiome-host cell communication has been reported to be mediated by secreted bacterial outer membrane vesicles (OMVs), which correlate with bacterial biomass (figure 2) \[8\]. We believe that identifying airway bacterial genera which elicit aberrant Th17 response among PLWHA with COPD could be a stepping stone towards developing strategies aimed at restoring microbial balance either through direct elimination of pathobionts by antibiotics or restoration of commensals through probiotics to promote colonization resistance at mucosal surfaces.

Based on this background, we aim to investigate if airway microbiome fuels Th17 mediated airway inflammation in COPD among HIV-infected individuals. We hypothesize that: (1) microbiome induces airway Th17 and suppresses Treg immune responses among HIV-infected individuals with COPD; (2) bacterial-driven Th17 response promotes expression of pro-inflammatory genes among individuals with COPD and (3)airway microbiome mediates its effect on Th17 immune response through secretion of bacterial outer membrane vesicles (OMVs).

In this study, we will: (1) establish a relationship between airway microbiome and Th17/Treg cellular phenotypes among HIV-infected individuals with COPD; (2) investigate bacterial-mediated Th17 upregulation of pro-inflammatory and pro-fibrotic genes among HIV individuals with COPD and (3) explore the role of bacterial outer membrane vesicles (OMVs) in mediating microbiome driven Th17 immune responses among HIV individuals.

Study aims and objectives

Study aim:

To investigate airway microbiome-driven Th17 mediated inflammation in COPD among HIV-infected individuals

Specific objectives

1. To establish a relationship between airway microbiome and Th17/Treg cellular phenotypes among HIV-infected individuals with COPD.
2. To investigate bacterial mediated Th17 upregulation of pro-inflammatory genes among HIV-infected individuals with COPD
3. To investigate the role of bacterial outer membrane vesicles (OMVs) in mediating dysbiosis driven Th17 immune responses among HIV individuals.

Problem statement Chronic obstructive pulmonary disease is increasing in prevalence among people living with HIV/AIDS (PLWHA) as widespread use of Antiretroviral Therapy (ART) has increased longevity in this population\[5\]. It is still unknown what drives chronic airway inflammation in PLWHA well controlled on ART. In spite of a well-established association between COPD and pulmonary tuberculosis, it does not fully account for the reported cases of COPD among PLWHA\[5\]. In our study population, neither smoking nor biomass exposure have been reported to be associated with COPD. There is need to explore factors driving chronic airway inflammation among PLWHA.

Justification Specific microbial communities capable of eliciting intense immune responses could determine the outcome of host-microbiome interaction, being either immune tolerance or inflammation. Whereas in-depth characterization of airway microbiome has been performed in COPD human studies and animal models\[5-8\], only associations have been reported and a few specific signatures have been implicated as predictors of COPD mortality\[9\]. Few animal-based, microbiome models have been translated into testable human airway or lung-based model to elaborate on the mechanistic role of microbiome in COPD. Deciphering which bacteria induce a strong immune response amidst a multitude of microbial communities remains a significant area of interest to sub phenotype and identify treatable traits. We believe that pursuit of this research question will provide a basis for immunophenotyping airway microbial communities as tolerogenic (Treg-inducing) or inflammatory (Th17-inducing), a potentially seminal scientific discovery in respiratory immunobiology.

Literature review Globally more than 80% of NCD-related deaths occur in Low-and-Middle-Income Countries (LMIC)\[10\]. Among NCDs, chronic lung disease is the fourth leading cause of death worldwide and is predicted to be the third-largest health problem in Sub-Saharan Africa by 2030\[11\]. COPD is a progressive life-threatening airflow obstruction that causes breathlessness initially with exertion and predisposes to exacerbations and serious illness\[12\]. In the general population, COPD had claimed a total of three million lives globally by 2015\[13\]. As a result of the global scale-up of antiretroviral therapy (ART) and cotrimoxazole prophylaxis, the incidence of pulmonary infections has declined dramatically but COPD is an increasingly recognized but poorly understood complication\[14\]. A meta-analysis on the epidemiology of COPD in the global HIV-infected population to date demonstrates COPD prevalence between 3 and 23%\[15\].

Until 2015, the prevalence and risk factors for COPD in Uganda had been largely uncharacterized. The prevalence of COPD was first estimated in a rural population in Uganda at 16% prevalence\[11\]. In this study, they did not compare prevalence by HIV status. The reported prevalence was higher compared to other studies in the region and may be explained by other factors. Siddharthan and colleagues reported a 6.1% prevalence of COPD in rural Ugandan communities of Nakaseke district compared to a 1.5% prevalence in urban communities of Kampala\[16\]. In this study, it was highlighted that the most important factor associated with COPD was rural residence, and HIV did not appear to be an important risk factor for COPD\[5\]. Nevertheless, this population-based study did not explore the effects of HIV viral load and CD4 count on COPD prevalence and lung function outcomes. Also, HIV status was self-reported at 8.1%. Similarly, in a recent study in Southwestern Uganda, North and colleagues did not report any significant association between HIV and respiratory symptoms nor lung function. However, the authors did not explore the effects of HIV-specific characteristics on COPD\[17\].

In a rural setting of ART clinics in Nakaseke district of Uganda, with participants having well-controlled HIV, we found a COPD prevalence of 6.22%\[5\]. History of pulmonary tuberculosis was strongly associated with COPD and reduced lung function. As expected, increasing age was associated with reduced lung function. However, HIV specific characteristics including use and duration of ART, current and baseline CD4+T cell counts and viral load were not associated with COPD or reduced lung function both in an adjusted and adjusted analysis\[5\]. Among those diagnosed, COPD was associated with reduced respiratory health status and severe respiratory symptoms. Currently, it's not fully known what drives chronic lung inflammation in our PLWHA population with over 90% virologically suppressed, cluster of differentiation 4 (CD4) count \>500, 80% non-smoker, with history of cure from pulmonary tuberculosis and no significant association between COPD and biomass exposure\[5\]. Evidence from recent studies suggests that bacterial communities inhabiting mucosal surfaces of airways drive chronic inflammation\[8\].

Under homeostatic conditions, airway mucosal surface is inhabited by commensal microbial communities. These organisms induce colonization resistance against pathobionts through several molecular mechanisms\[18\]. The most studied mechanisms involve microbe-microbe and microbe-host interactions. Commensals replicate very rapidly and subsist in densely occupied biological niches, limiting nutrient availability within these micro-habitats. This competition for resources such as amino acids, sugars, zinc, iron and oxygen limits colonization by pathobionts\[18\]. Thus, a higher microbial diversity implies more microbes utilizing a more versatile pool of metabolites, posing a challenge for any pathobionts to thrive. Therefore, any changes resulting in loss of microbial biomass or diversity destabilizes the microbial ecosystem, creating an opportunity for strains with increased fitness to proliferate.

Some commensals directly inhibit growth of pathobionts through secretion of antimicrobial peptides (AMPs). Such factors restrain the growth of adjacent bacteria by a plethora of mechanisms such as inhibition of cell wall synthesis, formation of pores, and nuclease activity. Other non-peptide molecules that inhibit proliferation of neighboring bacteria include short-chain fatty acids (SCFAs), hydrogen peroxide (H2O2), and secondary bile acids. Finally, bacterial secretion systems (especially Type VI secretion system) have been widely used by commensals to transport toxic substances into their environment or even nearby eukaryotic or prokaryotic cells\[18\].

Eligibility Criteria:
Inclusion Criteria:

* We will consider samples from Male and female HIV-seropositive and negative individuals ≥35 years who have been screened for COPD as per standard guidelines (European Respiratory Society, ERS, and American Thoracic Society, ATS) using the modified Burden of lung diseases (BOLD) questionnaire and spirometry.

Exclusion Criteria:

* We will exclude samples from subjects with asthma, significant chronic respiratory disease other than COPD or unable to give informed consent.