HIV Infects Bronchial Epithelium and Suppresses Components of the Mucociliary Clearance Apparatus

Recurrent lung infections and pneumonia are emerging as significant comorbidities in the HIV-infected population in the era of combination antiretroviral therapy (cART). HIV infection has been reported to suppress nasal mucociliary clearance (MCC). Since the primary components driving nasal MCC and bronchial MCC are identical, it is possible that bronchial MCC is affected as well. Effective MCC requires optimal ciliary beating which depends on the maintenance of the airway surface liquid (ASL), a function of cystic fibrosis transmembrane conductance regulator (CFTR) activity and the integrity of the signaling mechanism that regulates ciliary beating and fluid secretion. Impairment of either component of the MCC apparatus can compromise its efficacy and promote microbial colonization. We demonstrate that primary bronchial epithelium expresses HIV receptor CD4 and co-receptors CCR5 and CXCR4 and can be infected by both R5 and X4 tropic strains of HIV. We show that HIV Tat suppresses CFTR biogenesis and function in primary bronchial epithelial cells by a pathway involving TGF-? signaling. HIV infection also interferes with bronchial epithelial cell differentiation and suppresses ciliogenesis. These findings suggest that HIV infection suppresses tracheobronchial mucociliary clearance and this may predispose HIV-infected patients to recurrent lung infections, pneumonia and chronic bronchitis

TGF-β1 increases viral burden and promotes HIV-1 latency in primary differentiated human bronchial epithelial cells

Combination antiretroviral therapy (cART) has increased the life expectancy of HIV patients. However, the incidence of non-AIDS associated lung comorbidities, such as COPD and asthma, and that of opportunistic lung infections have become more common among this population. HIV proteins secreted by the anatomical HIV reservoirs can have both autocrine and paracrine effects contributing to the HIV-associated comorbidities. HIV has been recovered from cell-free bronchoalveolar lavage fluid, alveolar macrophages, and intrapulmonary lymphocytes. We have recently shown that ex-vivo cultured primary bronchial epithelial cells and the bronchial brushings from human subjects express canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. Together these studies suggest that the lung tissue can serve as an important reservoir for HIV. In this report, we show that TGF-β1 promotes HIV latency by upregulating a transcriptional repressor BLIMP-1. Furthermore, we identify miR-9-5p as an important intermediate in TGF-β-mediated BLIMP-1 upregulation and consequent HIV latency. The transcriptionally suppressed HIV can be reactivated by common latency reactivating agents. Together our data suggest that in patients with chronic airway diseases, TGF-β can elevate the HIV viral reservoir load that could further exacerbate the HIV associated lung comorbidities

NHBE redifferentiated at the ALI express HIV receptors and co-receptors and can support both R5 and X4-tropic infection.

<p><b>Panel A</b>: We analyzed expression of CD4, CCR5 and CXCR4 in NHBE by qRT-PCR. Total RNA was isolated from NHBE ALI cultures and expression of CD4 (Applied Biosystems # HS01058407-m1), CXCR4 (Applied Biosystems # HS00607978-s1) and CCR5 (Applied Biosystems # HS99999149-s1) was determined by qRT-PCR using Taqman probes. CFTR (Applied Biosystems # HS00357011-m1) expression was determined for comparison. NHBE ALI cultures express all canonical HIV receptors. <b>Panel B</b>: Western blot analysis from three different lungs demonstrates that NHBE ALI cultures show comparable expression of all HIV receptors. While RNA levels were significantly lower for CCR5 than CD4 and CXCR4, protein levels were comparable with CD4 and CXCR4. This could be due to inherent CCR5 mRNA instability due to a pseudoknot structure in the mRNA that promotes non-sense mediated decay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169161#pone.0169161.ref035" target="_blank">35</a>]. <b>Panel C</b>: To determine if bronchial epithelial cells express HIV receptors and co-receptors in vivo, Bronchial brushings were analyzed for expression of CD4, CCR5 and CXCR4 by qRT-PCR. Expression patterns of the three receptors were similar to that observed in our ex vivo model with maximal expression of CD4 followed by CXCR4 and significantly lower CCR5 expression validating the physiological relevance of our ex vivo NHBE ALI culture model. <b>Panel D and E</b>: NHBE ALI cultures can be infected with both R5 and X4-tropic strains of HIV. NHBE cultures redifferentiated at the Air-Liquid Interface were infected apically and basolaterally with either HIV IIIB (X4-tropic) or HIV BaL (R5-tropic) strains. After 16 hours cells were washed apically and basolaterally with PBS four times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as Day 0 to confirm that all input virus had been removed. No p24 was detected in the 4^th wash (Day 0) confirming that all input virus had been removed. We observe an initial spike in p24 output on Day 3 for both HIV IIIB and HIV BaL infections that declines gradually till Day 9 (panel D). Experiments were terminated and total RNA was isolated and cell associated HIV RNA was quantitated by qRT-PCR using Taqman probe (Applied Biosystems # PA03453409-s1). NHBE cells demonstrate cell associated viral RNA for both R5 and X4-tropic infections (panel E). <b>Panel F</b>: Another set of NHBE ALI cultures similarly infected in presence of with maraviroc (For HIV BaL infection) or AMD3100 (for HIV IIIB infection) which was retained for the remainder of the experiment. Maravoiroc was able to block infection of NHBE ALI cultures by the R5-tropic strain HIV BaL. Likewise, AMD3100 was able to block infection by the X4-tropic strain HIV IIIB. n = NHBE ALI cultures from 3 different lungs * = significant (p < 0.05).</p

HIV infects undifferentiated NHBE cells and inhibits ciliogenesis.

<p><b>Panel A</b>: NHBE ALI cultures were infected as described before on the day they were placed under Air-liquid culture conditions. Uninfected lung matched cultures were similarly differentiated as controls. At designated time points, culture supernatants were collected and analyzed for p24. Undifferentiated NHBE ALI cultures can support HIV infection. <b>Panel B</b>: On day 18 when ciliogenesis was observed in Lung-matched uninfected controls, experiments were terminated and total RNA was analyzed by qRT-PCR for presence of HIV RNA. HIV RNA was detected in both HIV IIIB and HIV BaL infected NHBE cells. n = NHBE ALI cultures from 3 different lungs. * = significant (p < 0.05). <b>Panel C</b>: Another set of cells were infected similarly was On Day 18 post-infection when ciliogenesis was observed in lung matched uninfected controls, Experiments were terminated, cells were fixed and stained for cilia as described by us earlier [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169161#pone.0169161.ref050" target="_blank">50</a>] and visualized using a Zeiss fluorescence microscope with high resolution Axiocam 506 mono microscope camera (Zeiss, Germany). HIV infection of NHBE ALI cultures impairs epithelial layer formation and interferes with ciliogenesis. Primary bronchial epithelium is pseudostratified. In a healthy well-differentiated epithelium, all ciliated cells will not be in the same plane. Hence some cilia are sharply focused in a particular plane while others, out of focus appear blurred. In infected cells, we do not observe the formation of the pseudostratified epithelium and the epithelial integrity is also impaired hence ciliated cells appear in a single plane.</p

HIV infects NHBE cells and undergoes reverse transcription to generate proviral DNA.

<p>HIV infects NHBE cells and undergoes reverse transcription to generate proviral DNA.</p

Long-term HIV infection in NHBE ALI cultures.

<p><b>Panel A and B</b>: NHBE cultures redifferentiated at the Air-Liquid Interface from three different lungs were infected apically and basolaterally with either HIV IIIB (X4-tropic) or HIV BaL (R5-tropic) strains. After 16 hours cells were washed apically and basolaterally with PBS four times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as Day 0 to confirm that all input virus had been removed. At designated time points, culture supernatants were collected and measured for p24 using the p24 ELISA kit. No p24 was detected in the 4^th wash (Day 0) confirming that all input virus had been removed. Maximal p24 is detected on Day 2 and then decreases gradually. Detectable p24 is observed up to Day 50 post-infection. Experiments were terminated on Day 50 due to excessive cell death. <b>Panel C</b>: qRT-PCR analysis of cell associated viral RNA from three independent lungs that were infected with HIV IIIB and HIV BaL. HIV RNA is detected in all cultures infected with HIV BaL and two of three cultures infected with HIV IIIB. n = NHBE ALI cultures from 3 different lungs * = significant (p < 0.05). ND = Not Done (due to cell death).</p

HIV Tat suppresses CFTR biogenesis and function.

<p><b>Panel A</b>: HIV Tat induces expression of TGF-β1 mRNA. NHBE redifferentiated at the ALI were treated with recombinant Tat (10nM) or heat inactivated Tat (as control) apically and basolaterally. After 24 hours, Medium was changed and replaced with fresh medium containing Tat. Forty-eight hours post- Tat treatment, total RNA was isolated and TGF-β1 and CFTR mRNA levels were quantitated by qRT-PCR. HIV Tat significantly induces TGF-β1 mRNA expression with a concomitant suppression of CFTR mRNA. <b>Panel B</b>: HIV Tat suppresses CFTR function via TGF-β signaling. NHBE ALI cultures grown on snapwells were treated to HIV Tat as mentioned. Separately, cells were treated with anti-TGFBR2 antibody (added 3 hours before treatment apically and basolaterally; 25 μg/ml) and retained for the remainder of the experiment. Cells were mounted in Ussing chambers and Cl^- efflux in response to albuterol addition was determined in the presence of amiloride. To ensure that change in short circuit current (ΔI_SC) is CFTR specific, Ussing chamber experiments were terminated by addition of CFTRinh172 (20 μM). HIV Tat decreases CFTR function. Anti-TGFBR2 neutralizing antibody caused a complete restoration of CFTR function in Tat treated cells. n = NHBE ALI cultures from 3 different lungs * = significant (p < 0.05).</p

A fluorescent HIV reporter demonstrates HIV undergoes reverse transcription and expresses viral genes in NHBE ALI cultures.

<p>A fluorescent HIV vector (RGH-WT) reported by Dahabieh et. al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169161#pone.0169161.ref043" target="_blank">43</a>] was packaged in HEK 293T cells using either R5-tropic env (pBal.01) or X4 tropic envelope (pHXB2 env). 10ngs p24 equivalent of both viruses was used to infect NHBE ALI cultures. On Day 6 and 12 cells were visualized using a Zeiss fluorescence microscope with high resolution Axiocam 506 mono microscope camera (Zeiss, Germany). GFP expression is observed in NHBE ALI cultures infected with RGH-WT virus enveloped with R5 envelope and X4 envelope. Expression of GFP was observed as distinct foci in the epithelium. Phase contrast microscope images demonstrate some cell death (yellow arrows) by Day 6 and significant cell death and disruption of epithelial barrier on Day 12 post-infection. Since this virus is env^-, cytotoxicity and disruption of epithelial layer could only be due to death of infected NHBE cells or a bystander effect of viral proteins (other than env) secreted by infected cells.</p