Gene-specific MicroRNA antagonism protects against HIV Tat and TGF-ß-mediated suppression of CFTR mRNA and function.

BACKGROUND: MicroRNAs play an important role in health and disease. TGF-ß signaling, upregulated by HIV Tat, and in chronic airway diseases and smokers upregulates miR-145-5p to suppress cystic fibrosis transmembrane conductance regulator (CFTR). CFTR suppression in chronic airway diseases like Cystic Fibrosis, COPD and smokers has been associated with suppressed MCC and recurrent lung infections and inflammation. This can explain the emergence of recurrent lung infections and inflammation in people living with HIV. METHODS: Tat-induced aberrant microRNAome was identified by miRNA expression analysis. microRNA mimics and antagomirs were used to validate the identified miRNAs involved in Tat mediated CFTR mRNA suppression. CRISPR-based editing of the miRNA target sites in CFTR 3'UTR was used to determine rescue of CFTR mRNA and function in airway epithelial cell lines and in primary human bronchial epithelial cells exposed to TGF-ß and Tat. FINDINGS: HIV Tat upregulates miR-145-5p and miR-509-3p. The two miRNAs demonstrate co-operative effects in suppressing CFTR. CRISPR-based editing of the miRNA target site preserves CFTR mRNA and function in airway epithelial cells INTERPRETATION: Given the important roles of TGF-ß signaling and the multitude of genes regulated by miRNAs, we demonstrate that CRISPR-based gene-specific microRNA antagonism approach can preserve CFTR mRNA and function in the context of HIV Tat and TGF-ß signaling without suppressing expression of other genes regulated by miR-145-5p.

Novel Approaches of CRISPR-Cas Technology in Airway Diseases.

Clustered regularly interspaced palindromic repeats (CRISPR) technique plays a vital role in preclinical modelling of many respiratory diseases. Diseases such as chronic obstructive pulmonary disease (COPD), asthma, acute tracheal bronchitis, pneumonia, tuberculosis, lung cancer, and influenza infection continue to significantly impact human health. CRISPR associated (Cas) proteins, isolated from the immune system of prokaryotes, are one component of a very useful technique to manipulate gene sequences or editing and gene expression with significant implications for respiratory research in the field of molecular biology. CRISPR technology is a promising tool that is easily adaptable for specific editing of DNA sequences of interest with a goal towards modifying or eliminating gene function. Among its many potential applications, CRISPR can be applied to correcting genetic defects as well as for therapeutic approaches for treatment. This review elucidates recent advances in CRISPR-Cas technology in airway diseases.

TGF-ß1 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-ß1 promotes HIV latency by upregulating a transcriptional repressor BLIMP-1. Furthermore, we identify miR-9-5p as an important intermediate in TGF-ß-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-ß can elevate the HIV viral reservoir load that could further exacerbate the HIV associated lung comorbidities.

Cigarette smoke promotes HIV infection of primary bronchial epithelium and additively suppresses CFTR function.

Recurrent lung infections are a common cause of morbidity and mortality in people living with HIV and this is exacerbated in smokers even when administered combination antiretroviral therapy (cART). The incidence of pneumonia is increased with smoking and treatment interruption and is directly dependent on viral load in patients when adjusted for CD4 counts. CFTR dysfunction plays an important role in aberrant airway innate immunity as it is pivotal in regulating mucociliary clearance (MCC) rates and other antibacterial mechanisms of the airway. In our earlier work, we have demonstrated that bronchial epithelium expresses canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. HIV Tat suppresses CFTR mRNA and function via TGF-ß signaling. In the present study, we demonstrate that cigarette smoke (CS) potentiates HIV infection of bronchial epithelial cells by upregulating CD4 and CCR5 expression. HIV and CS individually and additively suppress CFTR biogenesis and function, possibly explaining the increased incidence of lung infections in HIV patients and its exacerbation in HIV smokers.

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-ß 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.