Effect of HIF-1α expression on macrophage phenotype during infection with Mycobacterium tuberculosis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), presents a major global health challenge as antibiotic therapy is highly intensive and the occurrence of drug-resistant strains continues to increase. Therefore, there is a need for new adjunct therapies for TB. An interesting target for this is HIF-1α, a transcription factor that is involved in the cellular response towards hypoxia. Here, an in vitro model of human blood-derived macrophages infected with the avirulent mycobacterium strains Bacille Calmette Guérin (BCG) and H37Ra was used to study the effect of Mtb infection on the phenotype of macrophages, including polarization towards inflammatory (M1) or anti-inflammatory (M2) macrophages, as well as how this is affected by HIF-1α modulation. Additionally, the method of flow cytometry to measure HIF-1α expression was evaluated. Uninfected macrophages displayed a highly dominant M2 phenotype, which became a mixed with slightly dominant M1 phenotype after infection with BCG and H37Ra. While this seems favorable for Mtb killing, inhibitory molecules PD-L1 and IDO were upregulated, which could counteract the positive effect of M1 polarization. Stabilization or blockade of HIF-1α did not affect M1/M2 polarization of BCG-infected macrophages. However, expression of IL-1β was upregulated after stabilization of HIF-1α, which seems favorable for Mtb killing. The effect of HIF-1α stabilization and blockade could not be clearly demonstrated when visualizing HIF-1α expression with flow cytometry, but parallel western blot experiments revealed that at least HIF-1α stabilization was successful. Western blot seemed to have a higher sensitivity to detect differences in HIF-1α expression and less subject to donor variation compared to flow cytometry. All in all, additional research is needed to optimize the currently used flow cytometry protocol for the detection of HIF-1α, as well as to further characterize the effect of HIF-1α modulation on macrophage phenotype in the context of Mtb infection.