Consequences of CRISPR-Cas9-Mediated CFTR Knockout in Human Macrophages

Abstract

Macrophage dysfunction is fundamentally related to altered immunity in cystic fibrosis (CF). How genetic deficits in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to these defects remains unknown. Rapid advances in genomic editing such as the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) system provide new tools for scientific study. We aimed to create a stable CFTR knockout (KO) in human macrophages in order to study how CFTR regulates macrophage function. Peripheral blood monocytes were isolated from non-CF healthy volunteers and differentiated into monocyte-derived macrophages (MDMs). MDMs were transfected with a CRISPR Cas9 CFTR KO plasmid. CFTR KO efficiency was verified and macrophage halide efflux, phagocytosis, oxidative burst, apoptosis, and cytokine functional assays were performed. CFTR KO in human MDMs was efficient and stable after puromycin selection. CFTR KO was confirmed by CFTR mRNA and protein expression. CFTR function was abolished in CFTR KO MDMs. CFTR KO recapitulated known defects in human CF MDM (CFTR class I/II variants) dysfunction including (1) increased apoptosis, (2) decreased phagocytosis, (3) reduced oxidative burst, and (4) increased bacterial load. Activation of the oxidative burst via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assembly was diminished in CFTR KO MDMs (decreased phosphorylated p47 phox). Cytokine production was unchanged or decreased in response to infection in CFTR KO MDMs. In conclusion, we developed a primary human macrophage CFTR KO system. CFTR KO mimics most pathology observed in macrophages obtained from persons with CF, which suggests that many aspects of CF macrophage dysfunction are CFTR-dependent and not just reflective of the CF inflammatory milieu.

Enhancing Cystic Fibrosis Immune Regulation

“…Whole bone marrow aspirates, hMSC and Cftr sufficient macrophages provide clear implications for therapeutic development of immune support in managing CF lung infection and inflammation based upon the data we have presented in this manuscript. These observations are consistent with previous studies that have also pursued immune supportive therapeutic directives ( Weiss, 2008 ; Bruscia et al, 2009 ; Bonfield et al, 2012 ; Sutton et al, 2017 ; Duchesneau et al, 2020 ; Zhang et al, 2020 ). Follow-up studies will focus on delivering CF macrophages or hMSCs in the preclinical model and further investigate the functional insufficiency of CF derived cells.…”

CFTR Protein: Not Just a Chloride Channel?

“…Macrophages in CF show multiple defects, including an ineffective uptake of pathogens due to dysregulated phagocytic and/or signaling receptors and a decreased efferocytosis, phagocytosis, ROS production, and bacterial killing [ 64 ]. CFTR can actually also regulate the signaling of receptors involved in the recognition of microbial stimuli called pathogen-associated molecular patterns (PAMPs).…”

Anti-Inflammatory Influences of Cystic Fibrosis Transmembrane Conductance Regulator Drugs on Lung Inflammation in Cystic Fibrosis

“…In addition to promoting an inherent proinflammatory phenotype, CFTR-deficiency has also been found to implicate the microbicidal function of CF macrophages. Zhang et al utilised CRIPSR-Cas9-mediated CFTR KO macrophages to confirm that CF macrophage dysfunction is CFTR-dependent rather than a consequence of the CF inflammatory milieu [ 43 ]. CFTR-deficient macrophages possessed a defective microbicidal function though the inability to kill phagocytosed bacteria due to insufficient acidification of the late phagosome to a pH less than 5 [ 44 ].…”