A high-throughput CAMP assay for the evaluation of the vitamin D-dependent antimicrobial response

By Tarun Sai LomteMar 16 2023Reviewed by Danielle Ellis, B.Sc.

A recent study published in Nutrients developed a novel and high-throughput assay to evaluate cathelicidin antimicrobial peptide (CAMP).

Background

Cathelicidins exhibit broad-spectrum activity against gram-positive and -negative bacteria, viral pathogens, and fungi. The innate immune cells, such as dendritic cells, monocytes, neutrophils, and macrophages, express the human CAMP gene.

Vitamin D is crucial for CAMP expression, and deficient levels have been associated with higher rates of pneumonia, influenza, bacterial vaginosis, tuberculosis, and upper respiratory tract infection.

About the study

In the present study, researchers developed a novel high-throughput CAMP assay (HiTCA).

Clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated knock-in of the mCherry fluorescent gene reporter was performed at the 3’-end of the CAMP gene in the monocyte-macrophage SC cell line. Ribonucleoproteins with Cas9 and a modified single guide RNA (sgRNA) targeting the 3’-end of the CAMP gene were generated.

Additionally, donor plasmids with mCherry flanked by homologous arms of the target were also synthesized. Donor plasmids and Cas9-sgRNA complexes were electroporated into SC cells. Genomic DNA was isolated, amplified, and sequenced. Cell pools were seeded using a single-cell printer to generate monoclonal cell populations.

The researchers identified a monoclonal population of homozygous CAMPmCherry human SC cells (HiTCA cells). HiTCA and wild-type (WT) SC cells were cultured in media for maintenance and periodically tested for viability. HiTCA cells were incubated with human serum and treatment metabolites, such as vitamin D metabolites or ethanol. They were incubated for 24 hours, and high-throughput flow cytometry analysis was performed. WT cells served as negative controls.

The team tested the responsiveness of HiTCA cells to 25-hydroxyvitamin D3 (25(OH)D3) via its catalysis to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by cytochrome P450 family 27 subfamily B member 1 (CYP27B1)-hydroxylase. The effect of human serum on CAMP response was assessed by coincubation of HiTCA cells with pooled human sera. Further, the high-throughput assay was tested in a pilot experiment using sera from five white and five black males.

Findings

The researchers observed a dose-dependent response of HiTCA cells to 25(OH)D3. Ethanol- and 1 nM 25(OH)D3-treated cells showed baseline expression (10% CAMPmCherry). This increased to 50% and 100% with 10 nM and 100 nM 25(OH)D3, respectively, comparable to the levels observed in cells treated with 1,25(OH)2D3. This suggests that HiTCA cells could convert the inactive 25(OH)D3 to the active 1,25(OH)2D3.

Incubating HiTCA cells with human sera caused attenuation of CAMPmCherry expression in a dose-dependent manner. Response to sera was consistent across metabolite treatment conditions. In the pilot experiment with human donor sera, the team used 0.3% and 10% serum concentrations to examine whether differential CAMP expression occurred.

Likewise, HiTCA CAMPmCherry expression was attenuated at 10% donor serum concentration compared to 0.3%. Notably, at the lower concentration (0.3%), CAMPmCherry response was five to seven-fold elevated with 10 nM 1,25(OH)2D3 and 100 nm 25(OH)D3. CAMPmCherry response in sera from black males was higher than those from white males, even though serum levels of 1,25(OH)2D3 and 25(OH)D3 were lower in black samples.

Conclusions

In summary, the researchers developed HiTCA that addresses the limitations associated with a previous ex vivo CAMP assay in a fully scalable high-throughput workflow. CAMPmCherry response was strongly induced by exogenous 25(OH)D3 or 1,25(OH)2D3 without serum. Nonetheless, when serum was added, the CAMPmCherry response was strongly attenuated. Further refinement and testing of this assay may prove valuable in identifying serum factors that influence the immunoregulatory function of vitamin D.