Cell culture and co-culture with KUMAs15
The present study included two adherent human cell lines, keratinocytes (HaCaT) and liver epithelial cells (HepG2), obtained from the National Centre for Cell Science, Pune, India. The HaCaT cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; HiMedia) supplemented with high glucose, sodium pyruvate, and 10% fetal bovine serum (FBS; Gibco, USA), while the HepG2 cell lines were cultured in Eagle’s minimum essential medium (MEM; HiMedia) supplemented with 10% FBS. The cells were inoculated without antibiotics to prevent interference in microbial culture in co-culture experiments in subsequent assays. The cells were maintained at 37 °C with 5% CO2 in a standard CO2 incubator (Thermo Scientific, USA). The present study has been waived from ethical permissions according to the guidelines of the Indian Council of Medical Research (ICMR) for the biomedical research studies conducted in India [18].
Cellular toxicity assay
The cytotoxicity of Micrococcus sp. KUMAs15 on the HaCaT and HepG2 cells was determined by the MTT assay for the analysis of cellular viability. In the experiment, HaCaT and HepG2 cell lines treated only with PBS served as the negative control, whereas cells treated with Triton-X served as the positive control.
Cytokine assay
The inflammatory reactions of the HaCaT and HepG2 after the exposure to KUMAs15 were determined by cellular cytokine assay. ELISA analysis of the pro-inflammatory cytokines TNF-α, IL-8, IL-2, IL-6, IL-10, and IL-12p70 in the human cell lines HaCaT and HepG2 exposed to isolated Micrococcus sp. KUMAs15 was performed in a 4-h and 24-h time span. The PBS-treated human cell line not exposed to KUMAs15 served as the negative control group whereas cells co-cultured with E. coli served as the positive control due to its lipopolysaccharides, which elicit a cytokine response in these cell lines [19].
Transcriptional analysis of cytokine genes
The transcriptional expression of cytokine genes have been determined by semi-quantitative reverse transcriptase PCR. The HaCaT and HepG2 cells have been cultured and subjected to treatment with either PBS, served as the negative control, or the Micrococcus sp. KUMAs15 and have been incubated overnight. Total RNA isolation was carried out using Trizol reagent (Invitrogen, USA) following the standard protocol [20] and was eluted with 20 μL of RNase-free water (Thermo Scientific, USA) and quantified at 260 nm (Evolution 201 UV-Visible Spectrophotometer, Thermo Scientific, USA). The cDNA has been synthesized by reverse transcription, using the total RNA as the template with the RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific, USA) following the manufacturer’s protocol, followed by the PCR amplification of the genes of interest using the cDNA as the template and appropriate primers [21] and followed by the agarose gel electrophoresis. The constitutive expression of the housekeeping gene, β-actin, was used as the loading control in this experiment.
Translational analysis of cytokine genes
The expressional analysis at the transcriptional level of cytokine genes was further confirmed at the protein level by western blot analysis. The HaCaT and HepG2 were cultured and exposed to the isolated Micrococcus sp. KUMAs15 for 24 h followed by protein isolation and western blotting.
Statistical analysis
The statistical analysis was performed using GraphPad Prism 6 software. The GraphPad algorithm has been used to perform one-way ANOVA followed by Tukey’s multiple comparison tests. All the experiments were carried out in triplicate, and p < 0.05 was considered as statistically significant.