Cytokine-mediated inhibition of Staphylococcus aureus adherence and invasion into nonphagocytic cells

Abstract

Staphylococcus aureus is a major pathogen responsible for a wide range of infections, from minor skin diseases to life-threatening conditions, such as sepsis and pneumonia. Its ability to invade nonphagocytic cells, evading the immune system and persisting intracellularly complicates the treatment and contributes to recurrent infections. In this study, we investigated the role of cytokines in inhibiting S. aureus adherence and invasion into nonphagocytic human cells. Monomac-6 cells were stimulated with heat-killed S. aureus (HKSA) to produce a cytokine cocktail, which was used to treat various human cell lines, including HEK293, A549, HaCaT, and HT29. Our results demonstrate that cytokines significantly reduced S. aureus adherence and invasion into HEK293, HaCaT, and HT29 cells by altering the expression of key host cell receptors for S. aureus adhesins and invasion, such as integrins and heat shock proteins. These effects of cytokines were mediated via JAK-STAT pathway as tofacitinib supplementation, a JAK inhibitor, reversed the effects of cytokine cocktail. However, these effects were not observed in A549 cells, most likely due to their ability to actively internalize pathogens. These findings suggest that cytokines provide a crucial line of defense against the ability of S. aureus to invade nonphagocytic cells by modulating the expression of host cells receptors.

Supplementary Information: The online version contains supplementary material available at 10.1007/s00430-025-00840-4.

Keywords: Bacterial adherence; Bacterial invasion; Cytokines; Receptors for adhesin and invasin; Staphylococcus aureus.

Fig. 1

Heat-killed S. aureus (HKSA) USA300 LAC and Cowan induce the production of pro- and anti-inflammatory cytokines in Monomac-6 cells. Monomac-6 cells were treated with HKSA at an MOI of 30 and PBS (unstimulated) in the presence of dexamethasone (50 µg/ml) and incubated for 16 h, and cytokine production was measured using ELISA. We measured the production of proinflammatory cytokine (A) and anti-inflammatory cytokines (B). For all the graphs, each data point represents the mean value ± SD (n = 3). Two-way ANOVA was used to analyze the data, followed by the Bonferroni correction, with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001

Fig. 2

The supernatant of HKSA-stimulated Monomac-6 cells decreased bacterial adherence to and invasion into host cells. (A) The adherence and (B) invasion of S. aureus USA300 LAC and S. aureus Cowan in several cell lines (HEK293, A549, HT29 and HaCaT) were investigated after the cells were pretreated for 16 h with the supernatant of Monomac-6 cells. For all the graphs, each data point represents the mean value ± SD (n = 3). Unpaired t-test was used to analyze the data, with *p < 0.05 and **p < 0.01. Unstimulated: pretreated with supernatant of unstimulated Monomac-6 cells; HKSA: pretreated with supernatant of stimulated Monomac-6 cells with HKSA at MOI of 30; HKSA + Dexa: pretreated with supernatant of stimulated Monomac-6 cells with HKSA at MOI of 30 and dexamethasone (50 µg/ml)

Fig. 3

Overnight exposure of the cells to cytokines could hinder the adherence and invasion of S. aureus into host cells. (A) Adherence and (B) invasion assays were performed using S. aureus USA300 LAC on different types of cell lines (HEK293, A549, HT29, and HaCaT). The cells were pretreated for 16 h with individual cytokines (TNFα, IFNγ, IL-1β, IL-4, IL-6, IL-8, IL-10, and IL-13) at a concentration of 0.5 ng/ml prior to the assays. For all the graphs, each data point represents the mean value ± SD (n = 3). One-way ANOVA was used to analyze the data, followed by the Dunnett correction for multiple comparisons, with ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; and ^****p < 0.0001

Fig. 4

The supernatant of HKSA-stimulated Monomac-6 cells altered the gene expression of host cell receptors for S. aureus adhesins and invasins. The cells (HEK293, A549, HT29, and HaCaT) were treated with the supernatant of HKSA-stimulated Monomac-6 cells and the supernatant of unstimulated Monomac-6 cells and incubated for 16 h. Relative gene expression was then measured for the genes encoding receptors for S. aureus adhesins and invasions, namely, integrin beta 1 (ITGB1), integrin alpha 5 (ITGA5), integrin beta 3 (ITGB3), integrin alpha V (ITGAV), heat shock protein 90 (HSP90), annexin (ANXA2), von Willebrand factor (VWF), desmoglein (DSG1), heat shock protein 60 (HSP60), heat shock protein 70 (HSC70), and CD36. Beta-2-microglobulin (B2M) was used as a reference gene. For all the graphs, each data point represents the mean value ± SD (n = 3). An unpaired t test was used to analyze the data between unstimulated and HKSA-stimulated data with ^*p < 0.05

Fig. 5

Tofacitinib abrogated the effect of the cytokine cocktail produced by Monomac-6 cells. (A) Adherence and (B) invasion assays were performed using S. aureus USA300 LAC and overnight pretreated cells with the supernatant of HKSA-stimulated Monomac-6 cells and unstimulated one and with the supplementation of tofacitinib (200 nM). (C) qPCR gene expression analysis of ITGB1, ITGA5, ITGB3, ITGAV, HSP60, HSC70 and CD36 of HEK293 cells was conducted using the same conditions as in adherence and invasion assays. For all the graphs, each data point represents the mean value ± SD (n = 3). Ordinary one way ANOVA were used to analyze the data from the adherence and invasion experiments followed by the Dunnett correction for multiple comparisons, and two-way ANOVA was used to analyze the data from the qPCR gene expression analysis, followed by the Bonferroni correction for multiple comparisons: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; and ^****p < 0.0001. Unstimulated: pretreated with supernatant of unstimulated Monomac-6 cells; HKSA: pretreated with supernatant of stimulated Monomac-6 cells with HKSA at MOI of 30; HKSA + Tofacitinib: pretreated with supernatant of stimulated Monomac-6 cells with HKSA at MOI of 30 and tofacitinib (200 nM); Tofacitinib: pretreated with supernatant of unstimulated Monomac-6 cells treated with tofacitinib (200 nM)