Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases. It is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-γ. Under these conditions, isolates of several other major bacterial pathogens and microbiota of the skin were found to not have significant IL-18-maturing ability. These results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota

Bacterial activation of IL-18.

(A, B) IL-18 activation was measured in the supernatants from keratinocytes infected with the indicated gene knockouts of GAS strain 5448 or wild-type GAS, and after 4 h, bioactive IL-18 was measured with HEK-Blue IL18 reporter cells. (C) IL-18 activation was measured in the supernatants from keratinocytes infected with the indicated bacterial species and after 4 h, bioactive IL-18 was measured with HEK-Blue IL18 reporter cells. (D, E) IL-18 activation was measured in the supernatants from keratinocytes infected with the indicated gene knockouts of GAS strain 5448, L. lactis, or during treatment with 5 μM E-64. Spectinomycin and anhydrotetracycline to maintain SpeB expression from the indicated plasmids. Data represent at least 3 independent experiments with 4 replicates. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis compared to uninfected/untreated keratinocytes. Bars show median values ± standard deviation. *P P <0.005; ns, not significant.</p

Cytokine profiles of keratinocytes and related cell lines.

HaCaT, Detroit 562, HEp-2, A-431, primary keratinocytes, or HUVEC cells, were infected with 7.5 x 106 colony-forming units (CFU) of GAS for 6 h. (A) Relative abundance of select cytokines was examined by membrane-based antibody array. (B) Cytokine profiles of each cell were examined by multivariate (principal component analysis), of the total variance, PC1 explains 48.67% and PC2 20.87%, from the raw cytokine quantities tabulated in (A). Arrows indicate change in cells from uninfected to 6 h infection. (C) Graphical representation of the congruent cytokine profiles between cell types.</p

Examination of GAS requirements for IL-18 activation.

Primary human keratinocytes were infected with GAS at MOI 10 for 4 h, then (A, B) bioactive IL-18 was measured with HEK-Blue IL18 reporter cells. (C) SpeB activity was measured using specific substrate sub103 and IL-18 activity measured in the supernatants from keratinocytes treated with titrations of purified SpeB protein. (D) Recombinant human pro-IL-18 was purified and incubated with purified, active SpeB, then cleavage products were separated by SDS-PAGE and visualized by staining. (E) Coding sequence of human IL-18 with probable and known cleavage sites indicated; the largest and smallest confirmed by Edman sequencing. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. All data represent at least 3 independent experiments with 4 replicates. Bars show median values ± standard deviation. **P <0.005; ns, not significant.</p

Mouse IL-18 can be activated by SpeB but is not secreted under normal inert conditions.

(A) C57BL/6 wild-type or IL-18-knockout (il18-/-) mice were inoculated intradermally with 108 CFU of GAS 5448 or its DspeB mutant. After 72 h, mice were euthanized, and GAS CFU was enumerated at the infection site. Results are from 2 independent experiments with 5 mice in each. (B) Recombinant mouse pro-IL-18 was incubated with human Caspase-1 or SpeB and activation measured with HEK-Blue IL18 reporter cells. (C) Supernatants or lysates from mouse primary keratinocytes were examined for IL-18 by ELISA and (D) cell lysis confirmed by LDH release assay, or (E) incubated 4 h with SpeB, and active IL-18 was quantified with HEK-Blue IL-18 reporter cells. Data represent at least 3 independent experiments with 4 replicates. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. Bars show median values ± standard deviation. *P P <0.005; ns, not significant.</p

SpeB activation of IL-18 promotes antimicrobial IFN-γ responses.

(A) Diagram of primary keratinocyte/PBMC co-culture model; IFN-γ is a reporter of T cell activation, which can occur via IL-18 and additional mechanisms during infection. (B) Cocultured keratinocyte/PBMCs were infected with 105 CFU of GAS or treated with rSpeB. After 4 h, active IL-18 was quantified with HEK-Blue IL-18 reporter cells and IFN-γ by ELISA. (C, D) Cocultured cells were treated with SpeB as in (B), in combination or post-depletion of CD2+ T cells, or with treatments with IL-18 neutralizing antibody or 5 μM YVAD-cmk or 10 μM VX765 to inhibit inflammasome function. Data represent at least 3 independent experiments with 4 replicates. Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. Bars show median values ± standard deviation. *P P <0.005; ns, not significant.</p

Examination of IL-18 activation in GAS-infected keratinocytes.

Primary human keratinocytes were infected with GAS at a multiplicity of infection (MOI) of 10 for 4 h and (A) secreted IL-18 (total, pro- and mature forms) was measured by ELISA, (B) cell lysis measured by LDH release, (C) total IL-1β measured by ELISA, or (D) cells visualized by immunofluorescent microscopy with staining for cell death by propidium iodide uptake (red) and caspase-1 activation (green). (E) Secreted bioactive IL-18 measured with HEK-Blue IL18 reporter cells during infection as in (A). (F) Fresh or conditioned media were removed from primary human keratinocytes and incubated with GAS, then IL-18 activity was measured as in (E). Data were analyzed by 1-way ANOVA using Dunnett multiple comparisons analysis. All data represent at least 3 independent experiments with 4 replicates. Bars show median values ± standard deviation. **P <0.005; ns, not significant.</p

Images of cytokine arrays for quantification of secreted cytokines.

Images of cytokine arrays for quantification of secreted cytokines.</p