Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.

Amelia T Soderholm,Victor Nizet,Othmar Korn,Benjamin L Schulz,Matthew J Sweet,Mark J Walker,Tania Rivera-Hernandez,Lisa M Seymour,Christine A Wells,Timothy C Barnett

doi:10.3389/fcimb.2018.00160

Abstract

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.

Highlights

Streptococcus pyogenes (Group A Streptococcus; GAS) is the most common bacterial cause of acute pharyngitis (Bisno, 2001), with over 616 million incident cases of GAS pharyngitis predicted to occur worldwide each year (Ralph and Carapetis, 2013)
Different GAS serotypes have previously been shown to induce epithelial inflammatory responses of differing magnitudes (Klenk et al, 2007; Persson et al, 2015). To investigate whether this variation exists in primary cells and to identify potential mechanisms, we performed RNAseq on tonsil epithelial (TEpi) cells infected with GAS strains JRS4 or 5448, for 6 h at a multiplicity of infection (MOI) of 5, an MOI where no significant difference in cell death was observed between the two strains and mock-treated cells (Figure S1)
For JRS4infected TEpi cells, interacting genes were detected for activator protein-1 (AP-1), activating transcription factor 2 (ATF-2), and nuclear factor of activated T cells (NFAT) transcription factor networks, for transcription factors/signaling proteins (JUN, FOSB, FOSL1, ATF3, DUSP1, DUSP5, EGR2, EGR3), and inflammatory transcriptional targets of these pathways (TNF, IL8, IL6, IL1B, MMP1, CXCL1, CXCL2, CXCL3, CCL20)

Summary

Introduction

Streptococcus pyogenes (Group A Streptococcus; GAS) is the most common bacterial cause of acute pharyngitis (Bisno, 2001), with over 616 million incident cases of GAS pharyngitis predicted to occur worldwide each year (Ralph and Carapetis, 2013). In response to GAS infection, immortalized epithelial cell lines derived from epithelial cell cancers, such as Detroit 562 (cancer of the pharynx), HEp2 (HeLa derivative epithelial cells derived from the cervix), HaCaT (skin keratinocytes), and A549 (lung tissue derivative) cells have been shown to express and/or secrete a wide array of pro-inflammatory mediators, including chemokines (IL-8, CXCL2, CXCL3, CCL5/RANTES), cytokines (IL-1α, IL-1β, IL-2, IL-6, TNFα, TNFβ, IFN-γ, CSF2, IL-17A, IL-23), growth factors (IGF-II), and the eicosanoid prostaglandin E2 (Courtney et al., 1997; Wang et al, 1997; Nakagawa et al, 2004; Klenk et al, 2005, 2007; Tsai et al, 2006; Rizzo et al, 2013). While the limited studies utilizing human tonsil explants have been fairly consistent with these cell line results (Agren et al, 1998; Wang et al, 2010; Bell et al, 2012), as these studies did not isolate epithelial cells from the tonsil explant tissue, it is difficult to elucidate the contribution of tonsil epithelial cells to these immune responses during GAS infection

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