Table_1_Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.pdf

<p>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.</p

Image_4_Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.tif

<p>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.</p

Image_2_Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.tif

<p>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.</p

Image_1_Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.tif

<p>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.</p

Table_2_Group A Streptococcus M1T1 Intracellular Infection of Primary Tonsil Epithelial Cells Dampens Levels of Secreted IL-8 Through the Action of SpyCEP.pdf

<p>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.</p

Slfn4 over-expression caused splenomegaly.

<p>(A) Macroscopic appearance of spleens from <i>Slfn4</i> over-expressing mice (right) and from MacBlue littermate controls (left). (B) Organ weights are expressed as percentage of body weight. Data are combined from four independent experiments and are displayed as mean + SEM.</p

Increased macrophages and granulocytes in spleen from MacBlue/UAS-<i>Slfn4</i>-V5 mice.

<p>Splenocytes from MacBlue littermate controls (A) and MacBlue/UAS-<i>Slfn4</i>-V5 mice (B) were stained for the cell surface markers F4/80 (macrophage), Ly-6G (granulocyte), B220 (CD45R, B cell), and CD3ε (T cell). The samples were analysed by FACS and quadrants were set based upon isotype control profiles. Data are representative of two independent experiments.</p

Macrophage-specific expression of the UAS-<i>Slfn4</i>-V5 transgene.

<p>(A) Elements of the UAS-<i>Slfn4</i>-V5 transgene include six UAS, a kozak sequence, and the open reading frame of <i>Slfn4</i> followed by a V5-tag. Upon crossing of the UAS-<i>Slfn4</i>-V5 mouse with the MacBlue mouse, the offspring (MacBlue/UAS-<i>Slfn4</i>-V5 mice) contain the GAL4-expressing module, the GAL4-reporting module, and the UAS-<i>Slfn4</i>-V5 transgene. GAL4/VP16 protein binding to both the UAS induces the expression of ECFP and <i>Slfn4</i>-V5 specifically in cells of the myeloid lineage. (B and C) RNA from bone marrow (BM) or BMM from the offspring of four UAS-<i>Slfn4</i>-V5 founder lines (F1–F4) was extracted and cDNA was prepared. <i>Slfn4</i> mRNA levels relative to <i>hprt</i> were determined by quantitative real-time PCR. Data are combined from at least two independent experiments (mean + range) and are presented as expression relative to BM controls to enable normalization across different the transgenic lines (B), or as expression relative to <i>hprt</i> (no normalization across the transgenic lines) (C).</p

Putative regulatory elements within the <i>Slfn4</i> promoter.

<p>The <i>Slfn4</i> proximal promoter is a TATA-containing promoter with putative binding sites for STAT1, IRF family members, PU.1 and AP1. The TSS, designated +1, is marked with an arrow.</p

Induction of <i>Slfn</i> expression by LPS in BMM, and in the CIA mouse model.

<p>(A) BMM were stimulated with LPS over a time course (0h no treatment control, 2h, 4h, 8h, 24h), RNA was extracted and reverse transcribed, and the expression of <i>Slfn1</i>, <i>Slfn2</i>, <i>Slfn4</i>, <i>Slfn5</i>, <i>Slfn8</i>, and <i>Slfn9</i> was determined using quantitative real-time PCR. Data (mean + SEM) are combined from 3 independent experiments. * <i>P</i>&lt;0.05 compared to control; ** <i>P</i>&lt;0.01 compared to control; *** <i>P</i>&lt;0.001 compared to control. (B) RNA from whole joints (disease affected, score 3; or unaffected, score 0) from mice with CIA was used to synthesise cDNA. mRNA expression of <i>Slfn1</i>, <i>Slfn2</i>, <i>Slfn4</i>, <i>Slfn5</i>, <i>Slfn8</i>, <i>Slfn9</i>, <i>csf1r</i>, and <i>Emr1</i> was determined using real-time PCR. The data, displayed as fold induction relative to control (score 0), are combined from two independent experiments (mean + range).</p