Intracellular ROS produced following HSV infection is essential for cytokine expression.

<p>(A) RAW264.7 cells were seeded in chambered-cover slides, either pre-treated with 6.4mM LNAC or complete media for 30 min, and subsequently infected with HSV-2 for 1, 2, or 3 h. CM-H₂DCFDA (5 µM) was added immediately before imaging. ROS production was monitored in z-stack images (1 µm) and images were acquired with a 40x objective every 30 seconds by confocal microscopy. Images represent maximum intensity projections. Amount of ROS was determined and represented as mean fluorescence per cell with a minimum of 100 cells counted per condition. Data is presented as mean of 3 experiments for the untreated samples and of 2 experiments for the LNAC samples +/- sem. (B–D) Peritoneal macrophages were seeded and either left uninfected or infected with HSV-2 (3×10⁶ pfu/ml, MOI 3); each in the absence or presence of 10 µM hydrogen peroxide. Supernatants were harvested after 16 h for measurement of IFN-α/β, CXCL10, and CCL5. (E, F) Peritoneal macrophages were pre-incubated with increasing concentrations of PDTC or LNAC and subsequently infected with HSV-2 (3×10⁶ pfu/ml, MOI 3). Supernatants were harvested 12 h post infection and CCL5 protein was measured by ELISA. The data is shown as mean of 3 replicates +/- st. dev. and is representative of 3 experiments. CCL5 levels significantly different from those induced by HSV-2 alone (p<0.05) are indicated with *.</p

Identification of a conserved redox-sensitive surface-exposed cysteine in the TRAF C-terminal domain of TRAF2, 3, and 6.

<p>(A) Structure-based sequence alignment of a region of the TRAF domain of human and murine TRAF2, 3, 4, and 6. Secondary structures in the region and TRAF-motif interacting residues are indicated in the figure. (B) <i>Traf6-/-</i> MEFs were left untreated or treated with HSV-2 (3×10⁶ pfu/ml, MOI 3, 5 h) or poly(I∶C) (25 µg/ml, 2 h). Total cellular lysates we generated and phospho-IκBα was measured by Luminex. (C–F) Traf6-/- MEFs were transfected with empty vector (pCMV), WT TRAF6, or C390S TRAF6. The cells were treated with HSV-2 (3×10⁶ pfu/ml, MOI 3, 5 h), poly(I∶C) (25 µg/ml, 2 h), IL-1β (10 ng/ml), and LNAC (3.2 mM, 30 min prior to further treatment) as indicated. Total RNA and total cell lysates were isolated and analyzed for IFN-β mRNA and P-IκBα by RT-qPCR and Luminex, respectively. Data is shown as means of triplicates +/- st.dev. (G) <i>Traf3-/-</i> MEFs were transfected with empty vector (pRK5), WT TRAF3, or C455S TRAF3. The cells were treated with HSV-2 (3×10⁶ pfu/ml, MOI 3) and LNAC (3.2 mM, 30 min prior to further treatment) as indicated. Total RNA was isolated 5 h post-infection and analyzed for IFN-β mRNA by RT-qPCR. Data is shown as means of triplicates +/- st.dev. RU, relative units; NR, normalized ratio.</p

Redox-sensitive signaling following HSV recognition.

<p>(A) pDCs and (B) BMMs were treated with LNAC (3.2 mM) 30 min prior to infection with HSV-1 or 2 (MOI 3). Culture supernatants were harvested 16 h post infection, and levels of CCL5 were measured by ELISA. All data are shown as means of 3–5 replicates +/- st.dev. (C) MEFs were treated with LNAC (3.2 mM) 30 min prior to infection with HSV-1 or 2 (MOI 3). Total RNA was harvested after 6 h post-infection and IFN-β mRNA measured by real time PCR. Data is presented as means of triplicate measurements +/- st.dev. RU, relative units. (D, E) Mice were treated i.p. with LNAC (1.1 mmol/kg body weight) in saline or with saline alone followed 4 h later by infection with HSV-2 (5×10⁶ pfu). Eight and 24 hours post infection, serum and livers were harvested for measurement of type I IFN and viral load, respectively (n = 5). Data represents mean of 3–6 replicates +/- st.dev. (F, G) RAW264.7 macrophage-like cells were treated with LNAC (6.4 mM) for 30 min before infection with HSV-2 (3×10⁶ pfu/ml, MOI 3). (F) Total cell lysates were harvested at the indicated time points post infection, and phospho-IκBα was measured by Luminex. (G) The cells were fixed 3 h post infection, stained with anti-IRF-3 antibody and DAPI, and visualised by confocal microscopy. Cells were scored for nuclear IRF-3 staining. Data represent mean +/- st.dev. (H, I) Peritoneal macrophages from C57BL/6 WT and ASK1^-/- mice were cultured <i>in vitro</i> and infected with HSV-2 (3×10⁶ pfu/ml, MOI 3). Supernatants were harvested 16 h post infection, and the levels of CCL5 and IFN-α/β were measured. (J) C57BL/6 WT and ASK1^-/- mice were infected i.p. with 5×10⁶ pfu of HSV-2. Livers were isolated 3 days post infection and viral load in the organs was determined (n = 8). The data represent mean of multiple measurements +/- st.dev.</p

ROS and innate antiviral response: Cell-type and PRR dependence.

<p>(A, B) Macrophages were isolated from C57BL/6 WT, MAVS^-/- or double knock-out TLR2/9^-/- mice and cultured <i>in vitro</i>. Cells were treated with (A) HSV-1 KOS or Sendai Virus, or (B) HSV-1 KOS or ODN1826. Supernatants were harvested after 16 h and presence of type I IFNs were measured by bioassay. (C) pDCs were isolated from C57BL/6 WT or TLR9^-/- mice. Cells were treated with HSV-1 KOS or ODN1826. Supernatants were harvested after 16 h and presence of type I IFNs were measured by bioassay. (D) RAW264.7 macrophage-like cells were transfected with si-p204 or control siRNA and infected with HSV-1 KOS. Total RNA was harvested after 6 h post-infection and CCL5 mRNA measured by real time PCR. Data is presented as mean of duplicate measurements +/- st.dev. RU, relative units. (E) RAW264.7 cells were transfected with HSV1-60mer (2 µg/ml) following treatment with 6.4 mM LNAC or complete media. Culture supernatants were harvested 12 h p.i. and production of CCL5 was measured by ELISA. (F) RAW264.7 cells were seeded and treated with vehicle or LNAC (3.2 mM) 30 min prior to stimulation with Pam₃Csk₄ (200 ng/ml), ODN1826 (1 µM), and poly(IC):LyoVec (25 µg/ml). Culture supernatants were harvested 16 h post-stimulation, and levels of CCL5 were measured by ELISA. (G) RAW264.7 cells were incubated with poly(I∶C) (25 µg/ml) following treatment with 6.4 mM LNAC or complete media. Culture supernatants were harvested 16 h p.i. and production of CCL5 was measured by ELISA. Data represents mean of measurements from triplicate cultures +/- st.dev.</p

<i>S</i>. <i>pyogenes</i> rRNA induces cytokine production in a TLR13-dependent way and independently of TRIF, TLR3, TLR7, TLR8 and TLR9.

<p>BMDMs (A, B) or cDCs (C, D) from <i>Tlr8−/−</i>, <i>Trif−/−</i> or <i>Tlr379</i> triple-deficient mice as well as control mice (WT) were infected with <i>S</i>. <i>pyogenes</i> (MOI = 50) or left uninfected. At indicated time points, supernatants were collected and TNF (A, C) or IL-6 (B, D) release was measured by ELISA. (E, F) <i>S</i>. <i>pyogenes</i> cells were sonicated and the extracts were treated with either DNase I, RNase A, proteinase K, or left untreated (control extract). These extracts were delivered into BMDMs (E) or cDCs (F) using DOTAP. After stimulation for 6 h, supernatants were collected and TNF release was measured by ELISA. (G) RNA or DNA isolated from <i>S</i>. <i>pyogenes</i> was transfected into WT BMDMs or cDCs and after 6 h of stimulation supernatants were collected and TNF release was measured by ELISA. (H, I) BMDMs (H) and cDCs (I) from <i>Tlr8−/−</i>, <i>Trif−/−</i>, <i>MyD88−/−</i> and <i>Tlr379</i> triple-deficient mice as well as control mice (WT) were transfected with <i>S</i>. <i>pyogenes</i> or mammalian RNA or left untreated. Supernatants were collected after 6 h and TNF release was measured by ELISA. (J) <i>S</i>. <i>pyogenes</i> total RNA as well as the mRNA (isolated with the MICROBExpress Bacterial mRNA Enrichment Kit, contains 0.3% rRNA), rRNA and nt>200 fractions were used for transfection of BMDMs. TNF release was determined in supernatants of cells after 6 h of stimulation using ELISA. (K) <i>S</i>. <i>pyogenes</i> total RNA as well as the mRNA (isolated with the Ribo-Zero rRNA Removal Kit) were transfected into BMDMs. TNF release was measured 6 h after stimulation using ELISA. (L—O) BMDMs (L, M) and cDCs (N, O) from <i>Tlr13−/−</i> mice as well as control mice (WT) were transfected with <i>S</i>. <i>pyogenes</i> or mammalian RNA or left untreated. Supernatants were collected after 6 h and TNF (L, N) or IL-6 (M, O) release was measured by ELISA. Error bars in all panels represent SDs (n≥3).</p

Macrophage-like differentiated THP-1 cells respond to endosomal TLR ligands but not to the canonical TLR13 ligand.

<p>(A, B) THP-1 cells were either treated with 10 nM PMA or left untreated (as described in Material and Methods) before stimulation with R848 (5 μg/ml), LTA (5 μg/ml), LPS (10 ng/ml) or DOTAP-mediated delivery of poly(I:C) (5 μg). Supernatants were collected after 8 h and IL-8 (A) or TNF (B) release was determined by ELISA. Error bars represent SDs (n≥3). (C, D) RNA was isolated from differentiated or undifferentiated THP-1 cells treated as described in (A) and (B) for 4 or 8 h. <i>IL8</i> (C) and <i>TNF</i> (D) mRNA levels were determined by qRT-PCR (normalized to <i>HPRT</i>). Mean values ± SD are shown (n ≥ 3). (E) THP-1 cells stably expressing TLR13 (TLR13 THP-1) were stimulated with TLR13 ligand SA19, poor TLR13 ligand (mut-SA19), <i>S</i>. <i>pyogenes</i> RNA or LTA for 4 h. TNF was determined in supernatants by ELISA. Error bars in represent SDs (n≥3).</p

Human RNA-recognizing TLRs are not capable of sensing <i>S</i>. <i>pyogenes</i> RNA.

<p>(A, D) HEK293 stably expressing human TLR3 (HEK293-hTLR3) or control HEK293 cells (HEK ctl) were transfected with the TLR3 ligand poly(I:C) (5 μg), the Tlr13 ligand oligoribonucleotide SA19 (5 μg), <i>S</i>. <i>pyogenes</i> RNA (5 μg) using DOTAP or stimulated with human TNF (10 ng/ml). IL-8 release was measured in supernatants 24 h post stimulation by ELISA (A) or the levels of <i>IL8</i> mRNA were determined by qRT-PCR (normalized to <i>HPRT</i>) (D). Mean values ± SD are shown (n ≥ 3). (B, C, E, F) HEK293XL cells stably expressing human TLR7 (B, E) or TLR8 (C, F) or control HEK293XL cells were transfected with SA19 (5 μg), <i>S</i>. <i>pyogenes</i> RNA (5 μg) or stimulated with the TLR7 and TLR8 ligand R848 (5 μg/ml) or human TNF (10 ng/ml). IL-8 release was measured in supernatants 24 h post stimulation by ELISA (B, C) or the levels of <i>IL8</i> mRNA were determined by qRT-PCR (normalized to <i>HPRT</i>) (E, F). Mean values ± SD are shown (n ≥ 3).</p

mTORC1 activation promotes viral infection.

<p>(<b>A</b>) A549 cells were infected with WSN at MOI of 2 PFU/cell for 1 h and then treated with 250 nM Torin1 or DMSO for an additional 9 h followed by immunoblot analysis. (<b>B</b>) A549 cells were infected at MOI of 0.01 PFU/cell with WSN for 16 h in the presence of the depicted concentrations of Torin1 or DMSO. Viral titers were measured by plaque assay to yield plaque-forming units per mL (PFU/ml, plotted as % infection relative to DMSO control). Cell survival was also assessed at the depicted concentrations using the MTT assay. The following parameters were calculated: CC₅₀>500 nM; IC₅₀ = 0.46nM; and SI>1087. (<b>C</b>) Experiment was performed as in <b>B</b> except that cells were treated with 13.7 μM rapamycin or DMSO as control. Mean and standard error of the mean (SEM) are shown. <i>n</i> = 3, **p<0.01. (<b>D</b>) <i>Rictor</i>^<i>+/+</i> or <i>Rictor</i>^<i>-/-</i> MEFs were infected with WSN at MOI of 2 PFU/cell for 6 h and treated with 250 nM Torin1 or DMSO at 1 h post-infection. Immunoblot analyses were performed to detect viral proteins (HA, NP, PA, PB1, PB2, NS1, M1, M2 and NA) and host proteins (β-actin, Rictor, total and phosphorylated S6K and 4E-BP1). β-actin and S6K serve as loading controls. The upper band in the S6K/p-S6K blots is p85 S6K whereas the lower band is p70 S6K. (<b>E</b>) <i>Rictor</i>^<i>+/+</i> or <i>Rictor</i>^<i>-/-</i> MEFs were infected with WSN at MOI of 0.01 for 24 h and 48 h. Viral titers were measured by plaque assay to yield plaque-forming units per ml (PFU/ml). Mean and standard deviation (SD) are shown, <i>n</i> = 3, **p<0.01, *p<0.05.</p

<i>S</i>. <i>pyogenes</i> is recognized by a combination of Tlr2- and Tlr13-mediated sensing.

<p>(A) <i>S</i>. <i>pyogenes</i> RNA was either added directly to the BMDMs or transfected into BMDMs using DOTAP. Supernatants were collected after 6 h and TNF release was measured by ELISA. (B, C) BMDMs (B) or cDCs (C) from <i>Tlr2−/−</i> and <i>Unc93b1−/−</i> mice as well as control mice (WT) were infected with <i>S</i>. <i>pyogenes</i> (MOI = 50) or left uninfected, or treated with LTA or LPS as a control. At indicated time points, supernatants were collected and TNF release was measured by ELISA. (D) BMDMs were stimulated for 1 or 2 h with LTA, and after medium change cells were incubated without LTA for additional 5 or 4 h, respectively, As a control, BMDMs were treated with LTA for 6 h. TNF was determined in the supernatants by ELISA. (E) BMDMs from <i>Unc93b1−/−</i> mice as well as control mice (WT) were transfected with streptococcal- or mammalian RNA. Supernatants were collected after 6 h and TNF release was measured by ELISA. (F) BMDMs from <i>Tlr2−/−</i> as well as control mice (WT) were pre-treated for 30 min with cytochalasin D or left untreated prior infection with <i>S</i>. <i>pyogenes</i> (MOI = 50). At indicated time points, supernatants were collected and TNF release was measured by ELISA. (G) BMDMs were pre-treated with cytochalasin D (right panels) or left untreated (left panels) before infection with CFSE-labeled (green) <i>S</i>. <i>pyogenes</i> (MOI = 50). After 4 h cells were fixed and stained with anti <i>S</i>. <i>pyogenes</i> antibody (anti-S.p.) for extracellular bacteria (red). Immunofluorescence image depicts CFSE labeled bacteria (both extra- and intracellular) in green, extracellular antibody-stained bacteria in red, and a merge of the two channels in showing extracellular bacteria in yellow and intracellular bacteria in green. (H—J) BMDMs (H, I) and cDCs (J) from <i>Unc93b1Tlr2</i> double-deficient mice as well as control mice (WT) were infected with <i>S</i>. <i>pyogenes</i> (MOI = 50) or left uninfected. At indicated time points, supernatants were collected and TNF (H, J) or IL-6 (I) release was measured by ELISA. LPS treatment (6 h) served as specificity control. (K—M) BMDMs (K, L) and cDCs (M) from <i>Tlr13−/−</i> as well as control mice (WT) were incubated for 30–45 min with anti-TLR2, control IgG or left untreated prior to infection with <i>S</i>. <i>pyogenes</i> (MOI = 100) or stimulation with LTA. Supernatants were collected 4 h after infection and TNF (K, M) or IL-6 (L) release was measured by ELISA. Error bars in all panels represent SDs (n≥3).</p

Entire activation of the immune responses through both Tlr2- and Unc93b1-dependent pathways is required for protective defense against <i>S</i>. <i>pyogenes</i> in mice.

<p>(<b>A</b>—<b>C</b>) Kaplan-Meier survival curves of C57BL/6 and <i>Tlr2−/−</i>, <i>Unc93b1−/−</i> (8 mice per genotype) or <i>Tlr9−/−</i> mice (14 mice per genotype) after subcutaneous infection with 1×10⁸ CFU of <i>S</i>. <i>pyogenes ISS3348</i>. Survival was monitored for 6 days. Significance: * = p<0.05; ** = p<0.01.</p