Supplementary Figure 1 Production of cytokines and chemokines after vaginal HSV-2 infection. C57BL/6 mice were (a) treated intravaginally with 20 µl of PBS or infected with 6.7x10 4 pfu of HSV-2 in the same volume, or were (b) infected intravaginally with 6.7x10 4 pfu of HSV-2 produced in murine neurons. Vaginal washes were isolated 24 h p.i. and levels of CXCL10 were determined by ELISA. (c-k) C57BL/6 mice were infected intravaginally with 6.7x10 4 pfu of HSV-2. Vaginal washes were isolated at the indicated time points p.i., and analyzed for levels of (c) CCL2, (d) CCL5, (e) CXCL1, (f) IL-1β, (g) IL-10, (h) IL-12 p40, (i) IL-15, (j) IL-33, (k) TNF-α. Data are presented as means +/- st.dev. n = 6-8 per group. ** 0.001<p<0.01; *** p<0.001; ns, not statistically significant.
Supplementary Figure 2 An essential role for CXCR3 in early control of corneal HSV-1 infection. C57BL/6 and Cxcr3 -/- mice were infected in the cornea with 2x10 6 pfu of HSV-1 (McKrae). (a). Eye balls were isolated 24 h p.i. and analyzed for the levels of CXCL10 and bioactive type I IFN. n=8 per group. (b) Total RNA isolated from homogenized eyes of mice infected for 24 h was analyzed for gb and Gapdh mrna by RT-qPCR. Data are presented as means +/- standard deviation n=5-8 per group. * 0.01<p<0.05, ** 0.001<p<0.01, *** p<0.001.
Supplementary Figure 3 Type II and III IFNs are redundant for innate control of genital HSV-2 infection. C57BL/6, Il28ra -/-, and Ifngr -/- mice were infected intravaginally with 6.7x10 4 pfu of HSV-2. (a, c) Vaginal washes were isolated on day 1 and 2 points p.i., and analyzed for viral load by plaque assay on Vero cells. Data points represent individual mice (n= 5-10 per group). (b, d) The mice were scored daily for clinical signs of disease. Data are presented as mean disease score +/- standard deviation. ns, not statistically significant.
Supplementary Figure 4 Re-establishment of a mucosal layer by vaginal epithelial cells in vitro. Vaginal tissue was excised from female mice and treated with Collagenase and Dispase as described in the method section. The subepithelium was removed, and the epithelial cell layer was sat in culture for 24 h. (a) Total RNA was isolated and analyzed for expression of Muc1, 2, 5b, and Gapdh. For comparison, RNA from murine bone-marrow-derived dendritic cells (BMDCs) was analyzed in parallel. Data are presented as means +/- st.dev. n=3 per group. (b) Periodic acid Schiff staining of vaginal epithelial cells in culture after the treatment described above. As control is included a staining of a section from vaginal tissue treated with medium not supplemented with Collagenase/Dispase. The periodic acid Schiff stains glycans, which are abundantly present in the mucus layer (red).
Supplementary Figure 5 Expression of PRRs in the vagina of mice. (a) RNA was isolated from vaginas and bone-marrow-derived dendritic cells from C57BL/6 mice. Levels of Tlr2, Tlr3, Tlr9, Rig-I, Mda5, Mb21d1 (cgas) and β-actin mrna were measured by RT-qPCR. For each PRR, data was normalized to β-actin and expression levels are presented as mean of the ratio between vaginal epithelial cells and dendritic cells +/- standard deviation. n = 3. (b) The Ct values from the PCR data set on dendritic cells used for calculation of expression ratios.
Supplementary Figure 6 Development of disease after infection with infectious or attenuated HSV-2. C57BL/6 mice were infected intravaginally with 6.7x10 4 pfu of infectious or UV-inactivated HSV-2 or HSV-2 gd-r (revertant) or HSV-2 gd. (a) On subsequent days, the animals were scored for clinical signs of disease (n = 8). Data are presented as mean disease score +/- st. dev. (b) Tissue sections of the vagina from mice left untreated or infected for 24 h with HSV-2 or HSV-2 gd were stained with anti-hsv-2. Scale bar, 100 m.
Supplementary Figure 7 Characterization of HSV-2 devoid of O-linked sugars. (a-d) HSV-2 stocks were treated as indicated for 30 min at 37 o C and subjected to (a) SDS-PAGE and Western blotting using antibodies against gb and gd, (b, d) infection on MEFs followed by confocal microscopy (stainings: anti-vp5, anti-sting, and DAPI) and (c) viral plaque titration on Vero cells. (e) EdC-labeled HSV-2 grown in parental or Cosmc KO cells were incubated at 37 o C for 30 min. DNA and capsid were visualized with CLICK chemistry and anti-vp5 respectively. Data are presented as mean % CLICK+VP5- foci of the total number of counted VP5+ foci +/- st.dev. (f-g) C57BL/6 mice were infected intravaginally with 6.7x10 4 pfu of (f) HSV-2, which has been treated with buffer or an enzyme mix cleaving O-linked sugars before infection, or (g) HSV-2 grown in either parental or Cosmc KO HaCaT cells. Vaginal washes were harvested 48 hours p.i. for and levels of CXCL10 were measured (n= 8). (h) Virus titer for preparations of HSV-2 grown in parental or Cosmc KO HaCaT cells. Data are presented as means +/- st.dev. * 0.01<p<0.05, ** 0.001<p<0.01, *** p<0.001. ns, not statistically significant.
Supplementary Figure 8 Identification of NK cells and neutrophils by flow cytometry. C57BL/6 mice were infected intravaginally with 6.7x10 4 pfu of HSV-2 or left untreated. 24 hours p.i., single-cell leukocytes were isolated from the vaginal tissue and analyzed by flow cytometry (a) Ly6G+CD11b+ cells, and (b) CD45+ NK1.1+ cells. The plots represent one infected C57BL/6 mouse and one naive C57BL/6 mouse. (c) Vaginal washes were isolated 24 hours p.i. and analyzed for levels of CXCL10. Data are presented as means +/- standard deviation. n = 8 per group. ns, not statistically significant.
SupplementaryTable2. Expression of CLRs in the vagina CLR Mean Ct St.dev Dectin1 30.65 0.18 Dectin2 ND Cd209a 33.63 0.56 Cd209b 34.24 0.78 Cd209c 32.39 0.29 Cd209d 32.65 0.22 Cd209e 34.27 0.61 Cd209f 26.81 0.17 Cd209g 32.30 0.97 Man. rec 30.44 0.09 Mingle 27.48 0.15 Mdl1 31.05 0.42 Dngr-1 33.45 0.98 β-actin 17.81 0,01 Data are presented as mean Ct +/- standard deviation. Man. rec., mannose receptor; Mdl1, Myeloid DAP12-associating lectin.