Novel Role for Interleukin-17 in Enhancing Type 1 Helper T Cell Immunity in the Female Genital Tract following Mucosal Herpes Simplex Virus 2 Vaccination

Abstract

It is well established that interferon gamma (IFN-γ) production by CD4⁺ T cells is critical for antiviral immunity against herpes simplex virus 2 (HSV-2) genital infection. However, the role of interleukin-17A (IL-17A) production by CD4⁺ T cells in HSV-2 antiviral immunity is yet to be elucidated. Here we demonstrate that IL-17A plays an important role in enhancing antiviral T helper type 1 (T_h1) responses in the female genital tract (FGT) and is essential for effective protection conferred by HSV-2 vaccination. While IL-17A did not play a critical role during primary genital HSV-2 infection, seen by lack of differences in susceptibility between IL-17A-deficient (IL-17A^-/-) and wild-type (WT) C57BL/6 mice, it was critical for mediating antiviral responses after challenge/reexposure. Compared to WT mice, IL-17A^-/- mice (i) infected intravaginally and reexposed or (ii) vaccinated intranasally and challenged intravaginally demonstrated poor outcomes. Following intravaginal HSV-2 reexposure or challenge, vaccinated IL-17A^-/- mice had significantly higher mortality, greater disease severity, higher viral shedding, and higher levels of proinflammatory cytokines and chemokines in vaginal secretions. Furthermore, IL-17A^-/- mice had impaired T_h1 cell responses after challenge/reexposure, with significantly lower proportions of vaginal IFN-γ⁺ CD4⁺ T cells. The impaired T_h1 cell responses in IL-17A^-/- mice coincided with smaller populations of IFN-γ⁺ CD4⁺ tissue resident memory T (T_RM) cells in the genital tract postimmunization. Taken together, these findings describe a novel role for IL-17A in regulating antiviral IFN-γ⁺ T_h1 cell immunity in the vaginal tract. This strategy could be exploited to enhance antiviral immunity following HSV-2 vaccination.IMPORTANCE T helper type 1 (T_h1) immunity, specifically interferon gamma (IFN-γ) production by CD4⁺ T cells, is critical for protection against genital herpesvirus (HSV-2) infection, and enhancing this response can potentially help improve disease outcomes. Our study demonstrated that interleukin-17A (IL-17A) plays an essential role in enhancing antiviral T_h1 responses in the female genital tract (FGT). We found that in the absence of IL-17A, preexposed and vaccinated mice showed poor disease outcomes and were unable to overcome HSV-2 reexposure/challenge. IL-17A-deficient mice (IL-17A^-/-) had smaller populations of IFN-γ⁺ CD4⁺ tissue resident memory T (T_RM) cells in the genital tract postimmunization than did wild-type (WT) mice, which coincided with attenuated T_h1 responses postchallenge. This has important implications for developing effective vaccines against HSV-2, as we propose that strategies inducing IL-17A in the genital tract may promote more effective T_h1 cell immunity and better overall protection.

Keywords: CD4 T cell immunity; IL-17; genital tract immunity; herpes simplex virus; mucosal immunity; sexually transmitted diseases.

FIG 1

IL-17A^−/− mice demonstrate no significant difference in susceptibility to primary intravaginal HSV-2 infection. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 5 to 10/group) were infected intravaginally with sublethal doses of WT HSV-2 (10¹, 10², or 10³ PFU/mouse). Survival was monitored (A) and pathology scores were recorded on a scale of 0 to 5 (B) for 12 days postinfection. Data points superimposed on the x axes of panel B indicate mice without genital pathology, and the percentages represent maximum numbers of mice that demonstrated pathology. (C) Vaginal washes were collected daily for 6 days postinfection and HSV-2 shedding was assessed using a Vero cell-based assay. The bars in panel C indicate mean PFU per milliliter of shed virus. The dotted lines in panel C indicate the lower detection limit of the assay, and data points on this line indicate undetectable viral shedding. The percentages in panel C represent maximum numbers of mice that shed virus on any given day. Each symbol represents a single animal. The results are representative of those from two independent experiments.

FIG 2

Preexposed IL-17A^−/− mice are more susceptible to intravaginal HSV-2 reexposure. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 9/group) were intravaginally exposed to WT HSV-2 (10² PFU/mouse), and 6 weeks later, they were reexposed intravaginally with a higher dose of WT HSV-2 (5 × 10³ PFU/mouse). Survival was monitored (A) and pathology scores were recorded on a scale of 0 to 5 (B) for 12 days after reexposure. Significance in difference in survival (A) was calculated using the log rank (Mantel-Cox) test (*, P < 0.05). Data points superimposed on the x axes of panel B indicate mice without genital pathology, and the percentages represent maximum numbers of mice that demonstrated pathology. Vaginal washes were collected daily for 6 days after reexposure; HSV-2 viral shedding was calculated using a Vero cell-based assay (C and D), and cytokine and chemokine (IFN-γ, IL-6, TNF-α, RANTES, MCP-1, M-CSF, MIP-1α, and MIP-1β) concentrations were measured by multianalyte assays (E). The bars in panel C indicate mean PFU per milliliter of shed virus. The dotted lines in panel C indicate the lower detection limit of the assay, and data points on this line indicate undetectable viral shedding. The percentages in panel C represent maximum numbers of mice that shed virus on any given day. Data shown in panel D represent the viral loads (means ± SEMs) over 6 days. Each symbol represents a single animal. Data shown in panel E represent the means ± SEMs from two independent experiments, done in duplicate (n = 4 to 7/group). Data were analyzed using the unpaired, nonparametric, two-tailed Mann-Whitney test with 95% confidence interval, with the ROUT method used to identify outliers and the Bonferroni correction used to correct for multiple measures.*, P < 0.05; **, P < 0.01.

FIG 3

Preexposed IL-17A^−/− mice have lower proportions of IFN-γ⁺ T_h1 cells in the vaginal tract following intravaginal HSV-2 reexposure. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 4 to 6/group) were intravaginally exposed to WT HSV-2 (10² PFU/mouse) and 6 weeks later reexposed intravaginally with a higher dose of WT HSV-2 (5 × 10³ PFU/mouse). Vaginal tissue and lymph nodes were isolated at day 3 following reexposure, pooled, processed, stained with a panel of antibodies, and examined by flow cytometry. (A) CD4⁺ T cells were gated among total live CD3⁺ T cells in the vaginal tissue. (B) Isotype controls for intracellular staining of IL-17A and IFN-γ. (C) Vaginal cells were stimulated in vitro with a cell stimulation cocktail (CSC) containing Golgi inhibitors and PMA plus ionomycin for 16 h to detect intracellular staining of IL-17A and IFN-γ. (D) Intracellular staining for IFN-γ was used to examine the in vivo response to HSV-2 (without in vitro stimulation) and the differentiation of CD4⁺ T cells into T_h1 cells in the vaginal tract. (E) The differences in percentages and total cell numbers of IFN-γ-producing CD4⁺ T cells after HSV-2 reexposure in the vaginal tract were compared across five independent experiments (n = 4 to 6/group). (F and G) Intracellular staining for IFN-γ (F) and the differences in percentages and total cell numbers of IFN-γ-producing CD4⁺ T cells after HSV-2 reexposure in the lymph nodes (G) were compared across three independent experiments (n = 5 or 6/group). Data shown in panels E and G represent means ± SEMs. Significant difference in IFN-γ expression was calculated using the unpaired, two-tailed t test with 95% confidence interval. **, P < 0.01. ns, no significance.

FIG 4

Intranasally immunized IL-17A^−/− mice are more susceptible to intravaginal HSV-2 challenge. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 10/group) were immunized intranasally with TK⁻ HSV-2 (10² PFU/mouse) and 6 weeks later challenged intravaginally with WT HSV-2 (5 × 10³ PFU/mouse). Survival was monitored (A) and pathology scores were recorded on a scale of 0 to 5 (B) for 14 days postchallenge. Significance in difference in survival (A) was calculated using the log rank (Mantel-Cox) test (*, P < 0.05). Data points superimposed on the x axes of panel B indicate mice without genital pathology, and the percentages represent maximum numbers of mice that demonstrated pathology. Vaginal washes were collected daily for 6 days postchallenge; HSV-2 shedding was calculated using a Vero cell-based assay (C and D), and cytokine and chemokine (IFN-γ, IL-6, TNF-α, RANTES, MCP-1, M-CSF, MIP-1α, and MIP-1β) concentrations were measured by multianalyte assays (E). The bars in panel C indicate mean PFU per milliliter of shed virus. The dotted lines in panel C indicate the lower detection limit of the assay, and data points on this line indicate undetectable viral shedding. The percentages in panel C represent maximum numbers of mice that shed virus on any given day. Data shown in panel D represent the viral loads (means ± SEMs) over 6 days. Each symbol represents a single animal. Data shown in panel E represents means ± SEMs from two independent experiments, done in duplicate (n = 4 to 7/group). Data were analyzed using the unpaired, nonparametric, two-tailed Mann-Whitney test with 95% confidence interval, with the ROUT method used to identify outliers and the Bonferroni correction used to correct for multiple measures. *, P < 0.05; **, P < 0.01; ***, P <0.001.

FIG 5

Intranasally immunized IL-17A^−/− mice have lower proportions of IFN-γ⁺ T_h1 cells in the vaginal tract following intravaginal HSV-2 challenge. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 4 to 6/group) were immunized intranasally with TK⁻ HSV-2 (10² PFU/mouse) and 6 weeks later challenged intravaginally with WT HSV-2 (5 × 10³ PFU/mouse). Vaginal tissues and lymph nodes were isolated at day 3 postchallenge, pooled, processed, stained with a panel of antibodies, and examined by flow cytometry. Intracellular staining for IFN-γ was used to examine the in vivo response to HSV-2. (A and B) The differentiation of CD4⁺ T cells into T_h1 cells in the vaginal tract (A) and the differences in percentages and total numbers of IFN-γ-producing CD4⁺ T cells post-HSV-2 challenge in the vaginal tract (B) were compared across three independent experiments (n = 4 to 6/group). (C and D) The differentiation of CD4⁺ T cells into T_h1 cells (C) and the differences in percentages and total numbers of IFN-γ-producing CD4⁺ T cells after HSV-2 challenge in the lymph nodes (D) were compared across three independent experiments (n = 4 to 6/group). Data shown in panels B and D represent means ± SEMs. Significant difference in IFN-γ expression was calculated using the unpaired, two-tailed t test with 95% confidence interval. **, P < 0.01.

FIG 6

Phenotypic and functional characteristics of CD4⁺ T_RM cells localized in the vaginal tracts of WT and IL-17A^−/− mice following HSV-2 vaccination. OVX WT (C57BL/6) and IL-17A^−/− mice (n = 5 to 7/group) were vaccinated intravaginally with TK⁻ HSV-2 (10⁴ PFU/mouse), and 3 weeks later, vaginal tissues were collected, pooled, and processed. Vaginal cells were stimulated in vitro with a cell stimulation cocktail (CSC) containing Golgi inhibitors and PMA plus ionomycin for 16 h, stained with a panel of antibodies, and examined by flow cytometry. (A) CD4⁺ T cells were gated among total live CD3⁺ T cells in the vaginal tissue, and CD4⁺ T_RM cells were detected using surface markers CD44, CD103, CD69, and CD62L. CD4⁺ T_RM cells were defined as CD4⁺ CD44⁺ CD103⁻ CD69⁺ CD62L⁻. (B) The differences in percentages and total numbers of CD4⁺ T_RM cells postimmunization in the vaginal tract were compared across three independent experiments (n = 5 to 7/group). (C) IFN-γ⁺ CD4⁺ T_RM cells were further gated based on detection of surface markers associated with T_RM cells. (D) The differences in percentages and total numbers of IFN-γ⁺ CD4⁺ T_RM cells postimmunization in the vaginal tract were compared across three independent experiments (n = 5 to 7/group). Data shown in panels B and D represent means ± SEMs. Significant difference in IFN-γ expression was calculated using the unpaired, two-tailed t test with 95% confidence interval. *, P < 0.05; **, P < 0.01.