Il4ra-independent vaginal eosinophil accumulation following helminth infection exacerbates epithelial ulcerative pathology of HSV-2 infection

Abstract

How helminths influence the pathogenesis of sexually transmitted viral infections is not comprehensively understood. Here, we show that an acute helminth infection (Nippostrongylus brasiliensis [Nb]) induced a type 2 immune profile in the female genital tract (FGT). This leads to heightened epithelial ulceration and pathology in subsequent herpes simplex virus (HSV)-2 infection. This was IL-5-dependent but IL-4 receptor alpha (Il4ra) independent, associated with increased FGT eosinophils, raised vaginal IL-33, and enhanced epithelial necrosis. Vaginal eosinophil accumulation was promoted by IL-33 induction following targeted vaginal epithelium damage from a papain challenge. Inhibition of IL-33 protected against Nb-exacerbated HSV-2 pathology. Eosinophil depletion reduced IL-33 release and HSV-2 ulceration in Nb-infected mice. These findings demonstrate that Nb-initiated FGT eosinophil recruitment promotes an eosinophil, IL-33, and IL-5 inflammatory circuit that enhances vaginal epithelial necrosis and pathology following HSV-2 infection. These findings identify a mechanistic framework as to how helminth infections can exacerbate viral-induced vaginal pathology.

Keywords: HSV-2; IL-33; IL-5; Nippostrongylus brasiliensis; eosinophils; epithelial ulceration; helminths; systemic immunity; vagina.

Graphical abstract

Figure 1

Influence of N. brasiliensis exposure on uncolonized FGT, increase in FGT eosinophils following Nb exposure (A) Female mice were hormone-synchronized 7 days prior to Nb infection. (B) At day 9 post-Nb infection (Nb 9dpi), levels of IL-33, IL-4, and IL-5 in FGT homogenates or lavages were assessed by ELISA or Luminex. 3) of neutrophils (CD11b⁺Ly-6G⁺), Ly-6C^hi monocytes (CD11b⁺Ly-6C^hi), macrophages (CD11b⁺F480⁺), and eosinophils (CD11b⁺Siglec-F⁺SSC^hi) in the FGT of naive and Nb-infected mice. (F) Mean fluorescence intensity (MFI) of Siglec-F, CD11b, Gr-1, CD62L, and CD49d on lung (green, blue) and FGT (pink) eosinophils at Nb 9 dpi. Dotted line represents the MFI of CD45⁺ FGT cells. Data are representative of two independent experiments with 4–5 mice per group (mean ± SEM). Statistical significance was calculated by Mann-Whitney t test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001.

Figure 2

Prior Nb exposure results in earlier and exacerbated HSV-2 pathology (A) 7 days post-Nb infection, mice were infected intravaginally with 5 × 10⁵ plaque-forming units (PFUs) HSV-2. (B) Viral progression was determined by daily pathology scoring. (C) Viral shedding (PFU/mL) was measured by plaque assay of day 3 and 6 vaginal washes. (D) At day 6 post-HSV-2 infection, vaginal tissue was analyzed by H&E staining. Representative images (n = 4) were taken at ×50 and ×400 magnification. Magnified areas are indicated by yellow boxes. HSV-2 ulcerated epithelium is indicated by black dotted lines and qualified as percentage (%) of ulcerated epithelium. (E) At day 3 post-HSV-2 infection, vaginal tissue (n = 4) was analyzed by immunofluorescent (IF) staining for β-catenin (Bcat; white), α-smooth muscle actin (SMA; red), hoechst 33342 (blue), and c-Casp-3 (green). Yellow boxes identify magnified areas. Yellow arrowheads identify “necrotic” cells i.e., large Bcat-filled nuclei that are c-Casp-3 negative. (F) At day 3 post-viral infection, vaginal epithelial cells were isolated and analyzed by flow cytometry (CD45⁻CD90⁻EPCAM⁺): MFI of MHCI and MHCII on vaginal epithelial cells from virus-only and co-infected mice. Dotted line represents the MFI of uninfected epithelial cells. (G) Levels of IFN-γ in vaginal lavages at day 2 post-HSV-2 infection, determined by ELISA. (H) At day 2 post-viral infection, levels of STAT1 (87 kDa) and GAPDH (37 kDa) were determined in HSV-2-only (H) and co-infected (N + H) FGT homogenates, by western blot. Density of STAT1 was measured relative to GAPDH. (I and J) (I) Vaginal IL-33 measured by ELISA (day 2) and (J) IF staining of vaginal tissue (day 3; n = 4). Data are representative of two independent experiments with 4–6 mice per group (mean ± SEM). Statistical significance was calculated by two-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons and Mann-Whitney t test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ns, not significant.

Figure 3

Prior Nb exposure results in elevated inflammation, eosinophil infiltration, and ILC2 presence in genital tissue, following HSV-2 vaginal infection (A) At day 9 post-Nb infection (Nb only) and day 3 post-HSV-2 infection (HSV-2 only and Nb + HSV-2), numbers of FGT myeloid cells were analyzed by flow cytometry. (B) Vaginal tissue was analyzed by Sirius red staining. Representative images (n = 4) of virus-induced ulcers were taken at ×400 magnification. (C) Black boxes indicate magnified sections of (Ci) vaginal stroma (St) and (Cii) epithelium (Ep), taken at ×1,000 magnification. White arrows indicate eosinophilic cell infiltration and migration in ulcerated vaginal tissue. Black arrowheads indicate eosinophil presence in vaginal epithelial layer. (D) At day 2 post-HSV-2 infection, levels of MBP (25 kDa) and GAPDH (37 kDa) were measured in HSV-2-only and Nb + HSV-2 FGT homogenates. Density of MBP was measured relative to that of GAPDH. (E) MFI of Siglec-F, CD11b, Ly6G, Ly6C, CD62L, and CD49d on FGT eosinophils in Nb 9 dpi and Nb + HSV-2 mice. (F) Levels of IL-5 in vaginal lavages at day 2 post-virus infection, determined by Luminex. Dotted line represents LLOQ. (G) Frequencies (mean ± SEM) and numbers of Lin⁻IL-7Rα⁺ICOS⁺ST2⁺ cells (ILC2s) in the FGT of Nb 9 dpi, HSV-2-only and Nb + HSV-2 mice. (H and I) Frequency and number of (H) Lin⁻IL-7Rα⁺ICOS⁺ cells and (I) Ly6C^hi monocytes that are IL-5+ in the FGT, determined by flow cytometry. Data are representative of two independent experiments with 5–6 mice per group (mean ± SEM). (J) Nb-infected mice were treated with 20 μg α-IL-5 or isotype control, on day −2, 0, and 2 post-HSV-2 infection. Viral progression was determined by daily pathology scoring and HSV-2 ulcerated epithelium was qualified as percentage (%) of ulcerated epithelium. At day 6 post-HSV-2 infection, numbers of eosinophils in the iLN were determined by flow cytometry. Data are representative of two independent experiments with 4 mice per group (mean ± SEM). Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons and Mann-Whitney t test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001.

Figure 4

Nb-exacerbated HSV-2 pathology and FGT eosinophil infiltration is Il4ra independent WT and Il4ra^−/− mice were infected with HSV-2 following Nb exposure as previously described. (A) HSV-2 progression was determined by daily pathology scoring. (B) Viral shedding was measured by plaque assay of day 6 vaginal washes. (C) Genital levels of IL-5 and IFN-γ at day 2 post-HSV-2 infection, determined by Luminex and ELISA, respectively. Dotted line represents LLOQ of Luminex analysis. At 6 dpi, vaginal tissue was analyzed by H&E staining. (D) Representative sections (n = 3–4), displaying ulceration and inflammation of vaginal tissue. Images were taken at ×50 magnification. HSV-2-ulcerated vaginal epithelium is indicated by black dotted lines and qualified as percentage (%) of ulcerated epithelium. (E) Representative Sirius-red-stained sections (n = 3–4) of virus-induced (Ei) epithelial ulcers and (Eii) stromal inflammation. Black arrows indicate eosinophil presence in the vaginal epithelial layer. Images were taken at ×400 and ×1,000 magnification. (F) Numbers (×10³) of FGT eosinophils in WT and Il4ra^−/− co-infected mice compared with HSV-2-only controls. Data are representative of two independent experiments with 3–6 mice per group (mean ± SEM). Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001.

Figure 5

Epithelial IL-33-induced FGT eosinophil inflammation in the absence of Il4ra signaling (A) WT and Il4ra^−/− mice were treated intravaginally with 20 μg papain for 3 days. The next day, FGT cells were analyzed by flow cytometry. (B and C) Numbers of FGT (B) eosinophils (x10³) and (C) ILC2s (×10²) in papain-treated and untreated WT and Il4ra^−/− mice. Data are representative of two experiments with 3–4 mice per group (mean ± SEM). (D) To inhibit vaginal IL-33, co-infected mice were treated intravaginally with helminth-derived HpARI (day −3 to 3 post-HSV-2). (E) HSV-2 progression in WT and Il4ra^−/− co-infected HpARI-treated mice and BSA-treated controls was determined by daily pathology scoring (^∗WT Nb + HSV-2 versus WT Nb + HSV-2 + HpARI, ^#Il4ra^−/− Nb + HSV-2 versus Il4ra^−/− Nb + HSV-2 + HpARI). (F) Representative H&E-stained sections (n = 4) of ulcerated vaginal tissue. Images were taken at ×50 magnification. HSV-2-ulcerated vaginal epithelium is indicated by black dotted lines and qualified as percentage (%) of ulcerated epithelium. Data are representative of two independent experiments with 4–6 mice per group. Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons and Mann-Whitney t test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001, ns, not significant.

Figure 6

Depletion of eosinophils rescues HSV-2 pathology in co-infected mice (A) Co-infected mice were treated with 20 μg α-Siglec-F or isotype control antibody at days 5, 7, and 9 post-Nb infection. (B) Viral progression in HSV-2-only, α-Siglec-F-, and isotype-treated mice was determined by daily pathology scoring (^∗Nb + HSV-2 isotype control versus Nb + HSV-2 α-Siglec-F; ^#HSV-2 only versus Nb + HSV-2 isotype control). (C) Viral shedding was measured by plaque assay of days 3 and 6 vaginal washes. (D) Representative H&E-stained sections (n = 3) of vaginal tissue at day 6 post-HSV-2 infection. Images were taken at ×50 magnification. Ulcerated vaginal epithelium is indicated by black dotted lines and qualified as percentage (%) of ulcerated epithelium. Yellow boxes indicate magnified sections in (G). (E) IF analysis of day 6 post-HSV-2 vaginal tissue. White boxes indicate magnified sections. (F) Numbers of FGT eosinophils at day 3 post-virus infection in isotype control and α-Siglec-F-treated Nb + HSV-2 mice compared with Nb 9dpi and HSV-2 only controls. (G) Representative magnified sections (n = 3) of Sirius-red-stained vaginal tissue at day 6 post-HSV-2. Images were taken at ×1,000 magnification. (H) Numbers of FGT ILC2s at day 3 post-HSV-2 infection. (I) At day 2 post-HSV-2 infection, lavage or FGT levels of IL-33 and IFN-γ were measured by ELISA. Data are representative of two independent experiments with 3 mice per group (mean ± SEM). Statistical significance was calculated by two-way ANOVA with Bonferroni correction for multiple comparisons and Mann-Whitney t test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001.

Figure 7

Helminth-induced FGT eosinophils mediate exacerbated vaginal pathology during subsequent lytic viral infection (A) We hypothesize that Nb-induced infiltration of eosinophils and inflammatory monocytes in uncolonized FGT, results in epithelial stress and release of epithelial “alarmin” IL-33, which supports the local activation of ILC2 and release of IL-5, essential for eosinophil survival. (B) The consequence of this during a subsequent virus infection was exacerbated pathology caused by virus-induced epithelial necrosis. Type 2 immunity and inflammation is amplified following HSV-2 infection: (1) eosinophil release of granule proteins promotes epithelial necrosis and (2) further release of IL-33, which (3) expands ILC2s that are a source of IL-5, along with infiltrating monocytes. Impaired anti-viral IFN-γ responses associated with eosinophil accumulation in the FGT. (C) During late-stage disease, epithelial ulceration is increased, and tissue integrity is lost. Created with BioRender.com.