Interferon lambda restricts herpes simplex virus skin disease by suppressing neutrophil-mediated pathology

Abstract

Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1^-/-), both the IFN-λ and the IFN-αβ receptor (Ifnar1^-/- Ifnlr1^-/-), or IFN-λ cytokines (Ifnl2/3^-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions, independent of a direct effect on viral load. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity, and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils or blocking CXCL9 protected against severe HSV-1 skin lesions in Ifnlr1^-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection, and suggest potential applications for IFN-λ in treating viral skin infections.

Figure 1.. IFN-λ signaling protects against severe HSV skin disease without affecting viral replication.

A-E. 8–12 week-old male and female WT and Ifnlr1^−/− mice were infected with 10⁶ FFU of HSV-1 strain NS. A. Dermatome skin lesions were photographed 6 dpi. B. HSV-1 skin lesions were measured from photographs 6, 8, and 10 dpi using imageJ. C. Skin lesion severity was categorized based on 6 dpi lesion area. D. Skin viral loads were measured 2–10 dpi by focus-forming assay. E. Viral loads in ipsilateral and contralateral dorsal root ganglia (L2-L5 pooled for each mouse) were measured by qPCR. F-G. 8–12 week-old male and female WT and Ifnlr1^−/− mice were infected with 10³ FFU of HSV-2 strain 333. F. Dermatome lesions photographed 8 dpi. G. HSV-2 skin lesions were measured from photographs 6 and 8 dpi using ImageJ. H. 8–12 week-old female WT and Ifnlr1^−/− mice were infected with 10³ FFU of HSV-2 strain 333 intravaginally and HSV-2 vaginal disease was scored 6–8dpi as follows: 1: mild redness and swelling, 2: visible ulceration and fur loss, 3: severe ulceration and signs of sickness, 4: hindlimb paralysis, 5: moribund/dead. Differences in lesion area and viral load were compared by Mann-Whitney U test. Differences in categorical skin disease were compared by Cochran-Armitage test. P values are reported with P < 0.05 considered to be statistically significant.

Figure 2.. The protective effect of IFN-λ in the skin does not require IFN-α/β signaling.

8–12 week-old Ifnar1^−/− and Ifnar1^−/− Ifnlr1^−/− DKO male and female mice were infected with 10⁶ FFU of HSV-1. A. Lethality was monitored daily (n=15 Ifnar1^−/− and 10 DKO mice). B. Dermatome skin lesions were photographed 4 dpi. C. Skin lesion areas were measured from photographs 4 dpi using ImageJ. D. Skin lesion severity was categorized based on 4 dpi lesion area. E. Skin viral loads were measured 2 and 4 dpi by focus-forming assay. Survival differences were compared by Mantel-Cox Log-Rank test. Differences in lesion area and viral load were compared by Mann-Whitney U test and differences in categorical skin disease were compared by Cochran-Armitage test. P values are reported with P < 0.05 considered to be statistically significant.

Figure 3.. IFN-λ cytokines protect against severe HSV-1 skin disease.

A-D. 8–12 week-old WT, Ifnl2/3^−/−, and Ifnk^−/− male and female mice were infected with 10⁶ FFU of HSV-1. A. Dermatome skin lesions were photographed 6 dpi. B. Skin lesion areas were measured from photographs 6 dpi using ImageJ. C. Skin lesion severity was categorized based on 6 dpi lesion area. D. Skin viral loads were measured 6 dpi by FFA. E-G. 8 week-old WT and Ifnl2/3^−/− male and female mice were lethally irradiated and transfused with 10⁷ bone marrow cells from WT or Ifnl2/3^−/− donors. 10 weeks later, mice were infected with 10⁶ FFU of HSV-1. E. Skin lesion areas were measured from photographs 6 dpi using ImageJ. F. Skin lesion severity was categorized based upon 6 dpi lesion area. G. Skin viral loads were measured 6 dpi by FFA. H-I. 8–12 week-old WT male and female mice were depilated and then topically treated with 5 µg of recombinant murine IFN-λ3 or PBS. 24 hours later, mice were infected with 10⁶ FFU of HSV-1 at the treated site. H. Skin lesion areas were measured from photographs 6 dpi using ImageJ. I. Skin viral loads were measured 6 dpi by FFA. Differences in lesion area and viral load were compared by Mann-Whitney U test and differences in categorical skin disease were compared by Cochran-Armitage test. P values are reported with P < 0.05 considered to be statistically significant.

Figure 4.. IFN-λ signaling in both keratinocytes and leukocytes is necessary to restrict severe HSV-1 skin disease.

8–12 week-old male and female WT, Ifnlr1^−/−, K14-Cre-Ifnlr1^−/−, and Vav-Cre-Ifnlr1^−/− mice were infected with 10⁶ FFU of HSV-1. A. Dermatome skin lesions photographed were 6 dpi. B. Skin lesion areas were measured from photographs 6 dpi using ImageJ. C. Skin lesion severity was categorized based on 6 dpi lesion area. D. Skin viral loads were measured 6 dpi by FFA. Differences in lesion area and viral load were compared by Mann-Whitney U test and differences in categorical skin disease were compared by Cochran-Armitage test. P values are reported with P < 0.05 considered to be statistically significant.

Figure 5.. IFN-λ signaling in keratinocytes differentially regulates CXCL9 induction.

Primary keratinocytes were prepared from WT, Ifnlr1^−/−, Ifnar1^−/−, and Ifnar1^−/− Ifnlr1^−/− DKO mice and treated with IFN-λ3 (5 ng/mL), IFN-β (5 ng/mL), or media alone for 24 hours. A. RNA was extracted and IFN-stimulated gene expression was measured by qRT-PCR. Ifit1 expression is shown relative to ActB (housekeeping gene) and normalized to expression in media-treated WT cells. Results represent 6 samples from 2 independent experiments. B-F. RNA from 3 samples per genotype per treatment was analyzed by RNAseq (NovaSeq6000S4 XP Paired End 2×100). B. Principal component analysis for all analyzed samples. C. Volcano plots showing differentially expressed genes after IFN-λ or IFN-β treatment, compared to media-only treated keratinocytes. Differentially expressed genes were defined as having a |Log₂Fold Change| >1 and an FDR p-value ≤ 0.05. D. Venn diagram showing DEGs induced by IFN-λ and IFN-β in WT or knockout keratinocytes. E. Table of IFN-λ-specific DEGs, their Log₂Fold Change, and FDR p-value for IFN-λ and IFN-β-treated WT keratinocytes. F. Volcano plot showing DEGs after IFN-λ treatment compared to IFN-β treatment in WT and receptor knockout keratinocytes.

Figure 6.. IFN-λ signaling limits neutrophil abundance and severe skin pathology following HSV-1 infection.

8–12 week-old male and female WT and Ifnlr1^−/− mice were infected with 10⁶ FFU of HSV-1. A-B. Dermatome lesions and adjacent healthy skin were collected 4–8 dpi and analyzed by flow cytometry. A. Representative histogram of WT and Ifnlr1^−/− lesions showing Ly6G+ neutrophil gating strategy (CD45+, CD11b+, Ly6G+) at 6 dpi. B. Frequency of neutrophils (Ly6G+ out of CD45+ CD11b+ live cells) for WT and Ifnlr1^−/− dermatome lesions and adjacent healthy skin. Significant differences in neutrophil frequency were determined using an unpaired t test. P values are reported with P < 0.05 considered to be statistically significant. C-D. Inoculated flank skin was collected at 6 dpi and serial sections of the same skin lesion were processed for histology. C. H&E staining; black arrow denotes a neutrophilic pustule, red arrow denotes diffuse neutrophilic infiltrate. D. Anti-HSV-1 immunohistochemistry; viral antigen staining is denoted by black arrows. Scale bars are 100 µm.

Figure 7.. IFN-λ signaling suppresses neutrophil-mediated pathology to limit HSV-1 skin disease.

A. Experimental design for neutrophil depletions B-D. WT and Ifnlr1^−/− mice were infected with 10⁶ FFU of HSV-1. 0, 2, and 4 dpi mice were injected intraperitoneally with PBS, 250 µg of αIsotype control (IgG2a), or 250 µg of αLy6G (Clone 1A8). B. Spleens were harvested 6 dpi and analyzed by flow cytometry to confirm neutrophil depletion. C. Skin lesions were photographed 6 dpi and lesion areas measured ImageJ. D. Skin lesion severity was categorized based on 6 dpi lesion area. E-G. WT and Ifnlr1^−/− mice were infected with 10⁶ FFU of HSV-1 and 6 dpi skin lesions were photographed and analyzed via flow cytometry. E. Representative plots and gating strategy for neutrophil phenotyping markers LFA-1 and CD63 (% of CD45+ CD11b+ Ly6G+). F. Frequency of CD63+ neutrophils. G. Frequency of LFA-1+ CD63- neutrophils. Differences in lesion area and viral load were compared by Mann-Whitney U test and differences in categorical skin disease were compared by Cochran-Armitage test. Neutrophil frequencies were compared by unpaired t test. P values are reported with P < 0.05 considered to be statistically significant.

Figure 8.. IFN-λ signaling in neutrophils contributes to protection from severe HSV-1 skin disease.

8–12 week-old male and female WT, Ifnlr1^−/−, LysM-Cre-Ifnlr1^−/−, and Mrp8-Cre-Ifnlr1^−/− mice were infected with 10⁶ FFU of HSV-1. A. Dermatome skin lesions were photographed 6 dpi. B. Skin lesion areas were measured from photographs 6 dpi using image J. C. Skin lesion severity was categorized based on 6 dpi lesion area. Differences in lesion area and viral load were compared by Mann-Whitney U test. Differences in categorical skin disease were compared by Cochran-Armitage test. P values are reported with P < 0.05 considered to be statistically significant.

Figure 9.. IFN-λ signaling in keratinocytes restricts CXCL9 production to limit neutrophil recruitment and HSV-1 skin disease.

8–12 week-old male and female mice were infected with 10⁶ FFU of HSV-1 and skin lesions were analyzed 6 dpi. A. CXCL9 was measured by ELISA in homogenates of lesional skin or adjacent healthy skin from WT and Ifnlr1^−/− mice. B-D. WT and K14-Cre-Ifnlr1^−/− mice were infected with HSV-1 and injected intraperitoneally with anti-CXCL9 (300µg) or PBS 0, 2, and 4 dpi. Skin lesion areas were measured from photographs 6 dpi (B) and lesion severity categorized (C). D. Neutrophil frequency in skin lesions was measured by flow cytometry 6 dpi. Differences in lesion area and viral load were compared by Mann-Whitney U test. Differences in categorical skin disease were compared by Cochran-Armitage test. Differences in CXCL9 concentration and neutrophil frequency were compared by unpaired t test. P values are reported with P < 0.05 considered to be statistically significant.