Type I interferon-dependent CCL4 is induced by a cGAS/STING pathway that bypasses viral inhibition and protects infected tissue, independent of viral burden

Abstract

Type I interferons (T1-IFN) are critical in the innate immune response, acting upon infected and uninfected cells to initiate an antiviral state by expressing genes that inhibit multiple stages of the lifecycle of many viruses. T1-IFN triggers the production of Interferon-Stimulated Genes (ISGs), activating an antiviral program that reduces virus replication. The importance of the T1-IFN response is highlighted by the evolution of viral evasion strategies to inhibit the production or action of T1-IFN in virus-infected cells. T1-IFN is produced via activation of pathogen sensors within infected cells, a process that is targeted by virus-encoded immunomodulatory molecules. This is probably best exemplified by the prototypic poxvirus, Vaccinia virus (VACV), which uses at least 6 different mechanisms to completely block the production of T1-IFN within infected cells in vitro. Yet, mice lacking aspects of T1-IFN signaling are often more susceptible to infection with many viruses, including VACV, than wild-type mice. How can these opposing findings be rationalized? The cytosolic DNA sensor cGAS has been implicated in immunity to VACV, but has yet to be linked to the production of T1-IFN in response to VACV infection. Indeed, there are two VACV-encoded proteins that effectively prevent cGAS-mediated activation of T1-IFN. We find that the majority of VACV-infected cells in vivo do not produce T1-IFN, but that a small subset of VACV-infected cells in vivo utilize cGAS to sense VACV and produce T1-IFN to protect infected mice. The protective effect of T1-IFN is not mediated via ISG-mediated control of virus replication. Rather, T1-IFN drives increased expression of CCL4, which recruits inflammatory monocytes that constrain the VACV lesion in a virus replication-independent manner by limiting spread within the tissue. Our findings have broad implications in our understanding of pathogen detection and viral evasion in vivo, and highlight a novel immune strategy to protect infected tissue.

Fig 1. T1-IFN is not expressed within infected cells in vitro, but T1-IFN expression is required in vivo.

(A,B) Expression of IFN-β from a murine keratinocyte (A) or macrophage (B) cell line measured by RT-qPCR 6 hpi (left) or ELISA 24 hpi (right). (C-F) Tissue pathology of the ear pinnae after intradermal infection with VACV; ear thickness (C), lesion size (D), tissue loss (E), or total tissue damage (F), n = 8 mice per group. (G) Representative pictures of ear pinnae following intradermal VACV infection. (H,I) Expression of T1-IFN in ear tissue of wild-type mice at the indicated time post-infection measured by RT-qPCR (H, n = 3 naïve, 4 VACV-infected mice per group), or 5 dpi by ELISA (I).

Fig 2. A positive feedback loop is required for T1-IFN production in response to VACV.

(A) Expression of T1-IFN subtypes in ear tissue of wild-type mice 5 dpi measured by RT-qPCR. (B-D) Expression of early T1-IFNs in ear tissue of mice 5 dpi measured by RT-qPCR.

Fig 3. cGAS sensing of VACV-infected cells induces T1-IFN to limit local tissue pathology.

(A-D) Expression of IFN-β in ear tissue of mice 5 dpi measured by RT-qPCR. (E) Cryosections of ear tissue from wild-type mice 5 dpi with VACV-OVA stained with Wheat-Germ Agglutinin (Grey) to visualize cell membranes and DAPI (Blue) for nuclei. RNA fluorescence in situ hybridization was used to detect viral RNA (OVA-Green) and cellular IFN-β (Red). (F,G) Expression of IFN-β in ear tissue of mice 5 dpi measured by RT-qPCR (F) or ELISA (G). (H-K) Tissue pathology of the ear pinnae after intradermal infection with VACV; ear thickness (H), lesion size (I), tissue loss (J), or total tissue damage (K), n = 9–10 mice per group. (L) Representative pictures of ear pinnae following intradermal VACV infection.

Fig 4. T1-IFN does not control VACV replication in the skin.

(A) Expression of antiviral ISGs in ear tissue of wild-type mice 5 dpi compared to naïve controls measured using RT-qPCR array. (B) Expression of induced ISGs from (A) in ear tissue of wild-type and IFNαR^-/- mice 5 dpi measured using RT-qPCR array. (C) VACV titer in ears of mice 5 days post-intradermal infection with VACV. Dotted line indicates initial inoculum. (D) VACV titer in ears of mice at the indicated day post-infection with VACV by scarification, n = 5 mice per group.

Fig 5. T1-IFN induces ccl4 to recruit protective monocytes to the site of infection.

(A, B, C) Expression of cytokines and chemokines in ear tissue of mice 5 dpi measured using RT-qPCR array. Volcano plots demonstrate statistically significant points if above the p = 0.05 line. Gene expression changes in wild-type mice in response to VACV, with genes that are upregulated >2-fold by VACV infection in red in the upper right hand quadrant and genes that are downregulated > 2-fold by VACV infection in blue in the upper left hand quadrant (A). Gene expression changes in IFNαR^-/- mice in response to VACV, with genes that are upregulated >2-fold by VACV infection in red in the upper right hand quadrant and genes that are downregulated > 2-fold by VACV infection in blue in the upper left hand quadrant (B). Gene expression changes in VACV-infected wild-type mice compared to VACV-infected IFNαR^-/- mice with genes that are upregulated >2-fold in VACV-infected IFNαR^-/- mice relative to wild-type VACV infected in red in the upper right hand quadrant and genes that are downregulated > 2-fold in VACV-infected IFNαR^-/- mice relative to wild-type VACV infected in blue in the upper left hand quadrant (C). Ccl4 (in green) is the only gene that is upregulated upon VACV infection but is downregulated in IFNαR^-/- mice relative to wild-type mice. n = 6 naïve, 9 VACV-infected mice per group. (D) Expression of ccl4 in ear tissue of VACV-infected mice 5 dpi measured by RT-qPCR. (E) Chemokine receptor expression on inflammatory monocytes and Ly6G⁺ cells recruited into VACV-infected ears 5 dpi. Representative flow cytometry plots (left) and proportion of cells expressing each receptor (right). (F) Myeloid cell populations in VACV-infected ears 5 dpi measured by flow cytometry. Representative flow cytometry plots (left) and absolute number of cells (right). (G) Recruitment of inflammatory monocytes to the ear of uninfected, VACV-infected mice 5 dpi, or VACV-infected mice treated systemically with the CCR5 antagonist Maraviroc, 5dpi. (H) Immunofluorescence microscopy of ears 5 dpi with VACV-NP-S-eGFP reveal monocyte localization (Ly6C-Red) in relation to VACV-infected cells (GFP-Green). (I) Percentage of GFP⁺ cells in the ear 5dpi with VACV Early-GFP or VACV Late-GFP measured by flow cytometry. (J) Percentage of GFP⁺ cells in the ear 5dpi with VACV-NP-S-eGFP measured by flow cytometry.

Fig 6. ccl4 expression restores inflammatory monocyte recruitment and ameliorates tissue damage.

(A) Schematic of rVACV expressing ccl4 and mKate2. (B) Single step growth curve of wild-type VACV and VACV-CCL4 in 143B cells. (C) Multi-step growth curve of wild-type VACV and VACV-CCL4 in 143B cells. (D) Detection of mKate2 expression in 143B cells 6 hpi assayed using flow cytometry. (E) Expression of murine ccl4 in 143B cells 6 hpi measured by RT-qPCR. (F) Secretion of murine CCL4 from 143B cells 24 hpi measured using ELISA. (G) Monocyte recruitment into VACV-infected ears of IFNαR^-/- mice 5 dpi assayed by flow cytometry, n = 4 mice per group. (H) Ly6G⁺ cell recruitment into VACV-infected ears of IFNαR^-/- mice 5 dpi measured by flow cytometry, n = 4 mice per group. (I) Immunofluorescence microscopy of IFNαR^-/- ears 5 dpi with VACV-CCL4 or VACV-mCherry for monocyte localization (Ly6C-Blue) in relation to VACV-infected cells (mKate/mCherry-Red). (J-L) Tissue pathology of the ear pinnae of IFNαR^-/- mice after intradermal infection with VACV; ear thickness (J), lesion size (K), or total tissue damage (L), n = 6–7 mice per group. (M) Representative pictures of ear pinnae of IFNαR^-/- mice following intradermal VACV infection.