Immune Cell Targets of Infection at the Tick-Skin Interface during Powassan Virus Transmission

Abstract

Powassan virus (POWV) is a tick-borne flavivirus that can result in a severe neuroinvasive disease with 50% of survivors displaying long-term neurological sequelae. Human POWV cases have been documented in Canada, the United States, and Russia. Although the number of reported POWV human cases has increased in the past fifteen years, POWV remains one of the less studied human pathogenic flaviviruses. Ixodes ticks are the vectors for POWV, and the virus is transmitted to a host's skin very early during the tick feeding process. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. In our prior work, we examined the cutaneous immune gene expression during the early stages of POWV-infected Ixodes scapularis feeding. The present study serves to further investigate the skin interface by identifying early cell targets of infection at the POWV-infected tick feeding site. An in vivo infection model consisting of POWV-infected ticks feeding on mice for short durations was used in this study. Skin biopsies from the tick feeding sites were harvested at various early time points, enabling us to examine the skin histopathology and detect POWV viral antigen in immune cells present at the tick feeding site. The histopathology from the present study demonstrates that neutrophil and mononuclear cell infiltrates are recruited earlier to the feeding site of a POWV-infected tick versus an uninfected tick. This is the first report demonstrating that macrophages and fibroblasts contain POWV antigens, which suggests that they are early cellular targets of infection at the tick feeding site. These data provide key insights towards defining the complex interactions between the host immune response and early tick-mediated immunomodulation.

Fig 1. Histopathology of POWV-infected and uninfected Ixodes scapularis feeding sites, 3 and 6 hpi.

Biopsies from tick feeding sites were sectioned and stained with H&E as described in Materials and Methods section. (A). POWV-infected section at 3 hpi. (B). Uninfected section at 3 hpi. (C). POWV-infected section at 6 hpi. (D). Uninfected section at 6 hpi. In panel A, the red oval indicates the area with high levels of cellular infiltrates. In panels A, B, and D, the arrow is pointing to the tick hypostome. In panel C, the arrow indicates where the tick hypostome was located prior to being dislodged during sectioning. Scale bars represent 100 μm.

Fig 2. Histopathology of POWV-infected and uninfected Ixodes scapularis feeding sites, 12 and 24 hpi.

Biopsies from tick feeding sites were sectioned and stained with H&E as described in Materials and Methods section. (A). POWV-infected section at 12 hpi. (B). Uninfected section at 12 hpi. (C). POWV-infected section at 24 hpi. (D). Uninfected section at 24 hpi. In panels A, B, C, and D, the arrow is pointing to the tick hypostome. Scale bars represent 100 μm.

Fig 3. Immune cells detected at the POWV-infected Ixodes scapularis feeding sites, 3 and 6 hpi.

(A). Images of skin at the POWV-infected tick feeding site where macrophages are shown in orange and POWV-infected cells are shown in red. The F4/80 marker was used for macrophage detection. (B). Images of skin at the POWV-infected tick feeding site where fibroblasts are shown in orange and POWV-infected cells are shown in red. The vimentin marker was used for fibroblast detection. Scale bars represent 10 μm. DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear counterstaining. Open arrowheads indicate POWV-infected macrophages or POWV-infected fibroblasts. Closed arrowheads indicate other cells that are POWV-infected but that are not macrophages or fibroblasts.

Fig 4. Immune cells detected at the POWV-infected Ixodes scapularis feeding sites, 12 and 24 hpi.

(A). Images of skin at the POWV-infected tick feeding site where macrophages are shown in orange and POWV-infected cells are shown in red. The F4/80 marker was used for macrophage detection. (B). Images of skin at the POWV-infected tick feeding site where fibroblasts are shown in orange and POWV-infected cells are shown in red. The vimentin marker was used for fibroblast detection. Scale bars represent 10 μm. DAPI (4’,6-diamidino-2-phenylindole) was used for nuclear counterstaining. Open arrowheads indicate POWV-infected macrophages or POWV-infected fibroblasts. Closed arrowheads indicate immune cells that are POWV-infected but not macrophages or fibroblasts.