Abstract
Purpose: :
The autophagy response induced by HSV-1 is antagonized by the neurovirulence gene product, ICP34.5, which binds to the essential autophagy protein Beclin1 (Atg6) via the BBD. Because eyes infected with BBD-deficient HSV-1 (Δ68H) do not develop retinitis whereas eyes infected with the marker-rescued counterpart (Δ68HR) do, the purpose of this study was to determine if lack of the BBD affects the immune cell response during HSV-1 infection of the eye.
Methods: :
Female BALB/c mice were injected with 1×104 PFU of Δ68H or Δ68HR via the AC route. Injected eyes, spleen and superficial cervical and submandibular lymph nodes ipsilateral to the site of injection were collected at several times post inoculation (p.i.), single cell suspensions were prepared, and RBC were lysed by ammonium chloride lysis buffer (ACK) treatment. Cell preparations were incubated with FITC labelled anti-L3T4 (CD4), anti-Ly-3.2 (CD8), anti-pan-NK (DX-5) or anti-Mac-1(PharMingen) and examined by flow cytometry. RNA was extracted from additional HSV-1-injected eyes and from control eyes and 84 viral immune response genes were detected by real time PCR.
Results: :
At day 3 and 4 p.i., more Mac-1+ macrophage/microglia and DX5+ NK cells were observed in the injected eyes of Δ68H infected mice than in that of Δ68HR infected mice (Mac-1+ cells per eye: 1.7×105 vs 4.6×104 - day 3 p.i., 7.2×105 vs 5.0×104 - day 4 p.i., DX5+ cells: 9×104 vs 1.9×104 - day 3 p.i., 5.1×104 vs 3.2x104 - day 4 p.i). At day 2 p.i., more Mac-1+ cells were also detected in the spleens and lymph nodes of Δ68H infected mice than in that of Δ68HR infected mice. However, by day 5 p.i., more Mac-1+ cells were observed in Δ68HR injected eyes than from Δ68H injected eyes. There was no significant difference in either CD4+ and CD8+ cells between Δ68HR injected eyes and Δ68H injected eyes. Among 84 viral immune response genes detected by real time PCR, 43 genes were upregulated 3 or more fold during Δ68HR or Δ68H infection compared with medium-injected controls. However, several genes including imflammasome (NLRP3), chemokines (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11), IL12a, CD40 and TRIM25 were more upregulated in Δ68H injected eyes than in Δ68HR injected eyes.
Conclusions: :
These results suggest that during HSV-1 infection of the eye, BBD inhibits autophagy and the early innate immune response mediated by macrophage/microglia and NK cells in the injected eye which, in turn, allows increased virus replication and retinal damage.
Keywords: herpes simplex virus • immunomodulation/immunoregulation • retinitis