Immune Netw.  2015 Apr;15(2):91-99. 10.4110/in.2015.15.2.91.

Anti-herpes Activity of Vinegar-processed Daphne genkwa Flos Via Enhancement of Natural Killer Cell Activity

• Uyangaa E
• Choi JY
• Ryu HW
• Oh SR
• Eo SK

• KMID: 2168034
• DOI: http://doi.org/10.4110/in.2015.15.2.91

Abstract

Herpes simplex virus (HSV) is a common causative agent of genital ulceration and can lead to subsequent neurological disease in some cases. Here, using a genital infection model, we tested the efficacy of vinegar-processed flos of Daphne genkwa (vp-genkwa) to modulate vaginal inflammation caused by HSV-1 infection. Our data revealed that treatment with optimal doses of vp-genkwa after, but not before, HSV-1 infection provided enhanced resistance against HSV-1 infection, as corroborated by reduced mortality and clinical signs. Consistent with these results, treatment with vp-genkwa after HSV-1 infection reduced viral replication in the vaginal tract. Furthermore, somewhat intriguingly, treatment of vp-genkwa after HSV-1 infection increased the frequency and absolute number of CD3-NK1.1+NKp46+ natural killer (NK) cells producing interferon (IFN)-gamma and granyzme B, which indicates that vp-genkwa treatment induces the activation of NK cells. Supportively, secreted IFN-gamma was detected at an increased level in vaginal lavages of mice treated with vp-genkwa after HSV-1 infection. These results indicate that enhanced resistance to HSV-1 infection by treatment with vp-genkwa is associated with NK cell activation. Therefore, our data provide a valuable insight into the use of vp-genkwa to control clinical severity in HSV infection through NK cell activation.

Keyword

Daphne genkwa; Vinegar-processed flos; Herpes simplex virus; Genital infection; NK cells

MeSH Terms

Animals
Daphne*
Herpesvirus 1, Human
Inflammation
Interferons
Killer Cells, Natural*
Mice
Mortality
Simplexvirus
Therapeutic Irrigation
Ulcer
Interferons

Figure

• 

  Figure 1 Vp-genkwa treatment following mucosal HSV-1 infection reduces mortality and ameliorates clinical signs. (A and B) Effect of vp-genkwa treatment prior to HSV-1 infection. Groups of BALB/c mice received oral vp-genkwa twice, and were infected i.vag. with HSV-1 the next day. The infected mice were then examined daily until 15 dpi for survival (A) and for vaginal inflammation, neurological illness, and death (B). (C and D) Effect of vp-genkwa treatment after HSV-1 infection. Groups of BALB/c mice were infected i.vag. with HSV-1 and treated orally with vp-genkwa twice with a 1-day interval. The infected mice were examined daily until 15 dpi to assess survival (C), and vaginal inflammation, neurological illness, and death (D). Kaplan-Meiers survival curves were computed and analyzed using the chi-square test. Clinical severity was graded as follows: 0, no inflammation; 1, mild inflammation; 2, moderate swelling; 3, severe inflammation; 4, paralysis; 5, death. The graph of clinical scores represents the average clinical score of 10 mice per group.

• 

  Figure 2 Treatment with vp-genkwa after HSV-1 infection reduces viral replication. (A) Effect of vp-genkwa pretreatment on HSV-1 replication. (B) Effect of vp-genkwa posttreatment on HSV-1 replication. Groups of BALB/c mice (n=5) were infected i.vag. with HSV-1 after (A) and before (B) oral administration of vp-genkwa (10 mpk). Viral titers in vaginal lavages collected at 2 and 4 dpi were determined by plaque assay.

• 

  Figure 3 Enhancement of NK cell activity in vp-genkwa-treated mice following HSV-1 infection. (A) The frequency and absolute number of NK cells in the spleen of mice treated with vp-genkwa. The splenocytes were prepared from mice that were pre- and post-treated with vp-genkwa (10 mpk) 3 dpi, and used to enumerate NK cells (CD3+NK1.1+NKp46+) using FACS analysis. The left graph denotes the average percentage of NK cells in the spleen, and the right graph shows the absolute number of NK cells in the spleens of four mice per group. (B and C) The frequency and absolute number of NK cells producing IFN-γ and granzyme B. The splenocytes were prepared from mice pre- and post-treated with vp-genkwa 3 dpi and stimulated with PMA plus ionomycin. The frequency and absolute number of IFN-γ- (B) and granzyme B (C; GrB)-producing cells in NK cells (CD3-NK1.1+NKp46+) were determined by intracellular FACS staining. *,p<0.05; **, p<0.01; n.s., not significant between the indicated groups.

• 

  Figure 4 Treatment with vp-genkwa after HSV-1 infection increases vaginal IFN-γ. Groups of mice were treated with vp-genkwa (10 mpk) before (pre-treated) and after HSV-1 infection (post-treated). The levels of IFN-γ protein in vaginal lavages collected at 3 dpi were determined by ELISA. *, p<0.05; n.s., not significant between the indicated groups.

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