Immune Netw.  2012 Oct;12(5):196-206. 10.4110/in.2012.12.5.196.

Glutamine and Leucine Provide Enhanced Protective Immunity Against Mucosal Infection with Herpes Simplex Virus Type 1

• Uyangaa E
• Lee HK
• Eo SK

• KMID: 2150749
• DOI: http://doi.org/10.4110/in.2012.12.5.196

Abstract

Besides their role as building blocks of protein, there are growing evidences that some amino acids have roles in regulating key metabolic pathways that are necessary for maintenance, growth, reproduction, and immunity. Here, we evaluated the modulatory functions of several amino acids in protective immunity against mucosal infection of herpes simplex virus type 1 (HSV-1). We found that glutamine (Gln) and leucine (Leu) showed enhanced protective immunity to HSV-1 mucosal infection when two administration of Gln and single administration of Leu per day, but not when administered in combinations. Ameliorated clinical signs of HSV-1 challenged mice by the intraperitoneal administration of Gln and Leu were closely associated with viral burden and IFN-gamma production in the vaginal tract at 2 and 4 days post-infection. In addition, the enhanced production of vaginal IFN-gamma appeared to be caused by NK and HSV-1 antigen-specific Th1-type CD4+ T cells recruited into vaginal tract of mice treated with Gln and Leu, which indicates that IFN-gamma, produced by NK and Th1-type CD4+ T cells, may be critical to control the outcome of diseases caused by HSV-1 mucosal infection. Collectively, our results indicate that intraperitoneal administration of Gln and Leu following HSV-1 mucosal infection could provide beneficial effects for the modulation of protective immunity, but dosage and frequency of administration should be carefully considered, because higher frequency and overdose of Gln and Leu, or their combined treatment, showed detrimental effects to protective immunity.

Keyword

Glutamine; Leucine; Mucosal infection; Herpes simplex virus; Protective immunity

MeSH Terms

Amino Acids
Animals
Glutamine
Herpes Simplex
Herpesvirus 1, Human
Leucine
Metabolic Networks and Pathways
Methylmethacrylates
Mice
Polystyrenes
Reproduction
Simplexvirus
T-Lymphocytes
Viral Load
Amino Acids
Glutamine
Leucine
Methylmethacrylates
Polystyrenes

Figure

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  Figure 1 Gln and Leu, but not their combination, provide protection against mucosal infection of HSV-1. (A) In vivo anti-herpes activity of several amino acids. BALB/c mice (n=8) were infected with HSV-1 McKrae strain, and were then administered 4% solution of the indicated amino acid (200 µl/mouse) daily via intra-peritoneal injection. The proportion of survived mice was examined at day 6, 9, and 12 days post-infection. (B) Survival proportion of Gln and Leu-treated mice against HSV-1 mucosal infection. BALB/c mice were infected with HSV-1 McKrae strain, and were then administered a 4% solution of Gln, Leu, or their combined mixture (200 µl/mouse) daily via intra-peritoneal injection. The challenged mice were examined daily for any signs of inflammation, illness, and death until 15 days post-challenge. (C) The clinical severity of Gln and Leu-treated mice following HSV-1 mucosal infection. The clinical severity of Gln and Leu-treated mice were scored daily after HSV-1 mucosal infection. The data represent the average of clinical severity derived from each group.

• 

  Figure 2 Virus burden and intravaginal tract IFN-γ levels of Gln and Leu-treated mice following HSV-1 mucosal infection. (A) Virus titer in vaginal lavages of Gln and Leu-treated mice following HSV-1 mucosal infection. BALB/c mice were infected with HSV-1 McKrae strain, and were then administered a 4% solution of Gln, Leu, or their combined mixture (200 µl/mouse) via intra-peritoneal injection. The vaginal lavages collected at 2nd, 3rd, and 4th day post-infection were used for plaque assay to determine virus titer. Dots on the graph represent the individual levels of virus and the bold bar shows the median level of each group. (B) IFN-γ levels in vaginal lavages of Gln and Leu-treated mice following HSV-1 mucosal infection. The levels of IFN-γ in vaginal lavages collected at 2nd and 4th day post-infection were determined by ELISA. The data represent the average±SD of levels derived from each group. Statistical significance calculated using Student's t-test. *p<0.05; **p<0.01 compared with the levels of control group.

• 

  Figure 3 NK cell activity of Gln and Leu-treated mice following HSV-1 mucosal infection. (A) Frequency of NK cells (CD3-DX5+) in vaginal tract cells of Gln and Leu-treated mice. The vaginal tract cells were prepared from Gln and Leu-treated mice 2 days post-infection and were used for analysis of NK cell frequency. The values on dot-plots represent the average of NK cell frequencies derived from each group (n=4). (B) The absolute number of NK cells in spleen, iliac LN, and vaginal tract of Gln and Leu-treated mice. The absolute number of NK cells in spleen, iliac LN, and vaginal tract was determined by flow cytometric analysis 2 days post-infection. (C and D) The proportion and absolute number of granzyme B and IFN-γ-producing NK cells in spleen of Gln and Leu-treated mice. The proportion and absolute number of granzyme B (C) and IFN-γ producing cells (D) in NK cell population were determined by intracellular staining following PMA and ionomycin stimulation 2 days post-infection. The data represent the average±SD of levels derived from each group. Statistical significance was calculated using Student's t-test. *p<0.05; **p<0.01; ***p<0.001 compared with the levels of control group.

• 

  Figure 4 HSV-1 antigen-specific CD4+ T cell responses in Gln and Leu-treated mice following mucosal infection. HSV-1 antigen-specific CD4+ T cells were detected by combined intracellular staining of CD154, IFN-γ, and TNF-α in response to stimulation of UV- inactivated HSV-1 viral antigen 5 days after HSV-1 infection. The absolute number of IFN-γ+, TNF-α+, and IFN-γ+TNF-α+CD4+ T cells in spleen (A), iliac LN (B), and vaginal tract (C) were calculated after gated on CD4+CD154+ T cells. The data show the average±SD of levels derived from each group (n=4). Statistical significance was calculated using the Student's t-test. *p<0.05; **p<0.01; ***p<0.001 as compared with the levels of control group.

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