Recruited inflammatory monocytes stimulate antiviral Th1 immunity in infected tissue

Abstract

Monocytes patrol various tissues for signs of infection and inflammation. Inflammatory monocytes enter peripheral tissues at sites of microbial infection and differentiate into dendritic cells and macrophages. Here, we examined the importance of monocytes in primary mucosal infection with herpes simplex virus 2 (HSV-2), and demonstrate that monocyte-derived APCs are required to elicit IFN-γ secretion from effector Th1 cells to mediate antiviral protection. However, monocyte-derived APCs were dispensable for the generation of Th1 immunity and for the restimulation of memory Th1 cells during secondary viral challenge. These results demonstrate that distinct APC subsets are dedicated for CD4 T cell priming, elicitation, and memory recall responses to a given viral pathogen within the same mucosal tissue and reveal a specialized role for monocyte-derived APCs in the emergency response to infection.

Fig. 1.

CCR2 is required for the protection against intravaginal HSV-2 infection. C57BL6 mice (n = 9) or CCR2^−/− mice (n = 9) were challenged with WT HSV-2 (1.2 × 10² pfu per mouse). Virus titer in vaginal wash (A), average clinical score (B), and survival curve (C) were measured. These results are representative of three similar experiments (mean ± SEM). ***P < 0.001.

Fig. 2.

Ly6C^hiCD11b⁺ and CD11b⁺MHC class II⁺ cells failed to migrate into the infected tissue following intravaginal HSV-2 challenge. Rapid accumulation of total CD11b⁺ cells, Ly6C^himonocytes (CD11b⁺Ly6C^hiCD11c⁻MHC class II⁻), CD11b⁺ DC (CD11c⁺MHC class II⁺), CD11b⁻ DC (CD11c⁺MHC class II⁺) among 7-AAD⁻CD45⁺CD3⁻B220⁻NK1.1⁻DX5⁻Ly6G⁻CCR3⁻ cells, and granulocyte neutrophil (CD45⁺CD11b^hiCD11c⁻MHC class II⁻Ly6G⁺ cells) in vaginal tissues following genital herpes infection (WTHSV-2, 10⁴ pfu per mouse). Results are representative of three similar experiments (mean ± SEM).

Fig. 3.

CCR2 is required for tissue migration of monocytes following genital HSV-2 infection. Ly6C⁺ monocytes (2 × 10⁶ cells per mouse) isolated from the BM of CD45.1⁺CD45.2⁺ WT mice or CD45.2⁺ CCR2^−/− mice were adoptively transferred into CD45.1⁺ WT mice (n = 3) 1 d after ivag HSV-2 infection. Four days after ivag HSV-2 infection, recruitment of CD45.1⁺CD45.2⁺ CD11b⁺ monocyte-derived cells to the vagina (A) was analyzed. (B) Bar graphs represent absolute numbers of donor-derived total CD11b⁺ cells, CD11b⁺CD11c⁺, and CD11b⁺CD11c⁻ APCs in vagina. *P < 0.05, **P < 0.01. Results are representative of two similar experiments.

Fig. 4.

Type-I IFN-mediated up-regulation of CCR2 ligands is required for blood monocyte recruitment. (A and B) CD45.2⁺ WT mice (n = 3) or CD45.2⁺ IFN-αβR^−/− mice (n = 3) that had been transplanted with CD45.1⁺ CD11b⁺ monocytes (2 × 10⁶ cells per mouse) were challenged with HSV-2 ivag. Five days later, donor (CD45.1⁺)-derived CD11c⁺MHC class II⁺ cells and recipient (CD45.2⁺)-derived CD11c⁺MHC class II⁺ cells in vaginal tissues were counted (B). *P < 0.05. Results are representative of two similar experiments.

Fig. 5.

Monocyte-derived APCs reactivate Th1 cells to secrete IFN-γ within the virally infected tissue. (A) IFN-γ levels in vaginal wash in WT mice (n = 9) and CCR2^−/− mice (n = 9) was measured at the indicated days following genital herpes infection (WT HSV-2, 10⁴ pfu per mouse). (B) IFN-γ levels in tissue homogenates in WT mice (n = 4) and CCR2^−/− mice (n = 4) was detected at day 5 p.i. (C) Monocyte-derived APCs predominantly present viral antigen to effector CD4 T cells in the vaginal tissues. CD11c⁺CD11b⁺, CD11c⁻CD11b⁺, CD11c⁺CD11b⁻, and CD11c⁻CD11b⁻ subsets among live lineage⁻ cells were FACS-sorted from vaginal tissues at 5 d p.i. and cocultured with effector CD4⁺ T cells isolated from draining LN (day 7) in the presence of mock or viral antigen. IFN-γ secreted from T cells (Left) and T cell proliferation (Right) was measured by ELISA and thymidine incorporation, respectively. *P < 0.05, **P < 0.01, ***P < 0.001. These results are representative of three similar experiments (mean ± SEM).