IFN-gamma-producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection

Abstract

The importance of intraepithelial lymphocytes (IEL) in immunoprotection against orally acquired pathogens is being increasingly recognized. Recent studies have demonstrated that Ag-specific IEL can be generated and can provide an important first line of defense against pathogens acquired via oral route. However, the mechanism involved in priming of IEL remains elusive. Our current study, using a microsporidial model of infection, demonstrates that priming of IEL is dependent on IFN-gamma-producing dendritic cells (DC) from mucosal sites. DC from mice lacking the IFN-gamma gene are unable to prime IEL, resulting in failure of these cells to proliferate and lyse pathogen-infected targets. Also, treatment of wild-type DC from Peyer's patches with Ab to IFN-gamma abrogates their ability to prime an IEL response against Encephalitozoon cuniculi in vitro. Moreover, when incubated with activated DC from IFN-gamma knockout mice, splenic CD8(+) T cells are not primed efficiently and exhibit reduced ability to home to the gut compartment. These data strongly suggest that IFN-gamma-producing DC from mucosal sites play an important role in the generation of an Ag-specific IEL response in the small intestine. To our knowledge, this report is the first demonstrating a role for IFN-gamma-producing DC from Peyer's patches in the development of Ag-specific IEL population and their trafficking to the gut epithelium.

FIGURE 1

IFN-γ^−/− and pf^−/− mice are susceptible to oral E. cuniculi infection. A, IFN-γ^−/−, pf^−/−, and WT mice (n = 8 mice^/group) were orally infected with 2 × 10⁷ E. cuniculi spores. The animals were monitored daily for survival. The study was performed twice and data are representative of two separate experiments. B, Histopathology of infected mice. Spleen at necroscopy at magnification ×10 (i). WT mouse model demonstrating normal architecture. Spleen of IFN-γ^−/− mouse at magnification ×20 (ii) and ×40 (iii), demonstrating effacement of follicles and increase in red pulp. Liver at magnification ×20 (iv). WT mouse model demonstrating normal architecture. Liver IFN-γ^−/− mouse at magnification ×20 (v) and ×40 (vi), demonstrating inflammatory foci. Mononuclear cell infiltration into the liver (vi) associated with a slight increase in the number of Kupfer cells in the adjacent areas. PP in the intestinal track of mice are shown (vii–ix). There is some increased activity of lymphoid aggregates. However, no organism was detected. PP from IFN-γ^−/− mouse at magnification ×10 (vii) and ×40 (viii). PP at magnification ×20 (ix) from a pf^−/− mouse are shown.

FIGURE 2

Cytotoxic activity of IEL against E. cuniculi-infected target is perforin-dependent. IEL from WT mice were purified at day 7 p.i. (n = 6 mice/group). A, IELs were labeled for cytotoxic markers (perforin, CD95L, and granzyme B). Cells were isolated at day 7 p.i., and expression of perforin, granzyme B, and CD95L were analyzed by intracellular staining. B, Before incubation with ⁵¹Cr-labeled uninfected and infected macrophages (E:T ratio of 20:1), IEL were treated with either CMA or anti-FasL Abs. After 4 h of incubation, the cytolytic activity was determined by radioisotope release into the culture supernatant. The experiment was performed twice with similar results. Data are representative of one set of experiments.

FIGURE 3

Adoptive transfer of IEL isolated from pf^−/− and IFN-γ^−/− mice fails to protect SCID mice against E. cuniculi challenge. A, Adoptive transfer of IEL from IFN-γ^−/− and pf^−/− is unable to protect SCID mice against a lethal challenge with E. cuniculi. IEL from naive and infected pf^−/−, IFN-γ^−/−, and WT mice (n = 12 mice/group) were isolated at day 7 p.i. A total of 5 × 10⁶ IEL were injected i.p. into SCID mice (n = 6 mice/group). After 48 h, the mice were challenged orally with 2 × 10⁷ spores of E. cuniculi and the survival of the animals was monitored daily until the end of the experiment. B, IEL from WT mice were purified at day 7 p.i. CD8β⁺ and CD8β⁻ subset were separated and adoptively transferred to SCID recipients (n = 6 mice/group). The recipient animals were orally infected with 2 × 10⁷ E. cuniculi 2 days posttransfer. The survival of the animals was monitored daily until the end of the experiment.

FIGURE 4

IFN-γ^−/− mice exhibit a suboptimal IEL response against E. cuniculi infection. IEL from IFN-γ^−/− and WT mice (n = 6 mice/group) were isolated 7 days p.i. A, Induction of IEL response in IFN-γ^−/− mice following oral E. cuniculi infection. IFN-γ^−/− and WT mice orally infected with E. cuniculi were sacrificed at day 7 p.i., and IEL were purified (n = 4 mice/group). Isolated IEL were assayed for CD8αα, CD8αβ, TCRαβ, and TCRγδ expression by FACS analysis. Data represent the mean ± SD of two individual sets of experiments. B, Cytotoxic activity of IEL against E. cuniculi-infected macrophages. IEL from IFN-γ^−/− and WT mice were incubated with ⁵¹Cr-labeled uninfected and infected macrophages at different E:T ratio (20:1, 40:1). After 4 h of incubation, the cytolytic activity was determined by radioisotope release into culture supernatant. The experiment was performed twice with similar results and data are representative of one experiment.

FIGURE 5

DC from PP of IFN-γ^−/− mice are unable to stimulate naive CD8αβ IEL. Immature DC from naive IFN-γ^−/− and WT mice (n = 6 mice/group) were isolated, plated, and infected overnight with E. cuniculi. The next day, naive IEL purified from WT mice (n = 6 mice/group) were separated and CD8αβ IEL were added to the culture. A, Presentation was measured as induction of proliferation of CD8αβ IEL. B, DC were treated with anti-IFN-γ or isotype control Ab at the same time that E. cuniculi infection and for the remaining of the experiment. C, IL-12 production in the supernatants of E. cuniculi primed DC from WT and knockout animals, after overnight incubation, was measured by ELISA. Experiments were performed twice with similar results and data are representative of one experiment.

FIGURE 6

Splenic CD8αβ⁺ T cells primed with DC from IFN-γ^−/− mice have impaired ability to traffic to the gut. A and B, Immature DC from PP of naive IFN-γ^−/− and WT mice (n = 6 mice/group) were isolated and infected with E. cuniculi. After overnight incubation, purified CD8β⁺ splenocytes from naive WT mice (n = 4 mice) were added to the culture. Seventy-two hours later, cells were harvested and labeled with CFSE before transfer to WT recipient animals (n = 2 mice/group). Subsequently after 24 h, the recipients were sacrificed and cells from MLN, PP, spleen (A) and IEL (B) recovered and analyzed for the presence of CFSE-labeled CD3⁺ cells by flow cytometry. C–H, CD8β⁺ splenocytes isolated from naive mice were primed with PP DC from WT or IFN-γ^−/−. After 72 h, CD8β⁺ cells were labeled for CCR9 expression (C–E) or IFN-γ production by intracellular staining (F–H). Data in dot plots are gated on double positive CD8α⁺CD8β⁺ T cells primed with DC from WT (D–G) or IFN-γ^−/− (E–H) mice labeled with allophycocyanin-conjugated anti-CD8α and PE-conjugated anti-CCR9 (D and E) or IFN-γ (G and H). Percentage represents surface expression of CCR9 (D and E) and IFN-γ production (G and H) in CD8αβ cells. Experiments were performed twice with similar results and data are representative of one experiment.