Interferon consensus sequence binding protein confers resistance against Yersinia enterocolitica

Abstract

Interferon consensus sequence binding protein (ICSBP)-deficient mice display enhanced susceptibility to intracellular pathogens. At least two distinct immunoregulatory defects are responsible for this phenotype. First, diminished production of reactive oxygen intermediates in macrophages results in impaired intracellular killing of microorganisms. Second, defective early interleukin-12 (IL-12) production upon microbial challenge leads to a failure in gamma interferon (IFN-gamma) induction and subsequently in T helper 1 immune responses. Here, we investigated the role of ICSBP in resistance against the extracellular bacterium Yersinia enterocolitica. ICSBP(-/-) mice failed to produce IL-12 and IFN-gamma, but also IL-4, after Yersinia challenge. In addition, granuloma formation was highly disturbed in infected ICSBP(-/-) mice, leading to multiple necrotic abscesses in affected organs. Consequently, ICSBP(-/-) mice rapidly succumbed to acute Yersinia infection. In vitro treatment of spleen cells from ICSBP(-/-) mice with recombinant IL-12 (rIL-12) or rIL-18 in combination with a second stimulus resulted in IFN-gamma induction. In experimental therapy of infected ICSBP(-/-) mice, we observed that administration of rIL-12 induced IFN-gamma production which was associated with improved resistance to Yersinia. In contrast, treatment with rIL-18 failed to enhance endogenous IFN-gamma production but nevertheless reduced bacterial burden in ICSBP(-/-) mice. Although cytokine therapy with rIL-12 or rIL-18 ameliorated the course of Yersinia infection in ICSBP(-/-) mice, both cytokines failed to completely restore impaired immunity. Taken together, the results indicate that the transcription factor ICSBP is essential for efficient host immune defense against Yersinia. These results are important for understanding the complex host immune responses in bacterial infections.

FIG. 1

ICSBP^−/− mice are highly susceptible to extracellular Yersinia. Bacterial counts in spleens of ICSBP^+/+ (black symbols) and ICSBP^−/− (open symbols) mice on different genetic backgrounds (squares, C57BL/6; rhombi, C57BL/6 × 129/Ola) are shown. Mice were infected i.v. with 3 × 10³ CFU of Y. enterocolitica serotype O:8. On day 4 after infection, bacterial counts were determined. Each symbol represents one mouse.

FIG. 2

mRNA expression of cytokines in ICSBP^+/+ and ICSBP^−/− mice. Primer sequences and number of PCR cycles are listed in Table 1. Gene expression in the liver was determined by RT-PCR on day 4 after infection. Two representative results are shown for ICSBP^+/+ and ICSBP^−/− mice.

FIG. 3

Cytokine production of infected ICSBP^+/+ and ICSBP^−/− mice. (A) IL-12 and IFN-γ levels in sera of ICSBP^+/+ (black bars) and ICSBP^−/− (open bars) mice on day 4 after Yersinia infection. (B) IFN-γ and IL-4 production by HKY (10 μg/ml)-restimulated splenocytes from ICSBP^+/+ (black bars) and ICSBP^−/− (open bars) mice 4 days after Yersinia infection. Spleen cells were isolated as described in Materials and Methods, and cytokines were measured after 48 h in culture supernatants. ∗ indicates statistically significant differences (P < 0.05).

FIG. 4

Histological and immunohistological examination of liver tissue from ICSBP^+/+ (a and b) and ICSBP^−/− (c and d) mice 4 days after i.v. infection. Hematoxylin-eosin stains (a and c) or immunohistological stainings (b and d) of frozen sections with anti-Mac-1 antibodies (PAP method) and anti-Y. enterocolitica antibodies (APAAP method) are shown. Each photograph is a representative view of the entire section and for all mice studied. (a) Small granuloma-like lesion (arrowheads) in the liver of ICSBP^+/+ mice; hematoxylin-eosin stain. (b) Corresponding immunohistological staining with anti-Mac-1 and anti-Yersinia antibodies. The granuloma-like lesion (arrowhead) is composed of Mac-1⁺ cells. Few single Mac-1⁺ cells are visible. (c) Necrotic lesion in the liver of an ICSBP^−/− mouse. (d) Masses of Yersinia bacilli within the lesions and in Kupffer cells; no clear demarcation of the lesion by Mac-1⁺ cells. Many Mac-1⁺ cells are scattered throughout the liver tissue.

FIG. 5

IFN-γ production in spleen cells from uninfected ICSBP^+/+ and ICSBP^−/− mice after treatment with various stimuli. Supernatants of cultures were collected after 48 h and analyzed by ELISA as described in Materials and Methods. (A) Splenocytes from ICSBP^+/+ (black bars) and ICSBP^−/− (open bars) mice treated with medium alone, HKY (10 μg/ml), concanavalin A (ConA; 3 μg/ml), IL-12 (10 ng/ml), IL-12 (10 ng/ml) plus HKY (10 μg/ml), IL-18 (10 ng/ml), or IL-18 (10 ng/ml) plus HKY (10 μg/ml). (B) IFN-γ production in spleen cells of ICSBP^+/+ (black bars) and ICSBP^−/− (open bars) mice after treatment with various doses of IL-12 in combination with a constant dose of IL-18 (10 ng/ml). (C) IFN-γ production by splenocytes from ICSBP^+/+ (black bars) and ICSBP^−/− (open bars) mice after administration of different amounts of IL-18 in combination with a constant dose of IL-12 (10 ng/ml).

FIG. 6

rIL-12 induces IFN-γ production in Yersinia-infected ICSBP^−/− mice. (A) IFN-γ mRNA expression 4 days after Yersinia infection and experimental therapy with IL-12 (1 μg/day) or IL-18 (1 μg/day) in ICSBP^−/− mice. mRNA expression of β-actin serves as control. Results are shown for three separate ICSBP^+/+ and ICSBP^−/− mice. (B) Serum protein levels on day 4 in ICSBP^−/− mice after IL-12 (black bars) or IL-18 (open bars) treatment. ∗ indicates statistically significant differences (P < 0.05).

FIG. 7

Bacterial counts in ICSBP^−/− mice after i.v. Yersinia infection and cytokine therapy. Mice were treated i.p. for 5 days with a single dose of cytokine or PBS. Treatment was started 1 day before infection. Data are shown for three independent experiments. (A) Experiment 1. Mice were inoculated with 3 × 10³ CFU of Y. enterocolitica and sacrificed on day 4 after infection. (B) Experiment 2. Mice were inoculated with 3 × 10² CFU of Y. enterocolitica and sacrificed on day 5 after infection. (C) Experiment 3. Mice were inoculated with 3 × 10² CFU of Y. enterocolitica and sacrificed on day 4 after infection. ∗ indicates statistically significant differences (P < 0.05) compared to ICSBP^−/− mice treated with PBS (white bars). The ratio of surviving to infected mice is shown for each group.