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Comparative Study
. 1997 Mar 3;185(5):921-31.
doi: 10.1084/jem.185.5.921.

Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis

T Fehr  1 , G Schoedon, B Odermatt, T Holtschke, M Schneemann, M F Bachmann, T W Mak, I Horak, R M Zinkernagel
Affiliations
Comparative Study

Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis

T Fehr et al. J Exp Med. .

Abstract

Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF) 1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP-/- and to a lesser degree also IRF2-/- mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-gamma in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-gamma stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-gamma-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

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Figures

Figure 4
Figure 4
NO production and respiratory burst in PEM cultures. PEMs of mice deficient for different IFN-related transcription factors were cultured as described in Fig. 3. (A) After 42 h NO production was measured by determination of nitrite accumulation in the culture supernatants by using Griess reagent. Values are calculated as nmol nitrite per 105 cells. One of three independent and comparable experiments is shown. (B) Respiratory burst capacity of the same PEMs was determined by measuring H2O2 production by chemiluminescence after stimulation with PMA. Values were first calculated as nmol H2O2 per 105 cells, and then a stimulation index of stimulated versus unstimulated cultures was determined (stimulation index of unstimulated culture = 1). Error bars indicate standard deviation.
Figure 4
Figure 4
NO production and respiratory burst in PEM cultures. PEMs of mice deficient for different IFN-related transcription factors were cultured as described in Fig. 3. (A) After 42 h NO production was measured by determination of nitrite accumulation in the culture supernatants by using Griess reagent. Values are calculated as nmol nitrite per 105 cells. One of three independent and comparable experiments is shown. (B) Respiratory burst capacity of the same PEMs was determined by measuring H2O2 production by chemiluminescence after stimulation with PMA. Values were first calculated as nmol H2O2 per 105 cells, and then a stimulation index of stimulated versus unstimulated cultures was determined (stimulation index of unstimulated culture = 1). Error bars indicate standard deviation.
Figure 3
Figure 3
Elimination of Listeria from a PEM culture stimulated with LPS, IFN-γ or both. PEM of mice deficient for different IFN-related transcription factors were elicited with starch solution, harvested after 5 d and put into culture on cover slips under presence of the indicated stimulators. After 42 h (at time point t 0) they were infected with Listeria and allowed to digest the bacteria during 7 h. Then, cover slips were taken out, dried and May-Grünwald-Giemsa stained. The number of infected macrophages was counted under the microscope, compared to the infection rate at t 0 and expressed as difference in percentage of infected cells. Error bars represent standard deviation. Examples for absolute numbers: the number of infected macrophages at t 0 was between 80 and 106/200 for all strains; the number of infected cells in IFN-γ-stimulated cultures after 7 h of digestion was 19/200 for wild-type and 168/200 for ICSBP−/− mice.
Figure 1
Figure 1
Listeria titers in organs of different mouse strains deficient for molecules in the IFN signaling pathway. (A) Mice deficient for IFN- related transcription factors (ICSBP, IRF1, IRF2; filled diamonds) or (B) mice lacking functional IFN type I (A129; filled diamonds) or type II (G129; filled circles) receptors and control littermates (open diamonds) were infected with 5 × 103 CFU of Listeria intravenously. After 5 d bacterial titers were determined in liver and spleen. Groups of three to four mice were analyzed. Each symbol represents one mouse. One representative experiment of two is shown for each strain.
Figure 1
Figure 1
Listeria titers in organs of different mouse strains deficient for molecules in the IFN signaling pathway. (A) Mice deficient for IFN- related transcription factors (ICSBP, IRF1, IRF2; filled diamonds) or (B) mice lacking functional IFN type I (A129; filled diamonds) or type II (G129; filled circles) receptors and control littermates (open diamonds) were infected with 5 × 103 CFU of Listeria intravenously. After 5 d bacterial titers were determined in liver and spleen. Groups of three to four mice were analyzed. Each symbol represents one mouse. One representative experiment of two is shown for each strain.
Figure 2
Figure 2
Capacity of ICSBP−/− spleen cells to transfer Listeria resistance to RAG2−/− mice. Non adherent spleen cells of ICSBP+/+ (open diamonds) or ICSBP−/− (filled diamonds) mice were transferred to RAG2−/− mice. After 24 h the recipients as well as untreated RAG2−/− mice (open circles) were infected with 2 × 105 CFU of Listeria, and bacterial titers were determined on day 9 after infection to assess overall protection. Groups of four to five recipient mice were used. Each symbol represents one mouse. One of three comparable experiments is shown.
Figure 5
Figure 5
Listeria replication and iNOS expression in the liver after infection with Listeria. ICSBP−/− (D–H), IRF1−/− (L, M), IRF2−/− (N, O), and control mice (A–C, I, K) were infected with 5 × 103 CFU of Listeria. After 5 or 6 d liver and spleen were taken out. Conventional HE staining (A, D) and immunohistology for Listeria (B, E, G, I, L, N) and iNOS (C, F, H, K, M, O) was performed using polyclonal primary antibodies. Magnifications: (A, D), ×35, (B, C, E, F, I–O), ×60 (G, H), ×220.
Figure 6
Figure 6
Proposed possible signaling events in macrophages after Listeria infection. IFN-γ–induced ICSBP is of crucial importance for protection in murine listeriosis, probably partly via ROI production. G-CSF (66) plays a minor and IRF1-induced RNI (23) no limiting role for bacterial resistance. A potentiating effect or an additional factor is postulated. How ICSBP, NF-IL6 or other transcription factors are involved in TNF-mediated protection against Listeria remains to be determined.
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