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*Leishmaniasis
The Journal of Immunology, 2004, 172: 7574-7582.
Copyright © 2004 by The American Association of Immunologists

Protection against Progressive Leishmaniasis by IFN-{beta}1

Jochen Mattner2,*, Alexandra Wandersee-Steinhäuser*, Andreas Pahl{dagger}, Martin Röllinghoff*, Gerard R. Majeau{ddagger}, Paula S. Hochman{ddagger} and Christian Bogdan3,*,§

* Institute of Clinical Microbiology, Immunology and Hygiene and {dagger} Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany; {ddagger} Biogen Inc., Cambridge, MA 02142; and § Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Type I IFNs (IFN-{alpha}{beta}) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-{beta} protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (106) or low (104) number of Leishmania major parasites, respectively. IFN-{beta} treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-{gamma} and IL-12 in the draining lymph node. Low, but not high doses of IFN-{beta} caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-{beta}-induced increase of IFN-{gamma} production was dependent on STAT4. Protection by IFN-{beta} strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-{beta} led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-{beta} as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
L;-2qeishmania major is a protozoan parasite that in nature is transmitted by sand flies to various mammalian hosts, where it usually causes localized and self-healing cutaneous leishmaniasis. For several decades it has been used by immunologists as a prototype organism for the study of the immune response to intracellular pathogens. In the L. major mouse model macrophages, dendritic cells, NK cells, CD4+ Th1 cells, CD8+ T cells, IL-12, IFN-{gamma}, and inducible NO synthase (iNOS)4 were defined as the key components of the immune system that contribute to the control of the parasites in vivo (1, 2, 3, 4). Similar to humans, cutaneous inoculations of most mouse strains (e.g., C57BL/6, C3H/HeN, CBA, 129Sv) with low or high doses of L. major promastigotes led to transient papular or nodular swellings of the skin without ulceration that clinically resolves despite the life-long persistence of small numbers of parasites at the site of infection and in the draining lymph node (5). In a few mouse strains, however, local injections of L. major cause either chronic nonhealing skin lesions (e.g., DBA/2 mice) or progressive and ultimately fatal visceral disease (BALB/c mice). The detailed analysis of L. major infections in BALB/c mice provided insights into the genetic and immunological basis of their exceptional susceptibility (6, 7) and resulted in the development of two cytokine-based immunoprophylactic regimens that were able to confer protection against visceral leishmaniasis in this mouse strain, i.e., the neutralization (or genetic deletion) of IL-4 (8) and the application of recombinant murine (rm) IL-12 (9, 10). In contrast, the application of IFN-{gamma} alone was largely ineffective (8, 11).

Type I IFNs form an ancient and complex family of acid-stabile cytokines, which in the mouse consists of IFN-{beta} and at least 12 different subtypes of IFN-{alpha} (12). Originally identified and characterized in fibroblasts as antiviral proteins, it is now known that type I IFNs are produced by multiple types of cells, including T cells, NK cells, monocytes, macrophages, and, in particular, plasmocytoid dendritic cells (13, 14, 15, 16, 17). In addition to conferring resistance against lytic virus infections, type I IFNs exert antiproliferative and various positive or negative immunoregulatory effects, such as the stimulation or inhibition of the cytolytic activity and IFN-{gamma} production of NK cells; the rescue of activated or memory T cells from apoptosis; the induction or inhibition of T cell proliferation; the promotion or blunting of Th1 responses; the differentiation of monocytes into dendritic cells and the maturation of dendritic cells; and the up-regulation or suppression of iNOS in monocytes or macrophages (15, 16, 18, 19, 20). Consequently, type I IFNs are widely used for the treatment of viral infections, malignancies, and chronic inflammatory diseases such as relapsing multiple sclerosis (21, 22).

In the L. major mouse model, we recently presented a novel example for a protective effect of IFN-{alpha}{beta} in a nonviral infection. During the innate phase of response to infection, the expression of iNOS, the cytotoxic activity of NK cells, and the early production of IFN-{gamma} in self-healing mice was dependent on the endogenous release of IFN-{alpha}{beta} (23). L. major parasites triggered the release of IFN-{alpha}{beta} both in vitro and in vivo and L. major plus IFN-{alpha}{beta} activated macrophages for the production of NO (23, 24). In BALB/c mice, treatment with high doses of IFN-{alpha}{beta} 3–4 h before infection and along with the injection of L. major induced parasite containment and restored the NK cell cytotoxic activity in the draining lymph nodes of these mice at day 1 of infection (23). Based on these findings, we set out to investigate whether prolonged application of type I IFNs could induce a healing phenotype in otherwise highly susceptible BALB/c mice. Our results demonstrate that low doses of rmIFN-{beta} confer long-term protection against progressive cutaneous leishmaniasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c and C57BL/6 mice were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany). Breeding pairs of BALB/c STAT4–/– and C57BL/6 iNOS–/– were obtained from The Jackson Laboratory (Bar Harbor, ME). BALB/c IL-12p35–/– (25) were kindly provided by Dr. G. Alber (University of Leipzig, Leipzig, Germany) and C57BL/6 IL-12p35/p40–/– by Dr. H. Mossmann (Max Planck Institute of Immunobiology, Freiburg, Germany). The mice used were 6–12 wk of age and were age and sex matched. All mice were housed under specific pathogen-free conditions. The housing of the mice and the in vivo experiments were approved by the governmental animal welfare committee.

Preparation of rmIFN-{beta}

rmIFN-{beta} was produced in Chinese hamster ovary (CHO) cells at Biogen (Cambridge, MA). A murine IFN-{beta} cDNA (gift of Dr. J. Doly, Laboratoire de Regulation Transcriptionnelle et Maladies Genetiques, Université Paris V, Paris, France) (26) was inserted into a plasmid expression vector carrying a dihydrofolate reductase selection marker and an adenoviral promoter and was transfected into CHO cells by electroporation. High expressing cells were selected by using increasing concentrations of methotrexate. rmIFN-{beta} was purified from the culture supernatants of suspension-adapted CHO transfectants by precipitation of acid-labile proteins and two sequential affinity chromatography steps (with a blue Sepharose column and a Sepharose 4B column to which the monoclonal anti-mouse IFN-{beta} Ab MCA MB-7 (Yamasa Shoyu, Tokyo, Japan) was conjugated). Bioactivity assays (see below) or ELISA and 10–20% gradient SDS-PAGE followed by silver staining and Western blotting were performed to select the column fractions containing rmIFN-{beta}. The final rmIFN-{beta}-positive fractions were pooled, formulated with 1 mg/ml murine serum albumin (Sigma-Aldrich, St. Louis, MO; tested at Biogen to be low for endotoxin), passed through a 0.2-µm filter, aliquoted, and stored frozen at –70°C. The bioactivity of rmIFN-{beta} was measured in a virus protection assay using L929 cells and the encephalomyocarditis virus (1 U of activity is the concentration of rmIFN-{beta} that gave 50% protection in this assay; Ref 27). The lot of rmIFN-{beta} used in all experiments reported here had a specific activity of 2 x 109 U/mg and a LPS content of <10 pg/ml at 5.6 x 108 U/ml (280 µg/ml) as determined by a colorimetric Limulus amebocyte assay (Cambrex Bio Science Verviers, Apen, Germany). No antiviral bioactivity was detectable in the supernatant of confluent day 4 cultures of nontransfected CHO cells (data not shown).

L. major infection and IFN-{beta} treatment of mice

Origin and propagation of the L. major strain MHOM/IL/81/FEBNI were as reported (28). Unless otherwise indicated, mice were infected into the skin of the right hind footpad with 1 x 106 stationary phase L. major promastigotes in 50 µl of PBS. In the standard protocol established in this study, mice were injected with PBS or with rmIFN-{beta} i.p. 4–6 h before infection (1 x 102– 1 x 105 U/mouse in 0.5 ml of PBS) and into the footpad along with the parasites (1 x 102–1 x 105 U/mouse in 0.05 ml of PBS). Thereafter, PBS or rmIFN-{beta} was applied i.p. from day 2 of infection onward every other day until day 14 after infection. The measurement of the footpad swelling and the determination of the tissue parasite load by limiting dilution analysis was conducted exactly as described previously (29).

NK cell cytotoxicity of lymph node cells

Total cell suspensions were prepared from the popliteal lymph nodes of PBS- or rmIFN-{beta}-treated L. major-infected mice and analyzed for their NK cell cytotoxic activity against YAC-1 tumor target cells in a 4-h chromium release assay (23). Spontaneous release never exceeded 10–15% of the maximum release.

Cell proliferation and cytokine production

Popliteal lymph node or spleen cells from infected mice were restimulated with rmIFN-{beta} (500 U/ml), rmIL-12 (5 ng/ml; R&D Systems, Wiesbaden-Nordenstadt, Germany), rmIL-18 (10 ng/ml; R&D Systems), Leishmania Ag (freeze-thaw lysates of promastigotes; parasite:cell ratio = 5:1) or Con A (2.5 µg/ml, Sigma-Aldrich) in complete RPMI 1640 medium with 5% FCS for 24–72 h. For the determination of cell proliferation, the cultures were pulsed with 0.5 µCi (37 kBq) [3H]thymidine (New England Nuclear, Dreieich, Germany) per well for 24 h and processed by beta scintillation spectrophotometry. Culture supernatants were analyzed for their IFN-{gamma} and IL-4 content by capture ELISA (BD Biosciences, Heidelberg, Germany, and R&D Systems, sensitivity 50–150 pg/ml) (29).

FACS analysis and intracellular cytokine staining

Lymph node or spleen cells from infected mice were stained with fluorochrome (FITC-, PE-, or allophycocyanin-) labeled or biotinylated mAb against CD3 (BD Biosciences), CD4 (GK1.5; BD Biosciences), CD8 (CT-CD8{alpha}; Caltag Laboratories, Hamburg, Germany), F4/80 (CI:A3-1; Caltag Laboratories), CD11b (M1/70.15; Caltag Laboratories), CD11c (HL3; BD Biosciences), CD45R/B220 (RA3-6B2; BD Biosciences), and pan-NK cells (DX5; BD Biosciences) and subjected to flow cytometry. For the detection of biotinylated Abs, streptavidin-allophycocyanin, or, in the case of intracellular cytokine staining, streptavidin-PerCP (BD Biosciences) were used. The specificity of the staining was verified by the use of isotype control mAbs in each experiment (data not shown). Propidium iodide was included at 1 µg/ml in the final wash after immunofluorescent staining to detect dead cells. The analyses were performed on a FACSCalibur (BD Biosciences) using the CellQuest Pro software. The FL3 channel was used to exclude propidium iodide-positive dead cells.

For intracellular IFN-{gamma} staining, lymph node cells from infected mice were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 750 ng/ml ionomycin (Sigma-Aldrich) for 4 h at 37°C in the presence of 10 µg/ml brefeldin A, fixed in 2% formaldehyde, and stained in permeabilization buffer (PBS, 0.5% saponin, 2% FCS) with allophycocyanin- or PE-conjugated rat anti-mouse IFN-{gamma}. The specificity of the IFN-{gamma} staining was confirmed by the use of an isotype control mAb and by its complete blocking after preincubation of the cells with unconjugated anti-IFN-{gamma} mAb (data not shown).

Immunoprecipitation, SDS-PAGE, and Western blotting

For the detection of iNOS by Western blotting (24), total lymph node cells from L. major-infected mice (with or without IFN-{beta} treatment in vivo) were suspended in 0.5 ml of Tris buffer (40 mM, pH 8; 4°C) with protease inhibitors and lysed by sonication (30). To demonstrate equal loading of the lanes, the membranes were reprobed with an anti-{beta}-actin Ab (I-19; Santa Cruz Biotechnology, Santa Cruz, CA).

For immunoprecipitation of total cellular STAT1{alpha}, STAT4 and suppressor of cytokine signaling-1 (SOCS1), lymph node cells were lysed in 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM each of EDTA, EGTA, sodium orthovanadate, sodium pyrophosphate, sodium fluoride, and PMSF, 0.1 mM sodium molybdate, and 5 µg/ml each of pepstatin A, aprotinin, chymostatin, and leupeptin (all reagents from Sigma-Aldrich). The protein content of the lysates was determined by the Pierce BCA protein assay (KMF, St. Augustin, Germany). Per condition,2.5–3 mg protein of total cell lysate was immunoprecipitated with 1 µg of affinity-purified monoclonal mouse anti-mouse STAT1{alpha} IgG (C-111; Santa Cruz Biotechnology), 1.25 µg of polyclonal rabbit anti-mouse STAT4 IgG (C-20; Santa Cruz Biotechnology), or 0.6 µg of polyclonal goat anti-mouse SOCS1 IgG (N-18; Santa Cruz Biotechnology) using protein A/G-Plus-agarose (Santa Cruz Biotechnology). The immunoprecipitates were separated by 7.5% SDS-PAGE, transferred to nitrocellulose, and subjected to ECL-based Western blotting with mouse anti-phosphotyrosine IgG (PY-99; Santa Cruz Biotechnology), rabbit anti-mouse STAT1{alpha} IgG (M-23; Santa Cruz Biotechnology), goat anti-mouse SOCS1 (N-18; Santa Cruz Biotechnology), or rabbit anti-mouse STAT4 IgG (C-20; Santa Cruz Biotechnology) as described elsewhere (24, 31). For control purposes, equal amounts of protein of the different samples were subjected to an additional SDS-PAGE and Western blotting with an anti-{beta}-actin Ab (Santa Cruz Biotechnology).

Real-time PCR analysis

Total RNA was extracted from frozen tissue (lymph node and footpads at different time points after infection) using the RNAeasy kit (Qiagen, Hilden, Germany). For the quantitative determination of the mRNA of IFN-{gamma}, IL-4, IL-12p40, IL-12p35, and {beta}-actin, the reaction mixture in the one-tube RT-PCR (10-µl volume) was set up with the QuantiTect Probe RT-PCR kit (Qiagen) and contained 100 ng total RNA, 200 nM of each primer, and 100 nM probe. Primers (from MWG Biotech, Ebersberg, Germany) and probes (Eurogentec, Köln, Germany) were as follows: {beta}-actin, 5'-TCACCCACACTGTCCCCATCTATGA-3' (sense), 5'-GATGCCACAGG ATTCCATACCCA-3' (antisense), 5'-(FAM)-ACGCGCTCCCTCATGCCATCCTGCGT-(TAMRA)-3' (TaqMan probe); IL-12p35, 5'-CCACCCTTGCCCTCCTAAAC-3' (sense); 5'-GGCAGCTCCCTCTTGTTGTG-3' (antisense); 5'-(FAM)-ACCTCAGTTTGGCCAGGG-TCATTCCA-(TAMRA)-3' (TaqMan probe); IL-12p40, 5'-TTCAGTGTCCTGCCAGGA GG-3' (sense), 5'-CGGGTCTGGTTTGATGATGTC-3' (antisense), 5'-(FAM)-TGTCACCT GCCCACTGCCGAG-(TAMRA)-3' (TaqMan probe); IL-4, 5'-CCCCCAGCTAGTTGTCAT CCT-3' (sense), 5'-TGGTGTTCTTCGTTGCTGTGA-3' (antisense); 5'-(FAM)-CCAGGAG CCATATCCACGGATGCG-(TAMRA)-3' (TaqMan probe); and IFN-{gamma}, 5'-CLLACTACGGT CTCCAGCC-3'(sense), 5'-GCCTCTCCCCAGCAAAGTCT-3'(antisense), 5'- (FAM)-TCA GAGCTGCAGTGACCCCGGGAAG-(TAMRA)-3' (TaqMan probe). PCR amplification and detection were done on an ABI Prism 7900 sequence detector (Applied Biosystems, Darmstadt, Germany) with the following profile: 30 min at 50°C (reverse transcription reaction), 15 min at 95°C (activation of the polymerase, denaturation of the DNA), and 45 cycles of 30 s at 95°C (denaturation) and 60 s at 60°C (annealing and extension). mRNA levels (mean ± SD of triplicate samples for all mice per group) were calculated using the comparative cycle threshold method and normalized to {beta}-actin (32). All PCR products were confirmed by size and restriction enzyme digestion on agarose gels.

Immunohistology

Five- to 6-µm cryostat tissue sections from embedded skin lesions, lymph nodes, and spleens were fixed, blocked, and stained for iNOS, L. major, and cell types by immunoperoxidase staining (using 3-amino-9-ethylcarbazole as a substrate) and hematoxylin counterstaining as described previously (5).

Statistics

Statistical analysis was performed using the unpaired Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low doses of IFN-{beta} prevent progressive leishmaniasis in BALB/c mice

A single high dose (1–2 x 106 U) of purified mouse IFN-{alpha}{beta} up-regulated the NK cell cytotoxic activity and impeded the spreading of the parasites in L. major-infected BALB/c mice at day 1 of infection, but did not alter the course and outcome of infection (23) (C. Bogdan and J. Mattner, unpublished data). Likewise, systemic (i.p.) application of high doses of purified mouse IFN-{alpha}{beta} every day or every other day for a prolonged period of time after infection (14–19 days) did not cause reproducible protection against nonhealing, progressive leishmaniasis in BALB/c mice (data not shown). Considering that the composition of different batches of purified natural IFN-{alpha}{beta} might vary and that functional differences between subtypes of type I IFN do exist (33, 34, 35), we decided to use only recombinant mouse IFN-{beta} for all subsequent experiments.

Detailed dose-finding studies using a high parasite inoculum (1 x 106 stationary phase L. major promastigotes) revealed that the i.p. application of 5 x 103 U of IFN-{beta} every other day, starting 4 h before infection and lasting until 14 days after infection, was most effective in protecting BALB/c mice from progressive disease (Fig. 1A). One-third of the treated mice did not show any skin swelling or completely recovered, whereas another third of the mice developed persistent, but clinical stable lesions without ulceration or minimal, nonprogressing ulcers after day 100 of infection. In the remainder of the mice, the skin lesions progressed, but the occurrence of ulcers was strongly delayed compared with the control group (Fig. 1B and Table I). Surprisingly, the highest tested dose was the least effective one (Fig. 1A and Table I). The tissue parasite load paralleled the improved clinical course of infection. Although the parasite burden was up to 1000-fold lower in IFN-{beta}-treated compared with control animals, even mice with small lesions continued to harbor rather high numbers of Leishmania in the skin (Fig. 1C). Similar observations were previously made in BALB/c mice treated with IL-12 (9, 10). The protection achieved by IFN-{beta} was increased to 100% and lasted for at least 150 days, when the parasite inoculum was lowered to 1 x 104 L. major promastigotes (Fig. 1D). IFN-{beta}-treated BALB/c mice that had recovered from a high-dose infection with L. major were resistant to a challenge infection with 1 x 106 L. major in the contralateral footpad (data not shown).



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  		FIGURE 1. Effect of IFN-{beta} on the course of L. major infection in BALB/c mice. Groups of four to five BALB/c mice were infected with 1 x 106 (A–C) or 1 x 104 (D) L. major promastigotes into the right hind footpad. Different doses of IFN-{beta} (A), 5 x 103 U of IFN-{beta} (B–D), or PBS (A–D) were applied i.p. 4 h before infection s.c. along with the parasites and i.p. every 48 h thereafter until day 14 of infection. A, B, and D, The percent increase of footpad thickness was determined. A and D, mean ± SD. B, Same experiment as in A (5 x 103 U IFN-{beta}), but each mouse is shown individually. x depicts the occurrence of skin ulcers. A and D, Significant difference (p < 0.005 or smaller) between PBS control group and 5 x 103 U of IFN-{beta} group from day 20 or 25 onward, respectively. C, Tissue parasite burden at days 5, 15, and 50 of infection as determined by limiting dilution analysis (error bars represent the 95% confidence intervals). The experiments shown are representative for eight (A–C) and three (D) experiments.

 

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  		Table I. Titlea

 
During the 3-year study period, we also tested different schemes of application of IFN-{beta} (5 x 103 U). Neither the reduction (4 days) nor the prolongation of the treatment period (42 days) or an increase of the treatment frequency (i.p. application of IFN-{beta} every 24 h) led to the same degree of protection as the standard protocol. When the single dose of IFN-{beta} before infection was omitted, the development of the disease in the control and the IFN-{beta} group was almost indistinguishable (data not shown).

Together, these results show that a low dose of IFN-{beta} partially or completely protects against progressive leishmaniasis depending on the size of the parasite inoculum.

IFN-{beta} enhances NK cell cytotoxic activity in L. major-infected mice

As a first step to elucidate the mechanism(s) underlying the protective effect of IFN-{beta}, we analyzed the NK cell activity in the lymph nodes of L. major-infected mice. As published previously (36, 37), NK cell cytotoxic activity was only observed in infected mice (data not shown) and was clearly detectable in the popliteal lymph nodes of resistant C57BL/6 mice, but only barely present in susceptible BALB/c mice at day 1 of infection. However, after treatment with IFN-{beta} 4 h before and along with the infection, the NK cell activity in BALB/c mice was dose-dependently restored and became comparable to the levels seen in C57BL/6 mice (Fig. 2).



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  		FIGURE 2. IFN-{beta} enhances NK cell cytotoxic activity. The indicated IFN-{beta} doses or PBS were applied i.p. 4 h before infection and s.c. along with the parasites (1 x 106). Twenty-four to 36 h after L. major infection, popliteal lymph node cells (from groups of three C57BL/6 and three BALB/c mice) were prepared and NK cell cytotoxic activity was measured (*, p < 0.05; **, p < 0.02; ***, p < 0.005 compared with PBS control). One of eight similar analyses.

 
IFN-{beta} enhances lymphocyte proliferation and IFN-{gamma} production in L. major-infected BALB/c mice

Lymph node cells from L. major-infected BALB/c mice show an impaired response to L. major Ag, Con A, and to IL-12 compared with mouse strains with a healer phenotype (38, 39, 40). Since positive effects of type I IFNs on lymphocyte proliferation and/or IFN-{gamma} production have been described in various systems (20, 41, 42, 43), we tested whether IFN-{beta} treatment could reinstate a regular immune response in BALB/c mice. Indeed, IFN-{beta} treatment strongly enhanced the proliferation of lymph node cells in response to IFN-{beta}, IL-12 plus IL-18, Con A as well as L. major Ag at day 25 of infection (Fig. 3A). Comparable results were obtained at days 15, 40, and 50 of infection (data not shown). IFN-{beta} also increased the expression of IFN-{gamma} mRNA and decreased the level of IL-4 mRNA in the lymph nodes at early time points of infection (day 3, 5, 7, or 10 of infection; Fig. 3B and data not shown). In vivo treatment with IFN-{beta} restored or up-regulated the release of IFN-{gamma} protein by lymph node cells after stimulation with IL-12, IL-18, IL-12 plus IL-18, Con A, or L. major Ag (e.g., days 15, 25, 40, 50, and 60 of infection; Fig. 3C and data not shown). These striking effects were only seen in mice that clinically responded to the IFN-{beta} treatment (data not shown). Intracellular cytokine staining during days 15 and 55 of infection revealed that IFN-{beta} treatment caused a 60–80% increase in the percentage of IFN-{gamma}+CD8+ and IFN-{gamma}+CD4+ T cells in the draining lymph node (Fig. 4). Thus, IFN-{beta} clearly promotes the production of IFN-{gamma} in L. major-infected BALB/c mice.



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  		FIGURE 3. IFN-{beta} enhances lymphocyte proliferation and IFN-{gamma} production. BALB/c mice were infected with 1 x 106 L. major promastigotes and treated with PBS or IFN-{beta} (5 x 103 U/injection) following the standard protocol (see legend to Fig. 1). A, Proliferative response of total lymph node cells (day 25 of infection). B, IFN-{gamma} and IL-4 mRNA expression in popliteal lymph nodes from IFN-{beta}- or PBS-treated L. major-infected BALB/c mice as assessed by real-time RT-PCR analysis (day 10 of infection). C, IFN-{gamma} production of popliteal lymph node cells from PBS- or IFN-{beta}-treated BALB/c (day 25 of infection). One of 5 (A), 3 (B), or 14 (C) similar experiments.

 


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  		FIGURE 4. IFN-{beta} up-regulates the expression of IFN-{gamma} in CD4+ as well as CD8+ T cells. BALB/c mice were infected with 1 x 106 L. major promastigotes and treated with PBS or IFN-{beta} (5 x 103 U/injection) following the standard protocol (see legend to Fig. 1). At day 25 of infection, lymph node cells were analyzed for the expression of IFN-{gamma} by intracellular cytokine staining as described in Materials and Methods.

 
IFN-{beta} enhances the expression of iNOS in L. major-infected BALB/c mice

iNOS is essential for the control of L. major in the skin and lymph node (2, 23, 29). In self-healing C57BL/6 mice, the early expression of iNOS was dependent on IFN-{alpha}{beta} (23). Furthermore, IFN-{alpha}{beta} in combination with L. major promastigotes induced iNOS in macrophages (23, 24). Compared with C57BL/6 mice, L. major-infected BALB/c mice exhibited a reduced tissue expression of iNOS (44). We therefore investigated whether IFN-{beta} modulates the expression of iNOS and whether protection by IFN-{beta} can occur in the absence of iNOS. By immunohistology, low-dose IFN-{beta} treatment (5 x 103 U/ injection) up-regulated the expression of iNOS protein in the skin lesion and draining lymph node of L. major-infected BALB/c mice at days 15, 25, and 40 of infection, both with respect to the intensity of the iNOS staining and the number of iNOS-positive cell clusters; the latter was increased by a factor of 4- to 10-fold, depending on the organ and time point of infection (e.g., at day 15 of infection, there were 5 ± 1.6 vs 56 ± 10.8 iNOS+ clusters in the footpad of PBS- vs IFN-{beta}-treated BALB/c mice; mean ± SD of 5 sections) (Fig. 5 and data not shown). When single-cell suspensions were prepared from the lymph nodes of PBS- or IFN-{beta}-treated L. major-infected BALB/c mice, iNOS protein was readily detectable in the lymph node cells from low-dose IFN-{beta}-treated mice. In contrast, cells from PBS- or high-dose IFN-{beta}-treated mice required further stimulation with IFN-{gamma} in vitro to reveal the expression of iNOS by Western blotting (Fig. 6A).



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  		FIGURE 5. IFN-{beta} up-regulates the tissue expression of iNOS protein. At day 25 of infection with 1 x 106 L. major parasites, footpad (A and B) and popliteal lymph node sections (C and D) from BALB/c mice treated with PBS (A and C) or IFN-{beta} (B and D; 5 x 103 U/injection following the standard protocol, see legend to Fig. 1) were analyzed by anti-iNOS immunoperoxidase staining (red). Nuclei were counterstained with hematoxylin (blue). Magnification, x400 (A and B), x200 (C and D). One of eight experiments.

 


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  		FIGURE 6. IFN-{beta} enhances the expression of iNOS, up-regulates the expression and tyrosine phosphorylation of STAT1{alpha} and requires iNOS for conferring protection against L. major infection. A and B, BALB/c mice infected with L. major (1 x 106 parasites) were treated with PBS or the indicated doses of IFN-{beta} following the standard application scheme (legend to Fig. 1). A, At day 25 of infection, lymph node cells were cultured in medium alone (lanes NS) or in the presence of IFN-{gamma} (20 ng/ml; lanes I). After 24 h, cell lysates were analyzed for the expression of iNOS and {beta}-actin by sequential Western blotting. B, At day 5 or 45 of infection, equal amounts of total lymph node lysates (2.5 mg) were either immunoprecipitated with anti-STAT1 or anti-SOCS1 followed by anti-STAT1, anti-phosphotyrosine, or anti-SOCS1 Western blotting or directly analyzed by anti-{beta}-actin Western blotting. C, Groups of three BALB/c, C57BL/6 iNOS+/+, and C57BL/6 iNOS–/– mice were infected with 1 x 106 L. major promastigotes, treated with PBS or IFN-{beta} (5 x 103 U/injection, following the standard protocol), and followed for the development of skin lesions. x depicts the occurrence of skin ulcers. Significant difference (p < 0.005 or smaller) between the BALB/c PBS control group and the BALB/c 5 x 103 IFN-{beta} group from day 15 onward. One of three (A), six (B), and two (C) experiments.

 
As a molecular correlate for the differential regulation of iNOS by low and high doses of IFN-{beta}, the protein expression of STAT1{alpha} increased from days 5–45 of infection in the total lymph node cells from low-dose IFN-{beta}-treated mice, but decreased in the high-dose IFN-{beta}-treated mice and the control mice that received PBS (Fig. 6B). Furthermore, tyrosine phosphorylation of STAT1{alpha} was induced and the expression of SOCS1 protein was suppressed at both early and late time points of infection in the cells from the low-dose IFN-{beta} group, whereas no such changes were observed in mice treated with high doses of IFN-{beta} or PBS (Fig. 6B). In accordance with these data, the transcription factor STAT1{alpha} was shown to be essential for the IFN-{alpha}{beta}-mediated expression of iNOS in other systems, whereas SOCS1 was found to have an opposite effect (45, 46, 47).

To test whether IFN-{beta} can cause protection against progressive cutaneous leishmaniasis in an iNOS-independent manner, we treated iNOS+/+ and iNOS–/– mice on a genetically resistant background (C57BL/6) with IFN-{beta} using the standard low-dose protocol. As illustrated in Fig. 6C, IFN-{beta} treatment almost completely suppressed the skin swelling in iNOS+/+ mice, but was unable to prevent the development of ulcerated skin lesions in iNOS–/– mice.

From these data we conclude that the protective effect of IFN-{beta} in BALB/c mice is at least partly due to the up-regulation of iNOS and that IFN-{beta} is unable to confer protection against L. major in the absence of iNOS even in otherwise genetically resistant mice.

The role of STAT4 and IL-12 for the protective effect of IFN-{beta}

While this study was in progress, two groups working on unrelated mouse models showed that IFN-{gamma} production can be triggered by type I IFN via a STAT4-dependent, but IL-12-independent pathway. Whether this pathway is relevant for the control of infectious pathogens in vivo was not investigated (42, 43). We therefore tested whether the protective effect of IFN-{beta} in experimentalcutaneous leishmaniasis is mediated by STAT4 and/or IL-12.

In vivo treatment with low doses, but not with high doses of IFN-{beta}, up-regulated tyrosine phosphorylation of STAT4 in the lymph nodes of L. major-infected BALB/c mice as analyzed by direct ex vivo immunoprecipitation and Western blotting (Fig. 7A). Furthermore, the application of low doses of IFN-{beta} in vivo restored or enhanced the tyrosine phosphorylation of STAT4 in spleen cells after stimulation with IL-12 in vitro (data not shown). Analysis of BALB/c wild-type vs BALB/c STAT4–/– mice revealed that the IFN-{beta}-mediated enhancement of IFN-{gamma} production by lymph node or spleen cells in response to IL-12, IL-18, IFN-{beta}, L. major Ag, or combinations thereof was strictly dependent on the presence of STAT4 (Fig. 7B and data not shown). In STAT4–/– mice infected with a high dose of L. major promastigotes, IFN-{beta} treatment led to an improved clinical course of infection and a 102- to 103-fold reduced parasite burden in the skin, lymph node, and spleen. However, the number of residual parasites in IFN-{beta}-treated BALB/c mice remained high and IFN-{beta} was unable to prevent ultimate disease progression in the absence of STAT4 (Fig. 7, C and D, and data not shown). Thus, the restoration of the IFN-{gamma} production in BALB/c mice by IFN-{beta} requires STAT4, but in addition to that there is also a STAT4-independent protective effect of IFN-{beta}.



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  		FIGURE 7. IFN-{beta} enhances the tyrosine phosphorylation of STAT4 and requires STAT4 for the up-regulation of IFN-{gamma} production and for conferring full protection against progressive leishmaniasis. Groups of four BALB/c wild-type or BALB/c STAT4–/– mice were infected with L. major (1 x 106 parasites) and treated with PBS or IFN-{beta} (5 x 103 U/injection unless otherwise indicated) following the standard protocol (legend to Fig. 1). A, At day 5 or 45 of infection, total lymph node cells from BALB/c wild-type mice were analyzed for the expression and tyrosine phosphorylation of STAT4 by sequential immunoprecipitation and Western blotting. B, At day 25 of infection, total spleen cells were restimulated for 24 h as indicated and analyzed for the release of IFN-{gamma} by ELISA. C, Clinical course of infection as assessed by the increase of the footpad thickness (mean ± SD). Significant difference (p < 0.005 or smaller) between the PBS control mice and the IFN-{beta}-treated mice of both wild-type and STAT4–/– mice from day 25 onward. D, Tissue parasite burden at day 50 of infection as determined by limiting dilution analysis. One of six (A) or three (B–D) similar experiments.

 
Finally, we tested whether IFN-{beta} treatment enhances the production of IL-12 in L. major-infected BALB/c mice and whether endogenous IL-12 is needed for the protective effect of IFN-{beta}. At early time points of infection (days 3, 5, and 7), IFN-{beta} up-regulated the expression of IL-12p35 mRNA and, to a lesser extent, also of IL-12p40 mRNA. At later time points of infection (days 10 and 40), no significant differences were observed between PBS- or IFN-{beta} treated mice (Fig. 8A and data not shown). Infection of BALB/c IL-12p35–/– mice with high (106; Fig. 8B) or low (104; Fig. 8C) numbers of L. major promastigotes led to rapidly progressive and ulcerated skin lesions and death from visceral disease within 60–70 days. Treatment with IFN-{beta} markedly decreased the size of the cutaneous lesions and delayed the time point of ulceration (Fig. 8, B and C). IFN-{beta} led to a drastic reduction of the tissue parasite burden (103- to 105-fold), most notably in the spleen after low-dose infection, but ultimately was not able to prevent progressive visceral disease (Fig. 8, B and C, and data not shown). Very similar results were obtained with IL-12p35/p40 double knockout mice on a C57BL/6 background, in which IFN-{beta} treatment suppressed the development of ulcerated skin lesions for at least 100 days, whereas the control mice had died by day 60 of infection (data not shown). Thus, IFN-{beta} is able to improve the course of L. major infection in the absence of IL-12, but for its full protective effect endogenous IL-12 is required.



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  		FIGURE 8. IFN-{beta} enhances the expression of IL-12 and improves the clinical course of infection in BALB/c IL-12p35–/– mice. Groups of four BALB/c and BALB/c IL-12p35–/– mice were infected with 106 (A and B) or 104 L. major promastigotes (C) and treated with PBS or IFN-{beta} (5 x 103 U/injection) following the standard protocol (legend to Fig. 1). A, Real-time RT-PCR analysis of IL-12p35 and p40 mRNA expression in the draining lymph nodes from three BALB/c wild-type mice per group and time point. B and C, Lesion development and parasite burden (day 30 of infection) in BALB/c wild-type and IL-12p35–/– mice infected with the high (B) or low parasite number (C). Significant difference (p < 0.05 or smaller) between the PBS groups and the IFN-{beta} groups from day 25 (B) or 30 (C) onward. One of three (A) and two (B and C) similar experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report that IFN-{beta} can reverse the outcome of a long-term infection with an intracellular protozoan parasite. Our findings illustrate that IFN-{beta} stimulates multiple components of the innate and adaptive immune response and thereby protects otherwise highly susceptible BALB/c mice against a progressive infection with L. major. The results not only bear significance with respect to signaling pathways involved, but also reveal an unexpected dose dependency of the effect of IFN-{beta} with potential implications for future therapeutic applications of this cytokine.

IFN-{alpha}{beta} and infections

Type I IFNs are best known for their rapid induction after virus infections and their potent antiviral activities. The direct antiviral effects are attributable to the induction of proteins that promote mRNA degradation, inhibit mRNA translation, and block viral transcription (reviewed in Refs. 48 and 49). Additional, indirect antiviral effects presumably result from the numerous positive immunostimulatory functions of IFN-{alpha}{beta}, such as the activation of the cytolytic and/or proliferative capacity of NK cells and CD8+ CTLs (41, 50, 51, 52). Several nonviral pathogens (including Chlamydia trachomatis, Chlamydia pneumoniae, Mycobacterium avium, Mycobacterium tuberculosis, Listeria monocytogenes, Toxoplasma gondii, Trypanosoma cruzi, and L. major) have been described to induce the production of IFN-{alpha}{beta} in macrophages, dendritic cells, or fibroblasts in vitro and/or in mice in vivo (for a review, see Ref. 15 ; Refs. 53, 54, 55). However, only very few studies tested the effect of type I IFN when applied to mice infected with these pathogens. In mice infected with M. tuberculosis via the respiratory route, intranasal administration of 104 U of purified mouse IFN-{alpha}{beta} for 5 consecutive days per week for 4 wk led to increased numbers of bacteria in the lung, a 30–57% suppression of pulmonary IL-12 mRNA levels, and a reduction of the mean survival period of the mice from 112 to 35 days (56). In contrast, a partial protective effect of type I IFN was observed in M. avium-infected mice, in which the continuous infusion of rmIFN-{beta} (ca. 104 U/day) led to a one-log decrease in the bacterial burden in the liver and spleen (57). Similarly, T. cruzi-infected mice developed slightly (2- to 8-fold) reduced parasitemias after treatment with purified mouse IFN-{alpha}{beta} in seven daily doses starting on the day of infection, but the mortality rate of the disease remained unaltered (58). In mice infected with T. gondii the application of 104 U rmIFN-{beta} once before and once after infection protected 70% of the mice against early death during the brief observation period of 20 days, provided the parasite inoculum was not higher than 10 tachyzoites (59). In the short-term L. monocytogenes i.v. infection model, in which 100% of the mice died within 5–7 days of infection, a single injection of rmIFN-{beta} (106 U) conferred complete protection during the 14 days of the experiment (60). Together with our present analysis, this latter study is the only report that rmIFN-{beta} is able to completely reverse the course of a nonviral infection.

Mechanisms of action of rmIFN-{beta} in vivo

In the phenotypic studies discussed above, the mechanisms of the protective effect of type I IFNs in vivo were either not analyzed (58) or were assumed to result from the activation of macrophages (57, 59, 60) and/or the induction of IFN-{gamma} (59). The data in this article provide novel insights into the actions of IFN-{beta}. The protection conferred by IFN-{beta} most likely reflects pleiotropic effects on NK cells, macrophages, and T cells as revealed by 1) an enhanced NK cell cytotoxic activity; 2) an up-regulation of the production of IFN-{gamma} and suppression of IL-4; 3) an increased tyrosine phosphorylation of STAT1{alpha} and expression of iNOS; and 4) a restored responsiveness to IL-12. IFN-{beta} increased the expression of IL-12 at early time points of infection, up-regulated the tyrosine phosphorylation of STAT4, and induced IFN-{gamma} in a largely STAT4-dependent manner. The use of STAT4–/– and IL-12p35–/– mice demonstrated that long-lasting protection against L. major by IFN-{beta} requires IL-12 as well as STAT4. Although it remains to be elucidated whether IFN-{beta} acts via direct or indirect activation of STAT4 in vivo, the present study shows for the first time that STAT4 is an important component for the type I IFN-mediated control of an infectious pathogen. However, it should be noted that even in the absence of IL-12 or STAT4 IFN-{beta} led to a transient, but significant reduction of the lesion size and of the tissue parasite burden. This is likely to involve the activation of STAT1, the expression of iNOS as well as the induction of NK cell cytotoxicity, all of which can occur in the absence of STAT4 or IL-12 signaling (Ref. 61 and data not shown). Thus, the recently described STAT4-dependent (but IL-12-independent) pathway of induction of IFN-{gamma} by type I IFN (42, 43) is not solely responsible for the protective function of IFN-{beta} in the L. major model.

Dose dependency of the effect of IFN-{beta}

The finding that lower doses of IFN-{beta} were more effective in preventing progressive cutaneous leishmaniasis than higher doses was unexpected and raises the question as to possible underlying mechanisms. Our analyses of the phosphorylation of STAT1{alpha} and STAT4 and of the expression of SOCS1 and of iNOS provide molecular correlates for the superior clinical effect of 5 x 103 as compared with 1 x 105 U of IFN-{beta}. Furthermore, we previously observed that only intermediate, but not very high (>2000 U/ml) or very low (<100 U/ml) concentrations of IFN-{alpha}{beta} were able to synergize with L. major parasites for the induction of iNOS in murine macrophages (24). Interestingly, with respect to NK cell cytotoxic activity, higher doses of IFN-{beta} were more potent than lower doses (Fig. 2), indicating that the dose dependency of the effect of IFN-{beta} might vary with the cell type.

There are a few other examples for dose-dependent activities of type I IFNs. Maximal in vitro effects of IFN-{alpha}1 or IFN-{beta}4 on the differentiation of CTLs were seen with low concentrations in the range of 1–100 U/ml (51). High (≥100 U/ml), but not low concentrations of IFN-{alpha} or IFN-{beta} suppressed the Staphylococcus aureus-induced production of IL-12 and IFN-{gamma} by mixed splenocytes (62). Nonarray-based gene expression profiling of human vascular endothelial cells after stimulation with type I IFN revealed that low doses (50 pg/ml) of IFN-{alpha}2b induced only a subset of genes compared with high doses (5000 pg/ml). In the same in vitro system, a cluster of 220 cDNAs was found to be less efficiently induced by the highest doses of IFN-{beta}1a (1 or 5 ng/ml) compared with low doses (50 or 200 pg/ml) (63). Oral treatment of mice with purified IFN-{alpha}{beta} for 7 days reduced the number of B cells in the spleens. The optimal effect was achieved with 1 U of IFN-{alpha}{beta} per mouse and day, whereas both higher and lower doses showed less significant effects (64). The molecular basis for these striking dose-response profiles is currently unknown, but might involve negative feedback phenomena, inhibitory cross-talk between different components of the IFN signaling pathways as well as concentration-dependent occupancy of different ligand binding sites of the type I IFNR (63, 65).

In conclusion, the presented results show a potent protective effect of IFN-{beta} against an otherwise fatal L. major infection in the highly susceptible BALB/c mouse. They also unequivocally demonstrate that the immunoregulatory properties of IFN-{beta}, which in the previously studied viral infection models could not be reliably segregated from indirect effects resulting from its strong antiviral activities, are per se sufficient for the control of an infectious pathogen. Our study highlights the critical impact of the dosing and the treatment protocol. The observation that different doses of IFN-{beta} differentially affect important signaling and effector pathways provides a molecular explanation for the variable outcome of type I IFN therapies of autoimmune disorders and should receive particular attention during future clinical studies.


   Acknowledgments
 
We are grateful to Dr. Ion Gresser for his valuable advice throughout the years, to Andrea Hesse for technical help, to Drs. Gottfried Alber (University of Leipzig, Leipzig, Germany) and Horst Mossmann (Max Planck Institute, Freiburg, Germany) for providing knockout mice, and to Dr. Ulrike Schleicher for critical reading of this manuscript.


   Footnotes
 
1 This work was supported by a grant from the European Community (QLK2-CT-2001-02103) and the Research Program "Innate Immunity" of the German Research Foundation (Grant DFG Bo 996/3-1 to C.B.). Back

2 Current address: Department of Pathology, University of Chicago, 5841 South Maryland, MC 1089, Chicago, IL 60637. Back

3 Address correspondence and reprint requests to Dr. Christian Bogdan, Department of Medical Microbiology and Hygiene, Institute of Medical Microbiology and Hygiene, University Clinic of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany. E-mail address: bogdancn{at}ukl.uni-freiburg.de Back

4 Abbreviations used in this paper: iNOS, inducible NO synthase (NOS2); rm, recombinant murine; CHO, Chinese hamster ovary; SOCS1, suppressor of cytokine signaling-1. Back

Received for publication August 11, 2003. Accepted for publication March 31, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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