Inducible nitric oxide synthase-deficient mice develop enhanced type 1 cytokine-associated cellular and humoral immune responses after vaccination with attenuated Schistosoma mansoni cercariae but display partially reduced resistance

Abstract

High levels of nitric oxide (NO) are produced by inducible nitric oxide synthase (iNOS) in response to activating signals from Th1-associated cytokines and play an important role in cytotoxicity and cytostasis against many pathogenic microorganisms. In addition to its direct effector function, NO serves as a potent immunoregulatory factor. NO produced by gamma interferon-activated macrophages immobilizes and kills Schistosoma mansoni larvae, and several studies have indicated a role for this pathway in protective immunity against this parasite. The potential regulatory influence of NO in immunity to S. mansoni is less well understood. In this study, we have used iNOS-deficient mice to determine the role of NO in mice vaccinated with irradiated cercariae of S. mansoni. We show by enzyme-linked immunosorbent assay and reverse transcriptase PCR analysis that vaccinated iNOS-deficient mice develop exacerbated type 1 cytokine responses in the lungs, the site where resistance to infection is primarily manifested. In addition, parasite-specific immunoglobulin G2a (IgG2a) and IgG2b antibody responses were significantly increased in vaccinated iNOS-deficient animals and total IgE antibody levels in serum were decreased relative to those in wild-type controls. Surprisingly, since resistance in this vaccine model is largely Th1 dependent and since Th1-related cellular and humoral immune responses were found to be exacerbated in vaccinated iNOS-deficient mice, vaccine-elicited protective immunity against challenge infection was found to be reduced. These findings demonstrate that iNOS plays a paradoxical role in immunity to S. mansoni, both in the effector mechanism of resistance and in the down regulation of the type 1 cytokine response, which is ultimately required for NO production.

FIG. 1

Peritoneal macrophages from vaccinated iNOS-deficient mice fail to kill schistosomula in vitro. Larvicidal activity and NO production in SWAP-elicited macrophages isolated from vaccinated WT and iNOS-KO mice was evaluated. Mice were vaccinated with 500 cercariae irradiated with 40 kilorads and were injected intraperitoneally with 250 μg of SWAP 5 weeks later. The 18-h antigen-elicited cells were assayed for their ability to kill 3-h schistosomula and to secrete NO. Separate cultures also contained IFN-γ (100 U/ml) or a combination of IFN-γ and the inhibitor of NO synthase (l-NMMA). The data reported are the means and standard deviations of triplicate determinations and are representative of three experiments performed. The background spontaneous death of schistosomula in cultures without peritoneal cells added is indicated by the horizontal line in panel B.

FIG. 2

Vaccine-induced protection is reduced but not eliminated in iNOS-deficient mice. Groups of C57BL/6 × 129SvEv F₂ (WT, +/+) and iNOS-KO (−/−) mice were vaccinated (Vac) with 500 cercariae irradiated with 40 kilorads and were challenged percutaneously 5 weeks later (12 animals/group) with 120 nonattenuated cercariae. Nonvaccinated and challenged mice were included as controls. Worm recovery was assessed 6 weeks postchallenge and is illustrated as individual worm burdens. The average worm burden is indicated by the horizontal line in each group. Statistical comparisons were made by using Student’s t test. An almost identical level of protection was observed in a second experiment.

FIG. 3

Type 1-associated cytokine expression is increased in iNOS-deficient mice. C57BL/6 × 129SvEv F₂ (WT) and iNOS-KO vaccinated and control nonvaccinated mice were challenged with 500 unattenuated cercariae, as described in Materials and Methods. The animals were sacrificed on days 0, 10, 14, or 18 postchallenge. LALN were isolated, placed in culture at 3 × 10⁶ LALN cells/ml, and restimulated with medium alone, SWAP (50 μg/ml), or ConA (5 μg/ml). Culture supernatants were isolated at 72 h and assayed for production of IFN-γ (A) or IL-5 (B). The reactivities of lymph node cells pooled from four mice per time point are illustrated.

FIG. 4

IgE levels in serum are decreased in vaccinated iNOS-deficient mice. C57BL/6 × 129SvEv F₂ (WT) and iNOS-KO vaccinated and control nonvaccinated mice were challenged with 500 unattenuated cercariae. On days 0, 10, 14, and 18 postchallenge, four animals per group were bled and IgE levels in serum were determined by ELISA. The total IgE level in serum was recorded as the mean and standard error. Statistical comparisons were made by using Student’s t test, and the asterisk indicates that the groups are significantly different at that time point (P < 0.05).

FIG. 5

The Th1-associated humoral response is more prominent in vaccinated iNOS-deficient mice. C57BL/6 × 129SvEv F₂ and iNOS-KO vaccinated and control nonvaccinated mice were challenged with 500 unattenuated cercariae. On days 0 (solid circles), 10 (open circles), 14 (solid squares), and 18 (open squares) postchallenge, four animals per group were bled and SWAP-specific antibody isotypes were determined by ELISA. The serum from individual mice was diluted as illustrated in the figure, and the average optical density (O.D.) for each group is presented.

FIG. 6

The size of inflammatory foci induced by schistosomula in the lungs of iNOS-deficient mice are diminished and eosinophil accumulation in the lung is also reduced. C57BL/6 × 129SvEv F₂ (WT) and iNOS-KO vaccinated and control nonvaccinated mice were challenged with 500 unattenuated cercariae. On days 10, 14, and 18 postchallenge, the left lungs were placed in fixative and processed routinely to measure the size of inflammatory foci (A) and the degree of tissue eosinophilia (B). The diameters of all lesions (3 to 12 lesions) per lung per mouse (four mice per group) were measured with an ocular micrometer, and the volume of each focus was calculated by assuming a spherical shape. The averages for each group at each time point are shown. Statistical comparisons were made by using Student’s t test, and the asterisk indicates that the groups are significantly different at that time point (P < 0.05).

FIG. 7

Type 1 cytokine mRNA production is enhanced and type 2 cytokine mRNA levels are reduced in the lungs of iNOS-deficient mice. C57BL/6 × 129SvEv F₂ (WT) and iNOS-KO vaccinated and control nonvaccinated mice were challenged with 500 unattenuated cercariae, as described in Materials and Methods. Four animals per group were sacrificed on days 0, 10, 14, and 18 postchallenge, and total lung RNA isolated for RT-PCR analysis. Changes in cytokine-specific mRNAs were calculated from standard curves and are reported as fold increases in mRNA expression over the average background expression observed in untreated (unexposed WT control) mouse lungs ± standard error. Fold changes in mRNA levels which were significantly (P < 0.05) different between groups at the same time point are indicated with an asterisk.

FIG. 8

Thioglycolate-elicited peritoneal macrophages from vaccinated iNOS-deficient mice produce increased levels of TNF-α in response to IFN-γ and LPS. The production of TNF-α by PECs was evaluated in WT and iNOS-deficient mice. Mice were injected with 1.5 ml of sterile thioglycolate, and PECs were harvested 4 days later and placed in 24-well plates at 5 × 10⁶ cells/ml. Individual wells were stimulated with IFN-γ (100 U/ml), LPS (250 ng/ml), or both. Culture supernatants were collected at 3, 6, and 24 h postactivation and examined for TNF-α by ELISA. The data reported are the means ± standard deviations for four mice per group.