Tumor necrosis factor-alpha antagonism by the murine tumor necrosis factor-alpha receptor 2-Fc fusion protein exacerbates histoplasmosis in mice

Abstract

Treatment of some inflammatory conditions with tumor necrosis factor-alpha (TNF-alpha) antagonists is efficacious, but such treatments are associated with infections with intracellular pathogens, including Histoplasma capsulatum. We explored protective immunity to H. capsulatum in mice given a fusion protein consisting of TNF-alpha receptor 2 (TNFR2) bound to the Fc portion of mouse IgG1. Intraperitoneal administration of this inhibitor exacerbated primary or secondary pulmonary infection at dosages ranging from 1 to 5 mg/kg. All mice with primary infection given the inhibitor succumbed to infection within 10-21 days of treatment. In secondary histoplasmosis, mice receiving 1, but not 5, mg/kg survived treatment. Fungal burden was increased even if treatment with the inhibitor was initiated after the onset of infection. The inflammatory response of the lungs of mice given the inhibitor did not differ from that of mice given control vehicle. Susceptibility was not associated with major alterations in cytokines known to protect or exacerbate infection. However, expression of nitric oxide synthase 2 (NOS2) was depressed early in primary infection. These results demonstrate that antagonism of endogenous TNF-alpha by this fusion protein modulates susceptibility. Impaired immunity is not a result of altered cytokine responses or changes in the inflammation and may not be demonstrable in other murine strains.

FIG. 1

Treatment with p75-Fc exacerbates primary histoplasmosis in mice. Groups of mice were infected with 2 × 10⁶ yeasts i.n. and treated with p75-Fc or PBS on the day of infection and every 2 days thereafter. Mice were killed on day 7, and lungs (A) and spleens (B) were assessed for fungal burden. Results are expressed as means ± SEM of 6–8 animals. (C) Survival curve of mice treated with 1 or 5 mg/kg of p75-Fc or PBS. (D) Mice (6–8 per group) were treated with p75-Fc or PBS every 4 days. Animals were followed for survival. One of two experiments is shown. **p < 0.01 vs. controls.

FIG. 2

Effect of p75-Fc on secondary histoplasmosis. Mice were infected with 10⁴ yeasts i.n. and 8 weeks later challenged with 2 × 10⁶ yeasts i.n. and given 1 or 5 mg/kg of p75-Fc every 2 days. (A) Survival was assessed. (B and C) Mice were treated with 5 mg/kg of p75-Fc every 2 days, and fungal burden was assessed in lungs (B) or spleens (C) at day 7 of infection. Results represent mean ± SEM of 6–8 per group. **p < 0.01.

FIG. 3

p75-Fc alters fungal burden when given in active infection. Naïve mice (A and B) or immune mice (C and D) were infected with 2 × 10⁶ yeasts i.n. and treated on the day of infection or on day 2 or 4 after infection. The biological was administered every 2 days thereafter. Results represent mean ± SEM of 6 mice. *p < 0.05 vs. controls; **p < 0.01 vs. controls.

FIG. 4

Inflammatory cells in lungs of mice administered p75-Fc. Mice were treated with 5 mg/kg p75-Fc every 4 days beginning on the day of infection. Lung leukocytes (>95% CD45⁺) were analyzed on (A and C) day 3 and (B and D) day 7 for several cell subpopulations. Results represent mean ± SEM of 4–8 animals. *p < 0.05.

FIG. 5

qRT-PCR of cytokines and NOS2 for primary and secondary histoplasmosis. RNA from lungs of mice was prepared, and cDNA was synthesized. qRT-PCR was performed on lungs using commercially available primers. Expression in primary infection was compared with uninfected lungs and in secondary infection with lungs from mice that had received 10⁴ yeasts 8 weeks before rechallenge. Results represent mean ± SEM of 4–6 animals per group. *p < 0.05.

FIG. 6

Comparison of p75-Fc and mAb to TNF-α. Mice were infected with H. capsulatum and given p75-Fc every 4 days or 1 mg mAb to TNF-α at the time of infection. On day 7, mice were killed, and CFUs were determined in lungs (A and C) and spleens (B and D) of mice with primary (A and B) or secondary (C and D) histoplasmosis. Results represent mean ± SEM of 4–8 animals. *p < 0.05; **p < 0.01.