Identification of nitric oxide synthase as a protective locus against tuberculosis

Abstract

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Figure 1

Absence of NOS2 confers susceptibility to primary Mtb infection. (a) Survival curves of litter-matched NOS2^−/− (n = 15), wild-type (n = 11), and heterozygous mice (n = 4) mice injected i.v. with 10⁵ CFU of Mtb Erdman bacilli. Data are from two independent experiments. Differences between NOS2^+/+ or NOS2^+/− and NOS2^−/− were statistically significant (P < 0.0001, log-rank test). (b) Genotypic and haplotypic allele-specific PCR strategies. Primer positions (arrows) for murine NOS2 originate within the antisense phosphoglycerate kinase-Neo^r targeted insertion (hatched box) or 5′ untranslated region sequences. Terminal 8-bp template sequences are shown. Sizes of the expected amplificands are in brackets. NRAMP1 primer pairs flank intron 5, with the 3′ polymorphic substitution (nucleotide 596) underlined. (c) NRAMP1 haplotype distribution versus Mtb growth (mean ± SEM) in the lungs (○, •), livers (□, ▪), and spleens (⋄, ♦) of NOS2^+/+ (Bcg^r, n = 7; Bcg^s, n = 3) and NOS2^−/− mice (Bcg^r, n = 9; Bcg, n = 5), respectively. (Inset) PCR amplificands of inherited NRAMP1 variant alleles (r, Bcg^r; s, Bcg^s) and intact or targeted NOS2 alleles (arrows), the latter shown with a NOS2^+/− control.

Figure 2

Presence of AFB correlates inversely with NOS2 expression in NOS2^+/+ and NOS2^−/− hosts. Histological analysis of AFB in diseased organs at day 30 p.i. stained by the Ziehl–Neelsen method. Acid-fast bacterial rods appear red. Magnification 400× (lung) and 100× (spleen). Localized NOS2 expression in lung granulomas assessed by immunohistochemistry using anti-MuNOS2 antibody. Magnification 200×. (Inset) 1,000×. At least six mice of each group were examined.

Figure 3

Phenotypic similarity of NOS2-deficient and glucocorticoid-immunosuppressed mice infected with Mtb. (a) Survival times for Mtb Erdman-infected (10⁶ CFU i.v.) mice given 2.5 mg of HC s.c. on days 5 and 10 p.i. NOS2^+/+ (n = 16); NOS2^−/− (n = 18). Nonimmunosuppressed control groups received vehicle (0.2 ml PBS) alone. NOS2^+/+ (n = 17); NOS2^−/− (n = 19). Both HC-treated groups and NOS2^−/− mice were significantly different from PBS-treated wild-type controls (P < 0.0002, log rank). Data represent two independent experiments. (b) Organ mycobacterial burdens (means ± SEM) of infected mice (n = 5–10 per time point). HC-treated groups were examined earlier (day 20) due to overt illness. ∗, P < 0.0002 versus the PBS-treated wild-type group, ANOVA.

Figure 4

Suppressive effects of HC on inducible NO production and tuberculostatic cytokines. (a) Release of NO₂⁻ + NO₃⁻ and RSNO during the course of infection and the effects of HC on their secretion. Symbols are as in Fig. 3 and represent individual sera assayed in triplicate; horizontal bars denote group means. ∗, P < 0.01, ∗∗, P < 0.0001, unpaired t test. (b) Plasma TNF-α or IFN-γ responses (mean ± SEM) of PBS- and HC-treated mice determined in triplicate by ELISA. ∗, P < 0.048, ∗∗, P < 0.038.

Figure 5

NOS2 inhibition accelerates disease progression during the clinically quiescent phase of tuberculosis in wild-type mice. (a) Mortality in clinically stable wild-type mice (10⁵ CFU i.v.) receiving 4 mM l-NIL supplied within the period bracketed (n = 8) or parenteral HC treatment (2.5 mg) administered s.c. on days 45 (HC-1) and 50 p.i. (HC-2) (n = 8). Controls were untreated NOS2^−/− (n = 10) and NOS2^+/+ mice (n = 10) and NOS2^+/+ mice given 4 mM d-NIL (n = 8). Both l-NIL and HC-treated groups were significantly different from d-NIL-treated wild-type controls (P < 0.0005, log rank). (b) Mycobacterial titers (means ± SEM) at the start (day 41 p.i.), within (day 60 p.i.) and at the cessation (day 70 p.i) of inhibitor treatment (n = 4 per time point, with a common pretreatment group). The day 70 p.i. time point was omitted for the HC group due to earlier mortality. Isolated symbols represent recovered inocula on day 1 p.i. ∗, P < 0.01, ∗∗, P < 0.0001 versus the d-NIL group, ANOVA.