Nitric oxide-enhanced resistance to oral candidiasis

Abstract

A murine model of oral candidiasis was used to show that nitric oxide (NO) is involved in host resistance to infection with Candida albicans in infection-'resistant' BALB/c and infection-'prone' DBA/2 mice. Following infection, increased NO production was detected in saliva. Postinfection samples of saliva inhibited the growth of yeast in vitro. Treatment with NG-monomethyl-L-arginine (MMLA), an inhibitor of NO synthesis, led to reduced NO production, which correlated with an increase in C. albicans growth. Reduction in NO production following MMLA treatment correlated with an abrogation of interleukin-4 (IL-4), but not interferon-gamma (IFN-gamma), mRNA gene expression in regional lymph node cells. Down-regulation of IL-4 production was accompanied with an increase in IFN-gamma production in infection-'prone' DBA/2 mice. There was a functional relationship between IL-4 and NO production in that mice treated with anti-IL-4 monoclonal antibody showed a marked inhibition of NO production in saliva and in culture of cervical lymph node cells stimulated with C. albicans antigen. The results support previous conclusions that IL-4 is associated with resistance to oral candidiasis and suggest that NO is involved in controlling colonization of the oral mucosal surface with C. albicans.

Figure 1

Nitric oxide (NO) production in saliva from mice infected with Candida albicans. BALB/c and DBA/2 mice were orally infected with C. albicans. At various time-points after infection, saliva was collected for determination of nitrite concentrations by the Griess reaction. Results shown represent the mean values + standard error of the mean for five mice. *P < 0·05 compared with values from control mice.

Figure 2

Patterns of colonization with Candida albicans in BALB/c and DBA/2 mice following treatment with N^G-monomethyl-l-arginine (MMLA). Mice were injected intraperitoneally with an inhibitor of nitric oxide (NO) synthase, MMLA, every 2 days for 8 and 13 days in BALB/c mice and DBA/2 mice, respectively, after which the levels of colonization in the oral mucosa were assessed. Results shown represent the mean values + standard error of the mean for five mice. *P < 0·05, **P < 0·01 compared with values from control mice.

Figure 3

Effect of saliva on Candida albicans viability. BALB/c and DBA/2 mice were infected orally with C. albicans. At various time-points after infection, saliva collected from individual mice was tested for its effect on the growth of C. albicans in vitro. Results shown represent the mean values + standard error of the mean for five mice. *P < 0·05, **P < 0·01 compared with values from N^G-monomethyl-l-arginine (MMLA)-treated mice.

Figure 4

Inhibition of antifungal activity in saliva treated with a nitric oxide (NO) scavenger. DBA/2 mice were infected with Candida albicans in the oral cavity. At various time-points, saliva was collected and tested for its capacity to inhibit fungal growth in culture with or without haemoglobin (10% vol/vol). After incubation for 2 hr at 37°, the viability of C. albicans was assessed. Data shown represent the mean values + standard error of the mean for five mice. *P ≤ 0·05 compared with values from control mice.

Figure 5

mRNA gene expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in cervical lymph node (CLN) cells from mice infected with Candida albicans. BALB/c and DBA/2 mice were infected orally with C. albicans. At various time-points after infection, total RNA was extracted from CLN cells to determine the levels of cytokine gene expression by reverse transcription–polymerase chain reaction (RT–PCR) amplification. Equivalent loading of each sample was determined by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message shown. N, negative control; P, positive control.

Figure 6

Interleukin-4 (IL-4) and interferon-γ (IFN-γ) production by cervical lymph node (CLN) cells in culture stimulated with Candida albicans. BALB/c and DBA/2 mice treated or untreated with N^G-monomethyl-l-arginine (MMLA) were infected orally with C. albicans. At various time-points after infection, CLN cells were isolated and then stimulated in culture with C. albicans for 3 days, after which time the amounts of IL-4 and IFN-γ in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Data shown represent the mean values + standard error of the mean for five mice. *P < 0·05, **P < 0·01, ***P < 0·0001 compared with values from untreated mice.

Figure 7

Nitric oxide (NO) production in saliva and culture supernatant of cervical lymph node (CLN) cells from mice treated with anti-interleukin-4 (αIL-4). BALB/c mice, treated or untreated with αIL-4, were challenged orally with Candida albicans. At various time-points after oral challenge, CLN cells were isolated and then stimulated in culture with C. albicans for 3 days, after which the NO concentrations in saliva and in culture supernatants of CLN cells, stimulated or unstimulated with C. albicans antigens, were measured. Results shown represent the mean values + standard error of the mean for five mice. *P < 0·05 compared with values from control mice.