NOS2-derived nitric oxide regulates the size, quantity and quality of granuloma formation in Mycobacterium avium-infected mice without affecting bacterial loads

Abstract

Granuloma formation in response to mycobacterial infections is associated with increased expression of inducible nitric oxide synthase (NOS2) within granuloma macrophages and increased levels of nitrate/nitrite in the sera of infected mice. Continuous treatment with 5 mm or 10 mm l-N6-(1-imino-ethyl)-lysine (L-NIL), a selective NOS2-inhibitor, in acidified drinking water for up to 7 weeks consistently reduced infection-induced nitrate/nitrite to background levels in mycobacteria-infected BALB/c mice. Oral treatment with 5 mm L-NIL initiated at the time of infection significantly exacerbated growth of Mycobacterium tuberculosis, but had no effect on Mycobacterium avium colony-forming unit development in the liver, spleen and lungs of intravenously infected mice. In order to examine the role of nitric oxide in mycobacteria-induced granulomatous inflammation in the absence of any effect on the bacterial load, M. avium-infected mice were treated with 5 mm L-NIL from day 1 through 38 and the development of granulomatous lesions in the liver was assessed by histology, immunohistology and reverse-transcription-polymerase chain reaction (RT-PCR). Computer- and video-assisted morphometry performed at 4 and 7 weeks post-infection showed that treatment with L-NIL led to markedly increased number, cellularity and size of granulomatous lesions in infected mice regardless of the virulence of the M. avium isolate used for infection. Immunohistology of the liver revealed that in mice treated with L-NIL, the numbers of CD3+ T cells, CD21/35+ B cells, CD11b+ macrophages and RB6-8C5+ granulocytes associated with granulomatous lesions was increased. RT-PCR of the liver showed that in L-NIL-treated mice infected with M. avium, mRNA levels of tumour necrosis factor, interleukin-12p40, interferon-gamma, interleukin-10 and interferon-gamma-inducible protein-10 (IP-10) were up-regulated, while mRNA levels of interleukin-4, monocyte chemotactic protein-1 (MCP-1) and MCP-5 were similar to those in untreated control infected mice. When M. avium-infected mice were treated with 5 mm L-NIL between the 5th and 12th weeks of infection, similar changes in granuloma number and size were found in the absence of any effect on the bacterial load. These findings demonstrate that nitric oxide regulates the number, size and cellular composition of M. avium-induced granulomas independently of antibacterial effects by modulating the cytokine profile within infected tissues.

Figure 7

Bacterial loads and granuloma size and number in BALB/c mice infected with M. avium SE01 and treated with L-NIL in the chronic phase of infection. BALB/c mice were infected with M. avium SE01 and treated from day 35 to day 84 with 5 mm L-NIL (open columns) or left untreated (filled columns). Bacterial CFU (a), granuloma number (b) and granuloma area (c) were determined on liver tissue obtained at day 84 post-infection. Data represent the means±SD from five mice per group. *P<0·005.

Figure 6

Semi-quantitative RT-PCR determination of mRNA expression in the livers of M. avium-infected BALB/c mice following treatment with L-NIL. Mice infected with M. avium TMC724 were killed on day 28 post-infection, mice infected with M. avium SE01 were killed on day 38 post-infection. (a) Liver samples from four mice per group were individually processed for RT-PCR and amplified product was hybridized with a specific internal probe and exposed to X-ray film. Uninfected control mice were analysed in parallel, and a reference cDNA in twofold dilution steps was included in each run for calibration. (b) Evaluation of pixel values and calculated x-fold increases over pixel background values from original data shown in (a). Significant differences in pixel values from L-NIL treated (open columns) and untreated infected mice (filled columns) are indicated. Pixel values from uninfected control mice (hatched columns) are shown for comparison.

Figure 1

Nitrate/nitrite levels in mycobacteria-infected BALB/c mice following treatment with L-NIL. (a) BALB/c mice were infected with M. tuberculosis Erdman and treated with 5 mm L-NIL in acidified drinking water (+L-NIL) or were left untreated (control). Nitrite/nitrate levels were determined in mouse sera obtained 15 days post-infection. (b) BALB/c mice were infected with M. avium TMC724 and treated with 5 mm L-NIL (+L-NIL) or were left untreated (control). Nitrite/nitrate levels were determined in mouse sera obtained 28 days post-infection. (c) BALB/c mice were infected with M. avium SE01 and treated with 5 mm L-NIL (+L-NIL) or were left untreated (control). Nitrite/nitrate levels were determined in mouse sera obtained 38 days post-infection. The data represent the means±SD of four or five mice per group. The mean levels of nitrite/nitrate in the sera of uninfected mice are shown for comparison (n=6). *P<0·05, **P<0·01 (when compared to untreated controls and not statistically different from uninfected controls)

Figure 2

Mycobacterial CFU counts in the spleens and livers of infected BALB/c mice following treatment with L-NIL. (a) BALB/c mice were infected with M. tuberculosis Erdman and treated with 5 mm L-NIL in acidified drinking water (open columns) or left untreated (filled columns). Bacterial organ loads were determined on day 30 post-infection. (b) BALB/c mice were infected with M. avium TMC724 and treated with 5 mm L-NIL (open columns) or left untreated (filled columns). Bacterial organ loads were determined on day 28 post-infection. (c) BALB/c mice were infected with M. avium SE01 and treated with 5 mm L-NIL (open columns) or left untreated (filled columns). Bacterial organ loads were determined on day 38 post-infection. The data represent the means±SD of four or five mice per group. *P<0·001 when compared to untreated controls.

Figure 5

Immunohistological evaluation of the cellular composition of liver granulomas in M. avium-infected BALB/c mice following treatment with L-NIL. BALB/c mice were infected with M. avium TMC724 and treated with 5 mm L-NIL (b,d,f,h,j,l) or left untreated (a,c,e,g,i,k) and killed on day 28 post-infection. Liver sections were stained with mAb specific for CD3 (a,b), CD21/35 (c,d), Ki-67 (e,f), polymorphonuclear leucocytes (g,h), CD11b (i,j), and NOS2 (k,l). Immunoperoxidase stains, ×128. Identical results were obtained following infection with M. avium SE01.

Figure 3

Microscopic morphology of granulomas in the livers of mycobacteria-infected BALB/c mice following treatment with L-NIL. (a,b) BALB/c mice were infected with M. tuberculosis Erdman and treated with 5 mm L-NIL (b) or left untreated (a). Liver sections were prepared from mice killed on day 30 post-infection. HE×128. (c,d) BALB/c mice were infected with M. avium TMC724 and treated with 5 mm L-NIL (d) or left untreated (a). Liver sections were prepared from mice killed on day 28 post-infection. HE 128. (e,f) BALB/c mice were infected with M. avium SE01 and treated with 5 mm L-NIL (f) or left untreated (e). Liver sections were prepared from mice killed on day 38 post-infection. HE×128.

Figure 4

Granuloma number and size in the livers of M. avium-infected BALB/c mice following treatment with L-NIL. (a,c) BALB/c mice were infected with M. avium TMC724 and treated with 5 mm L-NIL (open columns) or left untreated (filled columns) and killed on day 28 post-infection. (b,d) BALB/c mice were infected with M. avium SE01 and treated with 5 mm L-NIL (open columns) or left untreated (filled columns) and killed on day 38 post-infection. (a,b) The number of granulomas per 0·25 cm² were determined in HE-stained sections from different areas of the liver (five sections per mouse, four mice per group); the data represent the means of 20 determinations±SD. (c,d) The granuloma area was determined by video-assisted morphometry on different, non-sequential HE-stained sections from different areas of the liver of four mice per group; the data represent the means of 100 determinations±SD. *P<0·005.