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Comparative Study
. 2008 Dec;125(4):522-34.
doi: 10.1111/j.1365-2567.2008.02865.x. Epub 2008 Jun 9.

Protective Role of Membrane Tumour Necrosis Factor in the Host's Resistance to Mycobacterial Infection

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Free PMC article
Comparative Study

Protective Role of Membrane Tumour Necrosis Factor in the Host's Resistance to Mycobacterial Infection

Nasiema Allie et al. Immunology. .
Free PMC article

Abstract

Tumour necrosis factor-alpha (TNF-alpha) plays a critical role in the recruitment and activation of mononuclear cells in mycobacterial infection. The role of membrane TNF, in host resistance against Mycobacterium bovis bacille Calmette-Guérin (BCG), was tested in knock-in mice in which the endogenous TNF was replaced by a non-cleavable and regulated allele (Delta1-12, TNF(tm/tm)). While 100% of mice with complete TNF deficiency (TNF(-/-)) succumbed to infection, 50% of TNF(tm/tm) mice were able to control M. bovis BCG infection and survived the experimental period. Membrane expressed TNF allowed a substantial recruitment of activated T cells and macrophages with granuloma formation and expression of bactericidal inducible nitric oxide synthase (iNOS). Using virulent Mycobacterium tuberculosis infection we confirm that membrane TNF conferred partial protection. Infection in TNF(tm/tm) double transgenic mice with TNF-R1 or TNF-R2 suggest protection is mediated through TNF-R2 signalling. Therefore, the data suggest that membrane-expressed TNF plays a critical role in host defence to mycobacterial infection and may partially substitute for soluble TNF.

Figures

Figure 1
Figure 1
Absence of soluble TNF after endotoxin or mycobacterial stimulation. (a) In vivo injection of 100 μg LPS i.p. in TNFtm/tm mice, TNF−/− mice and WT mice, serum was taken at 90 min and analysed for TNF by ELISA. (b) LPS and BCG induced TNF at 4 hr in the supernatant of cultured macrophages. Data are expressed as the mean ± SD (n = 4 mice). ND, not detected.
Figure 2
Figure 2
Survival of TNFtm/tm and TNF−/− mice infected with Mycobacterium bovis BCG. Body weight change (a) and survival (b) of TNFtm/tm (Δ), TNF-deficient mice (○) and WT mice (•) infected i.v. with 2 × 106 BCG bacilli and monitored for 3 months. Each group comprised 10 mice.
Figure 3
Figure 3
Lung pathology and uncontrolled bacterial growth in TNF−/− mice. TNFtm/tm mice (Δ), TNF−/− mice (○) and WT mice (•) were infected i.v. with 2 × 106 BCG M. bovis BCG. (a) Macroscopic lung changes in BCG infected mice at 2, 4 and 8 weeks post-infection. (b) Lung weights of BCG infected mice at 2, 4 and 8 weeks post-infection. (c) Bacterial load (CFU) was determined in lungs after 2, 4 and 8 weeks after BCG infection (2 × 106 BCG i.v.). The results are expressed as the mean ± SD of five mice.
Figure 4
Figure 4
Histopathology in lungs of M. bovis BCG infected TNFtm/tm mice, TNF−/− mice and WT mice. Mice (five mice per group)were killed at 2, 4 and 8 weeks post-infection and the sections stained with haematoxylin and eosin (a) (400×) or with Ziehl–Neelsen (b) (1000×) to detect the acid-fast bacilli.
Figure 5
Figure 5
Cellular recruitment in bronchoalveolar space in the lungs of M. bovis BCG-infected TNFtm/tm mice, TNF−/− mice and WT mice. BALF was collected from TNFtm/tm (line bar), TNF-deficient mice (open bar) and WT mice (black bar) infected i.v. with 2 × 106 BCG bacilli at 2, 4 and 8 weeks. Cells (200 cells/mouse) were stained using a Rapidiff staining kit (Clinical Sciences Diagnostics, Southdale, South Africa), the average differential cell count were analysed in triplicate and recorded as previously described. Mean values ± SD of total cell counts (a), macrophages (b), lymphocytes (c) and neutrophils (d) are given (n = 5).
Figure 6
Figure 6
Reduced pulmonary recruitment of CD4 and CD8 T cells in TNFtm/tm mice and TNF−/− mice. BALF was collected from M. bovis BCG-infected TNFtm/tm, TNF−/− and WT mice at 4 weeks post-infection as described in Methods. Four mice were individually analysed for each mouse strain. (a) Recruitment of CD4 and CD8 T cells in WT, TNFtm/tm and TNF−/− mice. (b) Expression of CD11a and CD44 on CD4 and CD8 cells in WT, TNFtm/tm and TNF−/− mice.
Figure 7
Figure 7
Reduced expression of iNOS in lungs of BCG-infected TNFtm/tm mice and TNF−/− mice at 4 weeks post-infection. Lung sections were incubated with iNOS antibody as described. Magnification 100×.
Figure 8
Figure 8
Cytokine and chemokine secretion in BALF of TNFtm/tm mice (line bar), TNF−/− mice (open bar) and WT mice (black bar). (a) IFN-γ and IL-12 were measured at 28 days, and (b) RANTES was analysed at 28 and 56 days. The results are expressed as the mean ± SD (n = 4).
Figure 9
Figure 9
Body weights and survival of TNFtm/tm, TNF−/− and WT mice exposed to Mycobacterium tuberculosis infection. TNFtm/tm mice (Δ), TNF−/− mice (○) and WT mice (•) were infected by aerosol (10–30 CFU/lung), and the survival monitored over 8 weeks. Bodyweight change is expressed as a ratio, relative to the bodyweight measured on the day of infection. Each group comprised 10 mice.
Figure 10
Figure 10
Mycobacterial burden in TNFtm/tm, TNF−/− and WT mice exposed to M. tuberculosis infection. (a) Lung (14, 28 and 45 days), (b) liver and spleen (45 days). TNFtm/tm (Δ, line bar), TNF-deficient mice (○, open bar) and WT mice (•, black bar) were infected by aerosol (10–30 CFU/lung). The results are expressed as the mean ± SD (n = 5).
Figure 11
Figure 11
Comparison of resistance to M. tuberculosis infection in TNFtm/tm and TNFR1−/− or TNFR2−/− × TNFtm/tm double transgenic mice. (a) Survival of mice infected by H37Rv i.n. (10–30 CFU per lung, n = 9–11 mice pooled from two independent experiments). (b) Mycobacterial burden in the lung (CFU) 6 weeks after infection. The results are expressed as the mean ± SD (n = 4–6 mice per group of transgenic mice).

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