Antigen-specific IFN-γ and nitric oxide release from splenocytes of <i>M. bovis</i> BCG-infected mice.

<p>IFN-γ and NO (nitric oxide) were evaluated in culture supernatant from cells incubated with antigens derived from <i>M. bovis</i> BCG (A and C) or with 10³ viable <i>M. bovis</i> BCG (B and D). Values are represented as mean ± <i>SEM</i> (<i>n</i> = 3 animals per group, assayed in triplicate). * p<0.04.</p

<i>M. tuberculosis</i> and <i>M. bovis</i> BCG infection studies performed in memTNF KI mice.

<p><i>M. tuberculosis</i> and <i>M. bovis</i> BCG infection studies performed in memTNF KI mice.</p

Decreased iNOS protein in memTNF KI but similar number of splenic CD11b⁺/TNF⁺ cells 4 weeks after <i>M. bovis</i> BCG infection.

<p>(A) Spleen iNOS protein expression was detected by western blot 4 weeks after <i>M. bovis</i> BCG infection. (B) iNOS protein was quantified by Image Quant software. Results are expressed as mean ± SEM of relative units of iNOS/actin (<i>n</i> = 4, except for TNFR1/TNFR2−/− mice <i>n</i> = 2 per group). *, p<0.04. FACS analyses showing the number of CD11b⁺ cells (C) and of CD11b⁺/TNF⁺ (D) in the spleen of uninfected, 2 and 4 weeks infected mice. Results are shown as mean ± SEM of positive cells per spleen (<i>n</i> = 4). *, p<0.01 compared to wild-type mice.</p

Hypothesis explaining differences between memTNF molecules.

<p>This figure presents the three situations explored in this work: wild-type TNF molecules which are digested by the TACE, and the two memTNF forms which cannot be digested and exert their activities by cell-to-cell contact. The difference between memTNF^Δ1–9,K11E and memTNF^Δ1–12 molecules may reside in the possibility of the TACE to bind but not cleave the memTNF ^Δ1–9,K11E, but in memTNF^Δ1–12 the TACE binding site is not accessible and the protease activity is exerted on TNFR2 which became more frequently soluble thus neutralising memTNF^Δ1–12 activities.</p

Lower number of macrophages expressing membrane bound TNFR2 but higher soluble TNFRs in memTNF^Δ1–12 KI mice.

<p>FACS analyses showing number of spleen CD11b⁺ cells expressing membrane TNFR1 (A) or TNFR2 (B) before or during <i>M. bovis</i> BCG infection. Results are shown as means ± SEM of spleen cell numbers (<i>n</i> = 3–4). *, p<0.02 compared to wild-type mice. Total amounts of TNFR1 (C) and TNFR2 (D) were evaluated on total spleen extracts during <i>M. bovis</i> BCG infection. Results are shown as means ± SEM of pg per spleen (<i>n</i> = 4–6). *, p<0.05 compared to wild-type mice. Amounts of solTNFR1 (E) and solTNFR2 (F) were assessed in the serum of mice before and after infection. Results as represented as means ± SEM of pg/ml of serum (<i>n</i> = 6–10). *, p<0.05 compared to wild-type.</p

BMDM nitric oxide, IL-6, RANTES production and p65 NF-κB activation by <i>M. bovis</i> BCG infection.

<p>NO (A), IL-6 (B) and RANTES (C) levels were assessed in supernatant of BMDM 24 hours after <i>M. bovis</i> BCG infection. These results are representative of two independent experiments and values are represented as mean ± <i>SEM</i> (<i>n</i> = 3 animals per group, assayed in triplicate) *p<0.05. (D) Phosphorylated and unphosphorylated p65 NF-κB proteins were detected by western blot in BMDM not infected or infected with <i>M. bovis</i> BCG at different time points. (E) Quantification of phosphorylated p65 NF-κB on western blot was done by Quantity One® analysis software on two different experiments. Results are expressed as mean ± SEM of relative units of phosphorylated p65 NF-κB/total p65 NF-κB (<i>n</i> = 2/group). *, p<0.05 compared to wild-type BMDM.</p

Histopathology of pulmonary lesions 4 weeks after <i>M. bovis</i> BCG infection.

<p>Wild-type mouse lung (A, E) shows small granulomas containing multinucleated giant cells, memTNF^Δ1–9,K11E KI mouse lung (B, F) shows larger granulomas compared with wild-type, also presenting multinucleated giant cells, and memTNF^Δ1–12 KI (C, G) and TNFR1/TNFR2−/− (D, H) mouse lungs exhibit large lesions containing numerous lymphocytes, neutrophils and monocytes, but lacking multinucleated giant cells. Regions of necrosis are observed in TNFR1/TNFR2−/− lungs. Figure is representative of two experiments (<i>n</i> = 6 per group). Magnification A–D 100× and E–H 400×.</p

Serum and lung levels of cytokines and chemokines after <i>M. bovis</i> BCG infection.

<p>IFN-γ levels were assessed in serum (A) and in lungs (B) at 2 and 4 weeks after <i>M. bovis</i> BCG infection. Serum (C) and lung (D) amounts of IL-12p40 were measured at 2 and 4 weeks after <i>M. bovis</i> BCG infection. Data are mean ± SEM and are representative of two independent experiments (<i>n</i> = 6–10 per group). *, p<0.03. RANTES (E, F) and MCP-1 (G, H) amounts were measured in serum (E, G) and in lungs (F, H) at 2 and 4 weeks after <i>M. bovis</i> BCG infection. Data are represented as means ± SEM from two independent experiments (<i>n</i> = 4–6 per group). *, p<0.04.</p

Decreased pulmonary phosphorylated p65 NF-κB in memTNF^Δ1–12 KI mice after 2 weeks of <i>M. bovis</i> BCG infection.

<p>(A) Phosphorylated and unphosphorylated p65 NF-κB and actin protein expressions were detected in lungs by western blot 2 weeks after <i>M. bovis</i> BCG infection. (B) Quantification of phosphorylated and unphosphorylated p65 NF-κB on western blot was done by Quantity One® analysis software on two different experiments. Results are expressed as mean ± SEM of relative units of phosphorylated p65 NF-κB/total p65 NF-κB (<i>n</i> = 4–5 mice/group) (*, p<0.02).</p

Survival curve, body weight and bacterial loads in lung and liver of mice infected with 10⁷CFU of <i>M. bovis</i> BCG.

<p>(A) Long-term survival of mice infected with living <i>M. bovis</i> BCG Connaught (10⁷). (B) Body weight change after <i>M. bovis</i> BCG infection. Wild-type and memTNF^Δ1–9,K11E KI, (<i>n</i> = 14 mice per group), memTNF^Δ1–12 (<i>n</i> = 10 mice), and TNF−/− mice (<i>n</i> = 6 per group). Data from two representative experiments are shown. CFU were determined in lungs (C, D) and liver (E, F) at 2 and 4 weeks after infection with 10⁷ CFU of <i>M. bovis</i> BCG. Data are represented as individual values and horizontal bars indicate mean (<i>n</i> = 4–6 mice per group). Asterisks indicate statistically significant differences between wild type and indicated group (*, p<0.03).</p