Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice.

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.

Protective role of membrane tumour necrosis factor in the host's resistance to mycobacterial infection.

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.

Long-term control of Mycobacterium bovis BCG infection in the absence of Toll-like receptors (TLRs): investigation of TLR2-, TLR6-, or TLR2-TLR4-deficient mice.

Live mycobacteria have been reported to signal through both Toll-like receptor 2 (TLR2) and TLR4 in vitro. Here, we investigated the role of TLR2 in the long-term control of the infection by the attenuated Mycobacterium, Mycobacterium bovis BCG, in vivo. We sought to determine whether the reported initial defect of bacterial control (K. A. Heldwein et al., J. Leukoc. Biol. 74:277-286, 2003) resolved in the chronic phase of BCG infection. Here we show that TLR2-deficient mice survived a 6-month infection period with M. bovis BCG and were able to control bacterial growth. Granuloma formation, T-cell and macrophage recruitment, and activation were normal. Furthermore, the TLR2 coreceptor, TLR6, is also not required since TLR6-deficient mice were able to control chronic BCG infection. Finally, TLR2-TLR4-deficient mice infected with BCG survived the 8-month observation period. Interestingly, the adaptive response of TLR2- and/or TLR4-deficient mice seemed essentially normal on day 14 or 56 after infection, since T cells responded normally to soluble BCG antigens. In conclusion, our data demonstrate that TLR2, TLR4, or TLR6 are redundant for the control of M. bovis BCG mycobacterial infection.

Toll-like receptor 2 is required for optimal control of Listeria monocytogenes infection.

The control of Listeria monocytogenes infection depends on the rapid activation of the innate immune system, likely through Toll-like receptors (TLR), since mice deficient for the common adapter protein of TLR signaling, myeloid differentiation factor 88 (MyD88), succumb to Listeria infection. In order to test whether TLR2 is involved in the control of infections, we compared the host response in TLR2-deficient mice with that in wild-type mice. Here we show that TLR2-deficient mice are more susceptible to systemic infection by Listeria than are wild-type mice, with a reduced survival rate, increased bacterial burden in the liver, and abundant and larger hepatic microabscesses containing increased numbers of neutrophils. The production of tumor necrosis factor, interleukin-12, and nitric oxide and the expression of the costimulatory molecules CD40 and CD86, which are necessary for the control of infection, were reduced in TLR2-deficient macrophages and dendritic cells stimulated by Listeria and were almost abolished in the absence of MyD88, coincident with the high susceptibility of MyD88-deficient mice to in vivo infection. Therefore, the present data demonstrate a role for TLR2 in the control of Listeria infection, but other MyD88-dependent signals may contribute to host resistance.

Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection.

Recognition of Mycobacterium tuberculosis by the innate immune system is essential in the development of an adaptive immune response. Mycobacterial cell wall components activate macrophages through Toll-like receptor (TLR) 2, suggesting that this innate immune receptor plays a role in the host response to M. tuberculosis infection. After aerosol infection with either 100 or 500 live mycobacteria, TLR2-deficient mice display reduced bacterial clearance, a defective granulomatous response, and develop chronic pneumonia. Analysis of pulmonary immune responses in TLR2-deficient mice after 500 mycobacterial aerosol challenge showed increased levels of interferon-gamma, tumor necrosis factor-alpha, and interleukin-12p40 as well as increased numbers of CD4(+) and CD8(+) cells. Furthermore, TLR2-deficient mice mounted elevated Ag-specific type 1 T-cell responses that were not protective because all deficient mice succumb to infection within 5 months. Taken together, the data suggests that TLR2 may function as a regulator of inflammation, and in its absence an exaggerated immune inflammatory response develops.

Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice.

Endotoxin from Gram-negative bacteria bound to CD14 signals through Toll-like receptor (TLR) 4, while components of Gram-positive bacteria, fungi, and Mycobacterium tuberculosis (M.tb.) preferentially use TLR2 signaling. We asked whether TLR4 plays any role in host resistance to M.tb. infection in vivo. Therefore, we infected the TLR4 mutant C3H/HeJ mice and their controls, C3H/HeN mice, with M.tb. by aerosol. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10-100 times higher CFU organ levels than the wild-type mice and succumbed within 5-7 mo, whereas most of the wild-type mice controlled infection and survived the duration of the experiment. The lungs of TLR4 mutant mice showed chronic pneumonia with increased neutrophil infiltration, reduced macrophages recruitment, and abundant acid-fast bacilli. Furthermore, the pulmonary expression of TNF-alpha, IL-12p40, and monocyte chemoattractant protein 1 was significantly lower in C3H/HeJ mice when compared with the wild-type controls. C3H/HeJ-derived macrophages infected in vitro with M.tb. produced lower levels of TNF-alpha. Finally, the purified mycobacterial glycolipid, phosphatidylinositol mannosides, induced signaling in both a TLR2- and TLR4-dependent manner, thus suggesting that recognition of phosphatidylinositol mannosides in vivo may influence the development of protective immunity. In summary, macrophage recruitment and the proinflammatory response to M.tb. are impaired in TLR4 mutant mice, resulting in chronic infection with impaired elimination of mycobacteria. Therefore, TLR4 signaling is required to mount a protective response during chronic M.tb. infection.