Helen Briscoe

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection.

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF−/−) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b+ macrophages and CD4+ T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF−/− mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF−/− mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF−/− mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.

Transmembrane TNF Is Sufficient to Initiate Cell Migration and Granuloma Formation and Provide Acute, but Not Long-Term, Control of Mycobacterium tuberculosis Infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF−/−, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF−/− mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF−/− mice, into either RAG−/− or TNF−/− mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.

Up-Regulation of VCAM-1 and Differential Expansion of β Integrin-Expressing T Lymphocytes Are Associated with Immunity to Pulmonary Mycobacterium tuberculosis Infection

Immune responses rely on an intricate system of adhesion molecules to coordinate the homing and retention of lymphocytes in both secondary lymphoid tissues and at sites of infection. To define the events associated with pulmonary immune responses, the expression of endothelial addressins and integrins on T cells was analyzed during Mycobacterium tuberculosis infection. In infected lung, expression of endothelial VCAM-1, but not mucosal addressin cell adhesion molecule-1, was up-regulated from 4 wk postinfection and persisted to at least 12 wk. Subsequent analysis of the corresponding integrins expressed on lung CD4+ and CD8+ T cells revealed an accumulation of β1high/β7−/low, and to a lesser extent β7high, integrin-expressing T cells during infection. Examination of integrin heterodimers showed that while α4 integrin was predominantly expressed on β1high/β7−/low cells, αE integrin was primarily associated with β7high. The majority of activated/memory T cells recruited during infection expressed high levels of β1 integrin and undetectable or low levels of β7 integrin. These T cells were capable of producing IFN-γ, a cytokine crucial for controlling M. tuberculosis infection. Rapid expansion of β1high, β7−, and β7high T cell populations in the lung upon secondary mycobacterial infection indicates the participation of these populations in the acquired immune response to the infection. Furthermore, treatment of infected mice with mAb to α4 or α4β7 integrin led to a reduction in lymphocytes and increase in granulocytes in the pulmonary infiltrate. These results reveal a crucial role for adhesion molecules in the generation of an effective pulmonary immune response to M. tuberculosis infection.

T cell‐derived tumour necrosis factor is essential, but not sufficient, for protection against Mycobacterium tuberculosis infection

Tumour necrosis factor (TNF) is critical for sustained protective immunity against Mycobacterium tuberculosis infection. To investigate the relative contributions of macrophage‐ and T cell‐derived TNF towards  this immunity T cells from wild‐type (WT) or TNF–/– mice were transferred into RAG–/– or TNF–/– mice which were then infected with M. tuberculosis. Infected RAG–/– mice and RAG–/– recipients of TNF deficient T cells developed overwhelming infection, with extensive pulmonary and hepatic necrosis and succumbed with a median of only 16 days infection. By contrast, RAG–/– recipients of WT T cells showed a significant increase in survival with a median of 32 days. Although initial bacterial growth was similar in all groups of RAG–/– mice, the transfer of WT, but not TNF–/–, T cells led to the formation of discrete foci of leucocytes and macrophages and delayed the development of necrotizing pathology. To determine requirements for macrophage‐derived TNF, WT or TNF–/– T cells were transferred into TNF–/– mice at the time of M. tuberculosis infection. Transfer of WT T cells significantly prolonged survival and reduced the early tissue necrosis evident in the TNF–/– mice, however, these mice eventually succumbed indicating that T cell‐derived TNF alone is insufficient to control the infection. Therefore, both T cell‐ and macrophage‐derived TNF play distinct roles in orchestrating the protective inflammatory response and enhancing survival during M. tuberculosis infection.

Differential Requirements for Soluble and Transmembrane Tumor Necrosis Factor in the Immunological Control of Primary and Secondary Listeria monocytogenes Infection

ABSTRACT The relative contributions of transmembrane tumor necrosis factor (memTNF) and soluble tumor necrosis factor (solTNF) in innate and adaptive immunity are poorly defined. We examined the capacities of wild-type (WT) mice, TNF−/− mice, and memTNF mice, which express only transmembrane TNF, to control primary and secondary Listeria monocytogenes infections. Soluble TNF was not required for induction or maintenance of protective immunity against a low-dose (200-CFU) Listeria infection. In contrast to TNF−/− mice, both WT and memTNF mice cleared the bacilli within 10 days and were fully protected against rechallenge with a lethal infective dose. Furthermore, T cells transferred from immune mice, but not from naïve, WT, and memTNF mice, protected TNF−/− recipients against an otherwise lethal infection. By contrast, infection with a higher dose of Listeria (2,000 CFU) clearly demonstrated that solTNF is required to coordinate an optimal protective inflammatory response. memTNF mice were more susceptible to a high-dose infection, and they exhibited delayed bacterial clearance, increased inflammation, and necrosis in the liver that resulted in 55% mortality. The dysregulated inflammation was accompanied by prolonged elevated expression of mRNAs for several chemokines as well as the macrophage effector molecules inducible nitric oxide synthase and LRG-47 in the livers of memTNF mice but not in the livers of WT mice. These data demonstrated that memTNF is sufficient for establishing protective immunity against a primary low-dose Listeria infection but that solTNF is required for optimal control of cellular inflammation and resistance to a primary high-dose infection. By contrast, memTNF alone is sufficient for resolution of a secondary, high-dose infection and for the transfer of protective immunity with memory T cells.

The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guèrin) in protecting against systemic Mycobacterim avium infection

Sub‐unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections. For example, immunization with DNA vaccines expressing the gene for the immunodominant 35 000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M. avium or M. leprae in mice. However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette–Guèrin (BCG). The cytokine, interleukin (IL)‐12, is essential for priming naïve CD4+ T lymphocytes to differentiate into interferon‐γ (IFN‐γ)‐secreting T cells. We have used a novel self‐splicing vector expressing both chains of murine IL‐12 to determine if plasmid IL‐12 would increase the efficacy of a vaccine expressing the M. avium 35 000 MW protein (DNA‐Av35). Co‐immunization with p2AIL‐12 and DNA‐Av35 led to a significant increase in the number of antigen‐specific IFN‐γ secreting cells and total amount of IFN‐γ released, but a concomitant fall in the antibody response to the 35 000 MW protein. This pattern of response was associated with enhanced clearance of M. avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA‐Av35. alone. Following M. avium challenge there was significant increase in the expansion of the 35 000 MW antigen‐reactive T cells in the coimmunized mice. Therefore, plasmid‐delivered IL‐12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M. avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M. leprae and M. tuberculosis.

Characterization of immune responses during infection with Mycobacterium avium strains 100, 101 and the recently sequenced 104

Mycobacterium avium strain 104 was chosen as the M. avium isolate to sequence, as it is virulent to humans, stable and readily transfectable. As this strain has not been widely studied we sought to investigate the pattern of 104 infection in mice. Bacterial growth and the immune response generated were compared with infection with the low virulence M. avium strain 100, and the high virulence common laboratory strain, 101. Mycobacterium avium strains 104 and 101 grew progressively within mice, while strain 100 was gradually cleared. Strains 104 and 101 induced strong T cell activation and spleen cell cultures produced similar levels of IFN‐γ. In mice infected with strain 100 no significant T cell activation or IFN‐γ production was measured. Further, mice infected with strain 104 or 101 also displayed comparable inflammatory responses and similar granuloma formation, while only minimal inflammation was seen in mice infected with strain 100. Strains 101 and 104 also grew in a similar fashion in bone‐marrow‐derived macrophages and induced significant levels of TNF and nitric oxide. Thus infection with M. avium strain 104 induced an immunological response comparable to M. avium strain 101 and, with the availability of its sequence, should be a useful tool for designing new vaccines or drugs therapies to treat the increasing incidence of M. avium infection in humans.

A novel tumor necrosis factor (TNF) mimetic peptide prevents recrudescence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in CD4+ T cell-depleted mice.

Tumor necrosis factor (TNF) is required to control mycobacterial infections, but its therapeutic value is limited by its in vivo instability and toxicity. The efficacy of a nontoxic TNF‐mimetic peptide (TNF70–80) was tested in mice infected with Mycobacterium bovis bacillus Calmette‐Guerin (BCG).In vitro TNF70–80 and recombinant human TNF (hTNF) acted with interferon gamma (IFN‐γ) to reduce bacterial replication and to induce synthesis of bactericidal nitric oxide (NO) in BCG‐infected, bone marrow‐derived murine macrophages. The dose‐dependent inhibitory effect on bacterial replication was blocked by neutralizing anti‐IFN‐γ and anti‐hTNF mAbs. Further,n‐monomethyl‐l‐arginine (n‐MMA) and a soluble TNF‐receptor I (TNFRI‐IgG) blocked bacterial growth and NO synthesis. Therefore, the peptide acted with IFN‐γ via induction of NO synthase and signaled through TNFRI receptors. Concomitantin vivo treatment with TNF70–80 or hTNF prevented reactivation of chronic BCG infection in mice depleted of CD4+ T cells by injecting anti‐CD4 antibodies. Granuloma number and bacterial load were comparable in treated, T cell‐depleted mice and in chronically infected, intact animals. Thus, TNF70–80 and hTNF can modulate recrudescent BCG infection in CD4+ T cell‐deficient mice.

LIGHT contributes to early but not late control of Mycobacterium tuberculosis infection

The TNF superfamily member, LIGHT, contributes to optimal T-cell activation in vitro through co-stimulation of dendritic cell cytokine production; however, its role in T-cell-mediated control of intracellular bacterial infections is unknown. Protective immunity against Listeria monocytogenes and Mycobacterium tuberculosis infection requires both antigen-specific CD4(+) and CD8(+) T cells. Using LIGHT-deficient mice we determined that LIGHT was necessary for optimal re-stimulation of anti-listerial CD8(+) T cells in vitro. By contrast, LIGHT(-/-) mice infected with L. monocytogenes generated equivalent T-cell responses and controlled the infection as effectively as normal C57BL/6 mice. Following M. tuberculosis infection, LIGHT(-/-) mice showed a significant increase in bacterial replication in the lungs at 4 weeks, but by 6 weeks had controlled the infection. Analysis of T-cell responses in vivo revealed that LIGHT was dispensable for the activation of primary T-cell responses and the production of IL-12 and IFN-gamma. In addition, LIGHT was not required for the induction of memory T-cell responses to anti-mycobacterial DNA or BCG vaccines and for subsequent protection against tuberculosis challenge. Therefore, LIGHT contributes to the optimal co-stimulation of anti-listerial CD8(+) T-cell responses in vitro and to the early control of M. tuberculosis infection; however, other mechanisms compensate for LIGHT deficiency in the control of these pathogens in vivo.