**6. Roles of cytokines, neutrophils, NK cells, NKT cells and γδT cells**

IFN-*γ* and TNF have long been implicated as regulators of T cell responses in mycobacterial disease [29]. The technique of gene targeting (knockout) has swept through biomedical research. IFN-γ, TNF-α, IRF-1, NF-IL6, NF-κB p50, STAT 1 and STAT 4 knockout mice succumbed to *M. tuberculosis* infection over time. There appears to be a cytokine and tran‐ scription factor hierarchy in experimental tuberculosis. The results indicate that these mole‐ cules play major roles in defense against the disease, IFN-γ and TNF-α being the leading players in this respect [30].

suppression by monocytes and IL-10. Recent studies have found that NK cells produce IL-22 [38], which was induced by IL-15 and DAP-10, an adaptor protein that is known to be involved in NK cell activation, in response to *M. tuberculosis.* Rohan Dhiman *et al*. also found that IL-22 can restrict growth of *M. tuberculosis* in macrophages by enhancing phagolysosomal fusion [39]. Nonetheless to fully understand the importance of NK cells in *M. tb* infection it may be

Pathophysiology of Tuberculosis http://dx.doi.org/10.5772/54961 133

Certain T subsets, such as NKT cells and *γδ* T cells, have features of innate immune cells including a partially activated phenotype, a rapid response following detection of infected cells, and the modulation of other cell types. Together with NK cells, these cell subsets are functionally defined as innate lymphocytes.CD1d-restricted invariant NKT (iNKT) cells are a conserved subset of T cells that express an invariant T cell receptor (TCR) α chain (Vα24-Jα18 in humans, and Vα14-Jα18 in mice) paired with TCR β chains encoded by one or a few Vβ gene segments (Vβ11 in humans, and predominantly Vβ2, 7 and 8 in mice). These cells show different phenotypes and functions [40].Many iNKT cells are CD4+, and they have been mainly associated with the induction of Th2 cytokines such as IL-4, IL-5, IL-13. This subset is believed to play a prominent role in suppression of autoimmune or chronic inflammatory diseases, and in promoting allergic conditions such as asthma. Few iNKT cells are CD8+, and most of those express only the CD8α subunit, which means that they likely express only CD8αα homodim‐ ers. An additional fraction of iNKT cells are negative for both CD4 and CD8 (DN T cells). They have been found to produce predominantly IFN-*γ* and other Th1-associated cytokines. Studies of human iNKT cells have shown that they have the ability to kill *M. tuberculosis* organisms within infected macrophages, possibly through their production of the peptide granulysin [41]. Jin S. Im *et al*. [42] found that the percentages of iNKT cells among total circulating T cells in TB patients were not significantly different compared to those in healthy controls. However, TB patients showed a selective reduction of the proinflammatory CD4−CD8− (DN) iNKT cells with a proportionate increase in the CD4+ iNKT cells. The mouse model of tuberculosis has been used by Sada-Ovalle et al to find that iNKT cells have a direct bactericidal effect on *M. tuberculosis*, and protect mice against aerosol *M.TB* infection [43]. Their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. In addition, pharmaco‐ logical activation of iNKT cells with the synthetic ligand aGalCer often enhances host resist‐ ance to infection. iNKT cell use several mechanisms to modify host immunity. These include induction of DC maturation, secondary activation of effector cells (NK cells) or recruitment of inflammatory cells to the site of infection [44, 45]. Thus, by being an early producer of IFN-*γ* and suppressing intracellular bacterial growth, iNKT cells function as an important part of the early immune response against *M. tb* that affect both the innate and the adaptive arms of the

Antigen-specific *γ/δ* T cells represent an early innate defense that may play a role in antimy‐ cobacterial immunity. Studies done in humans and animal models have demonstrated complex patterns of *γ/δ* T cell immune responses during early mycobacterial infections and chronic TB. Like *α/β* T lymphocytes, *γ/δ* T cells carry antigen TCR that vary in the physical properties of their ligand-binding sites. *γ/δ* T cells are frequently activated by a variety of pathogens including *M. tb* [46]. Mice lacking *γ/δ* T cells succumb more rapidly than control

necessary to differentiate their contributions at different stages of disease.

immune response.

The role of neutrophils in the development of tuberculosis remained unknown for a long time. We utilized LPS-induced transient neutrophilia in the lungs [31]. LPS (50μg/ml) was admin‐ istered intratracheally to male Fischer rats, which were then infected with *M. tuberculosis* via an airborne route. Intratracheal injection of LPS significantly blocked the development of pulmonary granulomas and significantly reduced the number of pulmonary colony-forming units (CFU). Treatment with amphotericin B (an LPS inhibitor) or neutralizing anti-rat neutrophil antibody reversed the development of pulmonary lesions. LPS-induced transient neutrophilia prevented early mycobacterial infection. The timing of LPS administration was important. When given intratracheally at least 10 days after aerial infection, LPS did not prevent the development of tuberculosis. Neutrophils obtained by bronchoalveolar lavage killed M. tuberculosis bacilli. These results indicate clearly that neutrophils participate actively in defense against early-phase tuberculosis.

Natural killer (NK) cells are innate lymphocytes which are a first line of defense against infection. NK cells can kill autologous infected cells without prior sensitization, and are believed to play a pivotal role in innate immunity to microbial pathogens. In mouse model, NK cells are activated and produce IFN-γ during the early response to pulmonary tuberculosis [31] and NK cell-produced IFN-γ regulates the anti-mycobacterial resistance mediated by neutrophils [32]. However animal models do not give a clear answer to whether NK cells is important in *M. tb* infection in vivo. Depletion of NK cells had no effect on bacterial replication in the lung of immunocompetent mice [33], suggesting that NK cells may be redundant in the presence of intact adaptive immunity. Surprisingly, IFN-γ knockout mice, which are impaired in their ability to clear mycobacteria, cleared them as effectively as wild-type mice when NK cells were depleted, suggesting that NK cells can inhibit protective immunity [34].

Human NK cells use the NKp46, the natural cytotoxicity receptors (NCRs) and NKG2D receptors to lyse *M. tuberculosis*-infected monocytes and alveolar macrophages [35], through damage of infected cells and secretion of cytokines, such as IFN-γ [36]. Inhibitory receptors of NK cells include killer immunoglobulin-like receptors (KIRs) and the NKG2A:CD94 dimer and NK cell activation can also be triggered by loss of inhibitory ligands from the cell surface. In addition, NK cells can also be activated by cytokines, including type I interferons, IL-12 and IL-18. NK cells are a potent and early source of cytokines, particularly IFN- γ, but they can also produce Th2-associated cytokines, such as IL-5 and IL-13, and the regulatory cytokine IL-10 [37]. NK cell NKp46 expression and cytotoxicity are reduced in freshly isolated peripheral blood mononuclear cells (PBMCs) from tuberculosis patients, which may be attributable to suppression by monocytes and IL-10. Recent studies have found that NK cells produce IL-22 [38], which was induced by IL-15 and DAP-10, an adaptor protein that is known to be involved in NK cell activation, in response to *M. tuberculosis.* Rohan Dhiman *et al*. also found that IL-22 can restrict growth of *M. tuberculosis* in macrophages by enhancing phagolysosomal fusion [39]. Nonetheless to fully understand the importance of NK cells in *M. tb* infection it may be necessary to differentiate their contributions at different stages of disease.

**6. Roles of cytokines, neutrophils, NK cells, NKT cells and γδT cells**

players in this respect [30].

132 Tuberculosis - Current Issues in Diagnosis and Management

in defense against early-phase tuberculosis.

IFN-*γ* and TNF have long been implicated as regulators of T cell responses in mycobacterial disease [29]. The technique of gene targeting (knockout) has swept through biomedical research. IFN-γ, TNF-α, IRF-1, NF-IL6, NF-κB p50, STAT 1 and STAT 4 knockout mice succumbed to *M. tuberculosis* infection over time. There appears to be a cytokine and tran‐ scription factor hierarchy in experimental tuberculosis. The results indicate that these mole‐ cules play major roles in defense against the disease, IFN-γ and TNF-α being the leading

The role of neutrophils in the development of tuberculosis remained unknown for a long time. We utilized LPS-induced transient neutrophilia in the lungs [31]. LPS (50μg/ml) was admin‐ istered intratracheally to male Fischer rats, which were then infected with *M. tuberculosis* via an airborne route. Intratracheal injection of LPS significantly blocked the development of pulmonary granulomas and significantly reduced the number of pulmonary colony-forming units (CFU). Treatment with amphotericin B (an LPS inhibitor) or neutralizing anti-rat neutrophil antibody reversed the development of pulmonary lesions. LPS-induced transient neutrophilia prevented early mycobacterial infection. The timing of LPS administration was important. When given intratracheally at least 10 days after aerial infection, LPS did not prevent the development of tuberculosis. Neutrophils obtained by bronchoalveolar lavage killed M. tuberculosis bacilli. These results indicate clearly that neutrophils participate actively

Natural killer (NK) cells are innate lymphocytes which are a first line of defense against infection. NK cells can kill autologous infected cells without prior sensitization, and are believed to play a pivotal role in innate immunity to microbial pathogens. In mouse model, NK cells are activated and produce IFN-γ during the early response to pulmonary tuberculosis [31] and NK cell-produced IFN-γ regulates the anti-mycobacterial resistance mediated by neutrophils [32]. However animal models do not give a clear answer to whether NK cells is important in *M. tb* infection in vivo. Depletion of NK cells had no effect on bacterial replication in the lung of immunocompetent mice [33], suggesting that NK cells may be redundant in the presence of intact adaptive immunity. Surprisingly, IFN-γ knockout mice, which are impaired in their ability to clear mycobacteria, cleared them as effectively as wild-type mice when NK

cells were depleted, suggesting that NK cells can inhibit protective immunity [34].

Human NK cells use the NKp46, the natural cytotoxicity receptors (NCRs) and NKG2D receptors to lyse *M. tuberculosis*-infected monocytes and alveolar macrophages [35], through damage of infected cells and secretion of cytokines, such as IFN-γ [36]. Inhibitory receptors of NK cells include killer immunoglobulin-like receptors (KIRs) and the NKG2A:CD94 dimer and NK cell activation can also be triggered by loss of inhibitory ligands from the cell surface. In addition, NK cells can also be activated by cytokines, including type I interferons, IL-12 and IL-18. NK cells are a potent and early source of cytokines, particularly IFN- γ, but they can also produce Th2-associated cytokines, such as IL-5 and IL-13, and the regulatory cytokine IL-10 [37]. NK cell NKp46 expression and cytotoxicity are reduced in freshly isolated peripheral blood mononuclear cells (PBMCs) from tuberculosis patients, which may be attributable to

Certain T subsets, such as NKT cells and *γδ* T cells, have features of innate immune cells including a partially activated phenotype, a rapid response following detection of infected cells, and the modulation of other cell types. Together with NK cells, these cell subsets are functionally defined as innate lymphocytes.CD1d-restricted invariant NKT (iNKT) cells are a conserved subset of T cells that express an invariant T cell receptor (TCR) α chain (Vα24-Jα18 in humans, and Vα14-Jα18 in mice) paired with TCR β chains encoded by one or a few Vβ gene segments (Vβ11 in humans, and predominantly Vβ2, 7 and 8 in mice). These cells show different phenotypes and functions [40].Many iNKT cells are CD4+, and they have been mainly associated with the induction of Th2 cytokines such as IL-4, IL-5, IL-13. This subset is believed to play a prominent role in suppression of autoimmune or chronic inflammatory diseases, and in promoting allergic conditions such as asthma. Few iNKT cells are CD8+, and most of those express only the CD8α subunit, which means that they likely express only CD8αα homodim‐ ers. An additional fraction of iNKT cells are negative for both CD4 and CD8 (DN T cells). They have been found to produce predominantly IFN-*γ* and other Th1-associated cytokines. Studies of human iNKT cells have shown that they have the ability to kill *M. tuberculosis* organisms within infected macrophages, possibly through their production of the peptide granulysin [41]. Jin S. Im *et al*. [42] found that the percentages of iNKT cells among total circulating T cells in TB patients were not significantly different compared to those in healthy controls. However, TB patients showed a selective reduction of the proinflammatory CD4−CD8− (DN) iNKT cells with a proportionate increase in the CD4+ iNKT cells. The mouse model of tuberculosis has been used by Sada-Ovalle et al to find that iNKT cells have a direct bactericidal effect on *M. tuberculosis*, and protect mice against aerosol *M.TB* infection [43]. Their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. In addition, pharmaco‐ logical activation of iNKT cells with the synthetic ligand aGalCer often enhances host resist‐ ance to infection. iNKT cell use several mechanisms to modify host immunity. These include induction of DC maturation, secondary activation of effector cells (NK cells) or recruitment of inflammatory cells to the site of infection [44, 45]. Thus, by being an early producer of IFN-*γ* and suppressing intracellular bacterial growth, iNKT cells function as an important part of the early immune response against *M. tb* that affect both the innate and the adaptive arms of the immune response.

Antigen-specific *γ/δ* T cells represent an early innate defense that may play a role in antimy‐ cobacterial immunity. Studies done in humans and animal models have demonstrated complex patterns of *γ/δ* T cell immune responses during early mycobacterial infections and chronic TB. Like *α/β* T lymphocytes, *γ/δ* T cells carry antigen TCR that vary in the physical properties of their ligand-binding sites. *γ/δ* T cells are frequently activated by a variety of pathogens including *M. tb* [46]. Mice lacking *γ/δ* T cells succumb more rapidly than control mice following intravenous challenge with virulent *M. tb*; however, such a difference has not been observed following infection by the aerosol route. *γ/δ* T cells constitute a whole system of functionally specialized subsets that have been implicated in the innate responses against tumors and pathogens, the regulation of immune responses, cell recruitment and activation, and tissue repair [47]. Human alveolar macrophages and monocytes can serve as antigen presentation cells (APCs) for *γ/δ* T cells. Furthermore, the predominance of V*γ*9V*δ*2 T cells in TB disease has been confirmed [48]. When MTB-activated CD4+ and *γ/δ* T cells from healthy tuberculin-positive donors were analyzed for cytokine production in response to MTBinfected monocytes, both groups secreted large amounts of IFN-*γ* [49]. Previous studies have also demonstrated an increased proliferative activity of V*γ*9V*δ*2 T cells from patients with TB [50], but reduced production of IFN-*γ*, compared with that of healthy tuberculin-positive donors [51]. Additionally, Dieli *et al.* reported that decrease of V*γ*9V*δ*2 T cell effector functions involves not only IFN-*γ* production but also expression of granulysin [52]. Fig. 3 shows interaction of cells and cytokines involved in tuberculosis.

of the immunopathogenesis of TB can facilitate the design of effective vaccines, new drug candidates and evaluation of their efficacy [53]. Understanding latent tuberculosis can also be

Pathophysiology of Tuberculosis http://dx.doi.org/10.5772/54961 135

1 Sino-US Tuberculosis Research Center and Clinical Laboratory Department of Henan

2 Center of Tuberculosis Diagnosis and Treatment, Shanghai Pulmonary Hospital, Tongji

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**Author details**

Ruiru Shi1

**References**

**Figure 3.** Cytokine and cellular network in tuberculosis +. Production of cytokine, ‐. No production of cytokine.
