Achieving global control of tuberculosis (TB) is a great challenge considering the current increase in multidrug resistance and mortality rate. Considerable efforts are therefore being made to develop new effective vaccines, more effective and rapid diagnostic tools as well as new drugs. Shortening the duration of TB treatment with revised regimens and modes of delivery of existing drugs, as well as development of new antimicrobial agents and optimization of the host response with adjuvant immunotherapy could have a profound impact on TB cure rates. Recent data show that chronic worm infection and deficiencies in micronutrients such as vitamin D and arginine are potential areas of intervention to optimize host immunity. Nutritional supplementation to enhance nitric oxide production and vitamin D-mediated effector functions as well as the treatment of worm infection to reduce immunosuppressive effects of regulatory T (Treg) lymphocytes may be more suitable and accessible strategies for highly endemic areas than adjuvant cytokine therapy. In this review, we focus mainly on immune control of human TB, and discuss how current treatment strategies, including immunotherapy and nutritional supplementation, could be optimized to enhance the host response leading to more effective treatment.
In the Western world, tuberculosis (TB) was long considered a disease of the past, controlled by effective public health systems that compensated for the relative shortcomings of the available intervention strategies. However, due to the lack of effective vaccines, early biomarkers, rapid diagnostic tools and novel therapeutic agents, the incidence rate is still high, with 1.4 million deaths due to TB in 2010, mostly in resource-poor areas . Of particular concern in sub-Saharan Africa is the high proportion of TB patients co-infected with the human immunodeficiency virus (HIV), further increasing morbidity and mortality. In most individuals, infection with the causative agent Mycobacterium tuberculosis (Mtb) remains latent without symptoms or transmission to others, but in approximately 5%–10% the infection progresses to active disease, killing many of those who are not diagnosed and effectively treated . TB has become the focus of attention in many parts of the world because of an alarming proportion of cases caused by drug-resistant bacteria. Nearly 50% of culture-positive patients in areas such as Belarus carry multidrug-resistant (MDR) TB, which is defined as resistance to the two most important antimycobacterial drugs isoniazid (INH) and rifampicin . Shortening the duration of TB treatment to increase compliance, development of new antimicrobial agents and treatment strategies to optimize the host response could all have a profound impact on the TB cure rate and the control of the infection, in particular as drug resistance is rapidly increasing. Considerable effort is now being made to develop new vaccines, but how bacteria surviving inside phagocytic cells can delay initiation of an effective immune response and how they can be detected and eliminated more efficiently remain to be determined.
Mycobacterium tuberculosis is the most successful human pathogen known, as indicated by its ability to infect up to one-third of the global population. The spread of the disease has not been limited despite the use of the Bacillus Calmette-Guérin (BCG) vaccine for more than 90 years. Mtb has achieved such success through evolving multiple mechanisms to avoid destruction and adapt to the host. Recent advances in understanding of the delicate balance between the host and Mtb have been reviewed extensively [4-7]. Despite increased knowledge of host defence mechanisms against Mtb, the optimal innate or cell-mediated immune (CMI) response to achieve immunity is unknown [5, 8]. It is evident that human TB is not as homogenous from the host response perspective as it is under strict experimental conditions using laboratory strains of Mtb [9-12]. Animal models are essential for understanding host immunity, especially in the early stages of innate response . However, it is relatively difficult to translate preclinical laboratory findings into novel treatment and diagnostic strategies.
From the clinical perspective, the importance of the CMI response is confirmed by several important observations. First, in HIV-infected patients, there is 10% yearly risk of developing TB compared with a 5% lifetime risk in immunocompetent individuals . Secondly, during treatment with tumour necrosis factor (TNF)-α inhibitors or other immunosuppressive drugs such as corticosteroids, there is an increased risk of developing active TB amongst latently infected individuals [14-17]. Moreover, individuals with decreased expression of T helper (Th)1-related cytokines, due to mutations in the genes of the interleukin (IL)-12/IL-23/interferon (IFN)-γ pathway are susceptible to mycobacterial infections [18-20]. Thus, the CMI response against TB plays a fundamental protective role against the development and outcome of active disease.
The most important areas to consider to achieve global TB control are: (i) improvement of vaccine efficacy; (ii) development of more effective and rapid diagnostic tools; (iii) establishment of shorter treatment regimens; and (iv) development of strategies for identifying patient groups at risk of developing active disease. Shortening the duration of TB treatment through revised regimens and/or modes of delivery of existing drugs, development of new antimicrobial agents and optimizing the host response via adjuvant immunotherapy could greatly improve TB cure rates. Of note, adjuvant cytokine immunotherapy is unlikely to be accessible for the majority of patients in highly endemic resource-poor areas. The priorities in such areas should be eliminating poverty, optimizing immunity by nutritional supplementation and treating HIV. In this review, we will mainly focus on the immune control of human TB, and discuss how the current treatment strategies could be optimized through the understanding of innate and adaptive immunity during TB infection.
Immunological ‘checkpoints’ of Mtb infection
The heterogeneity of human susceptibility to TB can be demonstrated by the Lübeck disaster in 1929 [21, 22] when 251 infants were accidentally challenged with an oral dose of virulent Mtb instead of the attenuated strain used in the BCG vaccine; 72 children died within 1 year, 135 developed TB but recovered and 44 became tuberculin-positive but remained well. The heterogeneity of human susceptibility to TB is also evident from studies of close (living in close household contacts) contacts of TB cases. Even after prolonged exposure, only 5%–10% of close contacts of patients with smear-positive TB develop active disease . It has been estimated that up to 50% develop latent TB as indicated by a positive tuberculin skin test . However, a fraction of exposed individuals do not show any signs of induction of systemic immunity to TB, indicating sufficient protection by innate immune mechanisms . This fact has long been disregarded, but knowledge of innate immune functions emerging during the last decade may explain how the host can clear or control TB infection without involvement of the adaptive immune response.
Much of the present understanding of host immunity is based on studies using inbred mouse strains. It is clear that there will be little variation in the observed immune response of the infected animals in such studies, with an initial increase in bacterial load in the tissues until the onset of adaptive immunity . However, recent studies in outbred monkeys  and rabbits  have shown that the heterogeneity of human TB may be better demonstrated using these models. In a study in macaques, it was shown that within the same TB-infected animal, a spectrum of different lesions is present, although with less advanced lesions in tissues from animals with latent TB .
On the basis of recent observations, we hypothesize that Mtb has to pass immunity ‘checkpoints’ to establish infection (Fig. 1) . The time it takes for the bacterium to pass the checkpoints, if it does, varies greatly between individuals ranging from a few weeks in patients with an impaired immune response, such as those infected with HIV, to decades in initially resistant hosts.
To pass the first checkpoint, Mtb must avoid being killed by the infected phagocytes, including neutrophils, macrophages and dendritic cells (DCs), of which macrophages are the primary target cells. It is likely that the interaction between these cells plays a key function in the early clearance of Mtb. However, Mtb has developed strategies to counteract innate immune mechanisms such as phagosomal maturation [28, 29] and intracellular killing. Apoptosis is also inhibited  and partly replaced by host detrimental necrosis [31, 32]. Failure of macrophages to eradicate the initial infection allows progression towards the next checkpoint and the spread of infection to distal sites . Such failure may be due to deficient phagolysosomal fusion or autophagy , impaired production of antimycobacterial peptides such as LL-37 or α-defensins  and other antimycobacterial compounds such as nitric oxide (NO). Of note, Mtb can be present in apparently normal lung tissue, indicating that the bacterium is able to coexist with its host without causing tissue damage and inflammation . The mechanisms by which macrophages are able to maintain a persister state of Mtb need further investigation, albeit acidification of phagosomes and phagolysosomal protease activity have been determined to play a role ; NO, which is produced by human macrophages at sites of Mtb infection, is also thought to be important . There is evidence that Mtb, if exposed to suboptimal doses of NO, can respond to this stressor by switching to an NO-tolerant phenotype .
The second checkpoint is passed when innate immunity fails to control the infection, allowing Mtb to start replicating to an extent that adaptive immunity is utilized. Enhanced inflammation caused by necrosis of infected host cells harbouring replicating bacteria is probably needed for the induction of antigen presentation to induce both cytotoxic T cell activity and an effective Th1 response to enhance macrophage activity via production of IFN-γ . Depending on host immunity and bacterial factors, the infection may progress to active disease. However, latent TB is observed in individuals who can control the infection by an effective adaptive immune response. It is also possible at this stage that the infection may be completely cleared. This is clinically important, as traditional tests for latent TB such as interferon IFN-γ assays measure the CMI response to Mtb, but do not determine whether the bacteria are still viable .
Progression to active disease occurs, due to insufficient immune control, when the third and final checkpoint is reached. Excessive inflammation contributes to the pathology; there is evidence that Mtb takes advantage of immune activation leading to necrosis , which is a prerequisite for the bacterium to spread to other individuals. Draining of the bacilli-containing necrotic cores of caseating granulomas or cavities into the bronchial tree is the major route by which Mtb is transmitted. A recent study of antigen variability in Mtb showed that in contrast to many other pathogenic microorganisms, which rely on antigen variability to evade specific immunity, Mtb has highly conserved antigen epitopes. This finding implies that at a certain stage of infection, Mtb manipulates the host immune response to take advantage of T cell-mediated immunity . Necrosis that is thus generated through host immunity is the platform from which Mtb can spread in the human population.
Details of the immune mechanisms that can be targeted to optimize host immune responses, according to the checkpoint model, will be discussed below. Through interventions targeting the immune system, Mtb could thus be prevented from passing checkpoint 3, allowing more efficient clearance of infection.
Innate immune mechanisms during TB infection
The fact that many individuals heavily exposed to Mtb do not develop infection despite not being immunized  suggests that several inherent and innate immune mechanisms are active before checkpoint 2. Alveolar macrophages represent the primary niche for replication of the bacillus, and macrophage functions such as phagosomal acidification  and autophagy  are important mechanisms by which these cells can control Mtb. The innate immune response to Mtb is triggered by several pattern recognition receptors, primarily Toll-like receptors (TLRs) 2 and 9 . Also, cytosolic innate receptor complexes, such as NOD2 and NALP3, recognize Mtb molecules and may affect phagosomal maturation and autophagy [7, 34]. A proinflammatory cytokine and chemokine response is mounted , recruiting neutrophils, DCs and macrophages to the site of infection. Recruited neutrophils may have several important functions during the early phases of infection. Initially, they may be beneficial to the host through phagocytosing and killing virulent Mtb via Nadph Oxidase 2 (Nox2)-generated reactive oxygen species (ROS) and other antimicrobial molecules (defensins, LL-37) , but also offer a niche that favours bacterial survival and growth. During this process neutrophils become apoptotic. Because apoptotic cells are cleared from the tissue by macrophages, Mtb-containing apoptotic neutrophils can be transferred to the macrophages and mount a proinflammatory response [42, 43]. This can also occur in DCs, where apoptotic neutrophils can facilitate DC maturation and augment subsequent Th cell activation . A well-recognized virulence-associated attribute of Mtb is its ability to impair apoptosis in macrophages [45, 46], as well as in neutrophils, thereby delaying the adaptive immune response  and forming an intracellular environment to survive and proliferate.
Although the early innate immune reaction can be seen as an infectious stage dominated by bacterial proliferation and spreading, it can also lead to a proper adaptive immune response in individuals in whom innate immunity is not limiting the infection (e.g. after checkpoint 1 in the model shown in Fig. 1). It has recently been suggested that neutrophils can form a link between innate and adaptive immune activation during the early phase of TB infection, by enhancing both a proinflammatory macrophage response and DC maturation .
Targeting host immunity for prevention and cure of TB
The rationale for treatment strategies that aim to strengthening the host immune response during TB is to reduce the risk of disease progression upon exposure and to optimize the treatment of active TB by stimulating the host immune response (Fig. 1). The timing for such interventions is important and should be considered carefully in relation to the immunological life cycle of Mtb. In terms of our model, this means the infection will be stopped at checkpoint 1 and extensive tissue damage prevented (Fig. 1). Interventions based on enhancing host immunity may cover both prevention and active disease such as HIV and chronic worm infection as well as optimizing nutritional status in the case of vitamin D and arginine supplementation. Such strategies can be cost-effective in reducing the risk of developing TB and preventing development of severe active disease in malnourished and HIV-infected individuals.
Nutritional supplementation in the treatment of TB
Before the availability of chemotherapy, nutritional supplementation was an important part of care in a Sanatorium, although the relative contribution of food supplementation to the cure of TB during this time is controversial [48, 49]. As care, diet and sanitation improved during late 19th and early 20th century in countries that today have a relatively low incidence of TB, the disease burden declined even before the introduction of the BCG vaccination and the development of antibiotics, underlining the importance of social factors and nutrition in reducing the spread of TB . In a large epidemiological study, being underweight (body mass index <18.5 kg m−2) and having a low serum albumin level (<3.5 g dL−1) were found to be strong risk factors for TB whereas low vitamin A levels and iron status were not . Decreased levels of vitamins and trace elements during active TB have been demonstrated in a number of clinical studies, but it is difficult to determine whether these deficiencies or malnutrition in general were the cause or result of TB . In a clinical trial conducted in Tanzania including 887 adult patients with pulmonary TB, multiple micronutrient supplementation (vitamins A, B-complex, C and E and selenium) reduced the risk of TB recurrence together with an increase in CD4 counts, but without any effects on mortality . The divergent results from nutritional intervention studies, for example investigating the effect of vitamin A [54-56], may be due to the composition and doses as well as inclusion criteria [52, 57].
What are the immunological mechanisms that underlie the effect on Mtb of nutritional interventions? The mechanisms of action of arginine, NO and antimicrobial peptides produced as a response to vitamin D are better understood than those of other micronutrients [58, 59]. The host response to TB involves production of molecules through protein synthesis and secretion, and the mediators produced also increase catabolism . Although the precise mechanisms of the dietary effect and which dietary factors are most important are unknown, impaired macrophage activation and reduced T cell-dependent production of protective cytokines are linked to malnutrition in general and protein calorie deficiency in particular .
Some recent interventional studies of patients with active TB investigating multivitamin/mineral supplementation as well as specific treatment with vitamin D and trace elements have shown promising results [62, 63]. Even though the contribution of dietary supplementation must be seen in the perspective of the effective TB drugs available [57, 64], supplementation of nutrients may shorten the duration of treatment, as well as increase extent of recovery in TB patients [53, 65]. Nutritional supplementation may also be important in highly endemic areas to prevent the development of active disease upon exposure (Fig. 1, I1); in such areas, the majority of patients are malnourished even before the onset of TB and thus immune defence is impaired.
The clinical relevance of vitamin D in TB
Interest in the possibility of vitamin D supplementation for patients with TB has increased since the finding in 2006 that TLR activation upregulates the expression of the vitamin D receptor (VDR) and the vitamin D hydroxylase genes in human macrophages, leading to induction of the antimicrobial peptide LL-37 and enhanced killing of intracellular Mtb . The active metabolite of vitamin D, vitamin D3 (also known as calcitriol), induces potent antibacterial effects including production of NO and antimicrobial peptides such as LL-37 as well as stimulation of autophagy . The effect of vitamin D3 may also be modulated by VDR polymorphisms involving the tt genotype of the TaqI allele .
Several randomized trials have been conducted to investigate the effect of vitamin D3 in patients with active TB [62, 67]. In one trial performed in the UK, patients with smear-positive TB (n =146) were assigned randomly to high-dose (2.5 mg) vitamin D3 or placebo every other week for 6 weeks. There was no effect on the primary outcome of time to culture conversion, but a significant effect was observed in the subgroup analysis of vitamin D3-supplemented patients with the tt genotype (5/12 patients) of TaqI in the VDR . In a recently published follow-up analysis, it was shown that vitamin D3 supplementation accelerated the resolution of inflammatory responses unrelated to the TaqI genotype . In a larger West-African trial, a lower vitamin D3 dose in patients with active TB had no effect on the reduction of a clinical severity score or mortality, which were the primary and secondary outcomes respectively . In addition, there was a trend towards an effect on the primary outcome in HIV-negative individuals and those with initially low vitamin D levels, as well as higher mortality in HIV-infected individuals. Vitamin D may be beneficial in some patients with active TB and vitamin D deficiency (VDD), in particular in those with certain VDR polymorphisms as well as in cases of MDR/Multidrug-resistant TB where it may contribute to the relatively weak efficacy of second-line drugs. Larger multicentre trials are urgently needed to verify these possibilities.
Association studies of VDD in TB patients and healthy control subjects have shown inconsistent results. A study from Guinea-Bissau showed a higher prevalence of VDD [25(OH)D(3) ≤ 25 nmol L−1] in healthy individuals relative to TB patients, but the mean serum level of vitamin D was lower in the TB group . In a recent trial conducted in South Africa, the number of TB cases was found to increase after the winter season, when the lowest mean serum levels of vitamin D are observed in the population. In this study, more than 60% of patients (n = 370) with active or latent TB had inadequate vitamin D levels, and VDD was strongly associated with HIV co-infection . Amongst healthy individuals with latent TB, 37%–52% exhibited VDD compared with 75%–86% in the active TB group depending on whether they were HIV positive or not . The findings of a study from Greenland demonstrated that both high and low serum vitamin D levels were associated with TB . Vitamin D levels increased during the first 2 months of treatment of active TB in a trial conducted in Tanzania, indicating that vitamin D levels may normalize spontaneously during treatment, at least in this setting . Although low serum levels of vitamin D may be a contributing risk factor for developing active TB at the population level, there are clearly large groups of healthy individuals with low levels of vitamin D who will not develop disease. Thus, in terms of prevention and causality, the relative importance of VDD for the risk of developing TB remains to be established.
The role of NO in resistance against and control of TB
In macrophages activated by Th1-mediated cytokines such as IFN-γ and TNF-α, NO is produced in large amounts from the amino acid arginine via the enzyme inducible NO synthase (iNOS) (Fig. 2) [72, 73]. On the other hand, transforming growth factor (TGF)-ß, IL-4 and IL-10 suppress the induction of iNOS [72, 74] and increase arginase activity in alternatively activated macrophages. Mtb may increase arginase activity, which would lead to competition with iNOS for its substrate arginine [75-77]. It was recently shown that, in addition to iNOS, arginase is present in biopsy material from human TB patients . Other Mtb defence strategies include inhibiting iNOS recruitment to the phagosomal membrane thereby avoiding NO exposure . In a mouse model, NO has been well established as a key molecule in host resistance against active and latent TB . Production of NO has been confirmed in human TB, but its relative importance is debated. The presence of iNOS and nitrotyrosine in macrophages has been shown at the site of TB infection in humans [37, 81-84]. Measurements of NO in exhaled air and NO metabolites in urine from TB patients indicate that human macrophages are also partly dependent on NO production for control of Mtb [82, 83]. Furthermore, it has been shown that polymorphisms in the promoter for iNOS may have a role in the regulation of NO production as well as in the host defence against the disease . The bicyclic nitroimidazole PA-824, the active metabolites of which generate reactive nitrogen intermediates (RNIs), was shown to largely mediate the anaerobic microbicidal effect against Mtb [85-88]. The results of recent clinical trials of this drug have been promising [87, 89].
Pathogenic mycobacteria have several antioxidant systems [90-92] and are inherently more resistant than nonpathogenic mycobacteria to RNIs. Clinical strains exhibit a variable susceptibility to NO although acidified nitrite was mostly used in early studies and modern NO-donating substances or macrophage-based assays may be more suitable for assessing this effect [90, 93, 94]. Moreover, recently published results indicate that part of the mechanism of action of INH may involve NO production from the bacteria . We have recently shown that clinical isolates have a variable resistance to NO, and that reduced NO susceptibility is correlated with INH resistance . Targeting bacterial factors that inhibit an effective antioxidative antimycobacterial response might lead to novel treatment of TB. Catalase peroxidase katG, the alkyl hydroperoxide reductase subunit C, superoxide dismutase, haem proteins, bacterial proteases, thioredoxin and lipoamide dehydrogenase can all scavenge ROS and RNS and reduce redox stress due to the host immune response [97-105].
It has been postulated that during the early phase of human TB exposure, an efficient innate response and high levels of NO from alveolar macrophages may kill Mtb even before infection occurs. In addition, other effector molecules such as granulysin from natural killer (NK) cells, LL-37 from macrophages and ROS from recruited neutrophils may contribute to this effect. In the host, if innate immunity and NO levels are inadequate to eradicate the bacteria (Fig. 1, checkpoint 1), a latent infection is established where cell-mediated immunity in most cases keeps the infection under control indefinitely. However, in latently infected individuals with malnutrition and l-arginine deficiency, as well as reduced levels of NO and subsequent loss of control of the infection, active disease may develop. In case of insufficient levels of NO production due to a poor CMI response or NO resistance, or because of the virulence of the bacterial strain, bacterial replication will continue and cause active disease. Some potential strategies for NO-based therapies are shown in Fig. 3.
To evaluate the role of NO, we initiated a double-blind randomized trial of arginine supplementation combined with chemotherapy in 120 smear-positive TB patients in Gondar, Ethiopia and observed an increased sputum smear conversion and reduced clinical symptoms in HIV-negative patients . In a subsequent clinical trial in the same area, we noticed that addition of a food supplement rich in arginine (peanuts) had a clinical effect in HIV-positive TB patients, and that nutritional supplementation per se was beneficial for the clinical outcome . Studies by Yeo et al. in patients with malaria indicate that the arginine dose can be increased considerably . l-arginine is a semi-essential amino acid which is taken up from arginine-rich food such as nuts and is obtained from endogenous sources such as citrulline . In TB patients, arginase activity may warrant high doses of l-arginine to overcome deficiency due to prediagnosis malnutrition and TB-related anorexia.
Interventions targeting cell-mediated immunity in TB patients co-infected with HIV and helminths
An obvious way to reduce the risk of developing active TB is early detection and treatment of HIV, the greatest risk factor for TB in highly endemic areas . It is important to note that TB is often unidentified in HIV-positive individuals due to atypical presentation [9, 109-111]. TB increases the replication of HIV, particularly locally in the lung, by activating CD4+ T cells and enhancing production of proinflammatory cytokines such as TNF-α. HIV in turn increases the susceptibility to TB by selectively depleting protective CD4+ T cells [112, 113]. The early initiation of antiretroviral therapy in TB/HIV co-infected patients with low CD4 counts is important as it has been shown to significantly reduce mortality [114, 115]. Clearly, early treatment of HIV is vital both to prevent development of active TB from latent infection, and reduce mortality in patients with established TB/HIV co-infection (Fig. 1).
In countries in which TB is common, helminth infections, which are often asymptomatic, are also highly prevalent. Similar to TB, it has been estimated that intestinal parasites infect up to one-third of the global population . It has been proposed that chronic worm infection might make the host more vulnerable to TB [117, 118]; however, there is a relative lack of clinical data to support this notion [119-121]. We have recently published a small observational study, demonstrating that asymptomatic worm infection affects host immunity in TB patients, and that approximately 30% of these patients have concurrent worm infection . Furthermore, in a case–control study at the same site, the prevalence of worms in patients with active TB was twice as high as in their healthy close (household) contacts. .
The issue of co-infection may be complicated by the fact that Mtb/helminth co-infected patients are often also HIV positive  The results of a recent meta-analysis indicated that deworming may slow progression of HIV ; in the few clinical trials conducted there was a trend towards reduced viral HIV loads and increased CD4 counts as the short-term outcome [125-127]. In one of these studies in Kenya, 299 of 1551 (19%) screened HIV-positive patients were also helminth (A. lumbricoides) co-infected. In the group of worm-infected patients treated with the anthelmintic albendazole, a significant effect on CD4 counts was observed after 3 month as well as a reduction in IL-10 production [126, 128]. A possible explanation of the beneficial effect of deworming may be that chronic helminth infection increases intestinal permeability which in turn increases bacterial translocation and subsequently increases immune activation and HIV replication .
The persistence and outcome of active TB in the host following helminth infection is considered to be linked to immunoregulatory mechanisms such as expansion of Treg cells producing IL-10 and TGF-ß, attenuation of Mtb-specific Th1 responses [117, 118, 121] and increased Th2 response with production of IL-4 which in turn could mediate alternative macrophage activation (Fig. 2) . A decreased Th1 response to TB and leprosy associated with worm infection has been shown in clinical studies [131, 132]. Furthermore, filarial infection was found to cause attenuated Th1 and Th17 responses in Mtb/helminth co-infected patients, which could be reversed by antifilarial treatment [133, 134]. With regard to the preventive effect of deworming, Elias et al.  showed in an Ethiopian study that albendazole treatment of PPD-negative and worm-positive, healthy individuals prior to BCG vaccination resulted in increased levels of the Th1-type cytokines IL-12 and IFN-γ, and reduced levels of the immunoregulatory cytokine TGF-ß. In conclusion, experimental and clinical data suggest that deworming can have a role both in the prevention and treatment of active TB, probably by redirecting the adaptive immune response to a CMI response and efficiently clearing Mtb from the tissues.
Therapeutic implications of effector mechanisms against Mtb induced by cytotoxic T lymphocytes
Experimental studies have established that cytotoxic T lymphocytes (CTLs) – primarily CD8+ T cells – are required for optimum host immunity against TB [136, 137]. However, supportive data from TB patients are scarce, and there have been no parallel observations of a link between CD4+ cells and HIV co-infection, confirming the relative importance of CD8+ T cells .
Mtb-infected macrophages can be recognized and killed by human CD8+ T cells following activation mediated by recognition of the class I MHC complex, and antigen-specific CD8+ T cells have been observed in patients with active TB [138, 139]. The major mechanisms of action of lymphocyte-mediated cytotoxicity include the release of cytotoxic granules containing perforin, granzyme proteases and granulysin, and the induction of apoptosis by the Fas ligand (CD95L) on the CTL and Fas on the target cell [137, 140-142]. The CTL-mediated lysis of infected cells results in the release of the pathogen into the extracellular environment, where the bacteria can be taken up by freshly recruited, activated macrophages, that are better able to kill them . Expression of membrane TNF, or secretion of TNF by CTLs, may promote TNF receptor-induced apoptosis of the target cells mediated by the TNF-related apoptosis-inducing ligand (TRAIL) , but data in human cells from TB patients are lacking. On the other hand, granulysin, produced from CD8+ CTLs and NK cells, has been shown to directly kill intracellular Mtb together with perforin [141, 142, 144]. Perforin induces a nonselective pore in the plasma membrane of infected cells and granulysin is subsequently delivered to the cytoplasm where it kills Mtb by attacking the bacterial membrane . In addition to the cytolytic capacity, CD8+ T cells can produce the proinflammatory cytokines IFN-γ and TNF-α, highlighting the overlapping effects of CD4+ and CD8+ T cells .
Findings from animal models lacking critical mediators of cytolysis, such as perforin, granzyme or CD95L, are inconsistent, which is partly due to the fact that these molecules are not specific for CD8+ T cells, but are also expressed by NK cells, γδ T cells and some CD4+ T cells [145, 146]. From studies of human biopsy material, it is clear that granulysin is produced in active TB, where it is mainly colocalized with perforin in CD8+ T cells surrounding the granuloma [147, 148]. In contrast to cells expressing granulysin, granzyme A-expressing CD3+ T cells were shown to be significantly upregulated in immunopathological studies of lesions from TB patients . Stimulation of T cells from tuberculin-positive individuals in vitro using Mtb to induce production of granzyme A and B, granulysin, perforin and CD95L (Fas ligand) in CD4+ as well as CD8+ T cells, and both cell subsets were able to lyse Mtb-infected monocytes .
Although it is possible to isolate Mtb-specific CD8+ CTLs in vitro, and their activity can be detected and localized in vivo, the relative importance of their cytolytic activity in patients with TB is largely unknown . This should warrant further clinical studies before CTL-based effector mechanisms such as granulysin can be translated into new therapeutic strategies. Pathways regulating CTL effector mechanisms may be important to consider in future vaccine development as well as for developing drugs that stimulate cytolytic CTL activity.
Cytokines as adjuvant immunotherapy in the treatment of active TB
Strategies for adjuvant immunotherapy have involved stimulation of a favourable immune response using Mycobacterium vaccae, immunosuppression to reduce inflammation with drugs that affect production of TNF-α and effector cytokines (IL2, INF) to enhance antimycobacterial effector mechanisms such as IL-2 and IFN-γ . The timing of immunotherapy needs to be considered carefully and should probably primarily have a sterilizing effect which could be maximized if it is initiated when the bacillary load has been reduced by effective antimycobacterial drugs. Of note, there are large gaps in the understanding of host immune mechanisms against TB, which may affect the use of cytokines as adjuvant immunotherapy . It is clear that mouse models have limited value regarding the development of the granuloma because of differences from human disease with regard to the organization of cells, lack of giant cells and lack of caseous necrosis . In the mouse model, IFN-γ, which increases the killing capacity of macrophages, and TNF-α, which is produced by T cells and macrophages, expressed in early-stage TB at the site of disease and essential for granuloma maintenance, are key molecules for host defence and control of Mtb in the T cell/macrophage immune axis . One of the first large-scale randomized trials of adjuvant IL-2 therapy in 110 Ugandan HIV-negative TB patients showed no effect on the sputum conversion which was the primary outcome. The rationale for the treatment relied mainly on animal models, in which IL-2 strongly induced IFN-γ production and was a potent growth factor in T cells . One possible cause of the unfavourable response is an unexpected increase in the activity of CD25+ Treg cells, which may counteract the proinflammatory reaction. IFN-γ administered by aerosol in five MDR patients showed some effect on sputum conversion, but results from follow-up trials were inconclusive . In most TB patients, single cytokine therapy is unlikely to be very effective, but may be essential for patients with defects in the IFNγ–IL-12 immune pathway . An approach to provide a stimulus that targets a suitable host response instead of using single cytokines, has been extensively investigated by therapeutic vaccination with M. vaccae. The rationale for this treatment, that it would enhance the Th1 response and suppress the Th2 response, has not yet been proven beneficial in clinical trials . Another possible strategy could be to downregulate the Th2 and Treg responses to gain a more effective Th1 response [107, 151, 155]. Adjuvant IL-7 therapy may impair TGF-ß production, as shown in HIV-positive patients . A novel strategy that has been investigated in several clinical trials is to administer high-dose corticosteroids or TNF-α inhibitors to induce therapeutic disruption of granulomas to increase the accessibility of antimycobacterial drugs [153, 157, 158].
Because of considerable biological variation, there might be a role for individualized treatment strategies in TB. The sources of variation include the virulence of Mtb, host response, nutrition and co-infection (e.g. with HIV), and pharmacokinetics of anti-TB chemotherapy. Individualized medicine is likely to have an impact on the outcome of TB treatment by optimizing antibiotic therapy through therapeutic drug monitoring and by enhancing the host immune response. A way of shortening the therapeutic regimen is to ‘hit hard and early’ with many bactericidal drugs when Mtb is at the extracellular stage. Such strategies combined with an improved host response through nutritional support, deworming and treatment of HIV, may reduce the long treatment period which is mainly designed to reduce the risk of relapse. In patients with resistant TB, it is important to consider strategies to optimize the immune response, as they might be at least as effective as some second-line drugs. In the future, with the availability of assays to measure nutritional status, host immunity and drug concentrations, it may be possible to tailor an individualized regimen to optimize both the host response and the effects of the available bactericidal drugs.
Conflict of interest statement
No conflict of interest was declared.
This work was supported in part by grants from the Swedish Research Council (ML and OS), the Heart-Lung Foundation (ML, TS and OS), the Swedish International Development Agency (OS), European developing countries clinical trial partnership (EDCTP), the Swedish Society of Medicine (TS), the Medical Research Council of Southeast Sweden (TS), the M&M Wallenberg Foundation (TS), the R Söderberg Foundation (OS) and the B&M Gates Foundation (ML).