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Immunotherapy for pulmonary TB: antimicrobial peptides and their inducers TB is an infectious disease that still has an enormous impact on public health worldwide. With the continuous increasing epidemic of multidrug-resistant TB, new drugs and vaccines are urgently needed. In the last decade there has been a broad advance in the knowledge of innate immunity in TB. Together with the growing research regarding immunomodulators, new promising insights have been developed that can contribute in the control of TB. This is the case of antimicrobial peptides, which can be potential therapeutic or adjuvant agents. The current high cost of antimicrobial peptide synthesis may be a current deterrent for treatment; antimicrobial peptide-inducers can be an alternative for low-cost treatment and/or adjuvants. KEYWORDS: antimicrobial peptides n defensins n HNP‑1 n IDRs n l‑isoleucine n LL‑37 n TB immunotherapy n vaccines n vitamin D

TB is an infectious disease caused by Mycobacterium tuberculosis. TB has existed in humanity for centuries. Despite this long relationship and the huge advance in the knowledge of the etiological agent, M. tuberculosis is still at the top of the list of pathogens that causes huge mortality worldwide; every time a new anti-TB drug comes aboard a new multidrug-resistant (MDR) M. tuberculosis strain emerges. This makes it a tough task for researchers to find novel anti-TB drugs. Vaccination is another strategy used to eradicate TB; however, the only licensed vaccine currently is BCG, which is about 100 years old and shows variable efficacy ranging from 0 to 80%. Altogether, these data suggest that present strategies to eradicate TB should be improved, and one promising approach is the use of immunotherapeutics, which together with conventional drugs and vaccines raise new hope. Within these immunotherapeutic agents, antimicrobial peptides (AMPs) are a very important group due to their versatile activity. This article will focus on the advances made in research of AMPs and AMP inducers, and their use in TB treatment.

Basic facts AMPs are small cationic molecules of a variable length. These peptides mainly constitute polar-hydrophilic, nonpolar-hydrophobic and positively charged amino acids. This special conformation gives the molecule amphipathic and cationic properties, which are very important for their bactericidal activity [1]. AMPs are broadly distributed in nature and all of them 10.2217/IMT.13.111 © 2013 Future Medicine Ltd

share fundamental structural characteristics that are very important for their broad-spectrum antimicrobial activity, such as length (less than 60 amino acids), amphipathic structure and the presence of cationic amino acids in their structure, providing them with a partial positive charge. However, due to their secondary structural characteristics, AMPs could be divided into subcategories that include: a‑helical, peptides enriched with repetition of one amino acid, and peptides with intramolecular bonding formed by cysteines. These features allow the AMPs to interact with most microorganisms’ lipid bilayer and eliminate them through membrane disruption or by translocating across the membrane and inhibiting cytosolic targets [2,3]. Recent findings show that the immunoregulatory activity is quite efficient since most of these activities have an effect at the nanomolar scale, acting over several innate immune response receptors. Although at the beginning it was thought that these functions were only focused to promote proinflammatory conditions, it is now well known that some peptides, such as cathelicidin have ambiguous functions promoting, in certain cases, an anti-inflammatory response as well [4–7].

Cesar Enrique Rivas‑Santiago1, Rogelio Hernández-Pando2 & Bruno Rivas‑Santiago*3 Rutgers University School of Public Health, Department of Environmental & Occupational Health, Center for Global Public Health, Piscataway, NJ, USA 2 Department of Experimental Pathology, National Institute of Medical Sciences & Nutrition “Salvador Zubirán”, Mexico City, Mexico 3 Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, 45 Zacatecas, cp.98000, Mexico *Author for correspondence: Tel.: +52 4929 226 019 [email protected] 1

Antimicrobial activity of AMPs As described above, AMPs share key biophysical properties that confer strong antimicrobial activity, such as the presence of a positive or cationic charge that permits chemoattraction and electrostatic attachment to the bacterial membrane. When the peptides are at a high Immunotherapy (2013) 5(10), 1117–1126

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concentration, they can insert into the bacterial membrane perpendicularly, causing alterations in the lipid bilayer, making it permeable and triggering bacterial death [3,8,9]. However, this is not the only mechanism of action known for AMPs; for example, it has been demonstrated that members of the buforines and cathelicidins family are able to cross the membrane and, in the cytosol, they can bind to DNA and RNA by electrostatic charges, interfering with vital processes [10]. However, nucleic acids are not the only intracellular target of AMPs; there are peptides such as mersacidin that inhibits cell wall synthesis by interaction with peptidoglycan precursors [11]. Some other peptides, such as PR‑39, HNP‑1 and -2, inhibit the synthesis of very important proteins for bacterial viability [12]. Finally, there is a group of AMPs described recently, such as hepcidin, that not only produce damage in the bacterial cell membrane [13], but also decrease the iron levels by negatively regulating its intestinal absorption [14].

Immune regulatory functions Proinflammatory AMPs have been shown to act as chemoattract ants for cells of innate and adaptive immunity, and in fact, many authors consider them as a bridge between innate and adaptive immunity [15–18]. One of the most studied peptides has been human cathelicidin LL‑37 because of its regulatory functions and being the only member of the cathelicidin family present in humans. LL‑37 is capable of attracting neutrophils, monocytes, T cells and mast cells using formyl peptide receptor-like 1, and a distinct Gi-coupled receptor at nanomolar concentrations [19,20]. Besides the remarkable chemotactic activity, it also induces several other responses in leukocytes and epithelial cells, modifying gene expression in order to improve or modulate immune response. It has been reported that stimulation of primary human monocytes and macrophages with LL‑37 led to the induction of a wide range of chemokines, chemokine receptors and other genes involved in cell adhesion, communication and motility [21]. In fact, most of the encounters between pathogenic microorganisms and cells lead to LL‑37 production, which in turn promotes proinflammatory chemokine production. Along with cathelicidin, defensins have a wide range of immunoregulatory activities owing to their ability to engage several cell surface receptors promoting chemotaxis, such as the recruitment of immature dendritic cells (DCs) and T lymphocytes after hbD‑2 engagement to 1118

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the CCR6 receptors [22]. Moreover, hbD‑3 can downregulate CXCR4, which in certain conditions, such as HIV‑1 infection, may contribute to avoiding virus infection [23]. In addition, APCs undergo maturation in the presence of hbD‑3 via Toll-like receptors (TLRs)‑1 and -2. Furthermore, murine b‑defensin and hbD‑3 have been shown to act directly on immature DCs as an endogenous ligand for TLR‑4, -2 and -3, inducing upregulation of costimulatory molecules and DC maturation, triggering robust Th1 polarized adaptive immune responses in vivo [24,25]. hbD‑3 also has a high affinity for interaction with CCR2 on myeloid cells resulting in chemoattraction in the absence of the natural ligand‑1/CCL2 [26]. Finally, hbD‑3 can compete with melanocyte-stimulating hormone a, the natural ligand of melanocortin (MC1R) in myeloid cells [27], which suggests that hbD‑3 may inhibit anti-inflammatory activity promoted by melanocyte-stimulating hormone a since this ligand has been shown to induce IL‑10 in cells expressing the ligand of melanocortin. Moreover, some peptides are capable of activating cells of the immune system through TLRs [22,24,25,28] or inducing the production of chemokines such as IL‑8 [28]. In fact, our group, based on this important characteristic, has found that b‑defensin‑2 binds to DCs during early stages of infection and promotes IFN‑g production in experimental TB [29], which has led us to design vaccines using defensins as an adjuvant and applying this vaccine to boost BCG with promising results [30]. These peptides with anti- and pro-inflammatory properties, such as b defensins, can induce the release of prostaglandin D2 and histamine in mast cells, which causes vascular permeability triggering the inflammatory process [31,32]. Anti-inflammatory Although many of the immunoregulatory effects of AMPs are related to proinf lammatory responses, in the past few years the anti-inflammatory aspect of AMPs has been demonstrated. For instance, defensins have powerful anti-inflammatory effects on human monocytes, human monocyte-derived macrophages and human myeloid DCs [33]. In fact, after phagocytic cells were treated with HNPs after exposure to lipopolysaccharides (LPS), it was observed that HNP‑1 blocked the release of IL‑1b from LPS-activated monocytes, but not the expression and release of TNF‑a [34]. It has been demonstrated that apoptotic and necrotic neutrophils inhibit the secretion of future science group

Immunotherapy for pulmonary TB: antimicrobial peptides & their inducers

proinflammatory cytokines from macrophages by releasing HNPs in the presence of both live and dead whole bacteria; thus, HNPs inhibit the LPS-mediated activation of macrophages without affecting the release of proinflammatory cytokines by macrophages. Pingel et al. found that hbD‑3 attenuates the IL‑6, IL‑10, GM‑CSF and TNF‑a response of human myeloid DCs [35]. Similarly, LL‑37 modulates host cell responses to stimuli and also affects the action of endogenous immune mediators: IL‑1b and GM‑CSF. This activity is dependent on the cell type and activation status, timing of exposure and the microenvironment. In certain cells, such as monocytes, macrophages, DCs and B lymphocytes, it inhibits cellular responses to interferon, showing suppression of cell activation and proliferation, and production of proinflammatory and Th1-polarizing cytokines and antibodies. It was further demonstrated in monocytes that the suppressive effects of LL‑37 were mediated through inhibition of STAT1-independent

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signaling events, involving both the p65 subunit of NF‑kB and p38 MAPK [7]. Another anti-inf lammatory function of AMPs is mediated by their ability to bind antigenic molecules preventing the activation of immune responses. One clear example of this is the binding of cathelicidins and defensins to LPS to prevent TNF‑a secretion [36]. Additionally, HNP‑1 binds to Bacillus anthracis lethal factor, inducing conformational changes that prevents enzymatic conversion and protects mice from B. anthracis lethal factor intoxication and death [37]. HNP‑1, HNP‑3 and HD‑5 bind to toxin B from Clostridium inhibiting glycosy lation in vitro of Rho guanosine triphosphatases [38]. Figure 1 outlines the different mechanisms of action described for AMPs.

AMPs in TB The pioneering studies to determine the relationship between AMPs and TB focused on the antimycobacterial activity of AMPs. These studies were performed using nonpathogenic strains,

Antimicrobial effect

Direct antimicrobial effect by pore formation in bacteria

Antimicrobial effect by inhibition of replication transcription and translation

AMPs

IL-1β TNF-α IL-6

IFN-γ

Chemoattraction of immune system cells; neutrophils,T cells, monocytes and DCs

Maturation of immature DC

Proinflammatory effects

Inhibit cellular response to IFN-γ

Block secretion of proinflammatory cytokines in monocytes and macrophages

Anti-inflammatory effects

Figure 1. Diverse mechanisms of action of antimicrobial peptides. (A) The different bactericidal effects observed in AMPs. Left: extracellular action; right: intracellular targets. (B & C) The inflammatory effects demonstrated by AMPs. AMP: Antimicrobial peptide; DC: Dendritic cell.

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observing an important decrease in the bacilli burden in an in vitro model [39]. However, it was not until the year 2000, when pathogenic strains were studied and an important effect against the bacilli was demonstrated [40,41]. Thereafter, Kisich et al. demonstrated that cellular synthesis of hbD‑2 after mRNA transfection to human macrophages conferred efficient mycobactericidal and mycobacteristatic activities [41]. Considering that the first cells that encounter M. tuberculosis during primary infection are epithelial cells, our group sought to determine whether these cells used AMPs to counterattack infection. Both in vitro and in vivo results showed that defensins are very important in killing mycobacteria, and a mouse strain that was susceptible to develop TB demonstrated markedly lower levels of defensins compared with the resistant strain. Working with this murine model, we demonstrated that during the early stages of disease there was high expression of b‑defensins, and this expression strikingly decreased during late active infection, which correlated with the severity of the disease and the increase of pulmonary bacillary loads in the mice. In a murine model of TB latent infection, a constant high production of defensins was observed, which could be associated with bacillary growth control due to reactivation induced by corticosterone and the production of defensins substantially decreased in coexistence with a high increase of mycobacterial pulmonary loads [42]. Another very important AMP that has been involved in the immunopathogenesis of TB is cathelicidin LL‑37, as well as its mice ortholog CRAMP. In the last few years our group has studied these peptides, observing that myco bacteria induce the production of LL‑37 in human alveolar macrophages and the over production of this AMP will lead to bacterial lysis during in vitro infection [43]; on the other hand, our studies in the murine model have shown that during M. tuberculosis infection, there are three peaks of cathelicidin expression at 1, 21 and 60 days postinfection. Intriguingly, cathelicidin is highly expressed once the pneumonia is established; however, this high production is not reflected by a decrease of bacillary loads in the lung, which probably means that cathelicidin is acting more as an immunomodulator rather than an antimicrobial, promoting an anti-inflammatory response [44]. Similar results were obtained in TB patients, in which they showed high production of cathelicidin during active disease, while in latent infected individuals LL‑37 levels were comparable with those found in healthy donors [45]. 1120


Another important factor related to innate immunity is that AMPs and TB control vitamin D and its receptors. It has been shown that stimulation of TLR‑2 and -1 increase the expression of these receptors as well as the enzyme Cyp27B1, which catalyzes the conversion to the active form of vitamin D, and finally leads to the induction of cathelicidin LL‑37, increasing the intracellular killing of the bacilli [46]. This group has also shown the direct participation of cathelicidin induction by vitamin D in the intracellular killing of M. tuberculosis, using interfering RNA specific for this AMP. They observed that macrophages with silenced cathelicidin had a higher bacillary burden compared with macrophages that did not receive the siRNA [47]. Conversely, M. tuberculosisinfected mice treated with 1 mg/kg of LL‑37 or CRAMP showed a modest decrease of bacilli burden but pneumonia was increased considerably [48]. Taken together these data suggest that cathelicidin is essential for innate immunity against mycobacteria; however, once the infection has been established cathelicidin promotes an anti-inflammatory response. Thus, LL‑37 has a dual effect in infection: during early infection it increases phagoc ytosis, expression of costimulatory molecules in DCs and induces a higher production of type Th1 cytokines [49]; and during late infection, it inhibits TNF‑a secretion from monocytes as well as some other proinflammatory cytokines [21]. Moreover, it reduces the response of monocytes, macrophages and DC to IFN‑g [7], which is the principal cytokine of the Th1 response. These characteristics show the duality of cathelicidin during the infectious process by avoiding inflammation and contributing directly to the elimination of the microorganism. Recently our group demonstrated that the addition of LL‑37 to noninfected macrophages led to the production of proinflammatory cytokines, but when macrophages were highly infected, it led to production of cytokines such as IL‑10 and TGF‑b [Rivas-Santiago B, Unpublished Data]. These experiments correlate with previous findings in experimental animal models [44]. Another potent AMP that has been studied in M. tuberculosis infection is hepcidin, an AMP produced in the liver in response to inflammation and high concentrations of iron, which is essential for bacilli growth and survival. Hepcidin is localized in the mycobacteriacontaining phagosome in macrophages and inhibits M. tuberculosis growth in vitro by causing bacterial structural damage [13]. However, macrophages are not the only cell of the innate future science group


immune system that overproduce hepcidin when infected by M. tuberculosis. Epithelial cells, such as pneumocytes type 2, or DCs in mice, are able to overexpress this AMP in response to some components of the bacilli [50]. There are other kinds of AMPs that are obtained synthetically by the fusion of different peptides [51], as well as by modification of some amino acids in the structure of wellknown peptides, with the purpose of increasing amphiphacity or by increasing their net positive charge [52]. These modifications have increased the efficiency of these AMPs against bacteria and fungi [51,52]. Recently our group has tested these semisynthetic peptides as promising antimycobacterial compounds in our mouse TB model. Some of these peptides showed magnificent activity to eliminate mycobacteria both in vivo and in vitro [48,53].

AMPs as therapeutics agents for pulmonary TB AMPs can be used as therapeutic agents administered as recombinant or synthetic protein, as well as using inductors, which stimulate specific cells for high AMP production and secretion. A good example of an inductor is l-isoleucine, which is an essential amino acid that has been reported as a strong inductor of b‑defensins in bovine kidney epithelial cells [54]. Our results showed that l-isoleucine significantly induced the production of these peptides in human pneumocyte cultures and this induction was specific for this amino acid as other amino acids with a similar structure, such as d-isoleucine, did not produce the same effects [54,55]. When l-isoleucine was used therapeutically in M. tuberculosisinfected mice with either a drug-sensitive or a drug-resistant strain over 2 months, a significant decrease of pulmonary bacilli burdens and reduction of tissue damage (pneumonia) was observed in correlation with high expression of b‑defensins [55]. As mentioned above, vitamin D is a cathelicidin inducer not only in TB but also in several infectious and noninfectious diseases. Indeed, it has been demonstrated that reduced serum levels of this vitamin are associated with the development of active TB [56,57], and in the presence of IFN‑g and TNF‑a, this vitamin induces the production of AMP and bacilli elimination [46,58]. Recent meta-analysis reports demonstrated that low serum vitamin D levels are associated with a higher risk of active TB [56]. However, the use of vitamin D as a supplement to increase the production of AMP was not as future science group

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effective as expected owing to a decrease in blood LL‑37 levels after vitamin administration [59]. The controversy among reports highlights the different doses used for each study. Thus, Mily et al. performed a dose-finding study and determined that 5000 IU once-daily vitamin D3 is the optimal dose for the induction of LL‑37 on macrophages and lymphocytes with efficient intracellular M. tuberculosis killing by macrophages, and the effect is synergically increased when vitamin D3 is coadministered with 500 mg of phenylbutyrate (PB) twice-daily [60]. The use of vitamin D as an immunomodulator is a promising therapy for the treatment of pulmonary TB; however, there are some issues that should be considered before clinical trials can start. As described above, cathelicidin induced by vitamin D may contribute to the production of anti-inf lammatory cytokines during active disease, which could be detrimental; however, further studies need to be carried out. Although vitamin D is the most significant immunotherapeutic candidate to use in TB, some other candidates have emerged. Previous experimental studies have shown that down regulation of the rabbit cathelicidin (CAP‑18) in the large intestine and lung can be counteracted by oral treatment with sodium butyrate, a shortchain fatty acid and PB [61,62].With this background, Raqib et al. developed a clinical trial in which sodium butyrate was used as an adjuvant in the treatment against shigellosis, finding that treatment with sodium butyrate led to a faster reduction of inflammation and that the decrease in inflammation and clinical illness could be associated with the induced expression of LL‑37 [63]. The same group further demonstrated that PB can induce LL‑37 expression synergistically with 1,25 dihydroxyvitamin D3, at both protein and mRNA levels in a lung epithelial cell line [64]. It is likely that an oral supplementation with PB will boost innate immunity in the lung mucosa by increasing expression of AMPs, and M. tuberculosis killing and autophagy, and is now being tested in a clinical trial of adults with active pulmonary TB [60]. Owing to the importance of vitamin D as an immunotherapeutic, different groups have developed different clinical trials in pulmonary TB. The results obtained are very variable; while some studies show the evident role of vitamin D, in others it is not that evident. Overal, it is probable that the administration of vitamin D as an adjuvant is helpful just for patients with the tt genotype of the TaqI www.futuremedicine.com

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vitamin D receptor polymorphism [65]. Besides that fact, the application of vitamin D could be helpful as prophylaxis in latent TB [66]; however, more clinical trials must be carried out taking into account the ethnic origin of the group and stage of the disease. Another interesting use of AMP is in vaccination. Our group recently showed that DNA vaccines containing coding sequences for b‑defensin‑2 induce a strong Th1 adaptive response against M. tuberculosis antigens, and when these DNA vaccines were used as a booster for immunization after BCG vaccination, a significant improvement of protection against M. tuberculosis strains was produced. Since BCG is the only vaccine for TB and has a reduced efficacy to protect against pulmonary TB, this new approach could greatly improve vaccination efficacy against highly transmissible and virulent strains. Thus, this study suggests that improvement of BCG vaccination combined with DNA vaccines in a prime-boost scheme is a good choice for the rational design of a more efficient vaccine against TB [30]. Further experiments need to be carried out to assess the efficacy of this vaccine as therapeutic rather than prophylactic. Lantibiotics are AMPs synthetized by Grampositive bacteria and these peptides are characterized by the presence of post-translationally modified amino acids in their structure, such as lanthionine and/or methyl lanthionine. The most studied lantibiotic is nisin A, a peptide

produced by Lactococcus lactis, which in 1988 was considered safe for use as a food preservative. The mechanism of action of this AMP is joining a cell wall precursor to lipid II, allowing pore formation and at the same time inhibiting biosynthesis of the bacterial cell wall [67,68]. Nisin A and its synthetic derivatives nisin S and nisin T are efficient lantibiotics against M. tuberculosis and non-TB bacteria, and they constitute interesting compounds for clinical studies [69]. In the last 5 years, the boom of innate defense regulator peptides (IDRs) has brought promising immunoregulatory alternatives for infectious disease including TB [53]. IDRs are synthetic immunoregulatory and anti-infective peptides that are based on the sequences of natural AMPs. IDRs were originally designed as antibiotics, but many of them also have immunoregulatory activities and their in vivo functions may ultimately be mediated through a combination of both functions [70–72]. In recent studies, it has been demonstrated that the protective activity of IDRs could be solely based on their immunomodulatory properties and that this protection is functional even in animals infected with MDR strains [73]. Besides this immunoregulatory property, the low potential of microbial resistance, lower toxicity and requirement of fewer doses, suggest that IDRs could be used as a treatment and as an adjuvant, as well as for conventional drug-sensitive, but

Table 1. Relevance of antimicrobial peptides in TB. Antimicrobial peptide

Source

Activity

Importance in TB

Human neutrophil Neutrophils peptides

Bactericidal effect, chemotaxis and activation of DCs and DNA-binding

Possible adjuvant in anti-TB chemotherapy

b‑defensin-2

Epithelial cells

Bactericidal effect, chemotaxis of a wide range of leukocytes through CCR2 and CCR6, and activation of DCs

Induction of this antimicrobial peptide reduce mycobacterial loads

[22,25,41,55,78]

b-defensin-3

Epithelial cells

Bactericidal effect, and chemotaxis of monocytes, macrophages and neutrophils through CCR2

Owing to its chemotactic effect, this antimicrobial peptide could be used as immunotherapy

[78,79]

LL‑37

Alveolar macrophages, MDM and alveolar epithelial cells

Bactericidal effect and modulation of immune response

A decrease in bacterial load was observed when applied as a therapy in infected mice

[7,48]

E2, E6 and CP26

Synthetic peptides

Bactericidal effect by disruption in cell wall of the bacteria

Application of these peptides significantly reduces the lung bacillary loads

Immunomodulatory functions by the induction of chemokines

In animal models, reduced bacillary loads as well as inflammation by decreased pneumonia

Innate defense Synthetic peptides regulator peptides

Ref. [74–77]

[48,80]

[53,70,81,82]

DC: Dendritic cell; MDM: Monocyte-derived macrophage.

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mainly MDR, strains combining their antimicrobial and wound healing properties to treat deep sores and ulcers [73]. Several in vitro and in vivo experiments have tested the efficacy of IDRs in experimental TB with pathogenic and MDR strains. In a murine model of progressive pulmonary TB, the intratracheal administration of the IDR peptides E2, E6 and CP26 during late disease in mice infected with drugsensitive M. tuberculosis or MDR strains significantly reduced lung bacillary loads. However, there was no reduction in the inflammatory infiltrate (pneumonia) compared with control nontreated mice [48]. Further experiments demonstrated that the use of some IDRs, such as HH2 or 1018, not only decreased bacillary loads but also pneumonic areas, therefore they are candidates for testing in humans [53]. Table 1 shows a summary of the different AMPs and their importance in TB.

Potential disadvantages of immunotherapy using AMPs for TB Although it seems that AMPs and their inducers could be used as a treatment against TB and that these options could help to eradicate this disease, currently this is quite far off. Even though there is greater knowledge of the relationship between TB and AMPs, there are many gaps that need to be filled before applying clinical approaches. For instance, it is still unknown which peptides could be used for each stage of the disease, since some peptides have antiinflammatory effects whereas others have proinflammatory effects. The induction of inflammation during progressive TB would accelerate the pneumonic process in the patient. Therefore, the design of precise AMPs for specific stages of TB must take place. Furthermore, it is known that M. tuberculosis develops resistance to antibiotics under evolutive pressure; AMPs would not be an exception and this would mainly be seen in developing countries where a medical prescription is not necessary to have access to antibiotics. In summary, basic investigation and new health policies are essential before AMPs are used as immunotherapy. Future perspective Although many issues remain to be studied regarding AMPs and their inducers for the immunotherapy of TB, this topic is a promising alternative for the control of M. tuberculosis infection mainly by MDR strains; therefore, for future studies it will be necessary to include these kinds of strains. Another important point to assess is future science group

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which peptides could be used for each specific stage of the pulmonary disease to avoid unnecessary inflammation that could impair patient health. In this case, IDRs are a good option, but further clinical studies are needed. It is widely demonstrated that vitamin D and l-isoleucine have undoubted in vitro effects, inducing AMPs that lead to M. tuberculosis killing. However, both these molecules must be studied further in clinical trials in people of different ethnic origins and it will be necessary to determine whether these molecules have synergic effects with convectional antimycobacterial drugs. Finally, the way AMPs or AMP inducers are delivered is likely to influence their effectiveness, thus efficient routes of delivery need to be studied.

Conclusion The possibility of the use of AMPs, AMP inducers and IDRs for the treatment of pulmonary TB is increasing, partly owing to the extensive research on innate immunity of TB. The improvement of low-cost peptide synthesis and peptide development companies are advancing therapeutic candidates that are relatively short-sequenced, do not require folding, such as disulfide bonds, and are required in low concentrations to be effective. The analysis of cost–benefit when compared with second- and third-generation antibiotics used for the treatment of MDR and extensively drug-resistant strains should be also considered. There is significant therapeutic potential for the use of AMPs. The issues that must be overcome are not trivial and include cost of goods, stability, toxicity and delivery. However, these issues are the same for any drug development candidate, but are likely to be overcome with advances in drug delivery, and with limitations as well as the attributes of innate immunity peptides. Acknowledgements The authors thank PA Cantarella IV (Rutgers University School of Public Health) for reviewing the manuscript.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. www.futuremedicine.com

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Executive summary TB is one of the most important infectious diseases and is responsible for 1.8 million deaths worldwide annually. In the last decade new multidrug-resistant strains have emerged, consequently making it a very hard task to eradicate TB. The use of antimicrobial peptides (AMPs) and AMP inducers are a promising option for the immunotherapy of TB by using them as direct antimicrobial molecules, as well as immunomodulators. AMPs possess both pro- and anti-inflammatory properties, which can be convenient for the immunotherapy of TB. The use of defensins as adjuvants in a prime-boost scheme is a good choice for the rational design of more efficient vaccines against TB. The use of innate defense regulator peptides can be a good option to promote Mycobacterium tuberculosis elimination; using them as immunotherapy avoids undesirable effects, such as inflammation. The use of AMP inducers, such as vitamin D and l-isoleucine, are a good option since all in vitro studies demonstrate their efficacy to induce AMPs and kill M. tuberculosis through several mechanisms; however, clinical studies are controversial, therefore further clinical studies are needed. LL‑37 modulates the effects of IFN-gamma on APCs. J. Immunol. 183(9), 5788–5798 (2009).

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