Review For reprint orders, please contact reprints@future-science.com Inhaled therapies for tuberculosis and the relevance of activation of lung macrophages by particulate drug-delivery systems Pathogenic strains of Mycobacterium tuberculosis (Mtb) induce ‘alternative activation’ of lung macrophages that they colonize, in order to create conditions that promote the establishment and progression of infection. There is some evidence to indicate that such macrophages may be rescued from alternative activation by inhalable microparticles containing a variety of drugs. This review summarizes the experience of various groups of researchers, relating to observations of induction of a number of classical macrophage activation pathways. Restoration of a ‘respiratory burst’ and upregulation of reactive oxygen species and nitrogen intermediates through the phagocyte oxidase and nitric oxide synthetase enzyme systems; induction of proinlammatory macrophage cytokines; and inally induction of apoptosis rather than necrosis of the infected macrophage are discussed. It is suggested that there is scope to co-opt host responses in the management of tuberculosis, through the route of pulmonary drug delivery. Inhalation therapy by means of particulate drug-delivery systems shows promise as an approach to combat tuberculosis (TB). Recent research authenticates the applicability of various inhalable drug-delivery systems such as microparticles [1–3] , nanoparticles [4] and liposomes [5,6] apart from drug powders by themselves (compounded with inhalable excipients) [7–9] , for TB therapy. Inhalations deliver therapeutic or prophylactic agents directly to the site of infection, specifically lung (alveolar) macrophages (AM). This noninvasive route of delivery also offers additional advantages: reduction in dose, frequency and duration of treatment, lower systemic toxicity, and improved patient compliance. Table 1 summarizes the drugs and delivery systems investigated for use as inhaled therapies for TB. Some research groups have experience with pulmonary delivery of drugs or drug-containing particulates or vesicles by nebulization of a suspension of the delivery system in a liquid medium, while others favor dry-powder inhalations as insufflations or ambient-pressure inhalations [1,7,10,11] . Even though nebulization is more familiar as a method of aerosol delivery for lung diseases (asthma, hay fever, COPD), drypowder inhalations can deliver larger amounts of payload to the deep lung. The optimum size range of particles for inhalation is usually considered to lie within 1–5 µm, but the key measurement of suitability for lung delivery is a particle’s median mass aerodynamic diameter. 10.4155/TDE.11.34 © 2011 Future Science Ltd Generally, particles below approximately 0.5 µm are exhaled undeposited as they do not have enough inertia to go through impaction or sedimentation in the lung, while particles larger than approximately 5 µm get entangled in the oropharyngeal and upper airway regions of the respiratory tree. Microparticles in the respirable size range deposit in the deep lung and are readily taken up by AM [12,13] . In recent years, evidence has been presented to demonstrate that AM infected with Mtb display markers of classical macrophage activation when they take up inhalable microparticles [14–15,201] . Some researchers have prepared microparticles incorporating a single anti-TB drug. Monotherapy, however, is not recommended in TB, so microparticles incorporating multiple drugs may be better suited to the task. Biodegradable microparticles composed of poly(lactide) and incorporating a high payload of isoniazid and rifabutin have been investigated in some detail, including their in vitro/ in vivo efficacy in experimental animals, pharmacokinetics and biodistribution of the incorporated drugs, ana lysis of innate effectors or inflammatory mediators induced as a result of inhalation or phagocytosis by macrophages in culture, induction of caspase-dependent and -independent apoptosis, autophagy, purinergic receptor activity, mitochondrial membrane stabilization and so on [1,3,10,16–18,201] . FiguRe 1 depicts three different states of macrophage activation, and Table 2 summarizes the status Therapeutic Delivery (2011) 2(6), 753–768 Rahul Kumar Verma1, Amit Kumar Singh1, Mradul Mohan1, Atul Kumar Agrawal1 & Amit Misra†1 1 Pharmaceutics Division, Central Drug Research Institute, CSIR, Lucknow, 226001, India † Author for correspondence: Tel.: +91 522 261 2411 Fax: +91 522 262 3405 E-mail: amit_misra@cdri.res.in ISSN 2041-5990 753 Review | Verma, Singh, Mohan, Agrawal & Misra Table 1. Drugs and delivery systems investigated as inhalation therapies for tuberculosis. Drug-delivery system Drug(s)/peptide Mode of administration Animal model Microparticles Rifampicin Rifampicin Rifampicin P-aminosalicylic acid Isoniazid + rifabutin Isoxyl Suspension nebulization Insufflation Intra-tracheal Insufflation Ambient pressure inhalation Insufflation Guinea pig Guinea pig Rat Rat Guinea pig, mouse Recombinant antigen-85 Capreomycin sulfate Capreomycin oleate Insufflation Inhalation Insufflation Guinea pig Guinea pig Isoniazid + rifampicin + pyrazinamide Antigen Rv1733c Rifampicin Capreomycin sulfate Nebulization Nebulization Nebulization Nebulization Guinea pig Mouse Guinea pig, mouse, rat Isoniazid + rifampicin Isoniazid + rifampicin + pyrazinamide Capreomycin sulfate Live-attenuated BCG + L-leucine P-824 Nebulization Nebulization Insufflation Insufflation Insufflation Guinea pig Guinea pig Guinea pig Mouse Guinea pig Nanoparticles Liposomes Solid-lipid particles Dry-powder inhalation Key Terms Mass median aerodynamic diameter: Median of the distribution of inhalable particle mass with respect to the aerodynamic diameter. A better indicator of lung and airway deposition and distribution than just the physical dimensions, since it takes relative density, aerosolization conditions and so on, into account. Apoptosis: Programmed cell death by which cells undergo an ordered sequence of events which lead to death of the cell. Autophagy: Extensive recycling of cytosolic contents through generation of Golgi vesicles in a section of the cytoplasm accomplished through self-digestion. Classically activated macrophages: Exhibit a Th1-like phenotype, promoting inlammation, extracellular matrix destruction and apoptosis. Alternative activated macrophages: display a Th2-like phenotype, promoting ECM construction, cell proliferation and angiogenesis. 754 of various markers associated with each of these activation states. The intention of this article is to adduce scientific rationale for incorporating into the standard treatment of TB, the objective of activating lung macrophages (e.g., by inhalable particles), so that host–defense responses may also be co-opted to combat infection. Induction of alternative activation of host macrophages by Mtb Mtb spreads through inhalation of moist droplets containing small numbers of bacilli, expelled in the cough of infected persons. Inhaled droplet nuclei lodge in the pulmonary alveoli and Mtb invades resident AM through a variety of cell-surface receptors. Other environ mental microorganisms or inert particles of similar size and composition are also phagocytosed by AM in the natural course of events. Particle phagocytosis is sufficient to induce a classical activation response in the AM, which mobilizes intracellular and extracellular calcium [19] , undergoes a respiratory burst, generates free radicals [14,20,201] and Ref. [114] [11] [115] [116] [1,3] [117] [118] [119] [120] [121] [122] [123] [124] [16] [4] [8,125] [126] [127,128] thereby destroy and digest the foreign particle. These responses have collectively been referred to as the ‘activation’ response [21,22] . Mtb has co-evolved with the human host to effect entry into the AM. Mtb enters macrophages through a variety of surface receptors including mannose receptors, complement receptors, Fc and scavenger receptors, which results in the delivery of mycobacterium into phagosomes [23] . Within the phagosome formed, as a result of phagocytosis of Mtb by AM of ‘susceptible’ individuals, the pathogen is able to evade or counteract the biochemical events that lead to destruction of phagocytosed particles. In general terms, the pathogen is able to subvert the activation response of the infected macrophage into a phenotype that is best described as alternative activation [21,24] . In individuals that are ‘resistant’ to TB, classical activation of the AM evidently ensues, resulting in containment or elimination of the infection. However, there is of course no clear dichotomy between ‘resistant’ and ‘susceptible’ individuals – nor is there any reason to expect that exclusively classical Figure 1. Classical, alternative and microparticle-mediated activation of macrophages (on facing page). Classical activation is initiated by IFN-g, subsequent to recognition of a ‘pathogenassociated molecular pattern’ by pattern-recognition receptors , which in turn induce the production of proinflammatory cytokines, such as TNF, IL-12, IFNs, IL-6 on the one hand, and direct-effector molecules such as ROS and nitric oxide. Alternative activation is initiated by IL-4 and IL-13 secreted as a consequence of mycobacterial infection, which enhance anti-inflammatory cytokines and inhibition of proinflammatory mediators. Microparticle-induced activation tends to reprogam alternatively activated macrophages to display markers of classical activation. RNI: Reactive nitrogen intermediate; ROS: Reactive oxygen species; TLR: Toll-like receptor. Therapeutic Delivery (2011) 2(6) future science group Inhaled therapies for TB Classical activation Ap opt osi s IL- Inflammasome activation Proteasome IFN-γ, TNF, LPS-TLR ligands 1β IL-1β Pro-IL-1β O2– TNF, IL-12, IFN, IL-6, MIP-1 p65 p50 Endoplasmic reticulum IκB p65 p50 AT ST TC R C-I I MHC-I ONOO TNF, IL-6, IL-12, IL-1β NO RNI, ROS iNOS n tio a z eri dim JAK TLR MH Proinflammatory cytokines NADPH oxidases Caspase-1 NF-κB | Review Activation of inflammasome Apoptosis IFN-γ TNF TCR C4+ T cells C8+ T cells Alternative activation Mannose receptor upregulation Scavenger receptor upregulation Ap opt osi Inhibition of inflammasome activation s Anti-inflammatory cytokines Arginase IL-10, AMAC-1 IL-4, IL-13 IL-10, IL-4, AMAC-1 NF-κB NO STAT6 Endoplasmic reticulum K JA JAK STAT3 iNOS T6 A ST Microparticle-mediated activation Scavenger receptor upregulation MHC-I Mannose receptor upregulation IL-4 IL-13 Ap Caspase independent opt osi 1β Pro-IL-1β Golgi bodies IL-1β O2– TNF, IL-12, IFN, IL-6, MIP-1 NF-κB p65 p50 Endoplasmic reticulum IκB p65 p50 AT ST MHC-I ONOO TNF, IL-6, IL-12, MIP-1α NO RNI, ROS iNOS n tio a z eri m i JAK d TLR TCR MHC-II upregulation Proinflammatory cytokines NADPH oxidases Caspase-1 Autophagy? s IL- Inflammasome activation Microparticles Humoral immunity IL-4Rα IL-13Rα MHC-II downregulation Arginase-1 Activation of inflammasome Apoptosis IFN-γ TNF Mycobacterium Microparticle Mycobacterium antigen C4+ T cells Therapeutic Delivery © Future Science Group (2011) future science group www.future-science.com 755 Review | Verma, Singh, Mohan, Agrawal & Misra Table 2. Molecular mediators in classical, alternative and microparticle-induced activation of macrophages. Classical activation Alternate activation Microparticle-induced activation Free radicals Inflammasome ↑ •NO, ↑ O2•, ↑ iNOS [136] Activation [47] IL-4, IL-13 [21,24] ↑ IL-10, ↑ IL-4, ↓ TNF, ↓ IL-6, ↓ IL-12 [64] ↑ AMAC-1 [131] ↓ MHC-II [135] , ↑ Mannose receptor [56] , ↑ CD86, ↓ CD18 [134] ↑ Arginase, ↓ •NO [30,60] Inhibition [51] Microparticles [14,201] ↑ TNF [3] , ↑ IL-6 [94] , ↑ IL-12 [16] Chemokines Marker molecules IFN-g [28] ↑ TNF [40] , ↑ IL-1b [47] , ↑ IL-6, ↑ IL-12 [46] ↑ MCP-1 [129] , ↑ MIP-1a [130] ↑ MHC-II [30] , ↑ CD86 [134] Key activation signal Cytokines or alternative activation phenotypes would be discernible in all the AM present in a single individual at any time. Instead there is evidence to indicate that in TB-infected mice, alternatively and classically activated AM are present in close proximity and in kinetically varying proportions at sites that are proximal or distal to granulomatous lesions [25] . „ Key Terms Th1 phenotype: Derived from the phenotype of T helper 1 lymphocytes that secrete signals (cytokines, chemokines, growth factors) favoring the development of inlammatory immune responses. These responses can kill intracellular pathogens or cancerous cells. Inlammasome: A multiprotein complex that is responsible for inlammatory diseases via activation of caspases. Th2 phenotype: deined by the set of cytokines/ chemokines/factors secreted by T helper 2 lymphocytes that promote the development of an immune response directed against ‘dangerous material’ in the systemic circulation. Phagosome maturation: Phagosomes steadily acidify and sequentially acquire markers of endosomes and lysosomes to kill engulfed microbe. 756 Classical bactericidal macrophage activation Classical activation of macrophages to combat foreign entities is extensively studied and well characterized [26] . Macrophage activation needs two steps: preliminary priming and a subsequent, secondary stimulus [27] . IFN-g and TNF are recognized as the foremost mediators of classical macrophage activation [28] . IFN-g produced by activated natural killer cells, macrophages, T helper cells and cytotoxic T cells, is usually the major stimulus which primes macrophage through the JAK-STAT pathway of transcriptional activation [29,30] . The second stimulus for classical activation is often provided by pathogen-associated molecular patterns or ‘dangerous material’ such as microbial lipopolysaccharide or peptidoglycans. Various surface receptors present on macrophages – Toll-like receptors (TLRs), mannose receptors, complement receptors, Fc receptors transduce the second signal. Stimulation of these receptors induces high levels of proinflammatory mediators such as Th1 cytokines, generation of reactive nitrogen intermediate (RNI) and reactive oxygen species (ROS) [31] . Classically activated macrophages with high levels of inflammatory mediators possess improved capacity of killing intracellular bacteria, such as Mtb [32] . With reference to TB, it is of interest to note that AM are a significant source of Th1 cytokines [33] and immunocompetent individuals may be expected to initiate their first encounter with Mtb by expressing some or all of these cytokines. Cytokine responses will be examined Therapeutic Delivery (2011) 2(6) ↑ MCP-1 [132] , ↑ MIP-1a [133] Unknown ↑ •NO, ↑ O2•, ↑ iNOS [102] Activation [137] in greater detail after the following sub-sections address other important components of classical activation. Effector molecules (ROS, RNI, host antimicrobial peptides) Reactive oxygen species and RNI generated as a consequence of phagocytosis are important components of the antimicrobial defense mechanism within macrophages or other phagocytic cells [34] . These are involved in direct killing of invading pathogens. Phagocytosis and internalization of Mtb into phagosomes induces macrophages to sharply increase their oxygen consumption. This phenomenon is called a respiratory burst, and is important in killing intracellular Mtb [35] . O2 acquired in the respiratory burst participates in a nonmitochondrial reaction in which it is reduced by an electron to superoxide radical (O2• ) through the NADPH oxidase enzyme complex [34] . O2• serves as precursor for highly reactive oxygen and halogen metabolites that are lethal to engulfed microbes. RNI are generated through downstream reactions with O2• following production of nitric oxide (NO), principally through the involvement of inducible NO synthetase (iNOS). Both NO and RNI are clearly mycobactericidal in mice [36] , but this activity is yet to be confirmed in human macrophages [37] . The mechanism of action of free radicals is related to applying oxidative stress on Mtb, which is a facultative anaerobe, and oxidizing key surface and cytosolic molecules of the bacillus. Several antimicrobial molecules produced by macrophages and other cells of the respiratory tract have been demonstrated to possess antimycobactericidal activity. These include host defense peptides suchas defensins a and b, cathelicidins, histatins hepcidin, collectins and other molecules such as lysozyme, complement, immunoglobulins, fibronectin, lactoferrin, transferrin, and so on [38] . These possess direct antimicrobial activity or facilitate the elimination of infectious pathogens by phagocytes [39] . future science group Inhaled therapies for TB Signaling molecules (cytokines, chemokines) Infected macrophages, and lymphocytes that interact with them, initially produce inflammatory cytokines that signal the presence of an intracellular pathogen. Th1 cells amplify phagocytemediated defense mechanisms against infection by secreting macrophage-activating cytokines such as IFN-g, TNF, IL-2, IL-12, and chemokines such as IL-18 [40] . These cytokines promote the ability of macrophages to phagocytose and destroy intracellular microbes. IFN-g and TNF are considered to play a central role in the activation of macrophages, with multiple effects on T-cell recruitment and phagocyte-mediated clearance of pathogens [41] . These cytokines potentiate the respiratory burst and NO generation [42] , activate professional phagocytes and upregulate MHC-II expression on surface phagocytic cells [43] . Aerosolized IFN-g as adjunct therapy in TB is under investigation [44] . TNF also plays a role as modulator of macrophage activation and contributes to the elimination of mycobacteria [45] . Another Th1 cytokine, IL-12 primarily produced by macrophages and dendritic cells, induces production of IFN-g and TNF which further enhance microbial killing [46] . Cell biological processes (inlammasome, autophagy, apoptosis) Recent evidence shows that stimulation of macrophages by cytokines such as IFN-g and TNF induces activation of inlammasomes [47] , autophagy [48] and apoptosis of the infected macrophage [49] . All three processes, by themselves or in coordination with each other, are extremely important in the killing and clearance of intracellular Mtb. Assembly of the inflammasome (a multiple protein complex) is linked to maturation of the Mtb-containing phagosome [50] , and is actively inhibited by a specific Mtb gene product [51] . If inflammasome assembly, activation and maturation proceeds as expected in classical activation of macrophages, IL-1b and caspase-1 are induced. Enhanced production of IL-1b directs the generation of ROS and RNI and secretion of proinflammatory cytokines, which results in a strong host response to the microorganism [47] . Autophagy is another phenomenon which plays an important role in the human innate defense mechanism in which a cell degrades its own intracellular compartments and removes unnecessary proteins and organelles by sequestering them into a vacuole which ultimately fuses with lysosomes and degrades [48] . Gutierrez et al. demonstrated that future science group | Review IFN-g induces autophagy in murine macrophages which is essential for antimicrobial action against Mtb [52] . In another study, Alonso et al. demonstrated mycobacterial killing by ubiquitin-peptide induced autophagy in infected macrophages [53] . Another potential mechanism involved in macrophage defense against mycobacterium is apoptosis. Host cell apoptosis is a last resort defense strategy to limit the growth of certain intracellular pathogens such as mycobacterium. It has been shown that this response sequesters bacilli within apoptotic cells and restricts their replication directly [54] , and is also thought to result in ‘appropriate’ presentation of Mtb antigens when such apoptotic bodies are picked up by bystander antigen-presenting cells (APCs). „ Alternative activation induced by bacteria A macrophage that has recently been exposed to IL-4 and IL-13 embarks on a program of activation that is markedly different from classical activation even if the stimuli provided are identical [21,24] . Consequently, alternatively activated macrophages are more susceptible to intracellular infections [55] . IL-4 [56] and IL-13 [57] directly induce alternative activation, while other Th2 cytokines such as IL-21, IL-25 and IL-33 further augment it [58,59] . Kaufmann and colleagues described critical disparity between transcription responses of alternatively versus classically activated macrophages. They demonstrated in mouse macrophages, that in contrast to macrophages activated ‘classically’ using IFN-g, alternatively activated macrophages (treated with IL-4) show differential regulation of genes relating to cell-surface expression of pathogen recognition receptors, production of cytokines and chemokines, proteins important for phagosome maturation and intracellular matrix remodeling, and so forth. Several key enzyme systems are also differentially regulated in alternatively activated macrophages. For instance, alternatively activated macrophages upregulate arginase activity which reduces NO production. It was concluded that alternatively activated macrophages support rather than inhibit growth of Mtb inside macrophages [60] . The Mtb-infected macrophage exhibits reduced generation of RNI and ROS, displays weaker antigen processing, decreased sensitivity to IFN-g and reduced secretion of proinflammatory cytokines [61,62] . Virulent Mtb also stimulates upregulation of the expression of anti-inflammatory cytokines, manipulates www.future-science.com 757 Review | Verma, Singh, Mohan, Agrawal & Misra signal transduction pathways and suppresses macrophage apoptosis [54] . In summary, Mtb can switch ‘classical activation’ of macrophages into ‘alternative activation’ in susceptible individuals. The ability of mycobacteria to persists in the host depends on evading or minimizing the induction of protective macrophages responses, as follows. Th2 rather than Th1 cytokines & anti-inlammatory chemokines Studies on immune responses and cytokine production by peripheral blood mononuclear cells isolated from TB patients illustrated elevated levels of IL-4 and reduced IFN-g [63] . Intracellular Mtb in macrophages downregulates proinflammatory modulators such as IL-12, TNF, IL-6 and so on and evokes antinflammatory cytokines such as IL-10, IL-13, IL-4 and IL-10 [64] . Furthermore, virulent strains seem to manipulate the activation of the eukaryotic superfamily of MAPKs to impair cytokine production or to stimulate anti-inflammatory response. Mtb inhibits IFN-signaling pathways in human macrophages and it directly or indirectly interrupts the association of STAT-1 with transcription co-activator CREB binding protein that are essential for the transcriptional response to IFN-g [29,30] . It is open to debate whether reactivation of latent TB is supported by a switch of Th1-polarized macrophages to the Th2 phenotype. Preferential antigen presentation on MHC class II rather than class I Mtb infection is intracellular in the strictest sense, but located in a unique, maturationarrested phagosome. It therefore typically presents an array of nonessential epitopes on MHC class II, for recognition by CD4 + T cells. Peptides derived from Mtb secretory proteins are loaded on MHC class I via proteasomal processing in the cytosol, but at much lower efficiency [65,66] . In susceptible individuals, therefore, recognition of Mtb-infected macrophages by CD4 + T cells predominates, typically leading to secretion of Th2 cytokines, which intensify alternative activation. Macrophages infected with Mtb, however, usually show a markedly reduced expression of MHC class II and hence decreased antigen presentation. Normally, expression of MHC class II on macrophages is unregulated by IFN-g in classical activation, but chronic TB subdues antigen presentation. Recent findings suggest that this may be related 758 Therapeutic Delivery (2011) 2(6) to attenuation of MHC gene transcription by a 19 kDa lipoprotein of Mtb, which could work by downregulating MHC class II synthesis, inhibiting vesicular trafficking or reducing antigen processing [67] . MHC class I-restricted CD8 + cytotoxic T lymphocytes play a crucial role against intracellular infections in general, and Mtb in particular [43,68–70] . Flynn demonstrated that mice lacking b-2-microglobulin (a component of MHC class I molecules) display greater vulnerability to TB, implying that MHC class I-restricted cells have a important function in protecting immunity against TB [43] . CD8 + T cells release IFN-g, lyse infected cells, and exert a direct antimicrobial activity through release of peptides, such as perforin and granulysin. Neutralization of free radicals & antimicrobial peptides Mtb produces antioxidant enzymes such as catalase-peroxidase systems and superoxide dismutase to scavenge or degrade ROS produced by macrophages as a defense mechanism. A variety of bacterial components are evolved to neutralize ROS, including sulfatides, lipoarabinomannan and PGL-1 [62,65,66] . Mtb-encoded methionine sulfoxide reductase confers resistance to the toxic effect of peroxynitrite [67] . Upregulation of arginase-1 which inhibits nitric oxide synthesis has been referred to earlier. Recent studies show that NoxR1 and NoxR3 expression confer resistance to the otherwise susceptible M. smegmatis [68,69] . Mtb-expressed peroxiredoxin alkyl hydroperoxide reductase subunit C (AhpC) protects bacteria from RNI toxicity [70] . Inlammasomes, phagosome maturation, autophagy & apoptosis Although numerous proinflammatory cytokines are initially secreted by Mtb-infected macrophages, IL-1b, an important multifunctional proinflammatory cytokine, is found at unexpectedly low levels. Generally, IL-1b requires proteolytic cleavage for its activation and processing which is carried by activation of inflammasomes (a caspase activating protein complex). Master et al. provided evidence that Mtb inhibits inflammasome assembly and activation and hence IL-1b processing. Several other reports indicate that Mtb infection induces assembly of inflammasomes, and macrophages infected in vitro with mycobacteria continue to release IL-1b, through an exocytosis pathway involving the pathogenencoded proteins of the ESX–ESAT system [71,72] . future science group Inhaled therapies for TB Furthermore, excessive inflammasome activation may be detrimental to the host, both in terms of immunopathology, as well as spread of disease [73] . Microparticle design aimed at modulating inflammasome assembly has not yet been reported in the public domain to our knowledge. Autophagy is also inhibited by Mtb, but the mechanism whereby this occurs is not yet known. Normally autophagy involves phagosome–lysosome fusion which depends on production of membrane lipids, phosphatidylinositol 3-phosphate (PI3P) [52] . Evidence indicates that Mtb inhibits PI3P to prevent autophagy [74] . Another mechanism by which Mtb evades autophagy is its translocation to cytoplasm from the phagosome using ESAT6 protein. Mtb infection is reported to prime human macrophages for TNF-mediated apoptosis. The virulent strains, however, induce significantly less apoptosis than avirulant or attenuated strains. Avirulant or attenuated strains of Mtb (H37Ra) induce apoptosis at even moderate multiplicities | Review of infection (MOI), while a closely-related virulent strain (H37Rv) inhibits apoptosis of host macrophages even at high MOI [54,75,76] . These observations suggest that apoptosis works as a host-defense mechanism which is suppressed by virulent mycobacterium [54] . Placido and colleagues found that using the virulent strain H37Rv, apoptosis was induced in a dosedependent fashion in BAL cells recovered from patients with TB, particularly in macrophages from HIV-infected patients [77] . Dual action of inhalable microparticles: drug delivery & macrophage activation Inhaled microparticles are recognized as alien by AM, which phagocytose them and initiate innate immune responses. Geiser et al. demonstrated an increase in numbers of airway/lung macrophages by approximately three times, immediately after inhalation of polystyrene microparticles by hamsters [78,79] . This was Table 3. Factors relevant to inhaled particles and their influence on macrophage activation. Factors affecting macrophage activation Outcome Particle-related factors 1–6 µm ↑ phagocytosis and macrophage activation >1–6 < ↓ phagocytosis and macrophage activation Rough surface, ↑ phagocytosis, ↑ inflammasome activation Smooth surface, ↓ phagocytosis Phagocytosis and macrophage activation: cationic > anionic > nonanionic† ↑ phagocytosis†, ↑ ROS inflammatory cytokines TNF, IL-1b, IL-6, IL-12 Phagocytosis and macrophage activation: opsonized particles > polysaccharide particles > polymeric > mineral† ↑ crystallinity, ↓ ROS ↑ phagocytosis and macrophage activation, ↑ ROS Size Surface topology Surface charge Surface coating/ opsonization Material of construction Crystallinity Hydrophobicity Cell-related factors Receptor interaction Cytoskeleton movement Macrophage status Ref. ↑ complement receptor ↑ mannose receptor ↑ scavenger receptor ↑ Fc receptor ↑ phagocytosis and macrophage activation Actin remodeling status Relaxed and thicker, ↓ phagocytosis Contracted and thinner, ↑ phagocytosis and activation Preclassically activated cell, ↑ phagocytosis and activation [12,14,138,139] [140] [12,141] [12,14,89,142] [14,139] [113] [12] [20,143–145] [146] [147] † These facts are not universal generalizations and not necessarily pertinent to all types of particles. Variations will be observed with composition of the particles, status of the macrophage and similar related conditions. ROS: Reactive oxygen species. future science group www.future-science.com 759 Review | Verma, Singh, Mohan, Agrawal & Misra attributed to chemotaxis following signaling by macrophages that had ingested the inhaled particles. The cell biological processes involved in microparticle-induced macrophage activation include interactions of the microparticle with the macrophage cell-membrane molecules, signal transduction down a variety of pathways and thus activation of gene transcription [80] . As the outcome of activation, macrophages release potent substances, proinflammatory mediators, growth factors and reactive oxygen or nitrogen intermediates and, failing all else, undergo programmed cell death. Cellular and molecular response after microparticle-induced macrophage activation depends upon the type of microparticles. Artusson et al. demonstrated that phagocytosis of polysaccharide microspheres induces macrophage activation: the production of cytokines and ROS, which are involved in host–defense against pathogenic micro-organisms [20] . Similar results were shown by Tabata and Ikada with polymeric microspheres [81] . Poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) microspheres have been extensively studied as polymers for encapsulation of a variety of drugs and vaccines for controlled delivery [1,2,7,11] . Prior and colleagues studied microparticles made of PLA and PLGA of different monomer ratios. Microparticle-induced macrophage activation as reflected in increased oxidative burst, correlated with increase in hydrophobicity of the polymer [14] . We too have observed that inhalable microparticles induce proinflammatory responses in macrophages [3,201] . Thus, in addition to selective drug delivery, the ingestion of microparticles may result in the activation of macrophages and, subsequently, enhance host–defense functions of the immune system. Terada et al., however, report that PLGA microparticles containing rifampicin do not induce responses such as TNF, NO, IL-10 and TGF-b1 in a rat macrophage line [2] . Induction of a proinflammatory macrophage response, however, has important implications for immunopathology. Generalized inflammation of lung and airway tissue as a consequence of inhaled material is unquestionably dangerous for the patient. Especially in the case of TB, a patient with already compromised lung function might be severely stressed if induction of inflammatory responses in lung macrophages progresses to full-blown inflammation. Thus, it is necessary to arrive at an optimal formulation that could work at or below a threshold of inducing proinflammatory responses; promoting innate 760 Therapeutic Delivery (2011) 2(6) bactericidal activity of infected macrophages, but not extensive inflammation involving mast cells or granulocytes and so on. „ Surface interaction & phagocytosis Phagocytic uptake of inhaled microparticles depends on particle geometry (size, shape, surface coating, hydrophilicity, stiffness), macrophage status (classical or alternative activation, synchronized cytoskeleton movements, phagosome position) and interaction of particles with macrophage membrane (nonspecifically by electrostatic contact or specifically by surface molecules such as the scavenger receptor, Fc receptor, complement receptor, mannose receptor, and so on (Table 3) . Some receptors also facilitate the phagocytosis of inert particles, for example scavenger receptors enhance uptake of polystyrene latex [82] , titanium dioxide, silica and iron oxide microparticles [83,84] and Fc receptors facilitate uptake of quartz particles [85] . Physical dimension-wise, there is significantly lower phagocytic uptake of large (>5 µm) polymer particles compared with particles of <5 µm of same aerodynamic diameter [13,86] . Coating the microparticle surface significantly influences the uptake of microparticles by macrophages. Stringer and colleagues demonstrated that a coating of surfactant protein over microparticles significantly enhances the uptake of polystyrene and TiO2 particles [87,88] whereas Evora and colleagues showed modestly decreased phagocytosis of 1,2-dipalmitoylphosphatidylcholine (surfactant phospholipid)-coated PLGA microparticles by rat lung macrophages [89] . Prior et al. demonstrated that phagocytosis was more efficient with increasing polymer hydrophobicity (PLA:PLGA [75:25] PLA:PLGA [50:50]) [14] . Disease is also an important factor for phagocytic uptake of inhaled particles. For example, Bennett et al. demonstrated improved particle uptake by macrophages in mild asthmatic conditions [90] . „ Release of cytokines, chemokines & growth factors Exposure of lung macrophages to microparticles can lead to the secretion of cytokine and chemokines which contribute to inflammation in lungs [91] . Among the important proinflammatory cytokines, TNF, IFN-g and IL-12 play an important role in the host-defense mechanism against TB. Due to the ability of TNF-a, IL-1b, and IL-6 to induce varied cellular events, these cytokines may play a role in inflammatory processes elicited by inhaled particulates. We future science group Inhaled therapies for TB demonstrated enhanced secretion of TNF by Mtb-infected macrophages when treated with microparticles [3,18,201] . Sharma et al. showed that TNF-a and IL-12 were significantly induced in response to treatment of macrophages infected with Mtb with inhalable microparticles in two experimental systems – cultured J774 cells and primary AM isolated from Swiss mice [3] . Yadav et al. demonstrated similar results with human monocyte-derived macrophages obtained by subjecting THP-1 cells to phorbol-induced differentiation, and also by peripheral blood monocytederived macrophages from five healthy volunteers. However, there was very wide variation in the extent of cytokine secretion by macrophages obtained from different individuals, and IL-12 upregulation was not observed in human macrophages [201] . An interesting observation that we consistently encounter is the simultaneous secretion of both Th1 and Th2 cytokines by murine, as well as human, macrophages infected with Mtb. Contrary to the well-known fact of crossregulation of Th1 and Th2 lymphocytes, macrophages evidently secrete both IL-10 and TNF/ IFN-g even under controlled culture conditions and using a clonal cell population. To some extent, inhalable microparticles apparently induce and sustain higher secretion of Th1 cytokines by infected macrophages in comparison to drugs in solution [3,92,201] . Some research groups have shown that micronsized asbestos and silica particles augment the release of TNF from macrophages in vitro and in vivo but IL-1b and IL-6 release was inconsistent [93,94] . Lemaire and co-workers demonstrated an enhanced release of IL-1b and IL-6 by lung macrophages upon exposure to asbestos particles [95,96] whereas Driscoll and colleagues did not observe augmentation of either cytokine [97] . Howard et al. showed that cells exposed to micron-sized mineral particles released chemokines such as MIP-la or MIP-2 which are monocyte and macrophage chemoattractants and can activate the NF-kB transcription factor [98] . The release of proinflammatory cytokines results in an array of events including recruitment of other inflammatory cells, stimulation of cell activation and proliferation and eicosanoid biosynthesis [97] . All of these are extremely important in macrophage activation and the elimination of intracellular pathogens. However, at this time, there is no clear consensus on the nature of cytokine and chemokine responses that can be evoked from Mtb-infected AM as a result of exposure to microparticles of diverse nature. future science group | Review „ Oxidative burst-generation RNI & ROS Upon phagocytosis, microparticles induce macrophages to generate ROS by stimulating NADPH oxidase, responsible for the production of superoxide anions. Prior et al. [14] and Sharma et al. [3] have demonstrated generation of oxidative radicals in response to biodegradable polyester microparticles, and numerous others report that AM release large amounts of superoxide anions upon exposure to micron-sized particulates such as silica [19] or asbestos [99] . Kagan and colleagues illustrated that exposure to micron-sized mineral particles such as crocidolite and chrysotile stimulates iNOS, which in turn generates NO and RNI, both in vivo and in vitro [100] . Primary macrophages exposed to inhalable microparticles containing isoniazid and rifabutin, upregulate NO production, regardless of whether or not they are infected with Mtb in vitro. Infection does, however, delay the peak NO response by approximately 9 h. Drug-containing microparticles also induced significant O2• production by infected macrophages in the same period [3] . As in the case of proinflammatory cytokines, enhanced production and release of ROS and RNI by AM upon exposure to various particulates may have both positive and negative effects. It may cause lung tissue damage, or damage the pathogen, or both. Indirectly elevated ROS and RNI activate signal-transduction pathways via kinases and transcriptions factors, processes that result in a complex cascade of events that may contribute to the eradication of pulmonary infection. „ Signal-transduction cascades Microparticles stimulate macrophages and activate signal-transduction cascades, but the specific receptors and pathways are inadequately understood. Microparticulates including biodegradable particles such as PLGA, polystyrene microparticles and inorganic micron-sized particles such as silica, asbestos, iron oxide, alum, monosodium urate, and so on, significantly augment the secretion of IL-1b and activation of caspase-1 in phagocytic cells [17,19,201] . The body of evidence suggests that this phenomenon is mediated by activation of inflammasomes (via lysosomal damage) by microparticles [101] . There are evidences that stimulation of macrophages with micronsized monosodium urate particles induces activation of TLR2 and TLR4 [102] , induces MyD88dependent IL-1 receptor signaling [102,103] , directs the activation of MAPK pathway (ERK1/2) [104] and chemokine secretion [105] . Several studies illustrate that mineral microparticles (asbestos, www.future-science.com 761 Review | Verma, Singh, Mohan, Agrawal & Misra quartz) enhanced the intracellular calcium concentration of macrophage in a dose-dependent manner, which in turn mediates a number of calcium-dependent cellular events that may affect cellular physiological functions including activation of tyrosine kinases, protein kinase and phospholipase [19,103] . Several studies illustrate that enhanced gene expression of inflammatory mediators upon exposure of particles is associated with an increased activity of transcription factors NF-kB and AP-1 [106] . „ Apoptosis Inhalable microparticles containing standard antituberculosis drugs, enhance both caspase-8 and -9-dependent apoptosis, while blank polymer microparticles (without drug) induced apoptosis through a caspase-independent pathway [17] . Crystalline mineral microparticles (mylonite, gabbro, basalt, feldspar, quart and hornfels) induce similar extents of macrophage apoptosis [107] . Kang et al. demonstrated that PLA–PLGA microparticles prepared by supercritical fluid technology were able to induce apoptosis in a lung cancer cell line A549 [108] , while Powell and colleagues demonstrated that dietary calcium phosphate and titanium oxide microparticles enhance apoptosis of intestinal macrophages [109] . As argued earlier, apoptosis is a last-resort response of the AM to Mtb infection. Apoptosis of the infected AM denies the pathogen its sanctuary, and also packages it in apoptotic bodies for APC uptake through pathways that a naked bacillus would not employ. It is speculated that processing and antigen presentation of Mtb taken up in an apoptotic body would result in presentation of appropriate, protective epitopes, rather than establishment of the pathogen in a maturation-arrested phagosome [201] . Conclusion Pathogenic strains of Mtb induce alternative activation of AM that they colonize, in order to create conditions that promote the establishment and progression of infection. A growing body of evidence suggests that such macrophages may be rescued from alternative activation by inhalable microparticles containing a variety of drugs. Biodegradable microparticles can be designed to mimic the surface properties of pathogens so that they can be easily phagocytized by AM. Besides providing efficient drug targeting and high chemotherapeutic potential, inhalable polymeric particles offer an additional potential to enlist the innate responses of the macrophage towards 762 Therapeutic Delivery (2011) 2(6) clearing the infection. Inhaled microparticles have exhibited the potential to reprogram the activation status of TB-infected macrophages [201] . Other researchers have reported observations of activation of a number of classical macrophageactivation pathways as a consequence of exposure to microparticles [14] . Our own findings point to restoration of a respiratory burst and upregulation of ROS and RNI through the phagocyte oxidase and NOS enzyme systems; induction of proinflammatory macrophage cytokines; and finally induction of apoptosis rather than necrosis of the infected macrophage [3,17,18,201] . There is evidence that particle-induced activation of macrophages depends upon particle size, surface properties and chemical composition of the particles. In an early study, Atwood et al. showed that carbon particles phagocytosed by Mycobacterium-infected macrophages resulted in a decrease of superoxide and hydrogen peroxide production and hence reduced killing of M. bovis by AM [110] . By contrast, our results illustrate that PLA microparticles enhance superoxide release and enhance in vitro killing of intracellular Mtb [3,201] . Prior et al. have indicated that physicochemical properties of the polymers used to prepare polyester microparticles had differential effects on macrophage uptake and activation [14] . By contrast, Terada et al. showed that molecular weight and monomer ratio of PLGA did not influence the phagocytosis activity [15] . Upon stimulation or during phagocytosis, monocytes produce reactive oxygen metabolites and other oxygen-independent products [111] . Terada and colleagues investigated the effect of mycobacterial infection on phagocytic activity of infected macrophages towards polymeric microparticles. Results showed that phagocytic ability of the macrophages is enhanced 1.3–1.5 times compared with uninfected macrophages up to 24 h [112] . This is an extremely encouraging finding in light of the suspicion that Mtbinfected AMs may lose their phagocytic ability. Biggs and colleagues investigated the cellular activation by PLA microparticle in vitro (in the rat AM cell line NR8383) and in vivo (by pulmonary delivery to guinea pigs) in terms of ROS and RNI production, and ascertained that as microparticle crystallinity increases, the activation level of the macrophage cells decreases [113] . This result suggests that immunomodulatory activity of the microparticles can be tailored by choice of polymer. Finally, inhaled particles containing anti-TB agents demonstrate surprisingly high efficacy against experimental animal TB [7,202] . This level future science group Inhaled therapies for TB of efficacy is not satisfactorily explained by arguments suggesting that high intra-cytosolic drug concentrations are sufficient to kill this recalcitrant pathogen. The evidence of macrophage activation offers room to formulate an alternative view: that of enlisting innate macrophage responses against Mtb infection. Given that a very small proportion of humans exposed to Mtb infection progress to active disease, it is likely that most of us are capable of eliminating or containing the infection through innate (macrophage) and acquired (lymphocyte) immune responses. It is also likely that augmentation of host macrophage responses may assist in clearing intracellular Mtb. It is, therefore, suggested that there is scope to co-opt host responses in the management of TB through the use of appropriately formulated inhalable microparticles. Future perspective There is every likelihood that the possibility of pharmacological intervention in the hostpathogen dialectic in TB will gain notice of the research community in the near future. Pulmonary delivery of the causative agent is a familiar technique for laboratory researchers addressing TB. Sufficient expertise and motivation is therefore available to investigate whether pharmacological or immunomodulatory agents | Review can play a role in managing experimental animal TB. Also, several groups have reached an advanced stage of preclinical testing of inhaled formulations prior to conducting clinical trials. The next 5 years should see publication of clinical data on the safety and efficacy of inhaled therapies for TB. It would be doubly exciting to see whether speculations on host macrophage activation can be borne out by clinical findings, (and the present authors are of the view that they definitely will), if only clinical studies also address host AM parameters in human subjects. Finally, a novel body of literature is likely to emerge on the use of agents that possess macrophage-activating activity for pulmonary delivery in TB. Financial & competing interests disclosure Work in our laboratory is funded by CSIR NWP0035. RK Verma, AK Singh and AK Agrawal acknowledge Research Fellowships from CSIR, M Mohan from ICMR. This is CDRI Communication Number 8082. All authors are employed by the Council of Scientific and Industrial Research, co assignee of patent US20070154408. The authors have no other 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 apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Executive summary „ Mycobacterium tuberculosis induces alternative activation of macrophages that it colonizes. „ Phagocytosis of particles is a signal for classical macrophage activation. „ „ „ „ „ „ Inhaled particles induce markers of classical activation in Mtb-infected macrophages, and possess extraordinarily high efficacy against TB in mice and guinea pigs. The physicochemical and pharmaceutical properties of inhalable particles strongly influence their ability to activate macrophages, and it is possible to engineer particles of ‘desired’ macrophage-activating capability. In general, hydrophobic, amorphous, multicomponent particles evoke higher levels of macrophage activation. There is no clear consensus on what degree of macrophage activation is necessary or sufficient to kill intracellular Mtb without inducing immunopathology. Nor is there sufficient understanding of inter-species or -individual differences in the amounts and kinetics of effector or signaling molecules that are produced following macrophage activation. Nevertheless, there is scope to co-opt innate host immune responses to eliminate Mtb from alveolar macrophages. Bibliography 2 Papers of special note have been highlighted as: „ of interest „„ of considerable interest 1 Muttil P, Kaur J, Kumar K, Yadav AB, Sharma R, Misra A. Inhalable microparticles containing large payload of antituberculosis drugs. Eur. J. Pharm. Sci. 32(2), 140–150 (2007). future science group „ Hirota K, Hasegawa T, Nakajima T et al. 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