Macrophages are the special cells of the immune system and play both immunological and physiological role. One of the peculiar characteristics of macrophages is that they are double-edged and highly plastic component of immune system. Due to this characteristic, they are responsible for both progressions as well control of a variety of inflammatory, infectious and metabolic diseases and cancer. These are found in the body in three major phenotypes, which are known as M0 (also known as naïve); M1 (classically activated macrophages); and/or M2 (alternatively activated macrophages) at normal physiological conditions. We have been exploring macrophages in context of bacterial infection and previously demonstrated that M2 polarization of M1 effector alveolar macrophages during chronic/persistent Chlamydia pneumonia, Mycobacterium tuberculosis and Helicobacter pylori pathogens are decisive for the infection induced cancer development in host. Since chronic infection with these pathogens has been associated with adenocarcinoma, therefore, we feel that disruption of macrophage plasticity plays crucial role in the host for the development of cancer. On the basis of this, we propose that in such pathological conditions, management of M1/M2 imbalance is paramount for minimizing the risk of developing cancer by chronic and persistent infection.
- metabolic programming
- sterile inflammation
Recent studies have demonstrated that macrophages display high grade of phenotypic plasticity due to which they can both enhance and inhibit immune response. This phenotypical plasticity of macrophages enables them to contribute to pathogenesis of large variety of diseases as well as homeostasis mechanisms. Due to this characteristic, these cells are now known as double-edge component of immunity as well. Many studies have demonstrated that these cells can enhance the progression as well as control many infectious and tumor  diseases. Both peripheral and tissue macrophages together constitute the reticuloendothelium system which plays a major role in both sensing microbial antigens and their subsequent eradication . Macrophages are recruited to the inflamed/infected tissues, react to a variety of stimuli, and acquire either classical phenotype also known as M1 or alternative phenotype also known as (AAM, M2). Classically activated macrophages are immunostimulatory in nature and have Th1-orienting capacity while M2 are immunoregulatory in nature and have Th2 programming capacity . The latter ones are anticipated to support the survival of various intracellular pathogens during persistency and believed to promote neoplastic transformation of infected tissue micromilieu (Figure 1). AAM accumulation in majority of adenocarcinoma (around 10% cases) confers poor prognosis during microbial persistency. Therefore in such abnormal pathological conditions, selective elimination of macrophages by ablating colony-stimulating factor 1(CSF-1) in LySMcre and op/op mouse model  or by the use of pharmacological drugs such as clodronate liposomes , which are among few possible modalities for mitigating macrophage-associated neoplasia. Within the frame of the above mentioned, this chapter will discuss various strategies to repolarize tumor-associated macrophages (TAM) during cancer development and uncover how selective activation of M1 macrophages could control infection-induced cancer but also existing anti-tumor immune therapies in both mouse and human model of tumors with special emphasis on gastric and lung tumors and inflammatory diseases like inflammatory bowel disease (IBD), which are responsible for global mortality. This may be achieved by targeting the major intracellular signaling component such as sphingolipids and Th2/Th17 responses, which promote M2 phenotype during persistent infection and potentially involve in the development of cancer.
2. Pathogens disrupt macrophage plasticity and effector response during persistency
Recent study has demonstrated that a bacterial product known as trabectedin is toxic to macrophages. This product inhibits NF-Y and KLF-2/4 which is important for the differentiation of macrophages in tumor micromilieu . Similarly mitigating NF-κB, STAT3 and HIF-1 are involved in the activation of naïve macrophage to M1 effector phenotype and hold tremendous therapeutic option for modulating macrophages activation. Histopathological analysis of persistently infected lungs reveals the infiltration by specialized macrophage known as foamy macrophages. These are lipid-loaded macrophages and quite refractory in their nature. These macrophages behave more like AAM and are actively involved in the clearance of cellular debris and dead bacteria containing neutrophils and DC . In some cases of coronary atheroma patients, these macrophages acquire phenotype similar to TAM (tumor-associated macrophages) and harbor dead bacteria in their endosome . The presence of these macrophages thus promotes non-immunogenic inflammation which is similar to cancer-associated inflammation and supports opportunistic survival of deadly pathogens. Both phenotypic and functional polarizations of M1/M2 effector phenotype of macrophages are believed to be one of the prognostic factors contributing to the development of tumor during persistent/latent infections (Figure 1) in host. Once infiltrated in the infected lungs, these AAM/foamy macrophages potentially modify effector T cells and predispose them also as refractory which are otherwise proficient in the killing of infected cells. These macrophages secrete a plethora of cytokines/growth factors like VEGF-β, TGF-β, hypoxia-inducible factor, and sphingolipids which altogether contribute to neoplastic transformation of infected tissue. High gradient of VEGF and TGF-β promotes the differentiation of regulatory T cells  and inhibits the effector response of CD8+ T cells . On the other hand, sphingolipids particularly S-1P/ceramide (either host or pathogen-derived) are known to promote mitophagy , M2 polarization of infiltrating M1, or naïve monocyte/macrophage populations . In view of this, and to restore Th1 effector immune response during latent infection, reactivation of M1 effector phenotype of macrophage thus represents the most suitable therapeutic interventions. Apart from this, modulating the cytokine network also seems to be the most effective strategy for boosting immunity for the management of latent/persistent infections.
3. Bacterial persistency hijacks programmed cell death and autophagy and promotes immune metabolic reprogramming
Pathogenic bacteria have evolved several ways to survive efficiently in the phagocytes during their dissemination across the lymphatic system. Various pathogens adapt various strategies to this purpose which range from conferring resistance to the apoptosis , immune evasion , and metabolic programming of myeloid cells  as shown in Figure 2. Of these, conferring resistance and insensitivity for cell death in the infected cell seems to be one of the most fundamental processes. A range of bacterial pathogens like
4. Human pathogens promote epigenetic changes in macrophages during persistency
At genome level,
5. Potential interventions for reactivating refractory macrophages for therapy outcome
While the application of antibiotics is sufficient to control acute infection however during persistent infection, the outcome of treatment mostly remains refractory. This is due to increased density of refractory macrophages in various affected tissues which resists many therapies as seen in many similar diseases like cancer and metabolic disease which is mediated with tissue accumulation of type 2 or tumor-associated macrophages. It is now well accepted by medical community that increased densities of these macrophages are associated with poor prognosis in many infectious, tumor, and metabolic disease. In such conditions antibiotics and/or chemotherapy would require an additional regimen for effective treatment. During past decades, the growing evidence suggested that TAMs clearly play an important role in tumor progression, metastasis, and resistance to available chemotherapies by modulating the microenvironment inside the tumor mass as well as in the stoma. Therefore, it is important to reeducate or target the TAMs (M2-like) to antitumor M1-like macrophage phenotype for successful treatment of several human malignancies. In the remaining sections of this chapter, we have discussed various macrophage-specific and nonspecific interventions for reactivating refractory population of macrophages for improving existing therapies.
5.1 Neoadjuvant for retuning refractory macrophages
Many interventions have been made to reactivate or retune the TAM, but most of them could not influence the disease outcome profoundly. In this context our recent studies have shown neoadjuvant impact of low-dose radiation for retuning TAM, T cell-aided therapy , and subsequent normalization of vasculature in solid tumor-bearing animals. Since infection induced adenocarcinoma is manifested with high grade infiltration of foamy macrophages, which are like M2 TAM, therefore, on the basis of our tumor studies, we propose low dose gamma irradiation as one of the non-specific therapeutic interventions for the management of persistent infection-induced tumor development.
5.2 Nanomedicine as immune adjuvant for refractory macrophages
Nanomedicine has emerged as one of the new modalities for reprogramming of both naïve as well as refractory macrophages toward their effector phenotype and thus represents one potential intervention for the management of latent infectious disease. We and others have recently demonstrated that due to their size and unspecific adjuvant properties, nanocarriers/nanocapsules can penetrate inflamed tissue microenvironment effectively and deliver drug in controlled and sustained rate for exerting adjuvant actions on macrophages in the inflamed and fibrotic lesions of infected tissue. Nanomedicine-based approaches may impact refractory macrophages at various levels, namely, (i) enhanced infiltration of fresh monocyte/macrophages, (ii) direct killing, and (iii) in situ polarization of AAM/foamy-like macrophages during chronic infection to assist clearing of infection. One of the interesting mechanisms by which nanoparticle may improve the therapy outcome is to control the differentiation of naive monocytes toward iNOS+ M1 effector macrophages and replace CD11b+/iNOS-/Arginase-1+ AAM during chronic infections. In this context, our recent work has shown that a certain biodegradable amino acid-based pNAPA nanocapsule can potentially stimulate naïve macrophage to the M1 effector phenotype. On the basis of these merits, the nanocapsules may be used as adjuvant for activating innate immune system for the management of infectious diseases and cancer. Another potential application of nanoparticles is to deliver drugs or biopharmaceuticals for preventing differentiation of effector phenotype of macrophage to refectory. In this context one study has shown that delivery of CCR2 and CCR5 siRNA-loaded nanoparticles was able to reduce the recruitment of monocytes to inflamed tissue . Nanocarrier-based approaches can be used for the direct killing of the refractory macrophages as well. For instance, liposomal formulations have been developed for the delivery of bisphosphonates such as zoledronates and clodronates. Subcutaneous/orthotropic injections of these nanocarriers result in the depletion of AAM accompanied with impaired angiogenesis and reduction in metastasis. Nonspecific targeting is the major issue with nanocarriers which can be addressed by tagging these nanocapsules with specific ligands such as LyP1 and mannose receptors (e.g., CD206) which are highly expressed by TAM/AAM  for effective targeting of macrophages. PLA-PEG nanoparticles, cyclodextrin nanoparticles, and liposomal formulations have been developed for loading drug cargoes such as sunitinib, IL-12 plasmids, TGF-β inhibitors, and VEGF siRNA for reprogramming of refractory macrophages for skewing in situ Th1 effector immune response against latent infections [51, 52, 53, 54].
5.3 Immunotherapeutics are the next-generation treatment modalities
One of the key characteristics of both AAM and TAM is to restrict Th1 immune response/T-cell programming by virtue of their releasing of Th2 cytokines and growth factors, which stimulate the neoplastic differentiation of inflamed fibroblast in tissue . One of the major mechanisms by which these cells limit effector T-cell response is to engage programmed cell death ligands 1 and 2 (PD-L1, PD-L2)  which are expressed by the AAM/TAMs. Pulmonary infiltration of lipid rich foamy macrophages is a typical evidence of persistent infection-induced non-immunogenic/sterile inflammatory immune response during persistent/latent
5.4 Antibody/small molecule inhibitor targeting polarization of refractory macrophages
Intracellular pathogens, during both acute and latent infection, fiddle with various signaling pathways which range from receptor-associated cell death and innate immune signaling, antigen presentation, vesicular transport, and phagocytosis pathways. Although we have disused these in earlier section, here we will discuss the pharmacological and clinical significance of various approaches which may be decisive for mitigating cellular perturbations in the host for restoring immune defenses of macrophage during persistency. In this context, our recent study  has proposed that Smac mimetic (IAP-specific inhibitors)-based strategy has potential for enhancing immunogenic cell death of infected cells and reactivating refractory macrophages for improved clearance. Due to these virtues, several Smac mimetics have entered in the second-phase clinical trial against cancer, and we anticipate that the same is expected to help immune system for the management of persistent bacterial infection as well. Other than this, many pathogens exploit MAPK pathways  for their benefits and induce production of IL-10 cytokines in the macrophages which further inhibits T-cell programming mainly by promoting T-cell exhaustion . Other than this, elevated levels of p38MAPK promote sterile/anti-inflammatory response, which supports opportunistic survival of pathogen inside macrophages. Likewise, many pathogens exploit cAMP/PKA pathways and acquire Th2 bias during their persistency  for securing their survival; TNF-α is a major and key cytokine responsible for receptor-mediated killing of infected cells. We (unpublished data) and others have shown that many intracellular pathogens, during persistency, potentially target this cytokine and inactivate cell death pathways in TACE- or ADAM-dependent manner. Pathogens like
5.5 Future perspectives: macrophage-based palliative strategies for tissue homeostasis post-antibiotic purging
Successful therapy post-antibiotic treatment infection should normalize the tissue microenvironment and restore homeostasis. This could be achieved by chelating oxidative stress and remnants of inflammatory response for the replenishment of tissue mass, which normally gets lost during various therapeutic procedures. Management of M1/M2 imbalance is believed to be the key for minimizing the risk of having cancer by chronic and persistent infection with intracellular pathogens. In the clinics, this can be achieved by exchanging refractory populations of macrophage with effector ones which can control the sterile reactions and tumorigenesis. However, due to the pro-inflammatory nature of iNOS+ effector macrophages, this may elicit another sequence of destruction, which alone may not be beneficial. Therefore in such delicate conditions, co-administration of M1 macrophage with mesenchymal stem cell regenerative approach seems to be optimum for reconstituting the affected tissues and organs. The potential inclusion of macrophage-mesenchymal cell-based therapeutic intervention could be categorized under prospective palliative therapies for restoration of physiological function post-treatment.
Since chronic infection with bacterial pathogens has been associated with adenocarcinoma, therefore, we believe that the management of M1/M2 imbalance is paramount for minimizing the risk of developing cancer by chronic and persistent infection of the lung, stomach, and cervix. This may be achieved by targeting major signaling pathways such as sphingolipids and Th2/Th17 responses which drive M2 phenotype and which are potentially involved in the development of cancer. In the light of the above, we propose that selective activation of M1 macrophages could improve existing antitumor immune therapies in both mouse and human models of tumors with special emphasis on gastric and lung tumors and inflammatory diseases like inflammatory bowel disease (IBD) which are responsible for global mortality.
S.K. acknowledges the grants from SERB-Department of Science and Technology (Project ECR/001173/2016) and Department of Biotechnology (Project BT/PR18562/BIC/101/424/2016), Government of India. This work was supported by Department of Biotechnology (DBT), under its Ramalingaswami Fellowship (BT/RLF/Re-entry/24/2014) and SERB-ECRA scheme (SERB File No. ECR/2016/001519) award to Dr. Manoj Garg.
Conflict of interest
The authors have no competitive/financial interest.
Acronyms and abbreviations
mitogen-activated protein kinase MAPK kinase MAPKK kinase nuclear factor κB signal transducer and activator of transcription toll-like receptor tumor necrosis factor-α
mitogen-activated protein kinase
nuclear factor κB
signal transducer and activator of transcription
tumor necrosis factor-α