Macrophage Polarization Is Decisive for Chronic Bacterial Infection-Induced Carcinogenesis

1. Introduction

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 [1] 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 [2]. 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 [3]. 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 [4] or by the use of pharmacological drugs such as clodronate liposomes [5], 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 [6]. 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 [7]. In some cases of coronary atheroma patients, these macrophages acquire phenotype similar to TAM (tumor-associated macrophages) and harbor dead bacteria in their endosome [8]. 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 [9] and inhibits the effector response of CD8+ T cells [10]. On the other hand, sphingolipids particularly S-1P/ceramide (either host or pathogen-derived) are known to promote mitophagy [11], M2 polarization of infiltrating M1, or naïve monocyte/macrophage populations [12]. 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 [13], immune evasion [14], and metabolic programming of myeloid cells [15] 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 Chlamydia trachomatis(C. tr), Chlamydia pneumonia(C. pn), and Helicobacter pylori(H. pylori) which are associated with the pathogenesis of lung [16] and stomach cancer [17] respectively, exploit death and immune signaling for surviving in the hostile environment of antigen presenting (Figure 2) and effector cells. We [18, 19] and others [20] have demonstrated that C. pnand C. trincrease the stability of various endogenous regulators of apoptosis proteins called IAP and inhibit the activity of both apoptotic and inflammatory Caspases 3, 8, and 11 during latent infection. Upregulation of CIAP2 and XIAP proteins [19] during acute and persistent/latent infection has shown the increase in noncanonical signaling of NF-κB which is a master transcription factor involved in both cell death inhibition and inflammatory programming and autophagy for Th1 effector response during infection. Our recent study has shown that C. pneumoniapotentially interfere with M1 programming of infected macrophages [21] when stimulated with their cognate innate and inflammatory stimuli which is due to increased expression of HIF-1 and p38MAPK proteins [22] which are known to promote unfolded proteins response (UPR) in the infected macrophages [23] which in turn predispose macrophages refractory to immune stimulation. Within macrophages, mitochondria is potentially involved in the innate immune response of macrophages against a variety of successful intracellular pathogens mainly by flushing catatonic peptides like LL37, CAP 12, and CAP 18 to cytoplasmic compartment for efficient capture and killing of pathogen in mature phagolysosomes [24]. Recent studies have amply demonstrated that most of the opportunistic pathogens interfere in the mitochondrial physiology by promoting mitophagy which jeopardizes innate immune defense of macrophages against pathogens. In such cases, tweaking mitochondria by using Smac mimetic-based interventions holds promises in the management of persistent infection. Although we have recently demonstrated that Smac mimicry [21] is capable of mounting an efficient immune response against mild pathogens, it fails to do so against pathogenic microbes like Leishmania donovani. Since pathogenic microbes enhance the expression of p38MAPK/HIF-1 pathways [25] for sabotaging macrophages functions, therefore we feel that at the moment targeting p38MAPK/HIF-1 in conjunction to Smac mimetics would be paramount for controlling pathogenic microbes. This is a quite intriguing aspect of the field and needs further in-depth investigation. In the same line, many pathogens are known to interfere with autophagy process which is yet another potential mechanism which influences antigen presentation by APC to T cells for the clearance of pathogens. Many pathogens attack mTORc1 complex and disrupt the autophagosome apparatus [26, 27] and inhibit their presentation by APC to T cells for immune surveillance during latent phase. Therefore pharmacological tweaking of autophagy may offer one potential strategy for clearing dead pathogens effectively from the lung tissue. Irrespective of acute or persistent phase, all pathogens utilize host metabolism for their survival inside the host. During persistency, many pathogens consume carbohydrate and protein reservoir of infected macrophages and alter their metabolic rates. l-Tryptophan [28] and glucose are critical for macrophages, and their fluctuation largely dictates the effector response of infected macrophages as well as the fate of pathogen in APC/phagocytes. During persistent infection, Chlamydiasp. utilize cellular depot of l-tryptophan amino acid by activating IDO gene and metabolize it to l-kynurenine which impedes glycolysis [29] in the macrophages. M1 effector macrophages rely on cellular depot of glucose for both activation and differentiation into effector phenotype; therefore, during latent infection, pathogens disrupt in the glucose metabolism and promote hypoglycemia rendering them refractory for optimum defense against pathogens.

4. Human pathogens promote epigenetic changes in macrophages during persistency

At genome level, C. trinfection causes global hypoacetylation and hypermethylation of lysine residues on core histones which alter histone post-translational modifications which differ between acute and persistent infections. Upregulation of pH2AX (Ser139) and H3K9me3 which are hallmarks of DNA double-strand breaks (DSBs) and senescence-associated heterochromatin foci (SAHF), respectively, during Chlamydophila trachomatis[30] infection suggested teratogenic manifestation of Chlamydiapersistency. This is largely due to increasing levels of reactive oxygen species (ROS) which is produced during latent infection. ROS contribute to DNA double-stranded breaks leading to persistent DNA damage, which in turn triggers SAHF formation in an ERK-dependent manner [30]. CPAF and CADD proteins from Chlamydophilapathogens are known to perturb host cell cycle machinery and inhibit recruitment of the DNA damage response proteins pATM and 53BP1 to damaged sites interfering with DNA repair mechanisms [30]. Despite impaired DNA repair, infected cells continue to proliferate which are in turn supported by enhanced oncogenic signals such as ERK, CyclinE, and SAHF. These changes altogether lead to the malignant transformation of infected tissue. Similarly, other pathogens like Campylobacter rectus[31], which is associated with oral cancer, downregulate Igf2 gene and enhance DNA methylation at its promoter which can be attributed to bacteria-mediated epigenetic modifications to the host genome. Other pathogens like Salmonella entericaserovar Typhi, which is one of the prognostic factors for the susceptibility for gallbladder carcinoma, exploit MAPK and AKT pathways [32] which initiate and sustain neoplastic transformation of infected host. Macrophages sense and trigger immune response against pathogens via TLR-linked signaling cascade. Under normal circumstance, almost all pathogens trigger TLR signaling pathways for activating macrophages; however, only few obligate intracellular pathogens, in hitherto, interfere with TLR signaling directly or indirectly and limit defense mechanisms [33] of effector macrophage like pattern of cytokines secretions, their uptake, and phagocytosis by macrophages. Although there are multiple ways how a pathogen can interfere with TLR signaling, so far TLR2/4 triggered hypoxia and associated sterile inflammatory response, and/or TLR masking/shedding mechanisms have been identified and proposed [34, 35]. Yersinia enterocoliticaand Candida albicansare known to induce immunosuppression through TLR2-mediated IL-10 release and differentiation of T-helper cells to CD4+CD25+ regulatory T cells [36]. Yersiniaspecies secrete a virulence (V) antigen, LcrV, which binds to CD14 and TLR2, trigger IL-10 secretion, and mediate immunosuppression. It has recently been shown that a particular residue in the N-terminal region of LcrV targets TLR2 and is required for altering IL-10 induction via TLR2 [37]. Likewise, H. pyloriescapes from recognition by the TLRs due to the removal of phosphate groups from the 1′ to 4′ positions of lipid A in LPS, which confers low negative charge to this molecule and increases the chance of escaping TLR recognition. The recognition of non-LPS ligands by TLR2 leads to anti-inflammatory responses that are associated with IL-10 production [38]. Flagellin of H. pyloriis one of the PAMP which potentially modifies the N-terminal recognition domain of TLR5 and helps in escaping the innate immune responses. Manipulation of amino acid 89–96 of the recognition domain of TLR5 results in low affinity to flagellin binding [39]. Under recurrent/latent infection state, TLR2-mediated signaling, hitherto, inhibits IFN-γ response and hijacks Th1 programming of macrophages. A pathogen like M. aviuminhibits IFN-γ signaling in TLR2-dependent manner where it enhances the expression of dominant-negative STAT1b. Similarly, 19KD protein of Mycobacterium tuberculosisinhibits IFN-γ-induced expression of HLA-DR and FcγR1 expression on human macrophages [40]. In addition to the induction of anti-inflammatory signals by TLRs, certain pathogenic microbes have developed strategies to either block or avoid their recognition by TLRs and subsequent activation of the innate defense. According to one recent study, phospholipids and Ypk protein of Treponema palliduminterfere in TLRs (TLR3, TLR4, and TLR9) signaling [41] by blocking the function of LPS-binding protein and CD14. Several bacterial pathogens have altered specific PAMP structures to circumvent recognition by TLR4 or TLR5; pathogens, such as Porphyromonas gingivalisor Leptospira, which have specialized LPS structures that only interact with TLR2; likewise, in Helicobacter pylori, the flagellin [39, 42] is not appropriately recognized by TLR5, approving the survival of the bacteria without loss of virulence. Virulent strains of Salmonella typhiescape from their recognition by host PRR by various mechanisms, which predominately include modifying their lipid A by various mechanisms including deacylation, palmitoylation, and the addition of aminoarabinose [43]. Pathogens have evolved in several ways of avoiding NO-mediated killing that plays a central role in effector response in phagocytes. Salmonella typhireside in a specialized membrane compartment called the Salmonella-containing vacuole (SCV), which is similar to inclusion in the case of Chlamydiasp. in macrophages, and use a T3SS called Salmonellapathogenicity island 2 (Spi2), which protects them from reactive nitrogen intermediates. Spi2-deficient strains of S. typhiget colonized in iNOS+ compartment efficiently [44] with the intracellular organisms in the SCV. Intracellular organisms have also developed mechanisms to detoxify NO-mediated effects. These include the ability to repair damage caused by reactive nitrogen intermediates and to detoxify these molecules. Pathogens have evolved the strategies of inhibiting iNOS activity which is the characteristic feature of M1 effector macrophages. Mucosal pathogen Citrobacterro dentiumcauses a marked reduction in the level of iNOS activity in macrophages [45]. There are many reported examples of bacterial pathogens altering inflammatory cytokines related to signaling. Staphylococcus aureusproteins A and M bind directly to the TNF-α receptor 1, on respiratory epithelium, which then potentiates a chemokine and cytokine cascade and subsequent disease [46]. Similarly, Shigella flexneri, through exploring type III effector, OspG, which is a protein kinase, activates ubiquitin-conjugated enzymes, thereby affecting phospho-ikBα degradation and subsequent NF-κB activation. Both Chlamydia pneumoniaand Chlamydia trachomatispromote shedding of TNF receptor 1 by activating TACE activity and shunt TNF signaling though TNFR2 [47] and resist antibacterial and inflammatory action of TNF which is major component of effector macrophages.

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.

6. Conclusion

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.

Acknowledgments

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

MAPK

mitogen-activated protein kinase