Indian Journal of Experimental Biology Vol. 55, March 2017, pp. 133-141

Mini Review

In vitro activation: A technique for screening anti-inflammatory, immunomodulatory and anticancer activity of phytomolecules

Paradkar PH1,2*, Mishra LS1, Joshi JV1, Dandekar SP2, Vaidya RA1 & Vaidya AB1 1Medical Research Centre (MRC), Kasturba Health Society (KHS), Vile Parle West, Mumbai, India 2Seth GS Medical College and KEM Hospital, Parel, Mumbai Received 19 January 2016; revised 28 December 2016

Macrophage activation plays a significant role in homeostasis of organisms. Various internal and external stress factors may affect their function, leading to adverse effects on the body. ‘In vitro macrophage activation techniques provide us with a window to understand the mechanisms of inflammation and response of to the modulating interventions. Apart from infectious diseases, inflammation is also the major culprit in pathogenesis of many noncommunicable diseases such as , obesity, metabolic syndrome, diabetes, cancer, cardiovascular disease etc. In vitro macrophage activation allows us to study the role of polarized macrophages in the process of pathogenesis. This emerging technique leads to newer diagnostics, understanding pathophysiological mechanism/s, drug development and management of chronic inflammatory diseases. We, at MRC-KHS, use this technique for screening of medicinal plant-derived phytomolecules for their anti-inflammatory, immunomodulatory and anticancer activities. This review briefly outlines the different experimental models of in vitro macrophage activation and their applications for understanding the pathophysiological mechanisms of underlying chronic inflammation and screening of therapeutic activity of plant-based phytomolecules.

Keywords: , , Herbal, Homeostasis, Inflammation, Tumor associated macrophages (TAMs)

Introduction Macrophages are a vital component of the immune The derived macrophages are flexible system and are characterized by functional and immune cell types, exhibiting a wide spectrum of morphological plasticity4. Recently, some phenotypes. These cells originate from circulating biotechniques have been evaluated to assess their role peripheral blood mononuclear cells, which develop in various pathophysiological states like inflammation, from hematopoetic stem cells in bone marrow and diabetes, metabolic syndrome, atherosclerosis, stroke, sequentially give rise to . They circulate and myocardial infarction and cancer5. The balance migrate to various tissues and get differentiated into between M1 and M2 types appears to decide the course tissue specific macrophages, which reside in the resting of a disease e.g. Repair or fibrosis in inflammation in phase unless stimulated. They rapidly respond to any any organ, regression or progression of tumours with or endogenous or exogenous stimuli which could arise without treatment. The detailed description of their from stress, tissue damage, infection or homeostatic functional and morphological attributes and variations processes by executing phagocytosis and clearance of in each disease state is beyond the scope of this article; infectious microorganisms, tissue debris and apoptotic however, considering the recent burst of new literature, cells. They also play a vital role in wound repair of we have tried to briefly summarize the various tissues. As a link between innate and adaptive techniques of evaluation of macrophage activation to immunity activated macrophages secrete small protein M1 or M2 types for potential investigational and molecules known as cytokines. These molecules therapeutic approaches in health and diseases, initiate the signaling cascade of immune system and including cancer6,7. exert the acute-phase response. These cytokines can influence the function of other cytokines in additive, Molecular mechanism of macrophage activation antagonistic or synergistic manner1-3. Macrophage activation refers to the changes in morphology, metabolism and function of resting —————— macrophages in response to any stimulation8. *Correspondence: Phone: +91 9702029317 Depending upon the surrounding microenvironment E-mail: [email protected] macrophages display polarized phenotype and 134 INDIAN J EXP BIOL, MARCH 2017

functions. There are 2 major phenotypes of through the cell membrane and cytoplasm receptors. macrophages, M1 and M2. T helper cells are Lipopolysaccharide (LPS) is a component of the outer responsible for the polarization of macrophages into membrane of gram negative bacteria. It binds to Classical (M1) or Alternative (M2) phenotype. membrane receptor CD 14 with LPS binding protein -2 (IL-2), gamma- -gamma (IFN-γ), (LBP)10. LPS is transferred inside the cell through and alpha (TNF-α) induce 96 (MD-2) and Toll-like receptor 4 (TLR-4). classically activated macrophage phenotype M1. They TLR4 after binding MyD88 and Receptor-interacting release proinflammatory cytokines (TNF-α, IL-1, IL-6, serine-threonine kinase 2 (RIPK2), initiates NF-kB- IL-12, IL-23). They also express high levels of MHC-I, activating pathway11. Activated NF-kB starts complementary factors and Class II for transcription of many proinflammatory phagocytosis. These pro-inflammatory cells promote genes such as TNF-α, IL-1β, IL-6, and IL-812. Fig. 1 arginine metabolism into Nitric oxide and Citrulline by illustrates the mechanism of LPS stimulated secreting inducible nitric oxide synthase (iNOS). macrophage activation. Measurement of these Conversely, Interleukin-4 (IL-4) and interleukin-13 cytokines released in culture medium gives a clue (IL-13) induce alternatively activated macrophage about the degree of inflammation. phenotype, M2. These anti-inflammatory M2 cells Ex vivo models for macrophage activation express IL-10, mannose receptor (MR) and scavenger Several models have been developed to understand receptor (SR). Their major functions are immune the mechanism of macrophage activation and evaluate modulation, tissue remodeling, parasite clearance, and their patho-physiological role in various diseases. 9 tumor progression . These models are briefly described below:

LPS stimulated macrophage activation PBMC derived macrophage activation Macrophages identify and act in response to the Peripheral blood mononuclear cells (PBMC) pathogen associated molecular patterns (PAMPs) primarily contain monocytes and lymphocytes. After

Fig. 1—Schematic representation of LPS induced macrophage activation pathway and expression of inflammatory cytokines. [LPS: lipopolysachharide, LBP: LPS binding protein, TLR:Toll like receptors, CD-14: Cluster of differenciation-14, TRIF: TIR-domain- containing adapter-inducing interferon-β, TRAM: TRIF-related adaptor molecule, MyD88: Myeloid differentiation primary response protein 88, MAL: MyD-88 adapter like, IRAK: interleukin-1 receptor-associated kinase, TNF-α: Tumor necrosis factor α, TRAF: TNF receptor associated factor, TAK 1: Transforming -beta-activated kinase 1, NFκB: Nuclear factor κB, IkB: Inhibitor of kB, IKK: IkB kinase, MAPK: mitogen activated protein kinase, MKK: MAPK kinase, MEKK: Mitogen/Extracellular signal- regulated Kinase Kinase, ERK: extracellular-signal-regulated kinases, AP-1: Activator protein-1] PARADKAR et al.: IN VITRO MACROPHAGE ACTIVATION: A TECHNIQUE FOR ACTIVITY ANALYSIS 135

density gradient centrifugation the buffy coat is Mycobacteria). These cells express macrophage like collected and seeded in culture vessels. Non adherent morphology, growth characteristics and cytochemistry cells (lymphocytes) are removed after overnight in vitro16. incubation and the remaining cells (monocytes) are treated with activating agents such as LPS, IFNγ. Mcrophage co-culture with other cells These cells can be further used for: (a) Phagocytosis Macrophage co-culture with other cells e.g. studies for engulfment of bacteria, yeast, etc.; Fibroblsts, adipocytes, etc., is used as a physiological (b) expression markers like CD64, CXCL10, CD23, model for that particular cell or tissue type. The CCL22 etc.; (c) Nitri Oxide (NO) production by functional phenotype of the macrophage depends spectrophotometry; and (d) cytokine and upon the surrounded microenvironment. This model is release in the culture supernatant such as IL1β, IL-6, employed to understand the cell-cell interactions TGFβ, TNFα, CX3CL1 CXCL 9, etc.13. which mimic the in vivo conditions17,18.

Primary murine macrophage activation Biomarkers for evaluation of inflammation Primary murine macrophages can be collected from Monocytes and activated macrophage the peritoneal cavity and the bone marrow. An subpopulations differ in terms of gene expression, inflammatory response is created using thioglycollate receptor expression, cytokine and chemokine broth or Bio-Gel polyacrylamide beads injection into production and effector function. Classically and the peritoneal cavity. These macrophages are Alternatively activated macrophages also express harvested and seeded into tissue culture vessels. After different markers, depending upon the surrounding 7 days these cells are labeled with specific antibody to environment. in vitro macrophage activation recognize the cell surface markers as above and 14 technique utilizes these markers, specific to M1 or M2 further sorted and identified by flow cytometer . phenotype, to evaluate the degree of inflammation.

Alveolar macrophage activation These are functional or phenotype markers. Some genomic variations of these cells like M2a, M2b, M2c Alveolar macrophages are present in the alveoli 9,19,20 and alveolar ducts. These macrophages are isolated and M2d are also reported (Table 1) . from broncho-alviolar lavage fluid. These cells are Inflammatory cytokines activated by IFNγ and used for phagocytosis, cytokine Cytokines are low molecular weight soluble protein release, and chronic inflammattory studies15. molecules for intercellular signalling. They are Macrophage cell lines effectors of macrophage function and transmit the Many macrophage like cell lines have been inflammatory signal to other cells. They can be developed by viral transformation to study a variety of divided into two major groups on the basis of functions such as cell mediated cytotoxicity, metabolic secretary cell type: pro-inflammatory and anti- studies, surface receptors etc. RAW 264.7 and U937 inflammatory. Cytokines released by classically and cell lines are primarily used for studying behaviour of alternatively activated macrophages are listed in monocytes and macrophages. They are activated by Table 1. In vitro secretion of cytokines is measured by 21 LPS or PPD (purified protein derivative from ELISA in media supernatant .

Table 1—Macrophage subsets, their activating agents and markers Cell type M1 M2a M2b M2c M2d Stimulatory factors IFN-γ and LPS or TNF-α IL-4 and IL-13 TLR/IL-1R IL-10 IL-6, MCF ligands Cytokines secreted TNF-α, IL-1β IL-6, IL-12, IL-23 IL-10, TGF-β TNF-α, IL-1 TGF-β IL-10 TNF-α, TGF-β IL-1Ra IL-6, IL-10 IL-10, IL-12 Chemokines and receptors CCL5, CXCL9, CXCL10, CCL17, CCL18, CCL1 CCL13, CCL16, CCL5, CXCL10, CXCL11, CXCL16, CX3CL1 CCL22, CCL24 CCL18 CXCL16 iNOS expression Yes No No No No Cells Surface markers CD80, CD86, CD-163, CD-206, CD86 CD163 VEGF TLR2, TLR4, IL1RI IL1RII, Decoy R TLR1, TLR8 TGM2 Adapted from Hao et al. 20129 and Duluc et al. 200720 136 INDIAN J EXP BIOL, MARCH 2017

Chemokines overlapping of the markers in macrophage subsets. Chemokines are small chemotactic protein Therefore, combination of these markers should be molecules from cytokine family. Their major role is, utilized for characterization and identification of trafficking immune cells through a concentration polerized macrophages24. gradient by activation of G protein coupled receptor.

These cells are accumulated at the site of chemokine Applications of in vitro macrophage activation production and exert their function. Chemokine Screening of Anti-inflammatory drugs function is essentially regulated by cytokines. Inflammation is a physiological response to Macrophage polarization intensely modulates the internal or external stimuli for protection. If this profile of chemokine production. response is not regulated, it results in chronic inflammation. A wide variety of medicinal plants Nitric oxide It is generated in activated macrophages by Inducible contain active phytochemicals which are believed to suppress inflammatory cascade at multiple levels. Nitric oxide synthase (iNOS) enzyme. Expression of this enzyme is regulated by cytokines and LPS. Once Veres has reviewed the anti-inflammatory activity of polyphenols which are metabolites of all traditional generated NO inhibits the activity of iron containg 25 enzymes due to its high affinity to iron molecule22. medicines and most natural products . They exert Nitric oxide can be measured in macrophage culture their anti-inflammatory activity through the employing various specrophotometric techniques. It modulation of MAPK, Akt and NF-κB pathways, by requires extra care and accurate assays as the half life of inhibition of NF-kB phosphorylation, thus NO is very short (few milliseconds). Measurement of transcriptional inactivation of proinflammatory NO concentration in cell culture supernatant, involves cytokines and chemokines. quantitation of Nitrate and nitrite, catalyzed by nitrate In vitro macrophage activation assay has been used reductase enzyme. for accessing anti-inflammatory activity of various compounds by inhibiting cytokine release. It may Tumor associated macrophages (TAMs) provide essential information about inflammation in Macrophages, present in the vicinity of tumor cells many diseases. Exposure of therapeutic compounds to are known as tumor associated macrophages. They play the macrophages, reduces the release of an important role in manipulating the tumor proinflammatory cytokines in response to LPS. This environment. At the primary stage of cancer, model is employed to study in vitro anti-inflammatory macrophages express proinflammatory phenotype (M1) activity of natural compounds such as alkaloids, and show tumoricidal activity, whereas at late stage of polyphenols, and other extracts26. They suppress the cancer, they express anti inflammatory phenotype (M2) activity of COX and iNOS and decrease the which provides favourable microenvironment to the production of ROS/RNS27. Biological modulation and tumor progression. They release immunosuppressive reduction of LPS-inducible inflammatory factors are mediators and Migration Stimulation Factor (MSF) to considered to be an effective strategy for promote the tumor growth. Presence of TAMs in high inflammatory diseases. numbers is associated with advanced stages of cancer. Polyphenols present in the Red wine are reported to This makes them a key prognostic marker for cancer have antioxindant and anti-inflammatory activity progression as well as targat for therapeutic 23 which reduce the risk of cardiovascular diseases. strategies . Bognar et al.28 has accessed the anti-inflammatory

Cell Surface Markers activity of Malvidin, a red wine polyphenol in RAW Macropahge subsets are also defined by expression 264.7 macrophages by LPS stimulation. In LPS of cell surface markers along with their functions. treated cells, Malvidin reduced phosphorylation of Cell surface markers are proteins specifically NF-kB p65 on Ser536 which is responsible to expressed in a particular cell type. In case of enhance its transcriptional activity but did not affect macrophages, these proteins are pathogen receptors, the expression of the protein. It also prevented nuclear cytokine receptors, chemokine receptors, adhesion translocation and DNA binding of NF-kB in LPS molecules or costimulators. stimulated cells. Malvidin suppressed LPS-induced As seen in Table 1 these markers singly cannot ROS production, MAPK and PARP activation in define the subset of macrophage. There is always an RAW 264.7 macrophages. PARADKAR et al.: IN VITRO MACROPHAGE ACTIVATION: A TECHNIQUE FOR ACTIVITY ANALYSIS 137

In vitro anti-inflammatory activity of 2-methoxy- with CurcuWIN™ (curcumin soluble in water)35. (2-MEO), a 17-estradiol metabolite was Water soluble curcumin significantly reduced TNF-α demonstrated by Shand et al.29. Antiproliferative and release in macrophage cells activated by LPS. antiangiogenic activity of 2-MEO is well characterized Similarly, we have assessed anti-inflammatory in vitro and in vivo30. It is also reported for the activity of Curcuma longa and Tinospora cordifolia prevention of arthritis and osteoporosis in extracts individually and synergistically using the postmenopausal women as an alternative for hormone same method. It was observed that the extracts replacement therapy31. Anti-inflammatory activity of significantly reduced IL-6 and TNF-α secretion in 2-MEO was seen in LPS activated human blood macrophages36. monocytes and mouse macrophage cell lines. It Assesment of Immunomodulation suppressed production of PGE2 and IL-6 in vitro as well Immunomodulation stands for stimulation or as in vivo. Whereas there was no effect on TNF-α and suppression of immune response in defense against nitrite production in vitro. pathogens or . The basic function of Molecular mechanism responsible for anti- immunomodulator is balancing and stabilizing the inflammatory activity of ethanolic extract of immune system. Devasagayam & Sainis37 have earlier Myagropsis myagroides (EMM) in LPS activated RAW 32 reviewed the mechanisms and studies based on 264.7 macrophages was investigated by Joung et al. . immunomodulatory and antioxidant activity of Indian Cells preincubated with extract were stimulated by LPS medicinal plants. Many of these studies are based on and concentrations of NO, PGE2, IL-1β, IL-6, and the macrophage activation technique. The mechanism TNF-α were measured in cultured media. EMM of immunomodulation involves many aspects of significantly reduced production of NO and immunity. These aspects can be studied to access in inflammatory cytokines in a dose dependent manner. It vitro immunomodulatory activity of compounds by also inhibited phosphorylation of MAPKs and Akt thus macrophage activation. preventing translocation of NF-kB in nucleus in LPS In a study to improve phagocytotic activity using stimulated cells. Similarly, Calendula officinalis BioBran, a modified arabinoxylan present in rice reduced levels of TNF-α in LPS stimulated bran, three in vitro macrophage activation models was macrophage culture supernatant as well as other utilized. Macrophages were activated by PMA and proinflammatory cytokines ie IL-1β, IL-6, IFN-γ and then incubated with BioBran and yeast. Percent CRP in serum of treated mice. It also inhibited 33 phagocytic cells, cytokine and NO production were expression of Cox-2 in treated mice . measured. Macrophages treated with BioBran showed Adipocyte-macrophage co-culture system was enhanced Spreading ability, increased production of utilized for the assessment of anti-inflammatory cytokines and nitric oxide38. activity of a known as nobiletin, present in The role of Myeloperoxidase in the in vitro release citrus fruit peels. It inhibited NO, TNF-α and MCP-1 of TNF-α by the peritoneal macrophages, has been secretion in a dose dependent manner and also blocked shown by Lefkowitz et al.39. It was suggested that many transcription factors expressed by adipocytes in possible mechanisms for the TNF release could be the interaction with macrophages. Thus, it breaks the radical produced by MPO or binding of MPO to the paracrine loop between macrophages and adipocytes 40 18 mannosylfucosyl receptor. Guan et al. employed and inhibits inflammatory changes . macrophage activation assay for measurement of In vitro anti-inflammatory of various medicinal nitricoxide production and Phagocytosis assay. They plants extracts has been studied by our group using demonstrated that crude polysaccharide fraction 34 human PBMC culture. Raut et al. have studied effect stimulated macrophage proliferation, phagocytosis of aqueous, hydroalcoholic and alcoholic extracts of and nitric oxide production in a dose dependent Commiphora wightii on monocyte derived macrophage manner. Orsi et al.41 evaluated immunomodulatry culture model. They observed a reduction in TNF-α activity of Propolis. It induces dose dependent and IL-1β levels in media supernatant. Commiphora elevation in H2O2 release and an inhibition in NO wightii is commonly used as an anti-inflammatory and generation by activated macrophages. Madaan et al.42 anti arthritic drug in Ayurvedic clinical practice. utilized dendritic cells from bone marrow and evaluated We have also investigated anti-inflammatory immunostimulatory activity of chyavanprash in vitro. activity of phytomolecule, curcumin and compared it Dendritic cells treated with chyavanprash solution 138 INDIAN J EXP BIOL, MARCH 2017

showed increased levels of IL-1β, IFN-γ and MIP-1α in the growth and progression or regression of tumours. without any other stimulation as compared to Recently, Sanchez-Reyes et al.49 have shown that controls. It also significantly enhanced phagocytotic supernatant from cervical cancer cells can change M1 activity of treated macrophages as compared to non to M2 types and increased expression of TLRs (Toll- treated macrophages42. like receptors). This has been confirmed for HPV 16 A three dimensional culture model was utilized by associated cervical cancer in vivo mouse model by Wang et al.43 to understand the immune mechanism Lepique et al.50 in studies utilizing the TAMs.

of Human Pappiloma Virus therapeutic vaccine. These aspects of TAMs are well utilized for Dendritic cells generated from PBMC were assessing in vitro anticancer activity of compounds by cocultured with T cells in the 3D culture model. The activation of macrophages. In vitro tumor promoting culture treated with HPV16mE7 increased activity of monocyte derived macrophages was proliferation of T-cells and IFN-γ expression. evaluated in two different culture conditions. Their 44 In a study by Rao et al. , α-linolenic acid morphologic variation, cytokine expression, phagocytic modulated the generation of reactive oxygen species activity, surface receptor expression and effect of in macrophages in vitro and in vivo. Rats were fed macrophage media supernatant on tumor cells were diet containing partially hydrogenated vegetable fat analyzed51. Investigation of cytotoxic effect of (PHVF) including elaidic acid and linseed oil which activated macrophages using the MTT method has contains α-linolenic acid. After 10 weeks, the been done in tumor cell line52. Antitumor activity of peritoneal macrophages of the PHVF fed rats Squalene-treated Cell Wall Skeleton of Nocardia rubra generated higher amount of reactive oxygen, NO and was pursued by activation of macrophages by peroxide species as as compared to the macrophages compound and culturing them with tumor cell lines of rats fed with linseed oil along with PHVF. FBL-3, and CEM53. Baay et al.54 have reviewed the potential targets and strategies for cancer therapy Cancer progression and targeted therapy associated with TAMs. They have focussed on the One of the primary functions of macrophages is secreted proteins which are responsible for interaction known to provide a defense mechanism against cancer between tumor cell and macrophage as intervention cells. They need to be stimulated for tumor targets.

cytotoxicity either by LPS, MDP or cytokines IFN-γ TAMs, therefore, are recognized as prospective and GM-CSF. TNF-α and NO release in response to 45 targets for therapy whereby their entry into tumours the stimulus have tumoricidal activity . may be blocked or M2 types may be converted to M1 Macrophages present inside the tumor are classified types for a favourable therapeutic outcome in cancer55. as Tumor Associated Macrophages (TAMs). These A recent study has reported that non classical cells display characteristics of M2 macrophages such patrolling monocytes play a key role in controlling as expression of anti-inflammatory molecules, (IL-10, tumor growth and in the lungs. The orphan TGF-β) and pro-angiogenic mediators (VEGF, COX-2- nuclear receptor Nr4a1 is abundantly expressed in derived PGE2, and matrix metalloproteinase). They these monocytes as compared to other myeloid cells also overexpress M2-specific genes, macrophage and regulates their function. These cells recruit and galactose type C-type lectin-2, ARG, etc. Depending activate Natural Killer (NK) cells to inhibit tumor upon the tumor stage, TAMs also express M1 specific growth. They also actively monitor the endothelial cells cytokines such as TNF-α, IL-6, IL-1β, IL-23p19, and 46 of vasculature and remove the dead cells and CXCL8 . TAMs are recruited at the malignant cite by microparticals. These properties facilitate the resolution tumor derived chemotactic factor CCL2 and cytokines 47 of inflammation and suppression of tumor growth and such as M-CSF, VEGF and PDGF . metastasis, which makes these cells a possible target Heusinkveld et al have observed that cervical cancer for cancer therapy56. tumour cells, which can produce PGE2 and IL-6, induce conversion of local macrophages to M2 type48. Limitations of macrophage activation techniques This promotes tolerance to tumours and allows tumour All experimental techniques of macrophage growth. Interaction of the M2 macrophages with TH1 activation in vitro or in vivo are likely to have cell types the M2 types can be converted to M1 type limitations with respect to species differences and indicating that immune system plays an important role artificial experimental conditions. PARADKAR et al.: IN VITRO MACROPHAGE ACTIVATION: A TECHNIQUE FOR ACTIVITY ANALYSIS 139

Though current in vitro models are easy, cost Examples of phytomolecules which have been effective and provide a direction for research, they may studied for anti-inflammatory,immunomodulatory or not give an accurate idea of the cell response in vivo. anticancer activity include curcumin, maldivin, The cell receives multiple positive and negative nobiletin, lycopene, and tyrosol, etc. However, feedbacks from several other cells in vivo and this more studies are required to understand the mechanisms microenvironment cannot be exactly replicated in vitro. by which macrophages regulate the diverse range of Macrophage co-culture and 3 dimensional culture biological systems in homeostatic and disease associated techniques solve this problem to some extent17. conditions. Thorough understanding of macrophage Moreover, minor changes can also alter the behavior of function may widen the scope of potential clinical monocytes and macrophages. 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