The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens

The cellular changes during ageing are incompletely understood yet immune system dysfunction is implicated in the age-related decline in health. The acquired immune system shows a functional decline in ability to respond to new pathogens whereas serum levels of cytokines are elevated with age. Despite these age-associated increases in circulating cytokines, the function of aged macrophages is decreased. Pathogen-associated molecular pattern receptors such as Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. Here we review the evidence for defective TLR signalling in normal ageing. Gene transcription, protein expression and cell surface expression of members of the TLR family of receptors and co-effector molecules do not show a consistent age-dependent change across model systems. However, there is evidence for impaired downstream signalling events, including inhibition of positive and activation of negative modulators of TLR induced signalling events. In this paper we hypothesize that despite a poor inflammatory response via TLR activation, the ineffective clearance of pathogens by macrophages increases the duration of their activation and contributes to perpetuation of inflammatory responses and ageing.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Characterization of tissue transglutaminase 2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in a number of cell functions. Up-regulation of TG2 is thought to be involved in monocyte to macrophage differentiation and defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be fully elucidated. Cell surface-associated TG2 is now recognized as being important in regulating cell adhesion and migration, via its association with cell surface receptors such as syndecan-4, ß1 and ß3 integrin, but its extracellular role in the clearance of apoptotic cells is still not fully explored. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function within the framework of apoptotic cell clearance. Both THP-1 cell-derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors (both cell permeable and impermeable, irreversible and active site directed) resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. Macrophage cell surface TG2 and, in particular, its cell surface crosslinking activity was found to be crucial in dictating apoptotic cell clearance. Our further studies demonstrate syndecan-4 association with TG2 and imply possible cooperation of these proteins in apoptotic cell clearance. Knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance which seems to involve protein cross linking and interaction with other cell surface receptors.

Analysis of Transglutaminase-2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Transglutaminase-2 (TG2) is a Ca2+-dependent protein crosslinking enzyme known to play an important role in apoptotic cell clearance by macrophages through an ill-defined mechanism. Several studies have implicated TG2 in the apoptosis programme e.g. raised TG2 levels in cells undergoing apoptosis; increased cell death with down-regulation of TG2; up-regulation of TG2 in monocytes upon differentiation into macrophages. Defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be elucidated. Here we aim to characterise the role of TG2 in macrophage function with a focus on apoptotic cell clearance. THP-1 monocytes were induced to differentiate to macrophage-like cells by three different stimulants and were analysed for the presence of TG2. Macrophage-apoptotic cell interaction studies in the presence and absence of irreversible TG2 inhibitors resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. TG2 was expressed at the macrophage cell surface and its association with ß3 integrin expression suggests the possible link between TG2 and ß3 integrins. Our current findings suggest that TG2 had got a crucial but yet to be defined role in apoptotic cell clearance.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes. © 2012 Macmillan Publishers Limited All rights reserved.

Characterization of tissue transglutaminase 2 in macrophage function

Apoptosis is a highly regulated process that removes damaged or unwanted cells in vivo and defective clearance of apoptotic cells by macrophages has significant immunological implications. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. TG2 as a guanosine triphosphate (GTP)-binding or GTP- hydrolyzing protein for mediating signal transduction and as a cell cycle regulator emphasized the importance of this enzyme in aging process. The ubiquitous presence of TG2 compared to the other organ-specific TGases has attracted special attention as a cellular aging device. TG2 activity and expression are known to increase in aging humans suggesting possible involvement in several age-related processes such as decrease in vascular compliance and increased stiffening of conduit arteries, cataract formation, Alzheimer's disease and senescent epidermal keratinocytes. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function. THP-1 cell derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors resulted in significant inhibition of interaction. Macrophage cell surface TG2 and, in particular, its cell surface cross linking activity was found to be crucial in apoptotic cell clearance. Syndecan-4 association with TG2 implies possible cooperation of these proteins and knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance.

Characterization of Transglutaminase 2 in macrophage clearance of apoptotic cells

Removal of dead or diseased cells is crucial feature of apoptosis for managing many biological processes such as tissue remodelling, tissue homeostasis and resolution and control of immune responses throughout life. Tissue transglutaminase (TG2) is a protein crosslinking enzyme that has been implicated in apoptotic cell clearance but also mediates many important cell functions including cell adhesion, migration and monocyte-macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4, ß1 and ß3 integrin. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise extracellular role of TG2 in apoptotic cell clearance remains ill-defined. This thesis addresses macrophage TG2 in cell corpse clearance. TG2 expression (cytosolic and cell surface) in human macrophages was revealed and data demonstrate that loss of TG2 activity through the use of inhibitors of function, including cellimpermeable inhibitors significantly inhibit the ability of macrophages to clear apoptotic cells (AC). This includes reduced macrophage recruitment to and binding of apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus it defines for the first time a role for TG2 activity at the cell surface of human macrophages in multiple stages of AC clearance and proposed that TG2, in association with heparan sulphates, may exert its effect on AC clearance via crosslinking of CD44.

The macrophage and the apoptotic cell:an innate immune interaction viewed simplistically?

Macrophages play important roles in the clearance of dying and dead cells. Typically, and perhaps simplistically, they are viewed as the professional phagocytes of apoptotic cells. Clearance by macrophages of cells undergoing apoptosis is a non-phlogistic phenomenon which is often associated with actively anti-inflammatory phagocyte responses. By contrast, macrophage responses to necrotic cells, including secondarily necrotic cells derived from uncleared apoptotic cells, are perceived as proinflammatory. Indeed, persistence of apoptotic cells as a result of defective apoptotic-cell clearance has been found to be associated with the pathogenesis of autoimmune disease. Here we review the mechanisms by which macrophages interact with, and respond to, apoptotic cells. We suggest that macrophages are especially important in clearing cells at sites of histologically visible, high-rate apoptosis and that, otherwise, apoptotic cells are removed largely by non-macrophage neighbours. We challenge the view that necrotic cells, including persistent apoptotic cells are, of necessity, proinflammatory and immunostimulatory and suggest that, under appropriate circumstances, persistent apoptotic cells can provide a prolonged anti-inflammatory stimulus.

Macrophage-mediated clearance of cells undergoing caspase-3-independent death

Little is known of the functions of caspases in mediating the surface changes required for phagocytosis of dying cells. Here we investigate the role played by the effector caspase, caspase-3 in this process using the caspase-3-defective MCF-7 breast carcinoma line and derived caspase-3-expressing transfectants. Our results indicate that, while certain typical features of apoptosis induced by etoposide – namely classical morphological changes and the ability to degrade DNA into oligonucleosomal fragments – are caspase-3-dependent, loss of cell adhesion to plastic and the capacity to interact with, and to be phagocytosed by, human monocyte-derived macrophages – both by CD14-dependent and CD14-independent mechanisms – do not require caspase-3. Furthermore, both etoposide-induced caspase-3-positive and -negative MCF-7 cells suppressed proinflammatory cytokine release by macrophages. These results demonstrate directly that cell surface changes that are sufficient for anti-inflammatory clearance by human macrophages can be regulated independently of stereotypical features of the apoptosis programme that require caspase-3.

Macrophage polarisation by fatty acids is PPARgamma-dependent

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. We investigated the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Palmitate increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300µM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300µM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis. We have also explored whether specific dietary fatty acids drive monocyte to macrophage polarisation via metabolic pathways. Here we show that monocytes pre-incubated with the saturated fatty acid palmitate increase production of inflammatory cytokines such as TNFa and IL-6 in response to a phorbol myristate differentiation trigger. This increases mitochondrial superoxide production, reduces dependency on oxidative phosphorylation through ceramide-dependent inhibition of PPARgamma activity and increases TNFa production, again via a mechanism that requires ceramide production.

Blockade of interleukin-6 effects on cytokine profiles and macrophage activation after spinal cord injury in mice

The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. MR16-1 antibodies versus isotype control antibodies or saline alone was administered immediately after thoracic SCI in mice. MR16-1-treated group samples showed increased neuronal regeneration and locomotor recovery compared with controls. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. MR16-1 treatment promoted arginase-1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site and enhanced positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

Macrophage migration inhibitory factor (MIF) family in arthropods : cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in Mud crabs, Scylla paramamosain

5. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (Nos. 31172438 and U1205123), the Natural Science Foundation of Fujian Province (No. 2012J06008 and 201311180002) and the projects-sponsored by SRF. TW received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions.