Cellular Immunology 291 (2014) 58–64

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Macrophages in renal transplantation: Roles and therapeutic implications Tony Kwan, Huiling Wu, Steven J. Chadban ⇑ Department of Renal Medicine, Royal Prince Alfred Hospital, Sydney, Australia Collaborative Transplant Research Group, Sydney Medical School, The University of Sydney, Sydney, Australia

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Article history: Received 5 February 2014 Accepted 27 May 2014 Available online 14 June 2014 Keywords: Macrophages Innate immunity Renal transplantation

a b s t r a c t The presence of macrophages within transplanted renal allografts has been appreciated for some time, whereby macrophages were viewed primarily as participants in the process of cell-mediated allograft rejection. Recent insights into macrophage biology have greatly expanded our conceptual understanding of the multiple roles of macrophages within the allograft. Distinct macrophage subsets are present within the kidney and these sub-serve discrete functions in promoting and attenuating inflammation, immune modulation and tissue repair. Unraveling the complex roles macrophages play in transplantation will allow identification of potential therapeutic targets to prevent and treat allograft rejection and maximize graft longevity. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Renal transplantation remains the optimal form of renal replacement therapy for those with end-stage renal failure. Despite technical and pharmacological breakthroughs that have improved short-term allograft outcomes, graft and patient attrition rates in the long term remain unchanged [1]. During the lifetime of an allograft it is vulnerable to numerous injurious processes that challenge its longevity. All transplanted organs undergo a period of ischemia–reperfusion injury (IRI) during the organ retrieval, preservation and transplantation procedures. As a consequence of this injury, an innate immune response is triggered, inflammatory cells are recruited within the allograft and parenchymal cells are also activated. The resulting inflammatory microenvironment has two key consequences: (a) allograft damage and repair; and (b) promotion of an adaptive allo-immune response, which may cause acute rejection. Whilst the vast majority of grafts survive IRI and acute rejection, ongoing risks of chronic rejection, recurrent disease and non-immune organ injury persist for the remainder of its life [2]. This review will focus on the role of macrophage/monocytes as mediators of allograft injury. Previous work on the pathogenesis of allograft rejection has focused on adaptive immunity and the role of T lymphocytes in this process is well documented [3]. The ⇑ Corresponding author at: Renal Medicine, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia. Fax: +61 2 9515 6329. E-mail address: [email protected] (S.J. Chadban). http://dx.doi.org/10.1016/j.cellimm.2014.05.009 0008-8749/Ó 2014 Elsevier Inc. All rights reserved.

increasing awareness of antibody-mediated rejection has provided additional insights into the role of B lymphocytes and plasma cells [4]. Macrophages have long been recognized within the graft during IRI, acute and chronic rejection [5]. Traditionally, these cells were viewed as contributors to T-cell mediated processes such as acute rejection, recruited into the graft under the influence of T cell derived chemokines to promote inflammation, cause tissue injury and act as antigen presenting cells (APCs) [6]. The more recent discovery of Toll-like receptors (TLRs) and their essential role as innate activators of macrophages during organ IRI has led to a growing appreciation of the role of macrophages and innate immunity in allograft responses and highlighted the importance of innate-adaptive cross-talk in the development of adaptive immune responses. In this brief review, we aim to provide an overview of monocyte/macrophage biology in the context of their potential roles in contributing to allograft damage. A summary of monocyte and macrophage subsets Monocyte and macrophage biology has recently been reviewed elsewhere in depth [5,7,8]. In brief, circulating monocytes arise from hematopoietic stem cells in the bone marrow. These stem cells subsequently undergo commitment to the myeloid lineage and pass through several stages of differentiation (the granulocyte/macrophage progenitor (GMP) and the macrophage/DC progenitor (MDP)) that incrementally restrict developmental potential. Both the growth factor macrophage colony-stimulating factor (M-CSF) and the transcription factor PU.1 are required for

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this process. The MDPs subsequently give rise to conventional dendritic cells (cDC), macrophages and monocytes. Phenotypic heterogeneity in both murine [9] and human monocytes [10] has been described. The murine monocyte subsets are classified by their expression of Ly6C. Ly6C+ monocytes also express high levels of the chemokine receptor CCR2 and low levels of the fractalkine receptor CX3CR1 (CCR2highCX3CR1low). These monocytes circulate in the blood and are selectively recruited to sites of active inflammation and produce high levels of TNF-a and IL-1b. Ly6C+ monocytes also have the capacity to differentiate into M1-type macrophages (discussed below) when they have migrated into inflamed tissue. Thus, the Ly6C+ subset are also called inflammatory monocytes on account of their numerous pro-inflammatory roles. In contrast, the Ly6C- monocytes are CCR2lowCX3CR1high and are usually found patrolling the vascular endothelium. They are termed ‘‘resident’’ monocytes because they are thought to maintain the population of resident tissue macrophages and dendritic cells. They also have the capacity to differentiate into M2 macrophages, and thus are involved in tissue healing and repair after the initial inflammatory process resolves. Recent studies of human monocyte subsets have expanded our understanding of the complexity in their phenotypic diversity. The original human monocyte subset classification is based on the expression of the low affinity Fcc receptor CD16. Approximately 85% of circulating monocytes are CD16- and constitute the ‘‘classical’’ subset. They are similar to murine Ly6C+ monocytes phenotypically in that they are also CCR2highCX3CR1low, but they are functionally distinct to CD16+ monocytes since they produce IL10 in addition to TNF-a upon LPS stimulation. The remaining 15% of the monocyte population is CD16+. This is further subdivided based on their expression of the LPS binding cofactor CD14: the ‘‘intermediate’’ subset is CD14+CD16+ whilst the ‘‘non-classical’’ subset is CD14-CD16++. The non-classical subset is functionally and phenotypically similar to the murine Ly6Cmonocytes. They are found patrolling the vascular endothelium and also respond poorly to LPS stimulation. The intermediate subset has a phenotype between that of the classical and non-classical subsets and originally was thought to represent a transitional stage between these two populations. Wong et al. [11] further clarified the distinct role of this monocyte subset using a combination of gene profiling, flow cytometry and cytokine analysis which revealed that the intermediate subset has strong pro-inflammatory roles and highly expresses genes required for T cell co-stimulation and antigen presentation. They also produce high levels of TNF-a, IL-1 and IL-6 upon LPS stimulation. Thus, this population most closely resembles the ‘‘inflammatory’’ murine monocytes. The disparity between phenotypic and functional correlation of human and murine monocytes highlights the complexities in this field and is an area of active investigation. Like monocytes, macrophages also demonstrate phenotypic and functional heterogeneity. Macrophages can be derived from in situ proliferation of resident tissue macrophages, or recruited as a result of differentiation from circulating monocytes that have migrated into the tissue. This process is dependent on the growth factor macrophage-colony stimulating factor M-CSF. Indeed, increased M-CSF has been demonstrated in association with increased macrophage and monocyte infiltration in numerous inflammatory kidney diseases [12,13]. Macrophages demonstrate significant plasticity and are able to change their phenotype and function in response to their surrounding microenvironment. M1 or classically activated macrophages are induced when monocytes are exposed to a combination of IFN-c, TNF-a and LPS. The M1 phenotype is pro-inflammatory, characterized by secretion of pro-inflammatory cytokines (TNF-a, IFN-c, IL-12 and IL-1b), enhanced phagocytic activity, and increased production of

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reactive oxygen species via up-regulation of inducible nitric oxide synthase (iNOS). M2 or alternatively activated macrophages encompass several phenotypes and are further classified into three subsets. The M2a phenotype is induced upon exposure to IL-4 or IL-13, the M2b phenotype is induced by LPS exposure in the presence of immune complexes, whilst the M2c phenotype comprise of a heterogeneous population that result from exposure to anti-inflammatory mediators such as IL-10, TGF-b and glucocorticoids. M2 macrophages tend to adopt a reparative or immunomodulatory role. They elaborate IL-10, demonstrate reduced phagocytic activity and up-regulate arginase rather than iNOS. The last action directs arginine and its metabolites towards biochemical pathways required for the synthesis of collagen (in particular the synthesis of proline). Although the M1/M2 classification system serves as a useful starting point to appreciate macrophage function according to propensity to induce or control inflammation, it does not adequately encompass additional diverse roles such as tissue homeostasis and immune regulation. An alternative classification system proposed by Mosser and Edwards [14] categorizes macrophages according to their function, namely: (1) classically-activated macrophages that participate in host defense; (2) wound-healing macrophages involved in tissue fibrosis and repair; and (3) regulatory macrophages responsible for modulation of the immune response. In summary, macrophages are phenotypically plastic cells that can tailor their function to the microenvironment in which they reside. Their functional diversity highlights their potential significance in a wide variety of pathologic processes. Under the influence of GM-CSF and IL-4, monocytes are capable of differentiating into dendritic cells [15,16]. Like macrophages, subsets of dendritic cells have been described [17]. Both DCs and macrophages are capable of processing and presenting antigen to T cells. However, DCs express high levels of co-stimulatory molecules on their cell surface and thus can effectively present antigen to both naïve and primed T cells. In the context of allograft rejection, it is likely that DCs initiate the process by presenting alloantigen to naïve T cells in the draining lymph nodes [3]. This is subsequently propagated and amplified through recruitment of activated macrophages to the graft which then interact with primed T cells. A certain degree of functional plasticity exists between DCs and macrophages, and differentiation of DCs to macrophages, and vice versa, have been described in vitro. The significance of these findings in vivo remains uncertain and it remains conceptually useful to recognize DCs and macrophages as distinct cell types in the context of allograft pathology.

Macrophages in ischemia–reperfusion injury The process of organ retrieval, preservation, transportation and implantation by necessity causes IRI to the transplanted organ. The severity of IRI incurred is dependent upon the duration and type of ischemic insult, with warm ischemia imparting greater damage than cold. The clinical impact is a delay in establishment of organ function following implantation: in the context of kidney transplantation, severe IRI causes delayed graft function requiring dialysis. Innate immunity plays a prominent role in mediating this process, particularly the TLRs [18]. TLRs are a family of germline encoded receptors that evolved to recognize molecular motifs that are ubiquitous to pathogenic micro-organisms (termed pathogenassociated molecular patterns or PAMPs). Thirteen TLRs have been characterized to date and their repertoire of ligands is diverse. These include bacterial cell wall components (diacyl- and triacyllipopeptides, LPS), flagellin, and genetic material unique to microorganisms (dsRNA, unmethylated CpG motifs). Of greater relevance

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to IRI, endogenous molecules produced or released as a result of tissue injury (termed damaged-associated molecular patterns or DAMPs) can also activate TLR signaling. Examples of DAMPs include hyaluronan, fibronectin, heat-shock proteins (HSPs), and high mobility group box-1 (HMGB-1). Macrophages express most of the TLRs and thus can be activated by PAMPs or DAMPs. As an example in kidney IRI, macrophage activation by HMGB-1 engagement of TLR4 has been well characterized in mice [19,20] and appears to be important in human [21]. TLR4 signals via the adaptor molecule MyD88, a pathway that ultimately results in activation of nuclear factor-jB (NF-jB). NF-jB is responsible for the transcription of several proinflammatory cytokines such as IL-1, IL-6, and TNF-a. Not only do these cytokines promote the recruitment of additional effector cells via production of chemokines and adhesion molecules, they also create a microenvironment conducive for induction of the M1 macrophage phenotype. Indeed, macrophage mediators of cell injury such as ROS and inflammatory cytokines such as IL-6 [20], TNF-a [22,23] and IL-18 [24] have been shown to be over-expressed in kidney during IRI. The importance of macrophage-derived cytokines in this context is underlined by studies demonstrating that absence of macrophage-derived IL-6 [20] and IL-18 [24] is protective in mouse models of renal IRI. In addition to causing organ damage, IRI may serve to modulate subsequent adaptive immune responses. This is critical in the context of transplantation as it potentially predisposes the allograft to acute and/or chronic rejection. Again, macrophage activation by TLRs appears to play an important role in this process. TLR activation leads to phagosome maturation, facilitating antigen processing and presentation on class II MHC. IFN-a and IFN-b are produced via MyD88-independent TLR signaling, which in turn induces macrophage up-regulation of co-stimulatory molecules such as CD40, CD80 and CD86. Taken together, these actions serve to enhance the capacity of macrophages to activate CD4+ T cells and thereby promote development of an adaptive immune response. Experimental evidence of innate modulation of adaptive allo-immunity was reported by Chalasani et al. [25] in a series of adoptive transfer studies. In a cardiac allograft model conducted in immuno-deficient mice, transfer of T effector cells caused acute rejection when administered to mice with freshly transplanted hearts but not when administered 6 weeks after cardiac implantation, by which time any IRI-mediated inflammation had subsided. The findings of acute rejection were recapitulated following delayed adoptive transfer if the allografts were subjected to IRI immediately prior to the transfer, implying that acute inflammation caused by IRI is required to facilitate effective, adaptive allo-immunity. The role of macrophages in IRI is further supported by findings in human allograft biopsy samples [26]. One study showed that the predominant cell type infiltrating post-reperfusion allografts (30– 60 min post-revascularization) were CD68+ macrophages, whereas T cells predominated in stable allografts. Quantitative PCR analysis supported the histologic findings, demonstrating up-regulation of genes involved in monocyte recruitment and acute inflammation in post-reperfusion biopsies, while genes involved in T cell function were dominant in stable grafts as compared to normal kidneys. Macrophages may also play a reparative role in IRI. Injection of M-CSF into kidneys subjected to ischemia enhanced proliferation of renal tubular epithelial cells and decreased collagen production. This was associated with accumulation of M2 macrophages [27]. In a murine model of renal transplantation, transcripts associated with alternatively activated macrophages were up-regulated in both isografts and allografts at 7 days post-transplantation, whereas transcripts associated with rejection were only upregulated in allografts. This suggests that IRI, which invariably

occurs during the transplantation procedure, is associated with recruitment of alternatively activated macrophages [28]. Macrophages have an important role in IRI, not only in causing tissue damage but also in facilitating the subsequent adaptive immune response. Macrophage activation via TLRs leads to enhanced inflammation, tissue damage as well as increased capacity to process and present antigen to T cells. This effect is further amplified as T cells recruited during IRI produce IFN-c, a potent inducer of M1 macrophage activation.

Macrophages in acute allograft rejection The allograft is subject to surveillance by the recipient immune system upon implantation. The degree of tissue injury sustained during the initial period of IRI may or may not facilitate this process. The first step of the allo-immune response involves presentation of donor antigen-derived peptide, bound to MHC II, to recipient TH cells. Normally, professional APCs such as DCs mediate this process. In a pro-inflammatory environment, such as an allograft subjected to recent IRI, additional cells such as macrophages and B cells can be induced to express MHC II and serve as APCs. Unlike DCs, they lack sufficient co-stimulatory capacity to activate naïve TH cells and instead preferentially target the activation of primed TH cells. Thus, macrophages may propagate and amplify an established allo-immune response. There are three pathways of antigen presentation. Shortly after the allograft has been implanted, donor APCs are capable of presenting allogeneic peptides to recipient TH cells via the direct pathway. As time passes, carrier APCs are gradually replaced by recipient bone-marrow derived APCs which are able to process donor antigens and present these to TH cells via the indirect pathway. A third, semi-direct pathway exists where donor MHC-peptide complexes are shed from the allograft and are phagocytosed by recipient APCs. The donor MHCalloantigen peptide complexes are subsequently externalized on recipient APCs and presented to TH cells. In vitro evidence for monocytes utilizing the semi-direct pathway has been provided by Xu et al. [29], who found that monocytes were able to phagocytose membrane fragments derived from allogeneic endothelial cells, via scavenger receptors, and subsequently presented the antigen/MHC complex to T cells to induce proliferation. Parenchymal cells of the allograft also contribute to the development of the allo-immune response. Endothelial cells are typically the first parenchymal cell encountered by circulating monocytes. In addition to contributing to the semi-direct antigen presentation pathway, endothelial cells elaborate various adhesion molecules that are required to for circulating monocytes to transmigrate into the allograft. The act of trasmigrating through the endothelial barrier may also endow monocytes with additional co-stimulatory capabilities which enhance their potential for T cell activation [30]. In the context of antibody-mediated rejection (ABMR), complement is deposited in peritubular capillaries and monocyte activation may ensue via their complement receptors. Allograft biopsies with ABMR which are C4d(+) in peritubular capillaries have increased numbers of intraglomerular monocytes [31] and macrophages [32] compared to those without peritubular capillary C4d staining. Proximal tubular epithelial cells (PTC) play an important role in the recruitment of circulating monocytes and proliferation of resident tissue macrophages. After endothelial transmigration, immune effector cells migrate within the allograft under the influence of numerous cytokines and chemokines. Chemokines that are involved in monocyte and macrophage recruitment include monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1a (MIP-1a), macrophage migration inhibitory factor (MIF), regulated on activation normal T cell expressed and

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secreted (RANTES, CCL5) and osteopontin. Increased PTC expression of these chemokines in biopsies obtained from cases of human and experimental acute rejection have been shown to be associated with increased monocyte and macrophage accumulation in the allograft [33–37]. Attempts to manipulate monocyte accumulation in rejecting allografts have generated mixed results in experimental animals. Blocking the RANTES/CCR5 pathway resulted in reduced allograft damage and prolonged survival in both renal [38,39] and cardiac [40] allografts, but MIF deficiency did not reduced the severity of allograft rejection [41]. Macrophage proliferation within the allograft is also an important contributor to macrophage accumulation during acute rejection. These are derived from both differentiated circulating monocytes and resident tissue macrophages, although Grau et al. [42] have shown that the former probably makes a more significant contribution. The process is primarily driven by M-CSF produced by both T cells and parenchymal cells and correlates with the degree of macrophage proliferation [43,44]. Blockade of the receptor for M-CSF, c-fms, in a murine model of renal allograft rejection reduced macrophage proliferation by 80% with a resultant 50% reduction in macrophage accumulation, but parameters of T cell involvement remained unchanged [45]. Serum levels of M-CSF in humans are significantly increased during acute allograft rejection [46]. Macrophages play a role in promoting inflammation within the allograft. M1 macrophages elaborate a variety of pro-inflammatory cytokines such as IL-1b, TNF-a, IL-18, IL-12, IFN-c and IL-6. IL-1b activates endothelial cells and induces the expression of various cytokines, chemokines and adhesion molecules. Firm adhesion of monocytes to endothelium mediated by the chemokine RANTES only occurs after endothelial cells are exposed to IL-1b [38]. Expression of high levels of the naturally occurring soluble IL-1 receptor antagonist has been found to be associated with a favorable prognosis in cases of cardiac and renal allograft rejection in humans [47,48]. TNF-a has broad pro-inflammatory actions and, like IL-1b, also promotes monocyte adhesion to endothelium. Both TNF-a protein and mRNA expression are increased in renal and cardiac allografts undergoing acute rejection [49,50]. Similarly, our group has described increased IL-18 mRNA expression in a rat model of renal allograft rejection. IL-18 expression is also associated with increased expression of other pro-inflammatory mediators such as IFN-c, TNF-a and iNOS [51]. Increased IL-18 mRNA expression has also been reported in human allograft biopsies undergoing acute rejection [52]. However, allograft survival in IL-18-/- mice or mice given IL-18-binding protein was not different compared to controls, despite a shift in the phenotype of the rejection response from TH1 to TH2 [53]. Both IL-12 and IFN-c can drive M1 activation of macrophages. As previously suggested, these cytokines may also act in concert with IL-18 to promote a TH1-type response. IL-12 also enhances the cytotoxic activity of CD8+ T cells. IFN-c has a broad array of actions, including iNOS activation, increased MHC I and MHC II expression on target cells, enhanced macrophage phagocytic activity, and promotion of leukocyte adhesion via induction of adhesion molecules. Macrophages may also contribute to tissue damage, and their association with severity of allograft rejection has been well documented in experimental and human cases [54]. Macrophage accumulation can be found early after transplantation [55] and its persistence in the allograft portends a poor prognosis [56]. Girlanda et al. demonstrated that both monocytes and T cell infiltrates are present in rejecting allografts, but allograft dysfunction was only quantitatively associated with monocyte infiltration [57]. The presence of a monocytic infiltrate also reported to discriminate between biopsies with clinical versus subclinical rejection [58]. M1

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macrophages express iNOS, which in turn generates reactive oxygen species that can cause tissue damage. Manipulation this pathway either by iNOS inhibition [59] or administration of a NO scavenger [59] in experimental cardiac allografts significantly prolonged survival and attenuated the allo-immune response. More direct evidence that macrophages mediate tissue damage has been provided via macrophage depletion studies. Our group utilized a rat model of renal allograft rejection whereby macrophages were selectively depleted with liposomal clodronate on days 1 and 3 post-surgery. Functional depletion of up to 75% of macrophages within the allograft was achieved resulting in substantial improvement in terms of allograft histology and a significant reduction in iNOS production [60]. T cell numbers remained unchanged and the cognate T cell response to alloantigen remained intact. The phenotypic plasticity of macrophages raises the possibility of these cells adopting multiple roles in different phases of the alloimmune response. The initial stages of the allo-immune response create a pro-inflammatory milieu that favors the adoption of the M1 phenotype. As inflammation subsides, this may alter the microenvironment to favor M2 differentiation. Definitive proof of existence for this in experimental transplantation is currently lacking, but some indirect evidence supports the possibility. In a study by Dehmel et al., CCR5-/- recipient mice receiving WT renal allografts exhibited reduced macrophage accumulation 42 days post-transplantation. Compared to controls, markers of M2 macrophage activation were up-regulated with reciprocal downregulation of M1 markers. The authors concluded that there was a shift toward M2 macrophage differentiation in CCR5-/- recipients [61]. Depriving cDCs of GM-CSF in cell culture provides a permissive signal for differentiation into immunosuppressive macrophages [62]. Whilst these studies raise the possibility of therapeutic manipulation of macrophage phenotypes, studies to dissect the different roles these macrophages play in transplantation are still required. At this time, the concept of non-specific targeting of macrophages as a therapeutic option to prevent or manage acute rejection should be approached with caution, since it carries a risk of attenuating potentially beneficial effects of selected macrophage populations in promoting tissue repair and immunomodulation. Macrophages in chronic allograft rejection Whilst acute rejection is a significant contributor to allograft loss within the first year after transplantation, chronic allograft nephropathy (CAN) is the main cause of graft loss overall [63]. The pathogenesis of CAN remains unclear, but it is postulated that cumulative injury from ongoing allo-immune and non-immune processes ultimately result in interstitial fibrosis and tubular atrophy [2]. Macrophages may contribute to this through continual induction of low-grade inflammation via mechanisms previously discussed, in addition to ongoing attempts at repair leading to fibrosis. The precise pathways through which this occur remains undetermined but could involve pro-fibrotic mediators such as platelet-derived growth factor and transforming growth factor-b, trophic support for myofibroblasts, and epithelial mesenchymal transition [64]. Chronic antibody-mediated rejection provides another possible pathway to injury. Whilst direct data supporting this is not extensive, multiple mechanisms are postulated based on macrophage biology. Complement fragments generated from fixation of donor-specific allo-antibodies may activate macrophages via complement receptors. Antibody-dependent cytotoxicity can be initiated upon direct binding of antibody to Fc receptors. Such mechanisms remain speculative at present. Experimental data also implicate a role for macrophages in the development of CAN and chronic rejection. Progressive monocyte

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and macrophage infiltration into the glomeruli [65], tubulointerstitum [66], and peritubular capillaries [67] is seen in chronically rejecting allografts in association with progressive glomerulosclerosis and tubulointerstital fibrosis. The pro-fibrotic role of macrophages in kidney disease is also supported by findings in a model of adriamycin nephropathy [68]. Adoptive transfer of M1 macrophages into severe combined immunodeficient mice (hence deficient in T and B cells) with adriamycin nephropathy significantly worsened histological and functional injury. In contrast these effects were attenuated when M2 macrophages were adoptively transferred. The data presented not only highlights the detrimental role macrophages play in chronic inflammation, but also emphasize the potential of anti-inflammatory macrophages as a therapeutic agent if properly manipulated in context. Supportive evidence is also found in human biopsy studies. The intensity of macrophage infiltration early in the course of transplantation is a predictive factor in the development of CAN. This association persists after adjustment for the severity of acute rejection, and allograft function at the time of the biopsy [69]. Another study by Croker et al. [70] attempt to evaluate predictive factors for graft survival in biospies with CAN. Only the degree of macrophage inflammation was predictive of graft survival throughout the life of the transplant. In addition, increased expression of thromboxane A synthase mRNA was found in association with the macrophage infiltrates. This implies ongoing persistence macrophages within the allograft may contribute to ongoing injury via elaboration of inflammatory effector molecules.

Therapeutic applications The many roles monocytes and macrophages play in the alloimmune response makes them an attractive target for therapeutic intervention. The challenge, however, is to selectively target those functions which are deleterious to graft function, whilst preserving or amplifying effects that may be beneficial. A further caveat is that macrophages are also involved in T cell activation, thus making it difficult distinguish direct from indirect effects of macrophage deletion. Macrophage deletion has been achieved using materials such as trypan blue, silica, carrageenan, and anti-macrophage serum. All universally provide some degree of protection against allograft rejection, but they all concurrently exhibit a profound reduction in T cells. Jose et al. successfully and selectively depleted macrophages and achieved significant protection from acute rejection in a rat model by administering liposomal clodronate [60], however such crude strategies are not suitable for use in humans. Recent work using immune-modifying microparticles to achieve selective monocyte removal holds significant potential in this area, having been recently demonstrated to be protective in mouse kidney IRI [71]. Another strategy involves manipulation of the IRI response prior to transplantation. As mentioned, the severity of IRI in part determines the outcome of the subsequent allo-immune response. One option to attenuate the severity of IRI is to subject the tissue to ischemic pre-conditioning. Indeed, remote ischemic pre-conditioning has been employed in cardiac surgery to reduce the incidence of post-operative acute kidney injury [72,73]. Experimental evidence demonstrating the benefit of ischemic pre-conditioning has been shown in a murine renal IRI model [74]. In that study, kidneys subjected to ischemic pre-conditioning had increased numbers of Tregs and reduced signs of histological damage. The protective effects of pre-conditioning were abrogated upon depletion of CD11c+ cells using liposomal clodronate. Administration of TLR ligands, such as HMGB1, to induce ischemic preconditioning is potentially a viable therapeutic option and was found to be effective in experimental kidney IRI [75].

The current standard of treatment for acute rejection is pulse corticosteroids. The drug exerts broad anti-inflammatory and immunosuppressive effects, but is also associated with myriad adverse events such as metabolic disturbances, osteoporosis, and increased risk of infection. Refining the therapeutic targets in acute rejection will both enhance efficacy as well as attenuate unwanted drug toxicity. Macrophages present an attractive option since it is integral in many of the events in the allo-immune response. Possibilities include TLR antagonists, macrophage proliferation inhibitors (specifically targeting M-CSF), chemokine antagonists, inhibitors of macrophage activation and macrophage depletion strategies. Indeed, manipulation of the CCR5/RANTES pathway has been met with mixed success in animal models [38,61,76]. However, this may shift the overall allo-immune response towards a TH2 phenotype, thus potentially exacerbating antibody-mediated rejection responses in the long term [61]. Selective inhibition of inflammatory monocyte recruitment via administration of CCR2 siRNA-laden nanoparticles was shown to be protective in mouse models of IRI and islet transplantation [77]. Administration of TNF-a has been shown to prolong survival of rat cardiac allografts, but this has not successfully been translated to humans. However, this may have resulted from FcR-dependent expansion of regulatory macrophages rather than blocking macrophage activation [78]. Advances in current technology have made cell-based therapies a potentially viable option. Macrophages can potentially be isolated from blood, expanded in vitro to the desired phenotype, and re-infused back into the patient. Hutchison et al. reported the successful outcomes of two live donor renal allografts using infusions of donor-derived regulatory macrophages [79]. These preliminary findings are promising but require confirmation in larger clinical trials. In summary, our understanding of macrophages has evolved from ‘‘merely a phagocyte’’ to a cell with diverse and complex functions. The allograft microenvironment plays a key role in determining whether the macrophage will have a detrimental or beneficial phenotype. Further understanding of macrophage biology will potentially facilitate the development of novel therapeutics for transplantation.

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