The Divergent Roles of Macrophages in Solid Organ Transplantation

Abstract

Purpose of review

This review summarizes the phenotype and function of macrophages in the context of solid organ transplantation and will focus on fundamental insights into their paradoxical pro-inflammatory versus suppressive function. We will also discuss the therapeutic potential of regulatory macrophages in tolerance induction.

Recent findings

Macrophages are emerging as an essential element of solid organ transplantation. Macrophages are involved in the pathogenesis of ischemia reperfusion injury, as well as both acute and chronic rejection, exacerbating injury through secretion of inflammatory effectors and by amplifying adaptive immune responses. Notably, not all responses associated with macrophages are deleterious to the graft, and graft protection can in fact be conferred by macrophages. This has been attributed to the presence of macrophages with tissue-repair capabilities, as well as the effects of regulatory macrophages.

Summary

The explosion of new information on the role of macrophages in solid organ transplantation has opened up new avenues of research and the possibility of therapeutic intervention. However, the role of myeloid cells in graft rejection, resolution of rejection and tissue repair remains poorly understood. A better understanding of plasticity and regulation of monocyte polarization is vital for the development of new therapies for the treatment of acute and chronic transplant rejection.

Introduction

Macrophages and their precursors, monocytes, constitute an essential component of the innate immune system and form the first line of defense against pathogens [1]. Macrophages have the capacity to differentiate into a variety of phenotypes in response to cues from the microenvironment, and it is this notable phenotypic plasticity that governs the expression of the broad range of inducible effectors [2]. In the transplant setting, macrophages can cause allograft injury, tissue remodeling or have immunoregulatory/suppressive effects depending on their state of activation [2–4]. In response to stimuli, infiltrating macrophages differentiate preferentially into “classically activated” or “alternatively-activated” subsets with markedly different functions [3, 4]. Classically activated macrophages, also referred to as M1 macrophages, develop in response to IFN-γ and engagement of Toll-like receptors (TLRs) by microbial products [2, 5]. They generally display a proinflammatory phenotype expressing high levels of CD86, iNOS, TNF-α, IL-1 and IL-6 (Figure 1a) [4]. In contrast, exposure to IL-4 or IL-13 leads to the development of “alternatively-activated” or “wound-healing” macrophages, also referred to as M2 macrophages, that display markers of alternative activation including CD206, the scavenger protein CD163, arginase-1, and IL-10 (Figure 1b) [6–8]. The M2 subset of macrophages is not a uniform population and is further subdivided into M2a, M2b, and M2c. Within this subset, M2a macrophages, generally referred to as alternatively activated macrophages, are induced by IL-4 and IL-13, with surface expression of CD163, CD206, CD209, IL-4, FcεR, and Dectin-1. Ligation of macrophage FcRs by IgG complexes coupled with TLR or CD40/CD44 engagement induces a Type II activation [2, 3, 9], which corresponds with an M2b phenotype. M2b macrophages are immunoregulatory and produce high levels of IL10, IL-1, IL-6 and TNF-α. M2c macrophages are referred to as deactivated macrophages given their role in down-regulation of pro-inflammatory cytokines, as well as tissue repair and remodeling. This macrophage subset is induced by IL-10, TGF-β, and glucocorticoids and in turn produces large amounts of IL-10 and TGFβ with surface expression of CD163, CD206, RAGE and other scavenger receptors [3, 10]. While these M2 variants have been explored in a variety of disease models [11, 12], they have yet to be characterized in the setting of solid organ transplantation. Regulatory macrophages (M regs), a less well-characterized subtype of macrophages, can suppress T cell function and have been utilized as therapeutic agents in transplantation (Figure 1c) [13, 14]. Regulatory macrophages express iNOS, MHC class II, and PD-L1, though little CD40 or CD86 [15]. M regs are fundamentally distinct and do not express most markers found on M1 or M2 macrophages and have been shown to mitigate acute and chronic inflammation in different disease models [16]. Though regulatory macrophages modulate inflammatory immune responses, these cells do not actively participate in wound healing [15]. Notably, peripheral blood monocytes have been divided into two subsets with distinct function and phenotype. The pro-inflammatory CD14+CD16+ subset exhibits high expression of proinflammatory cytokines [17], while the immunosuppressive monocytes are CD14+CD163+ and exhibit immunosuppressive mechanisms including IL-10 production [18]. The role of peripheral blood monocyte subsets in transplantation has been minimally studied, with contradictory findings, and requires further investigation [18, 19].

Figure 1. Macrophage plasticity and function in the context of allograft rejection.

a) M1 macrophages are classically activated, damage graft endothelium, recruit additional leukocytes, and mediate tissue injury. They are the dominant phenotype in acute rejection and their activity can be modulated by blockade of their activation or the factors they produce. b) M2 macrophages are alternatively activated, mediate tissue repair and injury resolution, and promote fibrosis. This subset is predominantly found in chronically-damaged allografts. c) Mregs are activated in a fashion distinct from the other two subsets. They modulate anti-inflammatory response, have T cell suppressive capacity, and are being investigated for use in cell-based therapy.

Macrophages in Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) is a multifactorial process, involving both innate and adaptive immunity, which impacts early and late graft dysfunction [20]. Cells of the innate immune system, particularly macrophages, are key potentiators of IRI, participating in both the early stages of injury and in late stage repair [21–25]. Animal models of IRI show that injury is associated with an influx of macrophages, implicating these innate immune cells in augmentation of ischemic injury [26]. One study demonstrated that knocking out CCR2, a receptor for monocyte chemo-attractant protein 1 (MCP-1), protected mice from kidney IRI, correlating with reduced macrophage infiltration [27]. In a murine model of liver IRI, blockade of TIM-1 on CD4 cells inhibited T cell mediated activation of macrophages and mitigated injury [28]. In a clinical study, using the selectin antagonist (rPSGL-1) reduced liver IRI with improved liver function and augmented cytoprotective IL-10, with a reduction in MCP-1, suggesting inhibition of macrophage infiltration [20]. Notably, while inflammatory macrophages contribute to the initial damage during IRI [21], alternatively activated macrophages promote repair following the injury. As such, Huen et al. showed that macrophages in the setting of kidney IRI can be skewed toward a distinct reparative phenotype which supports tubular proliferation and repair in response to GM-CSF [29, 30]. Similarly, a myeloid-specific PTEN knockout conferred protection from liver IRI by promoting development of M2 macrophages in response to TLR engagement. PTEN deficiency resulted in constitutive activation of the pro-survival PI3K pathway, which regulates macrophage differentiation by upregulating miR155. This M2 differentiation correlates with a decrease in expression of certain pro-inflammatory mediators and a marked increase in the anti-inflammatory cytokine IL-10 [31]. Moreover, over-expression of macrophage heme-oxygenase-1, an enzyme with anti-inflammatory properties, imposed an anti-inflammatory or M2 phenotype, selectively inhibiting M1 polarization. When adoptively transferred into mice, these macrophages mitigated injury and inflammation caused by ischemia reperfusion [32]. Collectively, these findings point to an instrumental role for macrophages in the pathophysiology of IRI depending on the nature of the macrophage subset during the time course of injury, as M1 macrophages can mediate the inflammatory process at the onset of ischemic injury, while M2 macrophages are involved in post-injury resolution. As IRI is an antigen independent event, macrophages involved in this process are activated through cytokines and/or engagement of TLRs or other pattern recognition receptors by endogenous ligands generated through cellular damage [33]. Consequently, mice deficient in TLR4 demonstrated reduced IRI after liver transplantation [34], while donor TLR4 was shown to contribute to renal allograft inflammation in humans [35]. A recent study revealed lipocalin2 (Lcn2), a defense mediator expressed in response to TLR activation, plays a crucial role in cardiac IRI, and neutralization of Lcn2 suppressed M1 macrophage polarization and instead mediated skewing of macrophages toward an M2 phenotype. Additionally, Lcn2 treatment suppressed infiltration of macrophages further limiting IRI [36].

Macrophages in Acute Allograft Rejection

Macrophages were first implicated in rejecting renal allografts over fifty years ago [37]. Macrophage accumulation in the allograft is associated with both acute antibody-mediated rejection (AMR), and acute cell mediated rejection [38, 39]. In instances of acute and chronic injury [39] in animal models as well as humans [40, 41], macrophages account for 38–60% of infiltrating leukocytes in rejecting organs [42–45]. Notably, a murine model of pancreatic islet grafts provided evidence of direct destruction of islet tissue by macrophages [46]. The presence of CD68+ macrophage infiltrates is associated with diagnosis of acute rejection in human renal allografts [39, 47–49]. Macrophage depletion has led to amelioration of graft injury and a reduction in pathological features of acute rejection in experimental models [40, 44, 50, 51]. Similarly, inhibition of macrophage accumulation and activation in murine cardiac allografts results in abrogation of graft injury and rejection [43, 52].

Macrophages propagate injury in the setting of AMR [53, 54] and are a distinguishing feature of graft pathology in AMR lesions [49]. In fact, one of the most important diagnostic criteria for AMR in cardiac transplantation is the presence of intravascular macrophages in the capillaries of endomyocardial biopsies [55]. In a clinical study, Kirk and colleagues found that there was a high incidence of AMR associated with infiltrating macrophages in renal transplant patients treated with Campath, a T cell-depleting drug [56, 57]. Similar detrimental effects were observed in a lymphocyte-deficient RAG−/− cardiac murine model of acute AMR [53, 54], adding support to the claim that macrophages are sufficient to induce allograft injury. In this study, passive transfer of anti-donor HLA antibodies induced accumulation of intravascular macrophages in heterotopic cardiac allografts, demonstrating pathological features of injury. In vitro, P-selectin blockade was shown to prevent antibody-mediated monocyte recruitment to endothelial cells, conferring protection from antibody induced damage. This was recapitulated in the above-mentioned murine model of acute AMR [53] and has had promising results in IRI [20], with potential for use in AMR in solid organ transplantation. In the setting of AMR, donor specific HLA IgG antibodies have been shown to recruit monocytes via an FcγR-dependent mechanism [9, 58]. Consequently, eliminating these antibody-FcγR interactions using EndoS, an endoglycosidase that modifies protein glycosylation, and IdeS, an IgG-degrading enzyme, was shown to significantly lessen monocyte recruitment to cardiac endothelium in vitro [58].

These combined findings implicate macrophages as an essential determinant in the induction of acute rejection. Though the exact mechanism by which macrophages mediate injury is not fully understood, in vitro and in vivo studies implicate the production of inflammatory mediators as a central mechanism whereby macrophages contribute to allograft injury [5]. Inside the graft, macrophages release inflammatory mediators such as nitric oxide (iNOS), IL-2, IL-6, IL-12, MCP-1, and TNF-α [40, 44], which activate and damage the microvasculature, recruit leukocytes, and induce donor-specific cytotoxic responses [1]. Studies where macrophages have been depleted, or receptors for leukocyte recruitment antagonized, confirmed the role of macrophage cytokine production and other pro-inflammatory mediators in graft rejection. For instance, chemical macrophage depletion results in a reduction in the severity of acute allograft rejection in rodent models of small bowel transplantation [44, 59]. The reduction in small bowel injury was attributed, in part, to lower expression of inflammatory genes including iNOS, MCP-1 and IL-6, factors associated with M1 macrophages. Blockade of inflammatory cytokines such as TNF-α and iNOS was shown to extend cardiac graft survival, underscoring the importance of macrophage-mediated-inflammation in heart transplant rejection [60, 61]. Similarly, administration of the chemokine receptor antagonist, Met-Rantes, inhibited monocyte adhesion to inflamed endothelium in a rat model of acute cellular renal injury in which monocytes constitute the majority of the infiltrating cells. Correspondingly, the treated animals displayed a decrease in the expression level of several pro-inflammatory cytokines [62, 63]. While M1 macrophages mediate injury, M2 macrophages are generally implicated in injury resolution and tissue remodeling, and therefore, they may promote allograft damage repair; though currently, their role in acute injury remains speculative. Histological studies of murine corneal allografts exhibiting acute rejection revealed the presence of M1 macrophages secreting proinflammatory mediators, while M2 macrophages were detected in the animals that did not reject the transplants [64]. An M1-dominant response was also observed in a rat model of acute renal AMR and in clinical biopsy samples of acutely rejecting kidney allograft recipients [65].

In light of these findings, selective depletion of macrophage subpopulations may be exploited to provide additional insight into the myriad functions of macrophages in the context of acute allograft injury and repair, more specifically targeting M1 macrophages as a therapeutic tactic. Albeit, it might be more prudent to target destructive macrophage subsets for manipulation, such as those skewed toward the M1 phenotype, for manipulation, rather than depletion, as studies suggest that macrophages are plastic and do not remain committed to a single phenotype/activation state [2, 3].

Macrophages in Chronic Allograft Rejection

Chronic rejection is the leading cause of long-term graft failure. The manifestations of chronic allograft rejection include vasculopathy and chronic vascular lesions, often accompanied by sub-endothelial leukocytes, and proliferation of vascular endothelial and smooth muscle cells [66]. Histological sections of chronically rejecting tissues stain positive for macrophage infiltrates, and macrophage labeling has been explored as a means of detecting chronic rejection prior to the onset of graft dysfunction [67]. Intragraft macrophages are associated with worse outcome in renal, liver, and cardiac transplantation in humans as well as animal models [68–70], and macrophages have been shown to directly cause tissue injury and fibrosis. Case studies focusing on the development of chronic allograft nephropathy have emphasized the pivotal role of macrophages in human biopsies culminating in end-stage renal failure [69, 71, 72]. Interestingly, monocytes have been shown to have altered activation levels, exhibited by enhanced TNF-α production, in patients undergoing chronic renal rejection [73].

As in the case of acute rejection, the current view is that macrophages promote worse graft outcome through the release of inflammatory mediators and regulation of cytokine dynamics. Studies conducted during the course of chronic rejection found up-regulation of MCP-1, RANTES, TNF-α, IFN-γ and iNOS among others, correlating with macrophage activation [74]. Yang et al. used a previously established rat renal allograft model to target a variety of macrophage-derived and macrophage-activating soluble mediators implicated in chronic graft rejection. Blocking the actions of TNF-α, IL-12, and IFN-γ reduced macrophage-mediated chronic injury [75]. Macrophage participation in chronically rejecting vascularized grafts can be further modulated by blockade of chemokine-chemokine receptor interactions, as administration of Met-RANTES, an agonist to the chemokine receptor CCR5, to transplant recipients has been successful in significantly lessening chronic injury in cardiac and renal grafts [76, 77]. A macrophage-specific inhibitor, gamma lactone, was successfully used to prevent murine chronic renal allograft nephropathy [68] with a correlative reduction in the levels of macrophage-produced inflammatory mediators. As in acute injury, the impact of macrophages in models of chronic rejection has been assessed through depletion strategies, demonstrating attenuation of chronic lesions and vasculopathy [78].

In patients presenting with chronic allograft nephropathy, mRNA levels of PAI-1, a glycoprotein which promotes fibrosis by inhibiting degradation of the extracellular matrix, were found to be increased in macrophages infiltrating the kidney [72]. These findings identify an additional mechanism where macrophages incite chronic rejection by promoting fibrosis. Fibrosis precedes clinical dysfunction of the allograft and the development of progressive fibrosis in turn has been attributed to M2 macrophages in the context of dysregulated inflammation [48]. Though the majority of M2 macrophages, including M2a and M2c macrophages, are generally considered to demonstrate beneficial reparative characteristics, with regard to ongoing injury, sustained activity may result in the continuous production of various wound-healing growth factors, ultimately becoming a pathological process leading to fibrosis [79]. Consequently, M2 macrophages were identified as the dominant macrophage subset found in chronic lesions [6]. Steroids and calcineurin inhibitors, used routinely in transplantation therapy, have been shown to induce CD163+ M2 macrophage polarization, with a correlative increase in mRNA levels of pro-fibrotic cytokines such as TGFβ-1 and connective tissue growth factor, thus promoting development of fibrosis and at times exacerbating rejection [6, 80]. These recent findings link progression of fibrosis to this subset of macrophages, suggesting that they may serve as a predictive biomarker of chronic rejection and that restricting their activity would serve as a potential therapeutic strategy to protect against macrophage-dependent mechanisms related to fibrosis. Fully understanding the function of the M2 macrophage subset in the setting of chronic rejection requires additional studies.

Macrophages as a therapeutic agent

Though much attention has been given to the detrimental role of macrophages in organ transplantation, limited studies have ascertained that regulatory macrophages have the potential to prolong allograft survival. M regs have been used in immunodeficient mice [81], and in non-immunosuppressed recipients of a mismatched heterotopic heart allografts, to ameliorate symptoms of rejection and prolong allograft survival [15]. Furthermore, administration of M regs to porcine recipients of single lung allografts improved graft prognosis [82].

Presently, it is not fully understood how M regs exert their immunosuppressive effects in vivo, though it is assumed it is controlled by multiple mechanisms. In principle, M regs could directly regulate and suppress polyclonal T cell proliferation and mediate T cell elimination through an iNOS-dependent mechanism and their ability to down-regulate L-selectin levels on T cells, which ultimately prevents T cell activation [15, 83]. Alternatively, M regs may secrete anti-inflammatory mediators, which help promote tissue repair. Consistent with this idea, the suppressive capacity of M regs has been attributed to IFN-γ-induced iNOS [15, 84], which has recently been implicated in macrophage-mediated immune suppression [15, 85].

From a therapeutic viewpoint, regulatory macrophages with the capacity to quell an aberrant inflammatory response could be used as a pharmacological agent for tolerance induction. A recent study showed that M regs can be generated from peripheral blood monocytes for potential use in solid organ transplantation [86]. Two human recipients of kidney allografts were adoptively transferred with donor-derived infusions of M regs and weaned to monotherapy [13]. No incidence of acute or chronic rejection has been observed at 5 years. The absence of acute rejection and lack of signs indicative of subclinical rejection suggested a lack of or attenuation of anti-donor reactivity [87]. In these studies, M regs demonstrated graft protective functions and pre-operative administration of M reg-based therapy was shown to mediate tolerance of the donor allograft. Donor M regs are used instead of recipient M regs, as a study by Riquelme et al. established that the graft-protective effect of M regs is specific to donor cells [15]. The described findings suggest there is a benefit to distinguishing between macrophage subsets present in allograft settings, as depletion of certain subsets of macrophages may prove more beneficial than total macrophage depletion.

Several key clinical concerns remain to be addressed regarding the translation of M reg therapy to clinical transplantation, such as the stability and safety of M regs in vivo and the efficacy of M reg usage in a wide and variable population. Some of these questions are now being addressed in the ONE Study consortium in Europe, aimed at determining the efficacy and safety of administering donor-derived M reg preparations to living-donor solid organ transplant recipients as a cellular immunotherapy, with the ultimate goal of reducing the need for conventional immunosuppression (NCT02085629).

The Effects of Immunosuppressives and therapeutics on Macrophages

Immunosuppressive drugs used routinely for the prevention of allograft rejection have been shown to affect the phenotype and function of macrophages. Macrophages treated with rapamycin, an inhibitor of the serine/threonine kinase mTOR, were impaired in their ability to present antigens and displayed a notable reduction in the expression of CD80 [88]. Rapamycin has also been shown to inhibit production of the inflammatory mediator iNOS in macrophage cell lines [89]. Bortezomib is a protease inhibitor mainly used in the treatment of AMR [90] and has also been found to block T-cell mediated responses [91, 92]. In a murine model of contact hypersensitivity, an inflammatory immune reaction mediated by T cells, Bortezomib treatment resulted in a noted reduction in macrophage infiltration [91]. Furthermore, Bortezomib has been shown to reduce inflammatory cytokine production in macrophages stimulated with LPS in vitro [91, 93]. Use of the calcineurin inhibitors CsA and FK506 has been shown to regulate TLR mediated pathways in myeloid cells and lead to macrophage activation by inhibiting the calcineurin/NFAT pathway. Blocking NFAT leads to activation of the downstream NF-κB and MAPK pathways, and to subsequent production of inflammatory mediators including IL-12 and TNF-α [94, 95]. As mentioned previously, calcineurin inhibitors have also been implicated in the promotion of M2 macrophage differentiation, as identified by the marker CD163 [6]. Butyric acid is used for treatment of auto-immune disorders and has been investigated for tolerance induction in allografts [96]. Butyrate treatment of monocytes in vitro was found to decrease their phagocytic capabilities and to reduce expression of markers including CD14, CD86 and MHCII [97]. In a separate study, butyrate prevented IL-12 production in human monocytes and promoted production of IL-10 [98], suggesting that it might play a role in the development of anti-inflammatory macrophages.

Conclusion

Modulation of graft homeostasis involves the interplay between the various subpopulations of macrophages, which can contribute allograft-destructive or protective mechanisms based on their phenotype and function. Though major advances have been made with regard to an improved understanding of the contribution of macrophages to graft outcome, there is a paucity of clinical data and further studies are warranted to establish a comprehensive understanding of their contribution to graft injury, repair and graft acceptance.

Key points.

• Based on cues from their microenvironment, macrophages differentiate into inflammatory (M1), wound healing (M2), or regulatory macrophages all with distinct functions and phenotypes.

• Macrophages generate inflammatory mediators that contribute to ischemia reperfusion injury and acute and chronic allograft rejection.

• Regulatory macrophages are an attractive candidate for use as an adjunct cell-based therapy to suppress allograft rejection in human transplantation.