Yianzhu Liu

Wenhao Chen

Chenglin Wu

Laurie J Minze

Jacek Z Kubiak

Xian C Li

Malgorzata Kloc
email: mkloc@houstonmethodist.orgrmghobrial@hostonmethodist.org

Rafik Mark Ghobrial

Macrophage/monocyte-specific deletion of RhoA down-regulates fractalkine receptor and inhibits chronic rejection of mouse cardiac allografts

Keywords: RhoA, chronic rejection, fractalkine, CX3CR1, macrophage, actin

Background:

The cellular and molecular mechanisms of chronic rejection of transplanted organs remain obscure. It is known however that macrophages play a critical role in the injury and repair of allografts. Among multiple factors influencing macrophage infiltration to allografts, the fractalkine CX3CL1/ CX3CR1 signaling pathway and actin cytoskeleton, which is regulated by a small GTPase RhoA, are of the utmost importance. To define the role of macrophage/RhoA pathway involvement in chronic rejection we generated mice with monocyte/macrophage specific deletion of RhoA.

Methods: Hearts from BALB/c (H-2d) donors were transplanted into RhoA flox/flox (no Cre) and heterozygous Lyz2 Cre+/-RhoA flox/flox recipients treated with CTLA4-Ig to inhibit early T cell response. Allografts were assessed for chronic rejection and monocyte/macrophage functions.

Results:

The deletion of RhoA inhibited macrophage infiltration, neointimal hyperplasia of vasculature and abrogated chronic rejection of the allografts. The RhoA deletion down-regulated G protein-coupled fractalkine receptor CX3CR1, which activates RhoA pathway and controls monocyte/macrophage trafficking into the vascular endothelium. This in turn promotes, through over-proliferation and differentiation of smooth muscle cells in the arterial walls, neointimal hyperplasia.

Conclusions:

Our finding of co-dependence of chronic rejection on monocyte/macrophage CX3CR1/CX3CL1 and RhoA signaling pathways may lead to development of novel anti-chronic rejection therapies.

Introduction

The chronic rejection of transplanted organs is the major and still unresolved problem in transplantation. Although phenomenon of chronic rejection, by virtue of its complexity and multifactoriality is still not well understood, it is known that monocytes and macrophages play an important role in this process [START_REF] Mannon | Macrophages: Contributors to allograft dysfunction, repair or innocent bystanders?[END_REF][START_REF] Salehi | The divergent roles of macrophages in solid organ transplantation[END_REF].

Mammalian organs (including heart) contain various subsets of macrophages with different origin (embryonic yolk sack versus blood or bone marrow-derived) and functions [START_REF] Davies | Tissue-resident macrophages: then and now[END_REF][START_REF] Epelman | Origin and functions of tissue macrophages[END_REF][START_REF] Murray | Protective and pathogenic functions of macrophage subsets[END_REF][START_REF] Sica | Macrophage plasticity and polarization: in vivo veritas[END_REF]. The injury or other organ/tissue stress, including transplantation, drives the production of bone marrow-derived monocytes, which via circulation enter target organ/tissue where they differentiate into dendritic cells and macrophages [START_REF] Geissmann | Development of Monocytes, Macrophages, and Dendritic Cells[END_REF]. The subset of these monocyte/macrophages, expressing high level of chemokine receptor CX3CR1 is specifically targeted to the endothelium of blood vessel walls, which produce high level of CX3CL1 chemokine (fractalkine; [START_REF] Geissmann | Development of Monocytes, Macrophages, and Dendritic Cells[END_REF][START_REF] Tacke | Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques[END_REF]. Interestingly, one of the hallmarks of chronic rejection is the remodeling (through the over-proliferation and differentiation of smooth muscle cells) of the arterial walls, resulting in neointimal hyperplasia, vessel occlusion and eventual failure of the graft [START_REF] Pilmore | Early up-regulation of macrophages and myofibroblasts: a new marker for development of chronic renal allograft rejection[END_REF][START_REF] Skaro | Donor versus recipient: Neointimal cell origin in allograft vascular disease[END_REF]. The smooth muscle over-proliferation and differentiation during neointimal hyperplasia as well as development of atherosclerotic plaque depend selectively on CX3CR1 expressing monocytes and CX3CR1/CXC3L1 pathway [START_REF] Tacke | Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques[END_REF][START_REF] Kumar | Bone marrow-derived CX3CR1 progenitors contribute to neointimal smooth muscle cells via fractalkine CX3CR1 interaction[END_REF][START_REF] Kumar | Role of CX3CR1 receptor in monocyte/macrophage driven neovascularization[END_REF][START_REF] Zhang | Chemokine CX3CL1 and its receptor CX3CR1 are associated with human atherosclerotic lesion vulnerability[END_REF]. The targeted deletion of CX3CR1 inhibits these processes and also prolongs cardiac allograft survival in mouse model system [START_REF] Haskell | Targeted deletion of CX(3)CR1 reveals a role for fractalkine in cardiac allograft rejection[END_REF]. In addition, it has been shown that CX3CR1/CX3CL1 response involves actin polymerization during cell movement [START_REF] Dichmann | Fractalkine induces chemotaxis and actin polymerization in human dendritic cells[END_REF][START_REF] Gevrey | Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine)[END_REF]. The CX3CR1 belongs to the family of G protein-coupled receptors [START_REF] Gilman | G proteins: transducers of receptor-generated signals[END_REF] , which upon ligand binding undergo conformational changes into the guanine nucleotide exchange factors (GEFs; 18; Fig. 8). The GEFs, in turn, activate RhoA and thus, indirectly, regulate actin polymerization pathway [START_REF] Bos | GEFs and GAPs: critical elements in the control of small G proteins[END_REF]. Previous studies on chronic rejection showed that RhoA/ROCK pathway interference (using Y-27632 inhibitor) disrupts actin and actin-related cell functions [START_REF] Allen | Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages[END_REF][START_REF] Kloc | RhoA Cytoskeletal Pathway to Transplantation[END_REF][START_REF] Ridley | Rho family proteins and cell migration[END_REF][START_REF] Vicente-Manzanares | Cell migration at a glance[END_REF], affects macrophages and abrogates chronic rejection of mouse cardiac allografts [START_REF] Liu | ROCK inhibition impedes macrophage polarity and functions[END_REF].

Recently, we also showed that RhoA deletion causes disregulation of RhoA pathway and changes actin organization in murine peritoneal macrophages [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF].

All these data prompted us to study how the targeted deletion of RhoA in the monocyte/macrophage lineage affects macrophages and chronic rejection of cardiac allografts in transgenic mouse model system. Here we show that deletion of RhoA down-regulates monocyte/macrophage CX3CR1/CX3CL1 signaling, inhibits macrophage infiltration and vessel occlusion, and abrogates chronic rejection of mouse cardiac allografts. (25 29). RhoA deletion in Lyz2 Cre+/-RhoA flox/flox mice is based on the principle behind the Lyz Cre system of deletion of floxed genes, and the fact that the activity of Lyz2 promoter is specific for monocyte/macrophage lineage [START_REF] Clausen | Conditional gene targeting in macrophages and granulocytes using LysMcre mice[END_REF].

Materials and Methods

Animals

Heart Transplantations

Heart allografts from BALB/c (H-2d) donors were heterotopically transplanted into Lyz2 Cre+/-RhoA flox/flox recipients (each experimental group consisted of 3-5 animals). Recipients received CTLA4-Ig (0.25 mg i.p. on day 2 and day 4 posttransplantation). Control groups consisted of RhoA flox/flox (no Cre) and heterozygous Lyz2 Cre+/-RhoA +/flox recipients treated with CTLA4-Ig. Additional control groups (described in chapter 4 of the Result section (dealing with the level of CX3CR1 expression in blood and bone marrow monocytes) included wild type and RhoA-deleted naïve (pre-transplantation, untreated) mice.

Histology, Neointimal index, collagen deposition and immunostaining

The paraffin sections of transplanted hearts were stained with VVG (Verhoeff- Van Gieson) and Trichrome Masson and immunostaining as described in Liu et al. [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF]. The VVG stained sections were used to calculate neointimal index (vessel occlusion). For comparison of vessel occlusion we inspected 5-8 vessels from each mouse (3 mice per experimental group), and 20 vessels were used to calculate neointimal indexes. Actin, vinculin, transferrin receptor and CX3CR1 immunostaining were performed as in Liu et al [START_REF] Liu | ROCK inhibition impedes macrophage polarity and functions[END_REF][START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF]. The following antibodies against macrophage markers were used: F4/80 (BioLegend, USA or Serotec, USA), Mac-2 (Cedarlane Laboratories, USA). Transferrin Receptor / CD71 was from ThermoFisher Scientific, USA, CX3CR1 (H-70) from Santa Cruz, USA and secondary antibodies were from ThermoFisher Scientific. In immunostained slides, cells were counted in 3-12 independent samples and statistical significance of cell percentage differences was evaluated using student t test.

Wild type and RhoA deleted mice (3-5 of each) were sacrificed after 30-50 days post-transplantation. The neointimal index and collagen occupied area in Masson stained samples was calculated (collagen/total area) using Image-pro plus 6.0 as decscribed previously [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF].

Apoptosis assay

Fixed heart sections were stained with Tunel-DeadEnd™ Fluorometric TUNEL System (Promega, USA) according to the manufacturer protocol and immunostained with Mac-2 primary antibody and Alexa Fluor 546 conjugated goat anti rat IgG (Life Technologies, USA) secondary antibody.

Peritoneal macrophage apoptosis assay was performed as described in Liu et al.

Monocyte isolation, flow cytometry, RTPCR and BRDU assay

Monocytes were isolated from peripheral blood collected form the mouse tails in the presence of heparin. For bone marrow monocyte isolation, mice were sacrificed and tibia and femur were removed. Bone marrow cells were flushed out bones with DMEM medium. Monocytes were used for flow cytometry with following antibodies (all from BioLegend, USA): anti-Cd45, CD11b, CD115, CX3CR1 and CCR2. Flow cytometry on blood and bone marrow monocytes and peritoneal macrophages and RTPCR were performed as described in Liu et al. [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF]. Flow cytometry on macrophages isolated from heart samples was performed as follows: Hearts were minced finely and digested with shaking in DMEM containing collagenase II for 30min at 37 0 C. The digested heart tissue was filtered through 70μM filters and pelleted by centrifugation in DPBS. For flow cytometry viable cells were stained using Zombie Aqua™(Biolegend) for 30min at 4 0 C. Nonspecific binding of antibodies was blocked by incubation with CD16, after which cells were labeled with CD45, CD11b, F4/80, CD206, CX3CR1 and CCR2 (all from BioLegend, USA). Antibodies were washed off and the samples were fixed/permeabilized in cyto fix/cytoPerm BD Bioscience). For proliferation assay the BRDU labeled DNA was digested for 1 hour with DNase (Sigma) at 37 0 C, and then samples were stained with anti-BRDU antibody (BioLegend).

Results

Deletion of RhoA inhibits chronic rejection and macrophage infiltration of cardiac allograft

We previously showed that macrophages isolated from Lyz2 Cre+/-RhoA flox/flox mice were deficient in expression of RhoA mRNA and its protein [START_REF] Leung | Modulation of Endocytic Traffic in Polarized Madin-Darby Canine Kidney Cells by the Small GTPase RhoA[END_REF]. Here we performed heterotopic heart transplantations into the Lyz2 Cre+/-RhoA flox/flox recipients. We found that treatment of cardiac allograft recipients (RhoA flox/flox (no Cre; further in the text and figures called the wild type), and heterozygous Lyz2 Cre+/-RhoA flox/flox (further called the RhoA KO) with CTLA4-Ig (0.25 mg i.p. on day 2 and 4 post-transplantation), which blocks early T cell response, prolonged graft survival to > 50 days (Fig. 1A) but histologically, the heart allografts demonstrated prominent vascular occlusion (Fig. 1B,D) and fibrosis (Fig. 1E,G), which are characteristic for chronic rejection, impaired heart muscle integrity (Fig. 1H), and heavy total and inflammatory macrophages infiltration (Fig. 2A,C,E). In contrast, cardiac allografts from Lyz2 Cre+/-RhoA flox/flox recipients treated with CTLA4-Ig showed not only prolonged survival (Fig. 1A) but also the abrogation of chronic rejection with low (statistically significant) vascular occlusion (Fig. 1C,D) and markedly improved heart muscle integrity (Fig. 1I). Histological examination showed that these allografts had reduced, although statistically nonsignificant, collagen deposition (Fig. 1F,G). The immunostaining with total (F4/80) and activated macrophage (Mac2) markers showed that allografts from Lyz2 Cre+/- RhoA flox/flox recipients had statistically significant reduction of infiltration with total and activated (inflammatory) macrophages (Fig. 2B,D,E).

Cardiac allografts show negligible apoptosis and RhoA deleted

macrophages are not more prone to apoptosis induction than control macrophages.

It is known that RhoA pathway regulates apoptosis [START_REF] Tsai | RhoA/ROCK1 signaling regulates stress granules formation and apoptosis[END_REF]. Thus, one of the explanations for low macrophage infiltration of the allografts from RhoA-deleted (Lyz2 Cre+/-RhoA flox/flox ) recipients is the possibility of heightened apoptosis of macrophages within the graft. To address this issue we performed apoptosis Tunel assay on transplanted hearts procured 50 days post-transplantation. treatment. Flow cytometry analysis showed (Fig. 3D) that apoptosis in untreated and TNF treated control peritoneal macrophages was comparable to apoptosis in RhoA-deleted peritoneal macrophages. This indicates that RhoA-deleted macrophages are not more prone to apoptosis induction than control macrophages.

RhoA deletion does not impair bone marrow proliferation and frequency of blood monocytes

Knowing that RhoA besides apoptosis also regulates cell proliferation [START_REF] Yang | Loss of RhoA expression prevents proliferation and metastasis of SPCA1 lung cancer cells in vitro[END_REF] we considered another possible explanation for low macrophage infiltration of the allografts from RhoA-deleted recipients. The possibility was that already in naïve mice the RhoA deletion caused reduction of macrophage precursors in the bone marrow, which would resulted in the lower number of monocytes released to the blood. The BRDU proliferation assay and flow cytometry of bone marrow of naïve (pre-transplantation) mice showed that there was no statistically significant difference in the bone marrow proliferation (Fig. 4A,B), and in the frequency of CD45 + /CD115 + /CD11b + bone marrow monocytes, which are potentially able to migrate into the blood i.e. are CCR2 positive (Fig. 4C,D) between control and RhoA-deleted naïve mice. Moreover, there was also no significant difference between the number of CD115 + /CD11b + monocytes in the blood of control and RhoA-deleted mice (Fig. 4E,F). These results strongly suggest that similar to lack of apoptosis, the reduction in macrophage precursors (monocytes) number is not a likely cause of low macrophage infiltration observed in the allografts of RhoAdeleted mice.

RhoA deletion down-regulates fractalkine receptor CX3CR1 in the monocytes and macrophages.

Another possible explanation for low macrophage infiltration of the allografts is that the RhoA-deleted mice were impaired in the chemotactic recruitment of monocytes/macrophages to the allograft. The most important pathways involved in the recruitment of monocytes/macrophages to the inflammation/injury site are CX3CL1/CX3CR1 and CCL2/CCR2 chemotactic pathways. The fractalkine (CX3CL1) -a chemokine abundantly expressed by endothelial cells of vasculature, chemoattracts monocytes/macrophages expressing CX3CR1 receptor, to the blood vessel walls. Thus, we checked what was the level of CX3CR1 expression in blood and bone marrow monocytes in control and RhoAdeleted naïve (pre-transplantation) mice. Flow cytometry analysis clearly showed that there was a statistically highly significant reduction of CX3CR1 expression in bone marrow and blood monocytes from RhoA-deleted mice (Fig. 5 A-D). For comparison we also checked the expression of CCR2 -the receptor of CCL2 chemokine and found that the frequency of CCR2 expressing blood monocytes was slightly lower (at the border of being statistically significant) in RhoA deleted mice (Fig. 5E,F). We also checked the level of CX3CR1 protein and mRNA expression in peritoneal macrophages. Western blot and RTPCR analyses showed that deletion of RhoA down regulated CX3CR1 protein expression in peritoneal macrophages while the expression of CCR2 receptor remained unchanged (Fig. 5). We also found that upon RhoA deletion there is up-regulation of CX3CR1 mRNA (Fig. 5 I). We believe that the up-regulation of CX3CR1 mRNA is a compensatory response to the insufficient level of receptor protein and faulty receptor recycling (see below and Discussion). Taken together all these results strongly suggest that the impairment of fractalkine (CX3CL1/CX3CR1) chemotactic pathway is the main reason for the low abundance of macrophages in the allografts transplanted into RhoA-deleted recipients.

RhoA deletion changes actin cytoskeleton and impairs CX3CR1 association with endosomes

Actin staining of RhoA-deleted macrophages showed profound changes in actin cytoskeleton organization caused by inability to disassembly focal adhesions and retract the tail (described in detail in Liu et al. [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF] and Fig. 6). Because it is known that receptor recycling i.e. translocation to the membrane and its post-activation internalization and degradation depend on endosome pathway, which in turn, is regulated by actin cytoskeleton [START_REF] Leung | Modulation of Endocytic Traffic in Polarized Madin-Darby Canine Kidney Cells by the Small GTPase RhoA[END_REF][START_REF] Garnacho | RhoA activation and actin reorganization involved in endothelial CAM-mediated endocytosis of anti-PECAM carriers: critical role for tyrosine 686 in the cytoplasmic tail of PECAM-1[END_REF] we wanted to see if the RhoA deletion influenced CX3CR1 association with endosomes. The co-immunostaining with antibodies against CX3CR1 and endosomal marker transferrin receptor (CD71) showed (Fig. 6) that while in control macrophages the CX3CR1 co-localizesd with endosomes, in RhoA-deleted macrophages the CX3CR1 association with endosomes was disrupted and it aggregated at macrophage membrane instead (Fig. 6 D, arrows, andE).

RhoA deletion decreases frequency of CX3CR1 + macrophages in the allograft

Because the deletion of RhoA down-regulated expression of CX3CR1 in blood and bone marrow monocytes we wanted to see if this had a bearing on identity of macrophages present in the allograft. Flow cytometry analysis showed that the allografts from RhoA-deleted mice had significantly less F4/80 + /CD11b + macrophages than allografts from control mice (0.67% versus 2.11%; Fig. 7 A-C), while the macrophage proliferation remained unchanged (Fig. 7 H,I). The analysis of the CX3CR1 and CCR2 expression in F4/80 + /CD11b + macrophages showed that both in control and RhoA deleted mice all these macrophages expressed high level of CX3CR1 and CCR2 receptors (Fig. 7 D-G). This indicates that only the monocytes/macrophages expressing high level of CX3CR1/ CCR2 were efficiently recruited to the allografts. Thus, the reduction of CX3CR1 expression in monocytes/macrophages in RhoA-deleted mice resulted in a very low abundance of CX3CR1 + monocytes/macrophages recruited into the allograft, which in turn drastically reduced the CX3CR1-dependent immune response.

Discussion

We showed here that the monocyte/ macrophage lineage-targeted deletion of RhoA down-regulates fractalkine receptor CX3CR1 in monocytes and macrophages, decreases macrophage infiltration and abrogates arterial neointimal hyperplasia and chronic rejection of cardiac allografts in transgenic mouse model system.

RhoA is a member of small GTPase protein family and regulates actin dependent cell functions including motility of monocytes and macrophages [START_REF] Cherfils | Regulation of small GTPases by GEFs, GAPs, and GDIs[END_REF][START_REF] Bos | GEFs and GAPs: critical elements in the control of small G proteins[END_REF][START_REF] Allen | Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages[END_REF]. During inflammatory response, the infiltration of the inflamed tissue/organ, depend on the subpopulation of chemokine receptors belonging to the G protein-coupled proteins family. These receptors (including fractalkine receptor CX3CR1), upon binding their chemokine ligands undergo conformational changes and become the GEFs [START_REF] Gilman | G proteins: transducers of receptor-generated signals[END_REF], which activate RhoA and regulate actin-dependent cell functions.

The fractalkine, which is expressed by endothelial cells of the blood vessel walls, recruits macrophage precursors (monocytes) and macrophages in the vicinity of blood vessels where they mount inflammatory responses , collagen deposition (fibrosis) and neointimal hyperplasia [START_REF] Kumar | Bone marrow-derived CX3CR1 progenitors contribute to neointimal smooth muscle cells via fractalkine CX3CR1 interaction[END_REF][START_REF] Kumar | Role of CX3CR1 receptor in monocyte/macrophage driven neovascularization[END_REF][START_REF] Apostolakis | CX3CR1 receptor is up-regulated in monocytes of coronary artery diseased patients: impact of pre-inflammatory stimuli and renin-angiotensin system modulators[END_REF][START_REF] Apostolakis | Chemokines and atherosclerosis: focus on the CX3CL1/CX3CR1 pathway[END_REF][START_REF] Liu | Fractalkine/CX3CR1 and atherosclerosis[END_REF]. Recent studies indicate that CX3CR1/CX3CL1 pathway is responsible for the monocyte/macrophage directed migration into artherosclerotic plaque [START_REF] Tacke | Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques[END_REF] and for smooth muscle differentiation and neiontima formation in vessel wall after the injury [START_REF] Kumar | Bone marrow-derived CX3CR1 progenitors contribute to neointimal smooth muscle cells via fractalkine CX3CR1 interaction[END_REF][START_REF] Kumar | Role of CX3CR1 receptor in monocyte/macrophage driven neovascularization[END_REF][START_REF] Zhang | Chemokine CX3CL1 and its receptor CX3CR1 are associated with human atherosclerotic lesion vulnerability[END_REF]. It has been also

shown that CX3CR1 knockout prolongs survival of cardiac allografts in mouse model system ( 14) Recently, we showed that RhoA pathway interference affected actin organization and macrophage phenotype [START_REF] Liu | ROCK inhibition impedes macrophage polarity and functions[END_REF][START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF]. However, we showed that in in vitro assay, the RhoA-deleted peritoneal macrophages were able to degrade extracellular matrix (a prerequisite for intra-and inter-tissue migration) and migrate normally [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF]. Although this observation does not prove that RhoA-deleted macrophages can migrate normally in vivo, it strongly suggests that low macrophage infiltration in the allografts in RhoA-deleted mice is not caused by the impairment of macrophage migration but rather a defect in their recruitment. In addition, although we have not directly proven that macrophages actually cause chronic rejection, our findings indicate that macrophages are crucial for development of chronic rejection.

Considering all these data we propose the following scenario of events (Fig. 8,9). Under naïve conditions (untransplanted mice) majority of bone marrow derived and blood monocytes and peritoneal macrophages express CX3CR1 receptors [START_REF] Tacke | Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques[END_REF]. The organ injury such as transplantation results in increased production and release of inflammatory chemokine (CX3CL1, fractalkine) and high recruitment of monocytes/macrophages to the allograft. The interaction of CX3CR1 receptor (which is the G coupled protein) with CX3CL1 causes conformational changes of the receptor into GEF (Fig. 8). The GEF activates RhoA, which in turn, changes actin polymerization/dynamics, which promotes monocyte/macrophage migration to the blood vessel wall (main source of CX3CL1), differentiation of smooth muscle cells in the wall and hyperplasia of neointima. This, over time, occludes blood vessels and leads to chronic rejection/organ failure (Fig. 8,9). The deletion of RhoA causes faulty RhoAdependent actin polymerization/dynamics, which interferes with normal recycling (and transcription/translation) of CX3CR1 receptors and leads to decreased expression of CX3CR1 in monocytes and macrophages (Fig. 8), which in turn reduces CX3CR1/CX3CL1-dependent recruitment to the allografts. Our study showed that deletion of RhoA caused decrease in CX3CR1 protein expression and increase in CX3CR1 mRNA level. It is known that the recycling/turnover of G protein coupled receptors, such as CX3CR1 occurs through endocytotic/exocytotic pathway and is actin-and RhoA-dependent (29, 30 34).

Proper receptor recycling (turnover), in turn, determines receptor's expression at transcriptional (mRNA) and translational (protein) level. We showed that RhoA deletion disrupted CX3CR1 association with endosomes. Thus, we believe that the increase in the expression of CX3CR1 mRNA, which we had observed in RhoA-deleted macrophages, is a compensatory response to the low expression of its protein resulting from a faulty CX3CR1 turnover (Fig. 8). All these defects, upon transplantation, lead to a dramatic decrease in the abundance of CX3CR1 positive macrophages in the allograft and its vasculature, decrease in CX3CR1/CX3CL1-dependent neointimal hyperplasia and resulted in abrogation of chronic rejection (Fig. 8,9).

It is known that the monocyte/macrophage inflammatory response does not depend exclusively on CX3CR1/CX3CL1 signaling but also, among multitude of other pathways, on the CCR2/CCL2 pathway [START_REF] Tacke | Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques[END_REF][START_REF] Fu | Monocyte chemoattractant protein-1/CCR2 axis promotes vein graft neointimal hyperplasia through its signaling in graft-extrinsic cell populations[END_REF]. The CCR2 receptor belongs to CC chemokine receptors family, which similar to CX3CR1 are the G protein-coupled proteins involved in regulation of, and regulated by, the RhoA pathway. Our study showed that while RhoA-deletion had impaired CX3CR1 it had only slight effect on CCR2 expression in blood monocytes. The question is why RhoA-deletion has different effect on two different receptors from the (broadly) same family of G protein-coupled receptors. Although answering this question requires further study, one of the possible explanations is that the changes caused by RhoA deletion are detrimental to the receptors with a specific structure only. Another explanation is that because the CCL2/CCR2 mediates RhoA activity through Smad3/ MAPK [START_REF] Fang | CCL2/CCR2 chemokine signaling coordinates survival and motility of[END_REF] and/or PKC signaling (37) while the CX3CR1/CX3CL1 interactions with RhoA pathway are more direct (12) the CX3CR1/CX3CL1 pathway is more "sensitive" to RhoA deletion.

Another fascinating possibility, which would explain the difference between the involvement and importance of CX3CR1 versus CCR2 pathways in chronic rejection may be related to selective utilization of CX3CR1 pathway, which was described in development of atherosclerotic plaque [START_REF] Tacke | Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques[END_REF] or the phenomenon of CCR2 to CX3CR1 receptor switching, which was described by Barlic et al. (38).

These authors showed that human macrophages present in human coronary artery have ability to switch CCR2 off and CX3CR1 on, which causes inhibition of CCR2-dependent migration in favor of CX3CR1-dependent macrophage accumulation in the vessel wall. If such switch also occurs during anti-allograft immune response than it would be quite understandable why the inhibition of CX3CR1 but not the CCR2 pathway had such profound inhibitory effect on neointimal hyperplasia and chronic rejection.

Recent study from our laboratory (25) also showed that RhoA deletion upregulates the activity of its downstream effector ROCK1 in peritoneal macrophages, and that this effect depends on non-apoptotic Caspase-3. Thus, in the future, it will be important to study if and how Caspase-3 regulates monocyte/macrophage CX3CR1/CX3CL1 pathway during chronic rejection.

  , generation of RhoA-deficient mice, and genotyping. Breeding and all experiments were performed according to The Methodist Hospital Research Institute's animal care and use NIH standards in concordance with the "Guide for the Care and Use of Laboratory Animals" (DHHS publication No. (NIH) 85-23 Revised 1985), the PHS "Policy on Humane Care and Use of Laboratory Animals" and the NIH "Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training." RhoA flox/flox mice (gift from dr Richard A. Lang from UC Department of Pediatrics and UC Department of Ophthalmology, Cincinnati Children's Hospital, Cincinnati, Ohio) were crossbred with B6.129P2-Lyz2tm1(cre)Ifo/J mice (JAX® Mice (Bar Harbor, Maine, USA) to generate Lyz2 Cre+/-RhoA flox/flox mice and genotyped as described in Liu et al.

Figure

  Figure 3A-C clearly shows that in contrast to high level of apoptosis in the acutely

Further

  studies are needed to eliminate a very remote possibility that the differences between the macrophage number in the grafts of RhoA-deficient and wild type recipients might in part reflect differences in severity of ischemia reperfusion injury of the grafts or in the properties of acute alloimmune responses. If these were true than the low number of macrophages in RhoAdeficient mice could then have reflected the lesser degree of the initial damage of allograft tissue. Although our findings can potentially lead to the development of novel anti-chronic rejection therapies in human transplantation, one has to remember, when extrapolating mouse model results to human situation, that human allograft vasculopathy (based on fibroblast functions and smooth muscle cells proliferation) differ from that in the mouse (based on smooth muscle cells proliferation only). Further studies are also necessary to establish what is the indirect effect of monocyte/macrophage specific RhoA-deletion on other types of immune cells participating in immune response toward the allograft. Conclusions: Our finding that monocyte/macrophage specific deletion of RhoA inhibits cardiac allograft chronic rejection via down-regulation of CX3CR1/CX3CL1 pathway may lead to development of novel anti-chronic rejection therapies. breast cancer cells through Smad3 protein-and p42/44 mitogenactivated protein kinase (MAPK)-dependent mechanisms. J Biol Chem. 2012; 287:36593-36608. 37. Stamatovic SM, Dimitrijevic OB, Keep RF, Andjelkovic AV. Protein kinase Calpha-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability. J Biol Chem. 2006; 281: 8379-8388 38. Barlic J, Zhang Y, Foley JF, Murphy PM. Oxidized lipid-driven chemokine receptor switch, CCR2 to CX3CR1, mediates adhesion of human macrophages to coronary artery smooth muscle cells through a peroxisome proliferator-activated receptor gamma-dependent pathway. Circulation. 2006; 114: 807-819.

Figure 1 .

 1 Figure 1. Macrophage specific RhoA deletion inhibits chronic rejection (A) Comparison of cardiac allograft survival in RhoA flox/flox (no Cre, labeled wild type, WT) and Lyz2 Cre+/-RhoA flox/flox (labeled RhoA KO) recipients. Recipients

Figure 2 .

 2 Figure 2. Macrophage specific RhoA deletion inhibits macrophage

Figure 3 .

 3 Figure 3. Apoptosis in cardiac allografts and peritoneal macrophages

Figure 4 .

 4 Figure 4. RhoA deletion does not impair bone marrow and blood

Figure 5 .

 5 Figure 5. RhoA deletion down-regulates expression of CX3CR1 receptor in

Figure 6 .

 6 Figure 6. RhoA deletion affects actin cytoskeleton and inhibits association

Figure 7 .

 7 Figure 7. RhoA deletion reduces number of CX3CR1 + macrophages in the

Figure 8 .

 8 Figure 8. Hypothetical model of the effect of RhoA, actin and

Figure 9 .

 9 Figure 9. Hypothetical model of the effect of monocyte/macrophage-

Acknowledgments/Funding

We are extremely grateful for the support from William Stamps Farish Fund and Donald D. Hammill Foundation.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose