MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

BACKGROUND: Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. METHODS: We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. RESULTS: Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. CONCLUSIONS: These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

Proteomics: Its Promise and Pitfalls in Shaping Precision Medicine in Solid Organ Transplantation.

Solid organ transplantation is an established treatment of choice for end-stage organ failure. However, all transplant patients are at risk of developing complications, including allograft rejection and death. Histological analysis of graft biopsy is still the gold standard for evaluation of allograft injury, but it is an invasive procedure and prone to sampling errors. The past decade has seen an increased number of efforts to develop minimally invasive procedures for monitoring allograft injury. Despite the recent progress, limitations such as the complexity of proteomics-based technology, the lack of standardization, and the heterogeneity of populations that have been included in different studies have hindered proteomic tools from reaching clinical transplantation. This review focuses on the role of proteomics-based platforms in biomarker discovery and validation in solid organ transplantation. We also emphasize the value of biomarkers that provide potential mechanistic insights into the pathophysiology of allograft injury, dysfunction, or rejection. Additionally, we forecast that the growth of publicly available data sets, combined with computational methods that effectively integrate them, will facilitate a generation of more informed hypotheses for potential subsequent evaluation in preclinical and clinical studies. Finally, we illustrate the value of combining data sets through the integration of 2 independent data sets that pinpointed hub proteins in antibody-mediated rejection.

Soluble HLA-G levels in heart transplant recipients: Dynamics and correlation with clinical outcomes.

PURPOSE: To describe the evolution of the serum levels of soluble HLA-G (s-HLA-G) during the first 12 months after heart transplantation (HT) and to correlate it with clinical outcomes. METHODS: Observational study based in a single-center cohort of 59 patients who underwent HT between December-2003 and March-2010. Soluble HLA-G levels were measured from serum samples extracted before HT, and 1, 3, 6 and 12 months after HT. The cumulative burden of s-HLA-G expression during the first post-transplant year was assessed by means of the area under the curve (AUC) of s-HLA-G levels over time and correlated with the acute rejection burden -as assessed by a rejection score-, the presence of coronary allograft vasculopathy (CAV) grade ≥ 1 and infections during the first post-transplant year; as well as with long-term patient and graft survival. Mean follow-up was 12.4 years. RESULTS: Soluble HLA-G levels decreased over the first post-transplant year (p = 0.020). The AUC of s-HLA-G levels during the first post-transplant year was higher among patients with infections vs. those without infections (p = 0.006). No association was found between the AUC of s-HLA-G levels and the burden of acute rejection or the development of CAV. Overall long-term survival, long-term survival free of late graft failure and cancer-free survival were not significantly different in patients with an AUC of s-HLA-G levels higher or lower than the median of the study population. CONCLUSIONS: Soluble HLA-G levels decreased over the first year after HT. Higher HLA-G expression was associated with a higher frequency of infections, but not with the burden of acute rejection or the development of CAV, neither with long-term patient or graft survival.

Investigation of Circulating MicroRNA Levels in Antibody-Mediated Rejection After Kidney Transplantation.

BACKGROUND: One of the most important possible complications determining long-term graft survival after kidney transplant is antibody-mediated rejection (ABMR). The criterion standard approach to recognize ABMR is currently the kidney biopsy with histopathologic analysis. However, this test has limitations because of difficulties in timing of sampling, the evaluability of histology because of the questionable representativeness of specimens, and the limited number of this intervention. Hence, new reliable, noninvasive biomarkers are required to detect the development of ABMR in time. METHODS: In this study, we analyzed the clinical data of 45 kidney transplant patients (mean age of 44.51 years, 20 male and 25 female subjects). These participants were recruited into 5 subcohorts based on their clinical status, histologic findings, and level of donor-specific anti-HLA antibodies. Circulating microRNAs (miR-21, miR-181b, miR-146a, miR-223, miR-155, miR-150) in plasma samples were quantified by quantitative polymerase chain reaction and their levels were correlated with the clinical characteristics in different subgroups. RESULTS: The relative expression of plasma miR-155 (P = .0003), miR-223 (P = .0316), and miR-21 (P = .0147) were significantly higher in patients who had subsequent histology-approved ABMR with donor-specific anti-HLA antibody positivity (n = 10) than in the "triple negative" group (n = 21), and miR-155 showed the highest sensitivity (90%) and specificity (81%) to indicate ABMR development based on receiver operating characteristic analysis. CONCLUSIONS: According to our preliminary data, plasma miR-155, miR-21, and miR-223 can indicate the development of ABMR after kidney transplant in correlation with classic clinical parameters. However, future studies with larger number of participants are necessary to further evaluate the diagnostic properties of blood miRNAs in prediction of this life-threatening condition.

The mounted alloimmunity of the iris-ciliary body devotes a hotbed of immune cells for corneal transplantation rejection.

Graft rejection is still the major obstacle causing corneal transplantation failure. However, the underlying pathogenesis remains largely unclear. The iris-ciliary body (I-C) is enriched with blood vessels and various immune cell populations, presumably predisposed to be involved in corneal transplantation rejection. After penetrating keratoplasty, compared to the normal (Nor) and syngeneic (Syn) groups, I-C tissues in the allogeneic (Allo) group displayed stronger alloimmune responses, with more infiltrations of CD45+ inflammatory cells and CD3+ lymphocytes, increased transcriptional levels of pro-inflammatory cytokines, and elevated NF-κB activity. This histopathology was similar to the pathological alterations of corneal allografts. Angiography analysis revealed the abnormal vasculature in the iris during allograft rejection, characterized by vasodilatation, increased vessel density, and vascular permeability. While, immunofluorescence staining showed the intact tight junction of the posterior iris epithelium. In vitro, human microvascular endothelial cells (HMECs) stimulated by tumor necrosis factor-α (TNF-α) showed an increased Evans blue (EB)-albumin leakage, with lower expression of zonula occludens-1 (ZO-1) and Occludin. The increased EB-albumin leakage, up-regulated NF-κB activity, and reduced expression of ZO-1 and Occludin could be partially reversed after cyclosporine A (CsA) administration. In contrast, the barrier function in primary mouse iris pigment epithelial cells (IPEs) after TNF-α treatment remained largely unchanged. These findings revealed the vigorous alloimmunity in I-C tissues, characterized with impaired vascularization but intact posterior epithelial barrier in the iris, which allowed proteins and immune cells to be exudated from the front surface of I-C tissues, and facilitated immune reaction in the anterior chamber, thereby contributing to aggravated corneal transplantation rejection.

Donor CYP3A5 Expression Decreases Renal Transplantation Outcomes in White Renal Transplant Recipients.

BACKGROUND After renal transplantation, immunosuppressants should be administered to prevent organ rejection and prolong graft survival. One of them is tacrolimus, which is metabolized by the CYP3A enzyme family. The variability of the CYP3A5 gene in renal transplant recipients has been previously studied for its correlation with acute rejection and allogeneic kidney function. CYP3A5 enzyme is also present in the renal tissue, and its relevance has not yet been extensively investigated. This study aimed to evaluate the effect of donor and recipient CYP3A5 expression status on early and long-term transplant outcomes. MATERIAL AND METHODS Single-nucleotide polymorphism in CYP3A5 (rs776746) was analyzed in 95 kidney transplant recipients and their grafts. The effect of donor and recipient genotypes on the primary endpoint, which was the loss of the renal graft over 5-year follow-up, was assessed. The secondary endpoints were biopsy-proven acute rejection, proteinuria, delayed graft function, and renal function. RESULTS Patients who received a CYP3A5*1 allele-carrying kidney (n=16) were at greater risk of graft loss (adjusted hazard ratio, 95% CI: 10.61, 2.28-49.42, P=.003) than those with the CYP3A5*3/*3 genotype (n=79). Renal CYP3A5 expression was also a predictor of acute rejection between the 2nd and 12th post-transplant months (adjusted odds ratio, 95% CI: 4.36; 1.08-17.6, P=.038) and proteinuria at different time intervals. No effect of the recipient CYP3A5 genotype was observed. CONCLUSIONS The donor CYP3A5 genotype is associated with inferior transplantation outcomes. Local renal tacrolimus metabolism is a potential target for improving long-term transplantation outcomes.

Effect of HO-1-modified BMMSCs on immune function in liver transplantation.

We examined whether haem oxygenase-1 (HO-1) could enhance the immunosuppressive effects of bone marrow mesenchymal stem cells (BMMSCs) on the rejection of transplanted liver allografts in rats. The animals were divided into three groups: the normal saline (NS) group, BMMSC group and HO-1/BMMSCs group. In vitro, the extraction, culture and HO-1 transfection of BMMSCs were performed. Mixed lymphocyte response (MLR) analysis of HO-1/BMMSCs efficacy was performed. The rejection model of orthotopic liver transplantation in rats was established when BMMSCs and HO-1/BMMSCs were transfused via the portal vein. To reduce research bias, we established an isogenic Liver transplantation model of (LEW → LEW) and (BN → BN), which can achieve tolerance. Changes in histopathology and liver function in the transplanted liver and changes in regulatory T cell (Tregs), natural killer (NK) cells and cytokines after transplantation were observed in the different groups. The severe acute rejection after liver transplantation on postoperative Day 10 was observed in the NS group. The BMMSC group showed strong protective effects against rejection within the first 10 days after transplantation, while HO-1/BMMSCs showed stronger effects on rejection than BMMSCs alone. In addition, the activity of natural killer (NK) cells decreased significantly, the levels of regulatory T cells (Tregs), interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) increased significantly and the levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased significantly in the HO-1/BMMSC group compared with the BMMSC group. HO-1/BMMSCs showed better immunosuppressive effects after liver transplantation than the other treatments. Our findings reveal that HO-1 can enhance the effects of BMMSCs on inhibiting acute rejection in orthotopic liver transplantation in rats.

Islet transplantation into brown adipose tissue can delay immune rejection.

Type 1 diabetes is an autoimmune disease characterized by insulin-producing ß cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.

Donor NKG2C homozygosity contributes to CMV clearance after haploidentical transplantation.

CMV infection remains an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Several investigators have reported that adaptive NKG2C+ NK cells persistently expand during CMV reactivation. In our study, 2 cohorts were enrolled to explore the relationships among the NKG2C genotype, NKG2C+ NK cell reconstitution, and CMV infection. Multivariate analysis showed that donor NKG2C gene deletion was an independent prognostic factor for CMV reactivation and refractory CMV reactivation. Furthermore, adaptive NKG2C+ NK cells' quantitative and qualitative reconstitution, along with their anti-CMV function after transplantation, was significantly lower in patients grafted with NKG2Cwt/del donor cells than in those grafted with NKG2Cwt/wt donor cells. At day 30 after transplantation, quantitative reconstitution of NKG2C+ NK cells was significantly lower in patients with treatment-refractory CMV reactivation than in patients without CMV reactivation and those with nonrefractory CMV reactivation. In humanized CMV-infected mice, we found that, compared with those from NKG2Cwt/del donors, adaptive NKG2C+ NK cells from NKG2Cwt/wt donors induced earlier and stronger expansion of NKG2C+ NK cells as well as earlier and stronger CMV clearance in vivo. In conclusion, donor NKG2C homozygosity contributes to CMV clearance by promoting the quantitative and qualitative reconstruction of adaptive NKG2C+ NK cells after haploidentical allo-HSCT.

Preincubation of donor tissue with a VEGF cytokine trap promotes subsequent high-risk corneal transplant survival.

AIMS: Pathological neovascularisation of the host bed and the transplant itself is the main risk factor for graft rejection after corneal transplantation. This study aims to prevent this process by preincubation of the corneal donor tissue ex vivo with an antivascular endothelial growth factor (VEGF) cytokine trap blocking additional postsurgical hemangiogenesis and lymphangiogenesis to promote high-risk graft survival. METHODS: The donor tissue was preincubated with a VEGFR1R2 cytokine trap for 24 hours prior to murine high-risk corneal transplantation (human IgG Fc was used as the control). The distribution of VEGFR1R2 Trap in the cornea was investigated by immunohistochemistry. Corneas were excised to quantify the blood vessels (BVs) and lymphatic vessels (LVs) and draining lymph nodes (dLNs) were harvested to analyse the phenotype of dendritic cells (DCs) and T cells at week 1, 2 and 8 post-transplantation. Graft survival was compared between preincubation with VEGFR1R2 Trap and human IgG Fc in high-risk recipients. RESULTS: VEGFR1R2 Trap was present in the graft for at least 2 weeks after surgery and additionally diffused into the corneal recipient. BVs, LVs and macrophages in the whole cornea were significantly decreased 1-week and 2-week post-transplantation (p<0.05). In dLNs the frequency of CD11c+DCs was significantly reduced, whereas CD200R+ regulatory DCs were significantly increased after keratoplasty (p<0.05). Furthermore, long-term high-risk graft survival was significantly improved (p<0.01). CONCLUSIONS: Preincubation of corneal donor tissue with a VEGFR1R2 cytokine trap can significantly promote subsequent high-risk corneal transplant survival and thereby opens new treatment avenues for high-risk corneal transplantation.

Integrative Omics Analysis Unravels Microvascular Inflammation-Related Pathways in Kidney Allograft Biopsies.

In solid-organ transplantation, microRNAs (miRNAs) have emerged as key players in the regulation of allograft cells function in response to injury. To gain insight into the role of miRNAs in antibody-mediated rejection, a rejection phenotype histologically defined by microvascular inflammation, kidney allograft biopsies were subjected to miRNA but also messenger RNA (mRNA) profiling. Using a unique multistep selection process specific to the BIOMARGIN study (discovery cohort, N=86; selection cohort, N=99; validation cohort, N=298), six differentially expressed miRNAs were consistently identified: miR-139-5p (down) and miR-142-3p/150-5p/155-5p/222-3p/223-3p (up). Their expression level gradually correlated with microvascular inflammation intensity. The cell specificity of miRNAs target genes was investigated by integrating their in vivo mRNA targets with single-cell RNA sequencing from an independent allograft biopsy cohort. Endothelial-derived miR-139-5p expression correlated negatively with MHC-related genes expression. Conversely, epithelial-derived miR-222-3p overexpression was strongly associated with degraded renal electrolyte homeostasis and repressed immune-related pathways. In immune cells, miR-150-5p regulated NF-κB activation in T lymphocytes whereas miR-155-5p regulated mRNA splicing in antigen-presenting cells. Altogether, integrated omics enabled us to unravel new pathways involved in microvascular inflammation and suggests that metabolism modifications in tubular epithelial cells occur as a consequence of antibody-mediated rejection, beyond the nearby endothelial compartment.

Small Extracellular Vesicles in Transplant Rejection.

Small extracellular vesicles (sEV), which are released to body fluids (e.g., serum, urine) by all types of human cells, may stimulate or inhibit the innate and adaptive immune response through multiple mechanisms. Exosomes or sEV have on their surface many key receptors of immune response, including major histocompatibility complex (MHC) components, identical to their cellular origin. They also exhibit an ability to carry antigen and target leukocytes either via interaction with cell surface receptors or intracellular delivery of inflammatory mediators, receptors, enzymes, mRNAs, and noncoding RNAs. By the transfer of donor MHC antigens to recipient antigen presenting cells sEV may also contribute to T cell allorecognition and alloresponse. Here, we review the influence of sEV on the development of rejection or tolerance in the setting of solid organ and tissue allotransplantation. We also summarize and discuss potential applications of plasma and urinary sEV as biomarkers in the context of transplantation. We focus on the attempts to use sEV as a noninvasive approach to detecting allograft rejection. Preliminary studies show that both sEV total levels and a set of specific molecules included in their cargo may be an evidence of ongoing allograft rejection.

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

Gut microbes enlarged the protective effect of transplanted regulatory B cells on rejection of cardiac allografts.

BACKGROUND: Regulatory B cells (Bregs) play an important role in maintaining immune homeostasis and have the potential to induce tolerance. Previous work has found that Breg cells are involved in heart transplantation tolerance. However, the effect of Breg on the transplantation tolerance and the underlying mechanisms remain to be clarified. METHODS: Using a within-species heart transplantation model, we aimed to investigate the role of CD19+CD5+CD1dhigh Bregs isolated from transplanted mice in preventing transplant rejection in vivo. We also explored the effects of CD40 and tumor necrosis factor receptor-associated factor 6 (TRAF6) ubiquitin ligase on Breg-mediated prolongation of survival in heart transplant (HT) mice, and the regulatory effects of downstream Cdk4 and Cdk6 proteins on dendritic cells (DCs), which clarified the function and molecular mechanism of Breg cells in HT mice. RESULTS: Our data suggest that adoptive transfer of the transplanted Bregs served as an effective tolerance-inducing mechanism in HT mice and was involved in the CD40-TRAF6 signaling pathway in DCs. Moreover, DCs collected from the Breg treated HT mice also prolonged the survival of HT mice. Furthermore, DC-specific knockout of TRAF6 diminished Breg-mediated prolongation of survival in HT mice. Interestingly, gut microbes from donors increased the survival of cardiac allografts both in both the absence and presence of Bregs but were not implicated in CD40-TRAF6 signaling. CONCLUSIONS: These findings reveal a role of Breg cells in the induction of transplantation tolerance through the blockade of the CD40-TRAF6 signaling pathway, which might be used in the treatment of HT in the clinic.

Chronic Active Antibody-Mediated Rejection Is Associated With the Upregulation of Interstitial But Not Glomerular Transcripts.

Molecular assessment of renal allografts has already been suggested in antibody-mediated rejection (ABMR), but little is known about the gene transcript patterns in particular renal compartments. We used laser capture microdissection coupled with quantitative RT-PCR to distinguish the transcript patterns in the glomeruli and tubulointerstitium of kidney allografts in sensitized retransplant recipients at high risk of ABMR. The expressions of 13 genes were quantified in biopsies with acute active ABMR, chronic active ABMR, acute tubular necrosis (ATN), and normal findings. The transcripts were either compartment specific (TGFB1 in the glomeruli and HAVCR1 and IGHG1 in the tubulointerstitium), ABMR specific (GNLY), or follow-up specific (CXCL10 and CX3CR1). The transcriptional profiles of early acute ABMR shared similarities with ATN. The transcripts of CXCL10 and TGFB1 increased in the glomeruli in both acute ABMR and chronic active ABMR. Chronic active ABMR was associated with the upregulation of most genes (SH2D1B, CX3CR1, IGHG1, MS4A1, C5, CD46, and TGFB1) in the tubulointerstitium. In this study, we show distinct gene expression patterns in specific renal compartments reflecting cellular infiltration observed by conventional histology. In comparison with active ABMR, chronic active ABMR is associated with increased transcripts of tubulointerstitial origin.

Transcription factor FOXF1 identifies compartmentally distinct mesenchymal cells with a role in lung allograft fibrogenesis.

In this study, we demonstrate that forkhead box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, was retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as key players in lung allograft remodeling and fibrosis. Using Foxf1-tdTomato BAC (Foxf1-tdTomato) and Foxf1-tdTomato Col1a1-GFP mice, we show that Lin-Foxf1+ cells encompassed the stem cell antigen 1+CD34+ (Sca1+CD34+) subset of collagen 1-expressing mesenchymal cells (MCs) with a capacity to generate CFU and lung epithelial organoids. Histologically, FOXF1-expressing MCs formed a 3D network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA-Seq confirmed distinct transcriptional signatures of Foxf1+ and Foxf1- MCs, with Foxf1-expressing cells delineated by their high expression of the transcription factor glioma-associated oncogene 1 (Gli1) and low expression of integrin α8 (Itga), versus other collagen-expressing MCs. FOXF1+Gli1+ MCs showed proximity to Sonic hedgehog-expressing (Shh-expressing) bronchial epithelium, and mesenchymal expression of Foxf1 and Gli1 was found to be dependent on paracrine Shh signaling in epithelial organoids. Using a murine lung transplant model, we show dysregulation of epithelial-mesenchymal SHH/GLI1/FOXF1 crosstalk and expansion of this specific peribronchial MC population in chronically rejecting fibrotic lung allografts.

Single-Cell RNA Sequencing Reveals the Immunological Profiles of Renal Allograft Rejection in Mice.

Allograft rejection is a common immunological feature in renal transplantation and is associated with reduced graft survival. A mouse renal allograft rejection model was induced and single-cell RNA sequencing (scRNA-seq) data of CD45+ leukocytes in kidney allografts on days 7 (D7) and 15 (D15) after operation was analyzed to reveal a full immunological profiling. We identified 20 immune cell types among 10,921 leukocytes. Macrophages and CD8+ T cells constituted the main populations on both timepoints. In the process from acute rejection (AR) towards chronic rejection (CR), the proportion of proliferating and naïve CD8+ T cells dropped significantly. Both B cells and neutrophils decreased by about 3 folds. On the contrary, the proportion of macrophages and dendritic cells (DCs) increased significantly, especially by about a 4.5-fold increase in Ly6cloMrc1+ macrophages and 2.6 folds increase in Ly6cloEar2+ macrophages. Moreover, myeloid cells harbored the richest ligand and receptor (LR) pairs with other cells, particularly for chemokine ligands such as Cxcl9, Cxcl10, Cxcl16 and Yars. However, macrophages with weak response to interferon gamma (IFNg) contributed to rejection chronicization. To conclude, reduction in CD8 T cells, B cells and neutrophils while increasing in Ly6cloMrc1+ macrophages and Ly6cloEar2+ macrophages, may contribute significantly to the progress from AR towards CR.

Variants in mycophenolate and CMV antiviral drug pharmacokinetic and pharmacodynamic genes and leukopenia in heart transplant recipients.

BACKGROUND: The objective was to assess the relationship between single nucleotide polymorphisms in mycophenolate and cytomegalovirus antiviral drug pharmacokinetic and pharmacodynamic genes and drug-induced leukopenia in adult heart transplant recipients. METHODS: This retrospective analysis included n = 148 patients receiving mycophenolate and a cytomegalovirus antiviral drug. In total, 81 single nucleotide polymorphisms in 21 pharmacokinetic and 23 pharmacodynamic genes were selected for investigation. The primary and secondary outcomes were mycophenolate and/or cytomegalovirus antiviral drug-induced leukopenia, defined as a white blood cell count <3.0 × 109/L, in the first six and 12 months post-heart transplant, respectively. RESULTS: Mycophenolate and/or cytomegalovirus antiviral drug-induced leukopenia occurred in 20.3% of patients. HNF1A rs1169288 A>C (p.I27L) was associated with drug-induced leukopenia (unadjusted p = 0.002; false discovery rate <20%) in the first six months post-transplant. After adjusting for covariates, HNF1A rs1169288 variant C allele carriers had significantly higher odds of leukopenia compared to A/A homozygotes (odds ratio 6.19; 95% CI 1.97-19.43; p = 0.002). Single nucleotide polymorphisms in HNF1A, SLC13A1, and MBOAT1 were suggestively associated (p < 0.05) with the secondary outcome but were not significant after adjusting for multiple comparisons. CONCLUSION: Our data suggest genetic variation may play a role in the development of leukopenia in patients receiving mycophenolate and cytomegalovirus antiviral drugs after heart transplantation. Following replication, pharmacogenetic markers, such as HNF1A rs1169288, could help identify patients at higher risk of drug-induced leukopenia, allowing for more personalized immunosuppressant therapy and cytomegalovirus prophylaxis following heart transplantation.

Phosphorylated S6 ribosomal protein expression by immunohistochemistry correlates with de novo donor-specific HLA antibodies in lung allograft recipients.

BACKGROUND: Per the ISHLT 2016 definition, a C4d-positive lung biopsy is required to meet criteria for definite antibody-mediated rejection (AMR). Unfortunately, C4d has poor sensitivity and specificity, and low inter-rater reliability. Phosphorylated S6 ribosomal protein (p-S6RP) expressed via the mTOR pathway has been shown to be a biomarker of AMR and correlates with donor-specific antibodies (DSA) in heart allografts. However, p-S6RP immunohistochemistry (IHC) in the setting of pulmonary AMR has yet to be evaluated. We sought to determine whether p-S6RP IHC performed on lung biopsies correlates with de novo DSA. METHODS: IHC for p-S6RP performed on 26 biopsies from lung transplant recipients with de novo HLA DSA (DSA+) and 28 biopsies from patients with no DSA (DSA-) were evaluated by 3 pathologists who independently scored the degree of alveolar macrophage and pneumocyte staining. Staining in ≥50% of the biopsy as determined by at least 2 pathologists was considered positive. RESULTS: Twenty-one (81%) DSA+ biopsies stained positive for p-S6RP in pneumocytes and 21 (81%) in macrophages. Six DSA- biopsies (21%) stained positive for p-S6RP in pneumocytes, 6 (21%) were positive in macrophages. Pneumocyte p-S6RP staining was 81% sensitive and 79% specific for DSA. Macrophage staining showed the same sensitivity and specificity but with lower inter-rater agreement (κ = 0.53 vs 0.68). CONCLUSIONS: This study demonstrates a positive relationship between de novo DSA and p-S6RP expression in pneumocytes and macrophages using IHC. p-S6RP is relatively sensitive and specific, and has superior inter-rater reliability compared to C4d.

IL-10 Mediated Immunomodulation Limits Subepithelial Fibrosis and Repairs Airway Epithelium in Rejecting Airway Allografts.

Interleukin-10 plays a vital role in maintaining peripheral immunotolerance and favors a regulatory immune milieu through the suppression of T effector cells. Inflammation-induced microvascular loss has been associated with airway epithelial injury, which is a key pathological source of graft malfunctioning and subepithelial fibrosis in rejecting allografts. The regulatory immune phase maneuvers alloimmune inflammation through various regulatory modulators, and thereby promotes graft microvascular repair and suppresses the progression of fibrosis after transplantation. The present study was designed to investigate the therapeutic impact of IL-10 on immunotolerance, in particular, the reparative microenvironment, which negates airway epithelial injury, and fibrosis in a mouse model of airway graft rejection. Here, we depleted and reconstituted IL-10, and serially monitored the phase of immunotolerance, graft microvasculature, inflammatory cytokines, airway epithelium, and subepithelial collagen in rejecting airway transplants. We demonstrated that the IL-10 depletion suppresses FOXP3+ Tregs, tumor necrosis factor-inducible gene 6 protein (TSG-6), graft microvasculature, and establishes a pro-inflammatory phase, which augments airway epithelial injury and subepithelial collagen deposition while the IL-10 reconstitution facilitates FOXP3+ Tregs, TSG-6 deposition, graft microvasculature, and thereby favors airway epithelial repair and subepithelial collagen suppression. These findings establish a potential reparative modulation of IL-10-associated immunotolerance on microvascular, epithelial, and fibrotic remodeling, which could provide a vital therapeutic option to rescue rejecting transplants in clinical settings.