Molecular Signature of Antibody-Mediated Chronic Vasculopathy in Heart Allografts in a Novel Mouse Model.

Cardiac allograft vasculopathy (CAV) limits the long-term success of heart transplants. Generation of donor-specific antibodies (DSAs) is associated with increased incidence of CAV clinically, but mechanisms underlying development of this pathology remain poorly understood. Major histocompatibility complex-mismatched A/J cardiac allografts in B6.CCR5-/- recipients have been reported to undergo acute rejection with little T-cell infiltration, but intense deposition of C4d in large vessels and capillaries of the graft accompanied by high titers of DSA. This model was modified to investigate mechanisms of antibody-mediated CAV by transplanting A/J hearts to B6.CCR5-/- CD8-/- mice that were treated with low doses of anti-CD4 monoclonal antibody to decrease T-cell-mediated graft injury and promote antibody-mediated injury. Although the mild inhibition of CD4 T cells extended allograft survival, the grafts developed CAV with intense C4d deposition and macrophage infiltration by 14 days after transplantation. Development of CAV correlated with recipient DSA titers. Transcriptomic analysis of microdissected allograft arteries identified the Notch ligand Dll4 as the most elevated transcript in CAV, associated with high versus low titers of DSA. More importantly, these analyses revealed a differential expression of transcripts in the CAV lesions compared with the matched apical tissue that lacks large arteries. In conclusion, these findings report a novel model of antibody-mediated CAV with the potential to facilitate further understanding of the molecular mechanisms promoting development of CAV.

Antibody-induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection.

HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68+ CD206+ CD163+ macrophages (M(HLA I IgG)), whereas HLA I F(ab')2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab')2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury.

Urinary-cell mRNA profile and acute cellular rejection in kidney allografts.

BACKGROUND: The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. METHODS: We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. RESULTS: A three-gene signature of 18S ribosomal (rRNA)-normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operating-characteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer-Lemeshow test indicated good fit (P=0.77). In an external-validation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P=0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti-interleukin-2 receptor antibodies from those who received T-cell-depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P=0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). CONCLUSIONS: A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts. (Funded by the National Institutes of Health and others.).

p40 homodimers bridge ischemic tissue inflammation and heterologous alloimmunity in mice via IL-15 transpresentation.

Virus-induced memory T cells often express functional cross-reactivity, or heterologous immunity, to other viruses and to allogeneic MHC molecules that is an important component of pathogenic responses to allogeneic transplants. During immune responses, antigen-reactive naive and central memory T cells proliferate in secondary lymphoid organs to achieve sufficient cell numbers to effectively respond, whereas effector memory T cell proliferation occurs directly within the peripheral inflammatory microenvironment. Mechanisms driving heterologous memory T cell proliferation and effector function expression within peripheral tissues remain poorly understood. Here, we dissected proliferation of heterologous donor-reactive memory CD8+ T cells and their effector functions following infiltration into heart allografts with low or high intensities of ischemic inflammation. Proliferation within both ischemic conditions required p40 homodimer-induced IL-15 transpresentation by graft DCs, but expression of effector functions mediating acute allograft injury occurred only in high-ischemic allografts. Transcriptional responses of heterologous donor-reactive memory CD8+ T cells were distinct from donor antigen-primed memory CD8+ T cells during early activation in allografts and at graft rejection. Overall, the results provide insights into mechanisms driving heterologous effector memory CD8+ T cell proliferation and the separation between proliferation and effector function that is dependent on the intensity of inflammation within the tissue microenvironment.

Men With Chronic Orchialgia Exhibit Differential Neuroinflammatory Gene Expression Relative to Asymptomatic Controls.

OBJECTIVE: To identify differences in neuroinflammatory gene expression in individuals with chronic orchialgia (CO) compared to asymptomatic controls. METHODS: Vas deferens, spermatic cord fascia, blood, and urine were collected from 9 men with CO at time of microscopic spermatic cord denervation and 7 asymptomatic controls at time of vasectomy. RNA was isolated and analyzed with the NanoString Human Neuroinflammation panel. Data were normalized, gene expression fold changes and enriched pathways relative to asymptomatic controls were determined. Gene expression was considered significantly different if there was a >2-fold change and P-value <.05 relative to controls. RESULTS: Mean patient age was 51 years and median symptom duration 12 months. There were 26 genes with significantly differential expression in vas deferens. cFos, a marker of nociceptive pain, had the greatest difference (30.2-fold change, P <.000001). Enriched pathways in vas deferens included nerve function, matrix remodeling, and innate immune responses. In fascia, cFos also had the greatest differential expression (38-fold, P = .000002), followed by S100A12 (11-fold, inducer of innate immune response). Enriched pathways in fascia included nerve function and inflammation. In blood, there were no differentially expressed genes, and in urine there were 95 differentially expressed genes. CONCLUSION: Men with CO have a diverse set of neuroinflammatory genes with differential expression in tissue and urine relative to healthy controls. These findings confirm pathologic changes in tissue targeted by denervation surgery, and suggest molecular changes in neuropathic pain that could lead to biomarker identification and novel treatment.

Linking erythropoietin to Treg-dependent allograft survival through myeloid cells.

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO's effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor-knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

Neuroinflammatory gene expression in chronic prostatitis/chronic pelvic pain syndrome patients: insights into etiology and phenotype biology.

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has diverse clinical phenotypes and its etiology is multifactorial. Studies to date of gene expression in humans have been limited to small numbers of target genes. NanoString can simultaneously measure hundreds of genes. We wished to study gene expression in blood and urine of CP/CPPS patients compared to controls for neuroinflammatory genes and characterize the results by patient phenotype. METHODS: Blood and urine were collected from 10 men with CP/CPPS and 7 asymptomatic controls. RNA was isolated from urine pellets using Qiagen RNeasy kits. Whole blood was collected and RNA isolated. 100 ng of RNA was used for gene expression analysis with the 770-gene NanoString Human Neuroinflammation gene panel. Data was imported into Rosalind (OnRamp Bioinformatics) for normalization, calculation of fold-changes and P values, and identification of enriched pathways. Gene expression was considered significantly different if there was a greater than 1.5× change compared to controls and corrected P was <0.05. RESULTS: Mean patient age was 42.2 years, median symptom duration was 15.5 months, median UPOINT domains was 3 and mean total National Institute of Health-Chronic Prostatitis Symptom Index Score was 28.8. In blood, there were 5 genes with significantly different expression to controls, the largest differences found in FOS1 (neuropathic pain control), PROS1 (blood clotting) and DDX58 (antiviral innate immunity). Gene set analysis showed differences in inflammation, angiogenesis and cytokine signaling. In urine there were 48 genes with significantly different expression including SLAMF8 (lymphocyte activation) and LAIR1 (inhibits B and T cell function). Gene set analysis showed differences in carbohydrate metabolism, neurons and neurotransmission, adaptive immunity and inflammatory signaling. Subgroup analysis by UPOINT domain showed unique gene expression in the Organ Specific and Neurologic/Systemic domains in both blood and urine for neurogenic pain and cytokine signaling associated genes. CONCLUSIONS: Men with CP/CPPS have a diverse set of neuroinflammatory genes with differential expression compared to controls. Clinical phenotypes have distinct patterns of gene expression. These findings could lead to novel biomarker development, emphasize the importance of multimodal therapy targeting diverse pathways and further validate the biologic basic of clinical phenotyping.

Neuroinflammatory gene expression analysis reveals potential novel mediators and treatment targets in interstitial cystitis with Hunner lesions.

BACKGROUND: We sought to study differential neuroinflammatory gene expression in men with interstitial cystitis (IC) with Hunner lesions compared with asymptomatic controls using NanoString, which uses barcoded probes to measure hundreds of genes. IC is a heterogenous condition lacking reliable biomarkers, and a subset of patients exhibits Hunner lesions, implicating the bladder as an inflammatory pain generator. METHODS: Blood, urine, and bladder biopsies were collected from 6 men with IC and Hunner lesions. 7 asymptomatic controls had blood and urine collected and 2 benign bladder biopsies were obtained from our tissue bank. RNA was isolated and analyzed with NanoString Human Neuroinflammation panel. Gene expression was considered significant if there was a >1.5-fold change and adjusted P value <0.05 compared with controls. RESULTS: Mean patient age was 61.5 years with 8 years median symptom duration. In bladder tissue, while many cytokine and chemokine genes had higher expression as expected (e.g., TNF, CXCL10), other significant genes included TRPA1 (1098-fold increased, expressed in pain sensing neurons) and TNFRSF17 (735-fold, B-cell related). In urine, there was 114-fold increase in S1PR4, which mediates pain via TRP-dependent pathways. A patient on cyclosporine had lower inflammatory gene expression levels relative to other IC patients, but no difference in TRPA1. CONCLUSIONS: Men with IC and Hunner lesions have a diverse set of neuroinflammatory genes with differential expression compared to controls. We identified genes linked to neuropathic pain through the TRP pathway and this expression was not reduced by cyclosporine. These findings open a new direction for biomarker and therapeutic discovery.

Human breast microbiome correlates with prognostic features and immunological signatures in breast cancer.

BACKGROUND: Currently, over half of breast cancer cases are unrelated to known risk factors, highlighting the importance of discovering other cancer-promoting factors. Since crosstalk between gut microbes and host immunity contributes to many diseases, we hypothesized that similar interactions could occur between the recently described breast microbiome and local immune responses to influence breast cancer pathogenesis. METHODS: Using 16S rRNA gene sequencing, we characterized the microbiome of human breast tissue in a total of 221 patients with breast cancer, 18 individuals predisposed to breast cancer, and 69 controls. We performed bioinformatic analyses using a DADA2-based pipeline and applied linear models with White's t or Kruskal-Wallis H-tests with Benjamini-Hochberg multiple testing correction to identify taxonomic groups associated with prognostic clinicopathologic features. We then used network analysis based on Spearman coefficients to correlate specific bacterial taxa with immunological data from NanoString gene expression and 65-plex cytokine assays. RESULTS: Multiple bacterial genera exhibited significant differences in relative abundance when stratifying by breast tissue type (tumor, tumor adjacent normal, high-risk, healthy control), cancer stage, grade, histologic subtype, receptor status, lymphovascular invasion, or node-positive status, even after adjusting for confounding variables. Microbiome-immune networks within the breast tended to be bacteria-centric, with sparse structure in tumors and more interconnected structure in benign tissues. Notably, Anaerococcus, Caulobacter, and Streptococcus, which were major bacterial hubs in benign tissue networks, were absent from cancer-associated tissue networks. In addition, Propionibacterium and Staphylococcus, which were depleted in tumors, showed negative associations with oncogenic immune features; Streptococcus and Propionibacterium also correlated positively with T-cell activation-related genes. CONCLUSIONS: This study, the largest to date comparing healthy versus cancer-associated breast microbiomes using fresh-frozen surgical specimens and immune correlates, provides insight into microbial profiles that correspond with prognostic clinicopathologic features in breast cancer. It additionally presents evidence for local microbial-immune interplay in breast cancer that merits further investigation and has preventative, diagnostic, and therapeutic potential.

Recipient myeloperoxidase-producing cells regulate antibody-mediated acute versus chronic kidney allograft rejection.

Antibody-mediated rejection (ABMR) continues to be a major problem undermining the success of kidney transplantation. Acute ABMR of kidney grafts is characterized by neutrophil and monocyte margination in the tubular capillaries and by graft transcripts indicating NK cell activation, but the myeloid cell mechanisms required for acute ABMR have remained unclear. Dysregulated donor-specific antibody (DSA) responses with high antibody titers are induced in B6.CCR5-/- mice transplanted with complete MHC-mismatched A/J kidneys and are required for rejection of the grafts. This study tested the role of recipient myeloid cell production of myeloperoxidase (MPO) in the cellular and molecular components of acute ABMR. Despite induction of equivalent DSA titers, B6.CCR5-/- recipients rejected A/J kidneys between days 18 and 25, with acute ABMR, whereas B6.CCR5-/-MPO-/- recipients rejected the grafts between days 46 and 54, with histopathological features of chronic graft injury. On day 15, myeloid cells infiltrating grafts from B6.CCR5-/- and B6.CCR5-/-MPO-/- recipients expressed marked phenotypic and functional transcript differences that correlated with the development of acute versus chronic allograft injury, respectively. Near the time of peak DSA titers, activation of NK cells to proliferate and express CD107a was decreased within allografts in B6.CCR5-/-MPO-/- recipients. Despite high titers of DSA, depletion of neutrophils reproduced the inhibition of NK cell activation and decreased macrophage infiltration but increased monocytes producing MPO. Overall, recipient myeloid cells producing MPO regulate graft-infiltrating monocyte/macrophage function and NK cell activation that are required for DSA-mediated acute kidney allograft injury, and their absence switches DSA-mediated acute pathology and graft outcomes to chronic ABMR.

Detection of SHV beta-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies.

BACKGROUND: beta-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, beta-lactam resistance most commonly results from the production of beta-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type beta-lactamases predominate. Therefore, new and accurate detection methods for these beta-lactamase producing isolates are needed. RESULTS: E. coli DH10B cells producing SHV-1 beta-lactamase and a clinical isolate of K. pneumoniae producing SHV-5 beta-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. E. coli DH10B cells without a blaSHV gene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV beta-lactamase that was sufficient for direct imaging. CONCLUSION: We developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.

Interleukin-27 promotes CD8+ T cell reconstitution following antibody-mediated lymphoablation.

Antibody-mediated lymphoablation is used in solid organ and stem cell transplantation and autoimmunity. Using murine anti-thymocyte globulin (mATG) in a mouse model of heart transplantation, we previously reported that the homeostatic recovery of CD8+ T cells requires help from depletion-resistant memory CD4+ T cells delivered through CD40-expressing B cells. This study investigated the mechanisms by which B cells mediate CD8+ T cell proliferation in lymphopenic hosts. While CD8+ T cell recovery required MHC class I expression in the host, the reconstitution occurred independently of MHC class I, MHC class II, or CD80/CD86 expression on B cells. mATG lymphoablation upregulated the B cell expression of several cytokine genes, including IL-15 and IL-27, in a CD4-dependent manner. Neither treatment with anti-CD122 mAb nor the use of IL-15Rα-/- recipients altered CD8+ T cell recovery after mATG treatment, indicating that IL-15 may be dispensable for T cell proliferation in our model. Instead, IL-27 neutralization or the use of IL-27Rα-/- CD8+ T cells inhibited CD8+ T cell proliferation and altered the phenotype and cytokine profile of reconstituted CD8+ T cells. Our findings uncover what we believe is a novel role of IL-27 in lymphopenia-induced CD8+ T cell proliferation and suggest that targeting B cell-derived cytokines may increase the efficacy of lymphoablation and improve transplant outcomes.

CD4+ T lymphocytes produce adiponectin in response to transplants.

Adiponectin is a pleiotropic cytokine with diverse immunomodulatory effects on macrophages and lymphocytes. In the current paradigm, lymphocytes and macrophages respond to adiponectin that is produced by adipocytes and other parenchymal cells. Using a model of chronic arterial inflammation in cardiac transplants, we found that T cells derived from the recipient migrate to the heart and produce adiponectin locally. The evidence that T cells produce significant amounts of adiponectin is based on 3 experimental approaches. First, CD4+ T cells isolated from the blood and spleen after cardiac transplantation express mRNA for adiponectin. Second, reconstitution of T cell-deficient recipients with transgenic CD4+ T cells that express receptors for donor antigens results in arterial infiltrates containing T cells and increased mRNA expression for adiponectin in cardiac transplants. Third, CD4+ T cells isolated from the allograft secrete adiponectin in vitro. Taken together, these data indicate that adiponectin-competent cells originating in the recipient migrate into the transplant. Establishing T cells as a source of adiponectin provides a new dimension, to our knowledge, to the modulatory effects of adiponectin on immune responses.