Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of HLA molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells produce IL-6 in the steady-state and this is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in endothelial cell immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered endothelial cell interactions with allogeneic PBMC and after anti-HLA or DSA binding to endothelial cells in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation, and C4d deposition were examined. Blockade of IL-6 reduced endothelial cell secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of endothelial cells by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Th17, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of human leukocyte antigen (HLA) molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells (ECs) produce IL-6 in the steady-state that is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in EC immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered EC interactions with allogeneic PBMC and after anti-HLA or DSA binding to ECs in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation and C4d deposition were examined. Blockade of IL-6 reduced EC secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of ECs by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

HLA-DQ alloantibodies directly activate the endothelium and compromise differentiation of FoxP3high regulatory T lymphocytes.

Development of donor-specific antibodies is associated with reduced allograft survival in renal transplantation. Recent clinical studies highlight the prevalence of human leukocyte antigen (HLA)-DQ antibodies amongst de novo donor-specific antibodies (DSAs), yet the specific contribution of these DSAs to rejection has not been examined. Antibody-mediated rejection primarily targets the microvasculature, so this study explored how patient HLA-DQ alloantibodies can modulate endothelial activation and so immunoregulation. HLA-DQ antibodies phosphorylated Akt and S6 kinase in microvascular endothelial cells. This activation prior to culture with alloreactive lymphocytes increased IL-6 and RANTES secretion. The antibody-mediated upregulation of IL-6 was indeed Akt-dependent. The binding of HLA-DQ antibodies to endothelial cells selectively reduced T cell alloproliferation and FoxP3high Treg differentiation. In clinical studies, detection of HLA-DQ DSAs with other DSAs is associated with worse graft survival than either alone. Endothelial cells stimulated with HLA-DR and HLA-DQ antibodies showed a synergistic increase in pro-inflammatory cytokine secretion and a decrease in Treg expansion. HLA-DQ antibodies strongly promote pro-inflammatory responses in isolation and in combination with other HLA antibodies. Thus, our data give new insights into the pathogenicity of HLA-DQ DSAs.

Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.

Inflammatory hepatocellular adenomas are benign liver tumours defined by the presence of inflammatory infiltrates and by the increased expression of inflammatory proteins in tumour hepatocytes. Here we show a marked activation of the interleukin (IL)-6 signalling pathway in this tumour type; sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene, which encodes the signalling co-receptor gp130. Indeed, 60% of inflammatory hepatocellular adenomas harbour small in-frame deletions that target the binding site of gp130 for IL-6, and expression of four different gp130 mutants in hepatocellular cells activates signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Furthermore, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by beta-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas fully explains activation of the acute inflammatory phase observed in tumourous hepatocytes, and suggests that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.

Adipocyte Glucocorticoid Receptor Activation With High Glucocorticoid Doses Impairs Healthy Adipose Tissue Expansion by Repressing Angiogenesis.

In humans, glucocorticoids (GCs) are commonly prescribed because of their anti-inflammatory and immunosuppressive properties. However, high doses of GCs often lead to side effects, including diabetes and lipodystrophy. We recently reported that adipocyte glucocorticoid receptor (GR)-deficient (AdipoGR-KO) mice under corticosterone (CORT) treatment exhibited a massive adipose tissue (AT) expansion associated with a paradoxical improvement of metabolic health compared with control mice. However, whether GR may control adipose development remains unclear. Here, we show a specific induction of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic vascular endothelial growth factor A (VEGFA) expression in GR-deficient adipocytes of AdipoGR-KO mice compared with control mice, together with an increased adipose vascular network, as assessed by three-dimensional imaging. GR activation reduced HIF-1α recruitment to the Vegfa promoter resulting from Hif-1α downregulation at the transcriptional and posttranslational levels. Importantly, in CORT-treated AdipoGR-KO mice, the blockade of VEGFA by a soluble decoy receptor prevented AT expansion and the healthy metabolic phenotype. Finally, in subcutaneous AT from patients with Cushing syndrome, higher VEGFA expression was associated with a better metabolic profile. Collectively, these results highlight that adipocyte GR negatively controls AT expansion and metabolic health through the downregulation of the major angiogenic effector VEGFA and inhibition of vascular network development.

Inflammation Determines the Capacity of Allogenic Endothelial Cells to Regulate Human Treg Expansion.

During allotransplantation, the endothelium acts as semi-professional antigen-presenting cells with the ability to activate proliferation and to promote differentiation of CD4+-T subsets. These abilities are dependent on the luminal expression of HLA class II antigens by microvascular endothelial cells, which is regulated by inflammatory cytokines. The upregulation of HLA-DR and HLA-DQ during rejection implies significant intragraft inflammation. Furthermore, the microvascular inflammation is an independent determinant for renal allograft failure. In this study, the potential of inflammation to modify endothelial regulation of peripheral CD4+ Treg cells was examined. Microvascular endothelial cells were exposed to pro-inflammatory cytokines for varying durations before co-culture with PBMC from non-HLA matched donors. Proliferation and expansion of CD4+Treg and soluble factor secretion was determined. Early interactions were detected by phosphorylation of Akt. Video microscopy was used to examine spatial and temporal endothelial-CD4+T interactions. Highly inflammatory conditions led to increased endothelial expression of HLA-DR, the adhesion molecule ICAM-1, the costimulatory molecule PD-L1 and de novo expression of HLA-DQ. Treg differentiation was impaired by exposure of endothelial cells to a high level of inflammation. Neither IL-6, IL-2 nor TGFß were implicated in reducing Treg numbers. High PD-L1 expression interfered with early endothelial cell interactions with CD4+T lymphocytes and led to modified TCR signaling. Blocking endothelial PD-L1 resulted in a partial restoration of Treg. The allogenic endothelial cell-mediated expansion of Treg depends on a critical threshold of inflammation. Manipulation of the PD-L1/PD-1 pathway or endothelial activation post-transplantation may promote or interfere with this intrinsic mechanism of allospecific Treg expansion.

Association of CYP1B1 germ line mutations with hepatocyte nuclear factor 1alpha-mutated hepatocellular adenoma.

Biallelic somatic mutations of TCF1 coding for hepatocyte nuclear factor 1alpha (HNF1alpha) are found in 50% of the hepatocellular adenoma (HCA) cases usually associated with oral contraception. In rare cases, HNF1alpha germ line mutations could also predispose to familial adenomatosis. In order to identify new genetic factors predisposing to HNF1alpha-mutated HCA, we searched for mutations in genes involved in the metabolism of estrogen. For 10 genes (CYP1A1, CYP1A2, CYP3A4, CYP3A5, COMT, UGT2B7, NQO1, GSTM1, GSTP1, and GSTT1), we did not find mutations nor differences in the allele distribution among 32 women presenting HNF1alpha-mutated adenomas compared with 58 controls. In contrast, we identified a CYP1B1 germ line heterozygous mutation in 4 of 32 women presenting HNF1alpha-mutated adenomas compared with none in 58 controls. We confirmed these results with the identification of four additional CYP1B1 mutations in a second series of 26 cases. No mutations were found in the control group, which was extended to 98 individuals, and only a known rare genetic variant was observed in two controls (P = 0.0003). We did an ethoxyresorufin O-deethylase assay to evaluate the functional consequence of the CYP1B1 mutations. We found reduced enzymatic activity in each CYP1B1 variant. In addition, an E229K CYP1B1 mutation was found in a woman with a germ line HNF1alpha mutation in a familial adenomatosis context. In this large family, all three patients with adenomatosis bore both HNF1 and CYP1B1 germ line mutations. In conclusion, our data suggested that CYP1B1 germ line-inactivating mutations might increase the incidence of HCA in women with HNF1alpha mutations.

Endothelial Cell Amplification of Regulatory T Cells Is Differentially Modified by Immunosuppressors and Intravenous Immunoglobulin.

Immunosuppressive treatment is a prerequisite for both organ transplantation and tolerance of the allograft. However, long-term immunosuppression has been associated with a higher incidence of malignancies and infections. Immunosuppressors mainly target circulating immune cells and little is known of their "off-target" effects, such as their impact on endothelial cells (ECs). In chronic antibody-mediated rejection (AMR), the allograft endothelium is a target of damage, histologically detected as transplant glomerulopathy, and which correlates with poor graft survival. Under inflammatory conditions, EC expression of HLA class II antigens can lead to CD4+-T lymphocyte alloactivation and selective expansion of pro-inflammatory Th17 and pro-tolerance Treg subsets. This response can be modified and preactivation of the EC by HLA-DR antibody binding promoted a proinflammatory Th17 response. However, whether or not immunosuppressors alter EC immunogenicity has not been examined. In alloimmunized patients with AMR, cyclosporine A (CsA) and mycophenolic acid (MPA) are often combined with intravenous immunoglobulins (IVIgs). This study reports changes in the microvascular EC phenotype and function after treatment with CsA, MPA, or IVIg. Both CsA and MPA decreased HLA-DR and increased CD54 expression, whereas IVIg increased HLA-DR expression. Interleukin 6 secretion was reduced by all three immunomodulators. Preincubation of ECs with CsA or MPA limited, while IVIg amplified, Treg expansion. Because CsA, MPA, and IVIg are known for their ability to act upon leukocytes, we confirmed that ECs maintained their immunoregulatory role when allogeneic leukocytes were pretreated with CsA, MPA, or IVIg. The results reveal that individual immunosuppressors, used in the induction and maintenance of renal allograft tolerance, had direct and distinct effects on ECs. Results of experiments associating IVIg with either CsA or MPA underlined the differences observed using individual immunosuppressors. Paradoxically, CsA or MPA may increase EC mediated inflammatory responses and long-term exposure may contribute to limitation of allograft tolerance. In contrast, IVIg interaction with the endothelium may mediate some of its immunosuppressive effects through promotion of Treg expansion, contributing to the maintenance of allograft tolerance.

Genomic profiling of hepatocellular adenomas reveals recurrent FRK-activating mutations and the mechanisms of malignant transformation.

Hepatocellular adenomas (HCA) are benign liver tumors predominantly developed in women using oral contraceptives. Here, exome sequencing identified recurrent somatic FRK mutations that induce constitutive kinase activity, STAT3 activation, and cell proliferation sensitive to Src inhibitors. We also found uncommon recurrent mutations activating JAK1, gp130, or ß-catenin. Chromosome copy number and methylation profiling revealed patterns that correlated with specific gene mutations and tumor phenotypes. Finally, integrative analysis of HCAs transformed to hepatocellular carcinoma revealed ß-catenin mutation as an early alteration and TERT promoter mutations as associated with the last step of the adenoma-carcinoma transition. In conclusion, we identified the genomic diversity in benign hepatocyte proliferation, several therapeutic targets, and the key genomic determinants of the adenoma-carcinoma transformation sequence.

Biochemical and functional analyses of gp130 mutants unveil JAK1 as a novel therapeutic target in human inflammatory hepatocellular adenoma.

Inflammatory hepatocellular adenomas (IHCAs) are benign liver lesions that can be characterized histologically by the presence of an inflammatory infiltrate and at the molecular level by the overexpression of acute phase inflammatory response genes. Recurrent somatic mutations of the interleukin-6 (IL-6) signal transducer (IL6ST) locus, encoding the critical component of the IL-6 signal transduction machinery gp130, are present in 60% of IHCAs and in a subset (2%) of hepatocellular carcinoma (HCCs). By screening of 256 human hepatic adenoma specimens (the largest genetic analysis of IL6ST performed to date in this setting), we identified 24 distinct somatic IL6ST mutations among 66 mutant adenomas. The functional analysis of nine different gp130 mutants expressed in hepatic cancer cell lines consistently revealed the constitutive and IL-6-independent activation of the JAK/STAT signaling pathway. We further demonstrated that the signaling activity of mutant gp130 in IHCA remains responsive to suppressor of cytokine signaling 3 (SOCS3), a physiological gp130 inhibitor. Specifically, cells expressing a double mutant variant of gp130 with a disrupted SOCS3-binding site at residue 759 (Y186/Y759F) displayed a hyperactivation of signal transducer and activator of transcription 3 (STAT3) as compared with cells expressing the endogenous IHCA-associated Y186 gp130 mutant. Notably, we identified that constitutive signaling via gp130 in IHCA requires the Janus kinase family member JAK1, but not JAK2 or tyrosine kinase 2. In support of this notion, AG490, a tyrosine kinase inhibitor that selectively blocks JAK2, had no effect on gp130 activity. In stark contrast, we showed that ruxolitinib, a JAK1/JAK2-selective tyrosine kinase inhibitor used to treat patients with myelofibrosis, dramatically impaired JAK1-STAT signaling downstream of all IHCA-associated gp130 mutants. In conclusion, our findings provide a rationale for the use of JAK1 inhibitors for the treatment of HCAs expressing mutant gp130 as well as a subset of HCCs that bear similar mutations.

Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas.

Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumors. 60% of these tumors have IL-6 signal transducer (IL6ST; gp130) mutations that activate interleukin 6 (IL-6) signaling. Here, we report that 12% of IHCA subsets lacking IL6ST mutations harbor somatic signal transducer and activator of transcription 3 (STAT3) mutations (6/49). Most of these mutations are amino acid substitutions in the SH2 domain that directs STAT3 dimerization. In contrast to wild-type STAT3, IHCA STAT3 mutants constitutively activated the IL-6 signaling pathway independent of ligand in hepatocellular cells. Indeed, the IHCA STAT3 Y640 mutant homodimerized independent of IL-6 and was hypersensitive to IL-6 stimulation. This was associated with phosphorylation of tyrosine 705, a residue required for IL-6-induced STAT3 activation. Silencing or inhibiting the tyrosine kinases JAK1 or Src, which phosphorylate STAT3, impaired constitutive activity of IHCA STAT3 mutants in hepatocellular cells. Thus, we identified for the first time somatic STAT3 mutations in human tumors, revealing a new mechanism of recurrent STAT3 activation and underscoring the role of the IL-6-STAT3 pathway in benign hepatocellular tumorigenesis.

Interactions between the rabbit CSN1 gene and the nuclear matrix of stably transfected HC11 mammary epithelial cells vary with its level of expression.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.