RESUMO
BACKGROUND AND AIMS: The etiology of biliary atresia (BA) is not known and is likely multifactorial, including a genetic predisposition, a viral or environmental trigger, an aberrant autoimmune response targeting cholangiocytes, and unique susceptibilities of the neonatal bile ducts to injury. Damaged cholangiocytes may express neo self-antigens and elicit autoreactive T-cell-mediated inflammation and B-cell production of autoantibodies. The aim of this study was to discover autoantibodies in BA that correlated with outcomes. APPROACH AND RESULTS: An autoantigen microarray encompassing approximately 9,500 autoantigens was used to screen for serum immunoglobulin M (IgM) and immunoglobulin G (IgG) autoantibodies in patients with BA or other liver disease controls. Validation of candidate autoantibodies by enzyme-linked immunosorbent assay on a second cohort of subjects (6-12 months following Kasai portoenterostomy) and correlations of autoantibodies with outcomes were performed (serum bilirubin levels and need for liver transplant in first 2 years of life). Mean anti-chitinase 3-like 1 (CHI3L1), anti-delta-like ligand (DLL-4), and antisurfactant protein D (SFTPD) IgM autoantibodies in BA were significantly higher compared with controls, and IgM autoantibody levels positively correlated with worse outcomes. Immunofluorescence revealed cholangiocyte-predominant expression of CHI3L1, DLL-4, and SFTPD. The humoral autoantibody response was associated with C3d complement activation and T-cell autoimmunity, based on detection of cholangiocyte-predominant C3d co-staining and peripheral blood autoreactive T cells specific to CHI3L1, DLL-4 and SFTPD, respectively. CONCLUSIONS: BA is associated with cholangiocyte-predominant IgM autoantibodies in the first year after Kasai portoenterostomy. Anti-CHI3L1, anti-DLL-4, and anti-SFTPD IgM autoantibody correlations with worse outcomes and the detection of C3d on cholangioctyes and antigen-specific autoreactive T cells suggest that autoimmunity plays a role in the ongoing bile duct injury and progression of disease.
Assuntos
Autoanticorpos/imunologia , Ductos Biliares Extra-Hepáticos/imunologia , Atresia Biliar/imunologia , Imunoglobulina M/imunologia , Ductos Biliares Extra-Hepáticos/citologia , Atresia Biliar/cirurgia , Linhagem Celular , Pré-Escolar , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Lactente , Masculino , Portoenterostomia HepáticaRESUMO
BACKGROUND: Biliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied. MATERIALS AND METHODS: Wild type or Ig-α-/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression. RESULTS: BA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1ß decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1ß increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors. CONCLUSIONS: Loss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA.
Assuntos
Ácidos e Sais Biliares/metabolismo , Atresia Biliar/imunologia , Colestase/imunologia , Inflamação/genética , MicroRNAs/metabolismo , Animais , Ductos Biliares/imunologia , Ductos Biliares/patologia , Atresia Biliar/patologia , Antígenos CD79/genética , Antígenos CD79/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Colestase/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Hepatócitos/metabolismo , Humanos , Inflamação/complicações , Inflamação/imunologia , Fígado/citologia , Fígado/imunologia , Fígado/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Biliary atresia (BA) is a neonatal T cell-mediated, inflammatory, sclerosing cholangiopathy. In the rhesus rotavirus (RRV)-induced neonatal mouse model of BA (murine BA), mice lacking B cells do not develop BA, and the lack of B cells is associated with loss of T-cell and macrophage activation. The aim of this study was to determine the mechanism of B cell-mediated immune activation (antigen presentation versus cytokine production) in murine BA. Normal neonatal B cells in the liver are predominantly at pro-B and pre-B cellular development. However, BA mice exhibit a significant increase in the number and activation status of mature liver B cells. Adoptively transferred B cells into RRV-infected, B cell-deficient mice were able to reinstate T-cell and macrophage infiltration and biliary injury. Nonetheless, neonatal liver B cells were incompetent at antigen presentation to T cells. Moreover, 3-83 immunoglobulin transgenic mice, in which B cells only present an irrelevant antigen, developed BA, indicating a B-cell antigen-independent mechanism. B cells from BA mice produced a variety of innate and adaptive immune cytokines associated with immune activation. In vitro trans-well studies revealed that BA B cells secreted cytokines that activated T cells based on increased expression of T-cell activation marker cluster of differentiation 69. Conclusion: Neonatal liver B cells are highly activated in murine BA and contribute to immune activation through production of numerous cytokines involved in innate and adaptive immunity; this work provides increased knowledge on the capacity of neonatal B cells to contribute to an inflammatory disease through cytokine-mediated mechanisms, and future studies should focus on targeting B cells as a therapeutic intervention in human BA.
Assuntos
Linfócitos B/metabolismo , Ductos Biliares/patologia , Atresia Biliar/imunologia , Citocinas/metabolismo , Imunidade Adaptativa/imunologia , Adolescente , Animais , Animais Recém-Nascidos , Linfócitos B/imunologia , Ductos Biliares/imunologia , Técnicas de Cultura de Células , Criança , Pré-Escolar , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Fígado/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência de RNA , Baço/imunologiaRESUMO
Apoptosis of alveolar macrophages and their subsequent clearance by neighboring phagocytes are necessary steps in the resolution of acute pulmonary inflammation. We have recently identified that activation of the Fas death receptor on the cell surface of macrophages drives macrophage apoptosis. However, the source of the cognate ligand for Fas (FasL) responsible for induction of alveolar macrophage apoptosis is not defined. Given their known role in the resolution of inflammation and ability to induce macrophage apoptosis ex vivo, we hypothesized that T lymphocytes represented a critical source of FasL. To address this hypothesis, C57BL/6J and lymphocyte-deficient (Rag-1(-/-)) mice were exposed to intratracheal lipopolysaccharide to induce pulmonary inflammation. Furthermore, utilizing mice expressing nonfunctional FasL, we adoptively transferred donor lymphocytes into inflamed lymphocyte-deficient mice to characterize the effect of lymphocyte-derived FasL on alveolar macrophage apoptosis in the resolution of inflammation. Herein, evidence is presented that lymphocytes expressing FasL enhance alveolar macrophage apoptosis during the resolution of LPS-induced inflammation. Moreover, lymphocyte induction of alveolar macrophage apoptosis results in contraction of the alveolar macrophage pool, which occurs in a FasL-dependent manner. Specifically, FasL-expressing CD8(+) T lymphocytes potently induce alveolar macrophage apoptosis and contraction of the alveolar macrophage pool. Together, these studies identify a novel role for CD8(+) T lymphocytes in the resolution of acute pulmonary inflammation.
Assuntos
Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteína Ligante Fas/imunologia , Macrófagos Alveolares/imunologia , Pneumonia/imunologia , Transferência Adotiva , Animais , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/imunologia , Proteína Ligante Fas/biossíntese , Proteínas de Homeodomínio/genética , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pneumonia/induzido quimicamenteRESUMO
Pathogenic fungi populate a wide range of environments and infect a diversity of host species. Despite this substantial biological flexibility, the impact of interactions between fungi and their hosts on the evolution of pathogenicity remains unclear. We studied how repeated interactions between the fungus Cryptococcus neoformans and relevant environmental and mammalian host cells-amoeba and mouse macrophages-shape the evolution of this model fungal pathogen. First, using a collection of clinical and environmental isolates of C. neoformans, we characterized a range of survival phenotypes for these strains when exposed to host cells of different species. We then performed serial passages of an environmentally isolated C. neoformans strain through either amoeba or macrophages for â¼75 generations to observe how these interactions select for improved replication within hosts. In one adapted population, we identified a single point mutation in the adenylyl cyclase gene, CAC1, that swept to fixation and confers a strong competitive advantage for growth inside macrophages. Strikingly, this growth advantage in macrophages is inversely correlated with disease severity during mouse infections, suggesting that adaptation to specific host niches can markedly reduce the pathogenicity of these fungi. These results raise intriguing questions about the influence of cyclic AMP (cAMP) signaling on pathogenicity and highlight the role of seemingly small adaptive changes in promoting fundamental shifts in the intracellular behavior and virulence of these important human pathogens.
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Background and Aims: Biliary atresia (BA) entails an inflammatory injury of the biliary tree, leading to fibrosis of the extrahepatic and intrahepatic bile ducts. The chronic inflammatory biliary injury may be due to lack of appropriate regulatory T cell (Treg) suppression of inflammation. The aims of the study were to characterize Treg deficits in human BA and to determine if Treg augmentation therapy improved outcomes in the rhesus rotavirus (RRV)-induced mouse model of BA. Methods: Immunophenotyping of human peripheral blood and liver Tregs was performed with flow cytometry, Vectra-6 multicolor immunohistochemistry (IHC), and real-time polymerase chain reaction. Measured outcomes of Treg augmentation with the interleukin-2 monoclonal antibody JES6-1/interleukin-2 in the RRV-induced mouse model of BA included survival, direct bilirubin, IHC, and liver flow cytometry. Results: Patients with BA had decreased peripheral blood Treg frequency and lack of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) upregulation despite a highly activated, effector Treg phenotype. IHC revealed decreased liver Treg frequency and Treg CTLA-4 expression. Treg augmentation in the murine model led to increased survival, decreased direct bilirubin levels and liver inflammation, and expansion of resident macrophages. In addition to the M2 phenotype of resident macrophages, these cells adopted an inflammatory M1 phenotype in response to RRV infection, which was inhibited with Treg augmentation. Conclusion: Patients with BA have Treg deficiencies associated with lack of sufficient CTLA-4 expression that is necessary for cell-cell contact inhibition of inflammatory responses. Treg augmentation therapy in murine BA protected from disease. Future treatment trials for BA should include agents that enhance Treg number or function, mimic CTLA-4 function, and promote anti-inflammatory M2 macrophage phenotypes.
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Environmental pathogens move from ecological niches to mammalian hosts, requiring adaptation to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. We demonstrate that the formation of a small C. neoformans morphotype-called "seed" cells due to their colonizing ability-is critical for extrapulmonary organ entry. Seed cells exhibit changes in fungal cell size and surface expression that result in an enhanced macrophage update. Seed cell formation is triggered by environmental factors, including C. neoformans' environmental niche, and pigeon guano with phosphate plays a central role. Seed cells show the enhanced expression of phosphate acquisition genes, and mutants unable to acquire phosphate fail to adopt the seed cell morphotype. Additionally, phosphate can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. Thus, C. neoformans' size variation represent inducible morphotypes that change host interactions to facilitate microbe spread.
Assuntos
Criptococose , Cryptococcus neoformans , Adaptação Fisiológica , Animais , Columbidae , Criptococose/microbiologia , Cryptococcus neoformans/genética , Mamíferos , Fosfatos/metabolismoRESUMO
Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.
Assuntos
Vasos Sanguíneos/fisiologia , Pulmão/fisiologia , Perfusão , Pressão , Alvéolos Pulmonares/fisiologia , Animais , Fixadores , CamundongosRESUMO
Biliary atresia is a progressive fibroinflammatory cholangiopathy of infancy that is associated with activation of innate and adaptive immune responses targeting bile ducts. A recently completed multicenter phase I/IIA trial of intravenous immunoglobulin in biliary atresia did not improve serum total bilirubin levels at 90 days after hepatoportoenterostomy or survival with the native liver at 1 year. A mechanistic aim of this trial was to determine if the peripheral blood immunophenotype was associated with clinical outcomes. Flow cytometry of peripheral blood cell markers (natural killer [NK], macrophage subsets, T- and B-cell subsets, regulatory T cells), neutrophils, and activation markers (clusters of differentiation [CD]38, CD69, CD86, human leukocyte antigen-DR isotype [HLA-DR]) was performed on 29 patients with biliary atresia at baseline and at 60, 90, 180, and 360 days after hepatoportoenterostomy. Plasma cytokines and neutrophil products were also measured. Spearman correlations of change of an immune marker from baseline to day 90 with change in serum bilirubin revealed that an increase in total bilirubin correlated with 1) increased percentage of HLA-DR+CD38+ NK cells and expression of NK cell activation markers CD69 and HLA-DR, 2) decreased percentage of regulatory T cells, and 3) increased interleukin (IL)-8 and associated neutrophil products (elastase and neutrophil extracellular traps). Cox modeling revealed that the change from baseline to day 60 of the percentage of HLA-DR+CD38+ NK cells and plasma IL-8 levels was associated with an increased risk of transplant or death by day 360. Conclusion: Poor outcomes in biliary atresia correlated with higher peripheral blood NK cells and IL-8 and lower regulatory T cells. Future studies should include immunotherapies targeting these pathways in order to protect the biliary tree from ongoing damage.