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1.
Cell ; 178(6): 1493-1508.e20, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31474370

RESUMO

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.


Assuntos
Doença de Crohn/terapia , Citocinas/imunologia , Intestinos/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Doença de Crohn/imunologia , Doença de Crohn/patologia , Humanos , Imunoterapia/métodos , Fagócitos/patologia , Análise de Célula Única , Células Estromais/patologia , Linfócitos T/patologia
2.
Nature ; 581(7808): 316-322, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433612

RESUMO

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus12-14.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Motivos de Aminoácidos , Animais , Feminino , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Transdução de Sinais
3.
Blood ; 142(3): 260-273, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37192303

RESUMO

Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study.


Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Animais , Camundongos , ADP-Ribosil Ciclase 1/metabolismo , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Linfócitos T/patologia
4.
Am J Physiol Gastrointest Liver Physiol ; 321(5): G500-G512, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34494462

RESUMO

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Animais , Disponibilidade Biológica , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/patologia , Doença de Crohn/enzimologia , Doença de Crohn/patologia , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Fezes/química , Humanos , Mediadores da Inflamação/metabolismo , Lipocalinas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas com Domínio T/genética
5.
Gastroenterology ; 156(4): 1082-1097.e11, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30452921

RESUMO

BACKGROUND & AIMS: Intestinal fibrosis is a long-term complication in inflammatory bowel diseases (IBD) that frequently results in functional damage, bowel obstruction, and surgery. Interleukin (IL) 36 is a group of cytokines in the IL1 family with inflammatory effects. We studied the expression of IL36 and its receptor, interleukin 1 receptor like 2 (IL1RL2 or IL36R) in the development of intestinal fibrosis in human tissues and mice. METHODS: We obtained intestinal tissues from 92 patients with Crohn's disease (CD), 48 patients with ulcerative colitis, and 26 patients without inflammatory bowel diseases (control individuals). Tissues were analyzed by histology to detect fibrosis and by immunohistochemistry to determine the distribution of fibroblasts and levels of IL36R ligands. Human and mouse fibroblasts were incubated with IL36 or control medium, and transcriptome-wide RNA sequences were analyzed. Mice were given neutralizing antibodies against IL36R, and we studied intestinal tissues from Il1rl2-/- mice; colitis and fibrosis were induced in mice by repetitive administration of DSS or TNBS. Bone marrow cells were transplanted from Il1rl2-/- to irradiated wild-type mice and intestinal tissues were analyzed. Antibodies against IL36R were applied to mice with established chronic colitis and fibrosis and intestinal tissues were studied. RESULTS: Mucosal and submucosal tissue from patients with CD or ulcerative colitis had higher levels of collagens, including type VI collagen, compared with tissue from control individuals. In tissues from patients with fibrostenotic CD, significantly higher levels of IL36A were noted, which correlated with high numbers of activated fibroblasts that expressed α-smooth muscle actin. IL36R activation of mouse and human fibroblasts resulted in expression of genes that regulate fibrosis and tissue remodeling, as well as expression of collagen type VI. Il1rl2-/- mice and mice given injections of an antibody against IL36R developed less severe colitis and fibrosis after administration of DSS or TNBS, but bone marrow cells from Il1rl2-/- mice did not prevent induction of colitis and fibrosis. Injection of antibodies against IL36R significantly reduced established fibrosis in mice with chronic intestinal inflammation. CONCLUSION: We found higher levels of IL36A in fibrotic intestinal tissues from patients with IBD compared with control individuals. IL36 induced expression of genes that regulate fibrogenesis in fibroblasts. Inhibition or knockout of the IL36R gene in mice reduces chronic colitis and intestinal fibrosis. Agents designed to block IL36R signaling could be developed for prevention and treatment of intestinal fibrosis in patients with IBD.


Assuntos
Colite Ulcerativa/metabolismo , Colágeno Tipo VI/metabolismo , Colo/patologia , Doença de Crohn/metabolismo , Interleucina-1/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Receptores de Interleucina-1/metabolismo , Actinas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Colite/induzido quimicamente , Colite/patologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Sulfato de Dextrana , Fibroblastos/efeitos dos fármacos , Fibrose , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Interleucina-1/farmacologia , Ligantes , Camundongos , Camundongos Knockout , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/genética , Transdução de Sinais , Transcriptoma , Ácido Trinitrobenzenossulfônico
6.
J Biol Chem ; 291(32): 16597-609, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27307043

RESUMO

Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in ∼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.


Assuntos
Proteína Acessória do Receptor de Interleucina-1 , Subunidade alfa de Receptor de Interleucina-18 , Polimorfismo Genético , Células HEK293 , Humanos , Proteína Acessória do Receptor de Interleucina-1/química , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Subunidade alfa de Receptor de Interleucina-18/química , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Domínios Proteicos , Transdução de Sinais , Relação Estrutura-Atividade
7.
J Biol Chem ; 290(39): 23997-4006, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26269592

RESUMO

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.


Assuntos
Endocitose/fisiologia , Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Humanos , Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/genética , Transporte Proteico/fisiologia , Receptores de Interleucina/genética
8.
Blood ; 117(21): 5683-91, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21487111

RESUMO

TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, with 2 different fluorophores [5(6) carboxytetramethylrhodamine and Alexa Fluor 488] and monitored their intracellular localization in human plasmacytoid dendritic cells (pDCs). All fluorescent compounds induced the production of IFN-α, TNF-α, and IL-6 and the up-regulation of CD80 and CD86 by pDCs showing they retained TLR7-stimulating activity. Confocal imaging of pDCs showed that, similar to CpG-B, all compounds concentrated in the MHC class II loading compartment (MIIC), identified as lysosome-associated membrane protein 1(+), CD63, and HLA-DR(+) endosomes. Treatment of pDCs with bafilomycin A, an antagonist of the vacuolar-type proton ATPase controlling endosomal acidification, prevented the accumulation of small molecule TLR7 agonists, but not of CpG-B, in the MIIC. These results indicate that a pH-driven concentration of small molecule TLR7 agonists in the MIIC is required for pDC activation.


Assuntos
Adenina/análogos & derivados , Aminoquinolinas/farmacocinética , Células Dendríticas/metabolismo , Corantes Fluorescentes , Genes MHC da Classe II/fisiologia , Imidazóis/farmacocinética , Receptor 7 Toll-Like/agonistas , Adenina/farmacocinética , Antineoplásicos/farmacocinética , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Humanos , Imiquimode , Macrolídeos/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Quinolinas/química , Quinolinas/farmacocinética , Receptor 7 Toll-Like/metabolismo
9.
J Immunol ; 186(7): 4213-22, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21383246

RESUMO

TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules. We have generated a mouse by N-nitrose-N'-ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7(rsq1). In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7(rsq1) localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7(rsq1) fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7(wt/wt) splenocytes. Thus, TLR7(rsq1) is a bona fide phenocopy of the TLR7 null mouse. Because TLR7(rsq1) binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7(rsq1) mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.


Assuntos
Mutação Puntual/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células HEK293 , Humanos , Imidazóis/farmacologia , Ligantes , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor 7 Toll-Like/deficiência
10.
Mol Immunol ; 156: 31-38, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36889184

RESUMO

Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases.


Assuntos
Ativação Linfocitária , Receptor de Morte Celular Programada 1 , Humanos , Células Jurkat , Quinases da Família src , Apoptose
11.
mBio ; 14(2): e0298122, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36976016

RESUMO

Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B. burgdorferi strain B31. However, B5's epitope has never been elucidated, despite widespread interest in OspC as a possible Lyme disease vaccine antigen. Here, we report the crystal structure of B5 antigen-binding fragments (Fabs) in complex with recombinant OspC type A (OspCA). Each OspC monomer within the homodimer was bound by a single B5 Fab in a side-on orientation, with contact points along OspC's α-helix 1 and α-helix 6, as well as interactions with the loop between α-helices 5 and 6. In addition, B5's complementarity-determining region (CDR) H3 bridged the OspC-OspC' homodimer interface, revealing the quaternary nature of the protective epitope. To provide insight into the molecular basis of B5 serotype specificity, we solved the crystal structures of recombinant OspC types B and K and compared them to OspCA. This study represents the first structure of a protective B cell epitope on OspC and will aid in the rational design of OspC-based vaccines and therapeutics for Lyme disease. IMPORTANCE The spirochete Borreliella burgdorferi is a causative agent of Lyme disease, the most common tickborne disease in the United States. The spirochete is transmitted to humans during the course of a tick taking a bloodmeal. After B. burgdorferi is deposited into the skin of a human host, it replicates locally and spreads systemically, often resulting in clinical manifestations involving the central nervous system, joints, and/or heart. Antibodies directed against B. burgdorferi's outer surface protein C (OspC) are known to block tick-to-host transmission, as well as dissemination of the spirochete within a mammalian host. In this report, we reveal the first atomic structure of one such antibody in complex with OspC. Our results have implications for the design of a Lyme disease vaccine capable of interfering with multiple stages in B. burgdorferi infection.


Assuntos
Borrelia burgdorferi , Doença de Lyme , Carrapatos , Humanos , Animais , Camundongos , Borrelia burgdorferi/metabolismo , Epitopos de Linfócito B/genética , Vacinas contra Doença de Lyme , Antígenos de Bactérias , Doença de Lyme/prevenção & controle , Proteínas da Membrana Bacteriana Externa/química , Mamíferos/metabolismo
12.
EBioMedicine ; 75: 103758, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34933179

RESUMO

BACKGROUND: Loss of intestinal epithelial barrier integrity is a critical component of Inflammatory Bowel Disease (IBD) pathogenesis. Co-expression regulation of ligand-receptor pairs in IBD mucosa has not been systematically studied. Targeting ligand-receptor pairs which are induced in IBD mucosa and function in intestinal epithelial barrier integrity may provide novel therapeutics for IBD. METHODS: We performed transcriptomic meta-analysis on public IBD datasets combined with cell surface protein-protein-interaction (PPI) databases. We explored primary human/mouse intestinal organoids and Caco-2 cells for expression and function studies of uPA-uPAR (prime hits from the meta-analysis). Epithelial barrier integrity was measured by Trans-Epithelial Electrical Resistance (TEER), FITC-Dextran permeability and tight junction assessment. Genetic (CRISPR, siRNA and KO mice) and pharmacological (small molecules, neutralizing antibody and peptide inhibitors) approaches were applied. Mice deficient of uPAR were studied using the Dextran Sulfate Sodium (DSS)-induced colitis model. FINDINGS: The IBD ligand-receptor meta-analysis led to the discovery of a coordinated upregulation of uPA and uPAR in IBD mucosa. Both genes were significantly upregulated during epithelial barrier breakdown in primary intestinal organoids and decreased during barrier formation. Genetic inhibition of uPAR or uPA, or pharmacologically blocking uPA-uPAR interaction protects against cytokine-induced barrier breakdown. Deficiency of uPAR in epithelial cells leads to enhanced EGF/EGFR signalling, a known regulator of epithelial homeostasis and repair. Mice deficient of uPAR display improved intestinal barrier function in vitro and during DSS-induced colitis in vivo. INTERPRETATION: Our findings suggest that blocking uPA-uPAR interaction via pharmacological agents protects the epithelial barrier from inflammation-induced damage, indicating a potential therapeutic target for IBD. FUNDING: The study was funded by Boehringer Ingelheim.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Células CACO-2 , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Humanos , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Junções Íntimas/metabolismo
13.
Front Immunol ; 12: 773445, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35095847

RESUMO

NFAT activating protein with ITAM motif 1 (NFAM1) is an ITAM bearing-transmembrane receptor that has been reported to play a role in B cell signaling and development. We performed expression analysis of NFAM1 using publicly available gene expression data sets and found that NFAM1 expression is significantly induced in intestinal biopsies from Crohn's disease (CD) and ulcerative colitis (UC) patients. At the cellular level, we further observed high expression of NFAM1 in monocytes and neutrophils, and low expression in B and T cells. To explore the role of NFAM1 in multiple immune cells and its potential role in IBD, we generated NFAM1-/- mice. In contrast with previous reports using NFAM1-transgenic mice, NFAM1-/- mice have no obvious defects in immune cell development, or B cell responses. Interestingly, NFAM1-/- monocytes produce reduced levels of TNF-α in response to activation by multiple IBD-relevant stimuli, including CD40L, TLR ligands and MDP. Additional cytokines and chemokines such as IL-6, IL-12, CCL3 and CCL4 are also reduced in CD40L stimulated NFAM1-/- monocytes. Collectively, these findings indicate that NFAM1 promotes monocyte activation, thereby amplifying the response to diverse stimuli. Similarly, we observed that deletion of NFAM1 in human monocytes reduces expression of CD40L-induced CCL4. Lastly, to assess the role of NFAM1 in IBD, we compared development of anti-CD40 induced colitis in NFAM1+/+ and NFAM1-/- mice. We found that although NFAM1 deletion had no impact on development of gut pathology, we did observe a decrease in serum TNF-α, confirming that NFAM1 promotes pro-inflammatory cytokine production in vivo. Taken together, we conclude that NFAM1 functions to amplify cytokine production and should be further evaluated as a therapeutic target for treatment of autoimmune disease.


Assuntos
Citocinas/imunologia , Inflamação/imunologia , Proteínas de Membrana/imunologia , Monócitos/imunologia , Animais , Linfócitos B/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Células Cultivadas , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Interleucina-12/imunologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Neutrófilos/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologia
14.
Respir Res ; 10: 43, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19486528

RESUMO

BACKGROUND: The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases. METHODS: TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C). RESULTS: There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFalpha were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy. CONCLUSION: These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.


Assuntos
Inflamação/induzido quimicamente , Poli I-C/farmacologia , Receptor 3 Toll-Like/fisiologia , Animais , Linhagem Celular , Citocinas/metabolismo , Feminino , Humanos , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pletismografia , Testes de Função Respiratória , Receptor 3 Toll-Like/deficiência , Receptor 3 Toll-Like/genética
15.
Front Immunol ; 10: 2903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921164

RESUMO

Genome-wide co-expression analysis is often used for annotating novel gene functions from high-dimensional data. Here, we developed an R package with a Shiny visualization app that creates immuno-networks from RNAseq data using a combination of Weighted Gene Co-expression Network Analysis (WGCNA), xCell immune cell signatures, and Bayesian Network Learning. Using a large publicly available RNAseq dataset we generated a Gene Module-Immune Cell (GMIC) network that predicted causal relationships between DEAH-box RNA helicase (DHX)15 and genes associated with humoral immunity, suggesting that DHX15 may regulate B cell fate. Deletion of DHX15 in mouse B cells led to impaired lymphocyte development, reduced peripheral B cell numbers, and dysregulated expression of genes linked to antibody-mediated immune responses similar to the genes predicted by the GMIC network. Moreover, antigen immunization of mice demonstrated that optimal primary IgG1 responses required DHX15. Intrinsic expression of DHX15 was necessary for proliferation and survival of activated of B cells. Altogether, these results support the use of co-expression networks to elucidate fundamental biological processes.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Imunomodulação , RNA Helicases/genética , Animais , Biópsia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Imunomodulação/genética , Camundongos , RNA Helicases/metabolismo
16.
MAbs ; 11(5): 956-964, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31068073

RESUMO

Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1 κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacocinética , Receptores de Interleucina-1/imunologia , Animais , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ratos , Receptores de Interleucina-1/metabolismo , Estudos Retrospectivos , Transcriptoma
17.
J Innate Immun ; 10(1): 56-69, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29176319

RESUMO

Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R.


Assuntos
Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Lisossomos/metabolismo , Psoríase/imunologia , Receptores de Interleucina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Endocitose , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-18/genética , Mutação/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
18.
Cell Immunol ; 248(2): 103-14, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18048020

RESUMO

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-kappaB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.


Assuntos
Anticorpos Monoclonais , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/imunologia , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular , Linhagem Celular Transformada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Projetos Piloto , Receptor 3 Toll-Like/metabolismo
19.
MAbs ; 9(7): 1143-1154, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28726542

RESUMO

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, ß and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.


Assuntos
Anticorpos Monoclonais/imunologia , Receptores de Interleucina/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Humanos , Camundongos , Psoríase/imunologia
20.
Physiol Genomics ; 26(2): 125-33, 2006 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-16554548

RESUMO

To gain global pathway perspective of ex vivo viral infection models using human peripheral blood mononuclear cells (PBMCs), we conducted expression analysis on PBMCs of healthy donors. RNA samples were collected at 3 and 24 h after PBMCs were challenged with the Toll-like receptor-3 (TLR3) agonist polyinosinic acid-polycytidylic acid [poly(I:C)] and analyzed by internally developed cDNA microarrays and TaqMan PCR. Our results demonstrate that poly(I:C) challenge can elicit certain gene expression changes, similar to acute viral infection. Hierarchical clustering revealed distinct immediate early, early-to-late, and late gene regulation patterns. The early responses were innate immune responses that involve TLR3, the NF-kappaB-dependent pathway, and the IFN-stimulated pathway, whereas the late responses were mostly cell-mediated immune response that involve activation of cell adhesion, cell mobility, and phagocytosis. Overall, our results expanded the utilities of this ex vivo model, which could be used to screen molecules that can modulate viral stress-induced inflammation, in particular those mediated via TLRs.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Indutores de Interferon/farmacologia , Leucócitos Mononucleares/metabolismo , Poli I-C/farmacologia , Análise por Conglomerados , Humanos , Inflamação , Interferons/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose , Receptor 3 Toll-Like/metabolismo
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