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1.
Blood ; 142(20): 1740-1751, 2023 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-37738562

RESUMO

Histiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation.


Assuntos
Histiocitose , Proteínas Quinases Ativadas por Mitógeno , Humanos , Histiocitose/genética , Histiocitose/patologia , Mutação , Receptores Toll-Like , Inflamação/genética , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo
2.
Eur J Immunol ; 51(4): 864-878, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616974

RESUMO

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.


Assuntos
Candida albicans/imunologia , Parede Celular/imunologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , beta-Glucanas/imunologia , Células CACO-2 , Candida albicans/metabolismo , Candida albicans/fisiologia , Linhagem Celular Tumoral , Parede Celular/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HT29 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mucosa Intestinal/microbiologia , Fosforilação/imunologia , Quinase Syk/imunologia , Quinase Syk/metabolismo , Zimosan/imunologia , Zimosan/metabolismo , beta-Glucanas/metabolismo
3.
Eur J Immunol ; 44(12): 3729-40, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25251945

RESUMO

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and may be involved in intestinal immune responses. Fungi are important components of the intestinal microflora. The potential role of fungi, and in particular their cell wall component ß-glucan, in modulating human intestinal epithelial responses is still unclear. Here we examined whether human IECs are capable of recognizing and responding to ß-glucans, and the potential mechanisms of their activation. We show that human IECs freshly isolated from surgical specimens, and the human IEC lines HT-29 and SW480, express the ß-glucan receptor Dectin-1. The ß-glucan-consisting glycans curdlan and zymosan stimulated IL-8 and CCL2 secretion by IEC lines. This was significantly inhibited by a Dectin-1 blockade using its soluble antagonist laminarin. Spleen tyrosine kinase (Syk), a signaling mediator of Dectin-1 activation, is expressed in human IECs. ß-glucans and Candida albicans induced Syk phosphorylation, and Syk inhibition significantly decreased ß-glucan-induced chemokine secretion from IECs. Thus, IECs may respond to ß-glucans by the secretion of pro-inflammatory chemokines in a Dectin-1- and Syk-dependent pathway, via receptors and a signaling pathway described to date only for myeloid cells. These findings highlight the importance of fungi-IEC interactions in intestinal inflammation.


Assuntos
Quimiocina CCL2/imunologia , Células Epiteliais/imunologia , Interleucina-8/imunologia , Mucosa Intestinal/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lectinas Tipo C/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/efeitos dos fármacos , beta-Glucanas/farmacologia , Linhagem Celular , Células Epiteliais/citologia , Feminino , Humanos , Mucosa Intestinal/citologia , Masculino , Transdução de Sinais/imunologia , Quinase Syk
4.
J Gastroenterol Hepatol ; 29(12): 1976-84, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25092526

RESUMO

BACKGROUND AND AIM: Crohn's disease (CD) is characterized by loss of tolerance to intestinal microorganisms. This is reflected by serological responses to fungal glycans such as mannan and ß-glucans. Fungal glycans have various effects on immune cells. However, the evidence for their effects in CD is vague. This study aimed to assess the effects of fungal cell wall glycans on human peripheral blood mononuclear cells (PBMCs) from CD and control patients. METHODS: Human PBMCs from CD and control patients were stimulated by fungal cell wall glycans. Cytokine secretion was detected by ELISA and glycan receptor expression by flow cytometry. RESULTS: Mannan, ß-glucans (curdlan), chitosan, and zymosan induced the secretion of interleukin (IL)-1ß, IL-6, IL-23, IL-10, and tumor necrosis factor-α by PBMCs. Spleen tyrosin kinase and Src tyrosine kinase were involved in the response to mannan and ß-glucans. Mannan and whole yeast cells induced a significantly higher pro-inflammatory cytokine response in CD compared with control patients. CONCLUSIONS: The results may suggest that CD is characterized by hyperresponsiveness to fungal glycans. Thus, glycans may potentially be triggering or perpetuating inflammation.


Assuntos
Doença de Crohn/imunologia , Leucócitos Mononucleares/imunologia , Mananas/imunologia , beta-Glucanas/imunologia , Adulto , Idoso , Quitosana/imunologia , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/fisiologia , Quinase Syk , Fator de Necrose Tumoral alfa/metabolismo , Zimosan/imunologia , Quinases da Família src/fisiologia
5.
bioRxiv ; 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38915605

RESUMO

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

6.
J Crohns Colitis ; 2024 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-39073573

RESUMO

BACKGROUND & AIMS: Janus kinase (JAK) inhibitors are used for treating inflammatory bowel diseases (IBD). We aimed to identify molecular effects of JAK inhibition in human intestinal mucosa, considering IBD location and phenotype. METHODS: Colonic and ileal explants from patients with ulcerative colitis (UC), Crohn's disease (CD), and non-IBD controls (NC) were assessed for phosphorylated signal transducers and activators of transcription (p-STAT) levels and Inflammatory genes expression panel in response to ex-vivo JAK inhibitor (tofacitinib). Cytokine production by lamina propria lymphocytes in response to tofacitinib was assessed. Human intestinal organoids were used to investigate JAK inhibitors' effects on iNOS expression. RESULTS: Explants were collected from 68 patients (UC=20; CD=20; NC=28). p-STAT1\3\5 inhibition rates varied, being higher in colonic compared to ileal explants. p-STAT1\3 inhibition rates negatively correlated with CRP levels. While significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including NOS2. JAK inhibition significantly decreased Th1\Th2\Th17-related cytokine production from lamina propria lymphocytes. Various JAK inhibitors reduced IFN-γ-induced increase in iNOS expression in organoids. CONCLUSIONS: Site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying personalized drug effects in patients with IBD.

7.
Front Immunol ; 14: 1142492, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36969163

RESUMO

Background: Intestinal epithelial cells (IECs) are the first to encounter luminal microorganisms and actively participate in intestinal immunity. We reported that IECs express the ß-glucan receptor Dectin-1, and respond to commensal fungi and ß-glucans. In phagocytes, Dectin-1 mediates LC3-associated phagocytosis (LAP) utilizing autophagy components to process extracellular cargo. Dectin-1 can mediate phagocytosis of ß-glucan-containing particles by non-phagocytic cells. We aimed to determine whether human IECs phagocytose ß-glucan-containing fungal particles via LAP. Methods: Colonic (n=18) and ileal (n=4) organoids from individuals undergoing bowel resection were grown as monolayers. Fluorescent-dye conjugated zymosan (ß-glucan particle), heat-killed- and UV inactivated C. albicans were applied to differentiated organoids and to human IEC lines. Confocal microscopy was used for live imaging and immuno-fluorescence. Quantification of phagocytosis was carried out with a fluorescence plate-reader. Results: zymosan and C. albicans particles were phagocytosed by monolayers of human colonic and ileal organoids and IEC lines. LAP was identified by LC3 and Rubicon recruitment to phagosomes and lysosomal processing of internalized particles was demonstrated by co-localization with lysosomal dyes and LAMP2. Phagocytosis was significantly diminished by blockade of Dectin-1, actin polymerization and NAPDH oxidases. Conclusions: Our results show that human IECs sense luminal fungal particles and internalize them via LAP. This novel mechanism of luminal sampling suggests that IECs may contribute to the maintenance of mucosal tolerance towards commensal fungi.


Assuntos
Células Epiteliais , Fungos , Fagocitose , beta-Glucanas , Humanos , Zimosan/farmacologia
8.
Nat Genet ; 53(4): 500-510, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33782605

RESUMO

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.


Assuntos
Artrite/genética , Colite/genética , Dermatite/genética , Linfoma Difuso de Grandes Células B/genética , Quinase Syk/genética , Adulto , Animais , Artrite/imunologia , Artrite/patologia , Artrite/terapia , Sequência de Bases , Transplante de Medula Óssea , Colite/imunologia , Colite/patologia , Colite/terapia , Dermatite/imunologia , Dermatite/patologia , Dermatite/terapia , Família , Feminino , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Lactente , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Linhagem , Inibidores de Proteínas Quinases/farmacologia , Quinase Syk/antagonistas & inibidores , Quinase Syk/deficiência
9.
J Cell Biochem ; 110(5): 1262-71, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20564222

RESUMO

Autophagy, a process of self-digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti-apoptotic proteins such as Bcl-2 via its BH3-like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST-Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST-Beclin 1. Further examination by cross linking and co-immunoprecipitation experiments confirmed that Beclin 1 self-interacts and that the coiled coil and the N-terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl-x(L), had no effect on Beclin 1 self-interaction. Moreover, this self-interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full-length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein-protein interactions.


Assuntos
Aminoácidos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Sirolimo/farmacologia , Aminoácidos/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Sítios de Ligação/genética , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Proteína bcl-X/metabolismo
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