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1.
EMBO Rep ; 25(7): 2914-2949, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38783164

RESUMO

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.


Assuntos
Alarminas , Catelicidinas , Armadilhas Extracelulares , Inflamação , Neutrófilos , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Inflamação/metabolismo , Inflamação/genética , Animais , Humanos , Camundongos , Alarminas/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Queratinócitos/metabolismo , RNA/genética , RNA/metabolismo , Psoríase/genética , Psoríase/metabolismo , Psoríase/patologia , Transdução de Sinais , Ativação de Neutrófilo/genética , Imunidade Inata/genética
2.
Gut Microbes ; 16(1): 2320291, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38417029

RESUMO

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.


Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Peptídeos , Policetídeos , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Microambiente Tumoral , Resistencia a Medicamentos Antineoplásicos , Mutagênicos/metabolismo , Recidiva Local de Neoplasia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Policetídeos/metabolismo , Lipídeos
3.
Gut Microbes ; 16(1): 2315631, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38385162

RESUMO

Immune checkpoint inhibitors (ICI) have been positioned as a standard of care for patients with advanced non-small-cell lung carcinomas (NSCLC). A pilot clinical trial has reflected optimistic association between supplementation with Clostridium butyricum MIYAIRI 588 (CBM588) and ICI efficacy in NSCLC. However, it remains to be established whether this biotherapeutic strain may be sufficient to heighten the immunogenicity of the tumor draining lymph nodes to overcome resistance to ICI. Herein, we report that supplementation with CBM588 led to an improved responsiveness to antibody targeting programmed cell death protein 1 (aPD-1). This was statistically associated with a significant decrease in α-diversity of gut microbiota from CBM588-treated mice upon PD-1 blockade. At the level of the tumor-draining lymph node, such combination of treatment significantly lowered the frequency of microbiota-modulated subset of regulatory T cells that express Retinoic Orphan Receptor gamma t (Rorγt+ Treg). Specifically, this strongly immunosuppressive was negatively correlated with the abundance of bacteria that belong to the family of Ruminococcaceae. Accordingly, the colonic expression of both indoleamine 2,3-Dioxygenase 1 (IDO-1) and interleukin-10 (IL-10) were heightened in mice with greater PD-1 blockade efficacy. The CBM588-induced ability to secrete Interleukin-10 of lamina propria mononuclear cells was heightened in tumor bearers when compared with cancer-free mice. Conversely, blockade of interleukin-10 signaling preferentially enhanced the capacity of CD8+ T cells to secrete Interferon gamma when being cocultured with CBM588-primed lamina propria mononuclear cells of tumor-bearing mice. Our results demonstrate that CBM588-centered intervention can adequately improve intestinal homeostasis and efficiently overcome resistance to PD-1 blockade in mice.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Clostridium butyricum , Microbioma Gastrointestinal , Neoplasias Pulmonares , Animais , Camundongos , Linfócitos T CD8-Positivos , Clostridium butyricum/fisiologia , Interleucina-10/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Receptor de Morte Celular Programada 1 , Linfócitos T Reguladores
4.
Sci Signal ; 17(820): eabg8145, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38261657

RESUMO

Inflammasomes are multiprotein complexes that drive inflammation and contribute to protective immunity against pathogens and immune pathology in autoinflammatory diseases. Inflammasomes assemble when an inflammasome scaffold protein senses an activating signal and forms a signaling platform with the inflammasome adaptor protein ASC. The NLRP subfamily of NOD-like receptors (NLRs) includes inflammasome nucleators (such as NLRP3) and also NLRP12, which is genetically linked to familial autoinflammatory disorders that resemble diseases caused by gain-of-function NLRP3 mutants that generate a hyperactive NLRP3 inflammasome. We performed a screen to identify ASC inflammasome-nucleating proteins among NLRs that have the canonical pyrin-NACHT-LRR domain structure. Only NLRP3 and NLRP6 could initiate ASC polymerization to form "specks," and NLRP12 failed to nucleate ASC polymerization. However, wild-type NLRP12 inhibited ASC inflammasome assembly induced by wild-type and gain-of-function mutant NLRP3, an effect not seen with disease-associated NLRP12 mutants. The capacity of NLRP12 to suppress NLRP3 inflammasome assembly was limited to human NLRP3 and was not observed for wild-type murine NLRP3. Furthermore, peripheral blood mononuclear cells from patients with an NLRP12 mutant-associated inflammatory disorder produced increased amounts of the inflammatory cytokine IL-1ß in response to NLRP3 stimulation. Thus, our findings provide insights into NLRP12 biology and suggest that NLRP3 inhibitors in clinical trials for NLRP3-driven diseases may also be effective in treating NLRP12-associated autoinflammatory diseases.


Assuntos
Doenças Hereditárias Autoinflamatórias , Inflamassomos , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal , Peptídeos e Proteínas de Sinalização Intracelular , Leucócitos Mononucleares , Proteína 3 que Contém Domínio de Pirina da Família NLR , Síndrome
5.
Front Immunol ; 14: 1224383, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38146368

RESUMO

Chronic obstructive pulmonary disease (COPD) is a major health issue primarily caused by cigarette smoke (CS) and characterized by breathlessness and repeated airway inflammation. NLRP6 is a cytosolic innate receptor controlling intestinal inflammation and orchestrating the colonic host-microbial interface. However, its roles in the lungs remain largely unexplored. Using CS exposure models, our data show that airway inflammation is strongly impaired in Nlrp6-deficient mice with drastically fewer recruited neutrophils, a key cell subset in inflammation and COPD. We found that NLRP6 expression in lung epithelial cells is important to control airway and lung tissue inflammation in an inflammasome-dependent manner. Since gut-derived metabolites regulate NLRP6 inflammasome activation in intestinal epithelial cells, we investigated the link between NLRP6, CS-driven lung inflammation, and gut microbiota composition. We report that acute CS exposure alters gut microbiota in both wild-type (WT) and Nlrp6-deficient mice and that antibiotic treatment decreases CS-induced lung inflammation. In addition, gut microbiota transfer from dysbiotic Nlrp6-deficient mice to WT mice decreased airway lung inflammation in WT mice, highlighting an NLRP6-dependent gut-to-lung axis controlling pulmonary inflammation.


Assuntos
Microbioma Gastrointestinal , Pneumonia , Receptores de Superfície Celular , Poluição por Fumaça de Tabaco , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/genética , Pneumonia/microbiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/patologia , Fezes/microbiologia , Bactérias/classificação , Bactérias/metabolismo , Biodiversidade , Expressão Gênica
6.
Front Immunol ; 14: 1252979, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37876927

RESUMO

Background: Crohn's disease (CD) is a complex and poorly understood myeloid-mediated disorder. Genetic variants with loss of function in the NOD2 gene confer an increased susceptibility to ileal CD. While Nod2 in myeloid cells may confer protection against T-cell mediated ileopathy, it remains unclear whether it may promote resolution of the inflamed colon. In this study, we evaluated the function of Nod2 in myeloid cells in a model of acute colitis and colitis-associated colon cancer (CAC). Methods: To ablate Nod2 specifically within the myeloid compartment, we generated LysMCre/+;Nod2fl/fl mice. The role of NOD2 was studied in a setting of Dextran Sodium Sulfate (DSS)-induced colitis and in azoxymethane (AOM)/DSS model. Clinical parameters were quantified by colonoscopy, histological, flow cytometry, and qRT-PCR analysis. Results: Upon DSS colitis model, LysMCre/+;Nod2fl/fl mice lost less weight than control littermates and had less severe damage to the colonic epithelium. In the AOM/DSS model, endoscopic monitoring of tumor progression revealed a lowered number of adenomas within the colon of LysMCre/+;Nod2fl/fl mice, associated with less expression of Tgfb. Mechanistically, lysozyme M was required for the improved disease severity in mice with a defect of NOD2 in myeloid cells. Conclusion: Our results indicate that loss of Nod2 signaling in myeloid cells aids in the tissue repair of the inflamed large intestine through lysozyme secretion by myeloid cells. These results may pave the way to design new therapeutics to limit the inflammatory and tumorigenic functions of NOD2.


Assuntos
Colite , Doença de Crohn , Macrófagos , Proteína Adaptadora de Sinalização NOD2 , Animais , Camundongos , Azoximetano , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Macrófagos/metabolismo , Muramidase/genética , Proteína Adaptadora de Sinalização NOD2/genética
7.
Gut Microbes ; 15(2): 2265138, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37842920

RESUMO

Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here, we characterize and investigate one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we show that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylation alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra's possible role in the carcinogenic process.


Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Humanos , Microbioma Gastrointestinal/genética , Firmicutes/genética , Bactérias , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia
8.
Gut Microbes ; 15(2): 2249960, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37655966

RESUMO

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.


Assuntos
Infecções por Caliciviridae , Colite , Gastroenterite , Microbioma Gastrointestinal , Proteína Adaptadora de Sinalização NOD2 , Animais , Camundongos , Infecções por Caliciviridae/imunologia , Colite/induzido quimicamente , Colite/virologia , Gastroenterite/imunologia , Gastroenterite/virologia , Proteína Adaptadora de Sinalização NOD2/metabolismo
9.
Front Immunol ; 14: 1181823, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37415975

RESUMO

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.


Assuntos
Doença de Crohn , Monócitos , Animais , Camundongos , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Doença de Crohn/genética , Doença de Crohn/patologia , Macrófagos , Inibidores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa
10.
PLoS Pathog ; 19(3): e1011192, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36888688

RESUMO

Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Camundongos , Animais , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo
11.
RMD Open ; 9(1)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36963782

RESUMO

OBJECTIVE: To assess whether gut microbiota composition is associated with patient characteristics and may have predictive value on the response to TNF inhibitor (TNFi) treatment in axial spondyloarthritis (AxSpA). METHODS: The study involved 61 patients fulfilling the Assessment of SpondyloArthritis International Society classification criteria for AxSpA. All patients had active disease despite non-steroidal anti-inflammatory drugs intake and were eligible for treatment with a TNFi. At baseline, the mean Ankylosing Spondylitis Disease Activity Score was 2.9±1 and mean C reactive protein (CRP) level 9.7±11.4 mg/L. Bacterial 16S ribosomal RNA gene sequencing was performed on stool samples collected at baseline (month 0 (M0)) and 3 months after TNFi initiation (month 3 (M3)). Alpha and beta diversity metrics were calculated on the relative abundance of core operational taxonomic units (OTUs). RESULTS: The HLA-B27 status affected at least in part the global composition of faecal microbiota at M0 as well as the abundance/prevalence of several anaerobic bacteria in the families Oscillospiraceae, Lachnospiraceae and Bifidobacteriaceae. In contrast, smoking affected the global composition of faecal microbiota at both M0 and M3. The prevalence/abundance of seven bacterial OTUs at M0 was associated with response to TNFi treatment. One of the candidates, present only in non-responders, is the genus Sutterella, and the other six candidates are in the class Clostridia. CONCLUSIONS: Several SpA patients' characteristics modulate the composition of gut microbiota as did TNFi treatment. Moreover, the abundance/prevalence of seven OTUs at baseline may be used as a novel non-invasive index that predicts the response to TNFi with greater accuracy than HLA-B27 status, CRP level and measures of disease activity.


Assuntos
Microbioma Gastrointestinal , Espondilite Anquilosante , Humanos , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Antígeno HLA-B27/genética , Resultado do Tratamento , Fator de Necrose Tumoral alfa , Espondilite Anquilosante/tratamento farmacológico
12.
Cancer J ; 29(2): 98-101, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36957980

RESUMO

ABSTRACT: Although antibiotic is a major contributor to shifts in the intestinal flora that may persist for up to several months after cessation, it is now increasingly recognized that its prescription may differentially influence clinical outcome of different anticancer treatments. Intense clinical and basic research efforts aim then at gaining sufficient insights about how the cooperative action between the intestinal ecosystem and immune surveillance modulates the efficacy of anticancer treatments. In this review, we summarize multiple levels of knowledge between vancomycin exposure, the gut microbiota, and a meaningful therapeutic response. Furthermore, we discuss the mode of action of antibiotic therapy that is prescribed for prophylaxis of bacteremia and neutropenia and outline the opportunity for judiciously improving the efficacy of anticancer drugs.


Assuntos
Microbioma Gastrointestinal , Vancomicina , Humanos , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Ecossistema , Antibacterianos/efeitos adversos
13.
Science ; 379(6634): 826-833, 2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36821686

RESUMO

The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.


Assuntos
Microbioma Gastrointestinal , Crescimento , Intestinos , Lactobacillaceae , Desnutrição , Proteína Adaptadora de Sinalização NOD2 , Animais , Camundongos , Parede Celular/química , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Microbioma Gastrointestinal/fisiologia , Vida Livre de Germes , Transtornos do Crescimento/fisiopatologia , Transtornos do Crescimento/terapia , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Intestinos/microbiologia , Intestinos/fisiologia , Lactobacillaceae/fisiologia , Desnutrição/fisiopatologia , Desnutrição/terapia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Crescimento/efeitos dos fármacos , Crescimento/fisiologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico
14.
Mol Ther ; 31(4): 970-985, 2023 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-36641622

RESUMO

Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftr gene in vivo in a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTR gene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.


Assuntos
Códon sem Sentido , Fibrose Cística , Camundongos , Animais , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Códon de Terminação/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética
15.
PLoS One ; 18(1): e0279028, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662875

RESUMO

Nod-Like Receptor Pyrin domain-containing protein 6 (NLRP6), a member of the Nucleotide-oligomerization domain-Like Receptor (NLR) family of proteins, assembles together with the ASC protein to form an inflammasome upon stimulation by bacterial lipoteichoic acid and double-stranded DNA. Besides its expression in myeloid cells, NLRP6 is also expressed in intestinal epithelial cells where it may contribute to the maintenance of gut homeostasis and negatively controls colorectal tumorigenesis. Here, we report that NLRP6 is very faintly expressed in several colon cancer cell lines, detected only in cytoplasmic small dots were it colocalizes with ASC. Consequently, it is very hardly detected by standard western-blotting techniques by several presently available commercial antibodies which, in contrast, highly cross-react with a protein of 90kDa that we demonstrate to be unrelated to NLRP6. We report here these results to caution the community not to confuse the 90kDa protein with the endogenous human NLRP6.


Assuntos
Inflamassomos , Neoplasias , Humanos , Inflamassomos/metabolismo , Homeostase , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular
16.
Gut ; 72(6): 1115-1128, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36175116

RESUMO

OBJECTIVE: In the management of patients with IBD, there is a need to identify prognostic markers and druggable biological pathways to improve mucosal repair and probe the efficacy of tumour necrosis factor alpha biologics. Vnn1 is a pantetheinase that degrades pantetheine to pantothenate (vitamin B5, a precursor of coenzyme A (CoA) biosynthesis) and cysteamine. Vnn1 is overexpressed by inflamed colonocytes. We investigated its contribution to the tolerance of the intestinal mucosa to colitis-induced injury. DESIGN: We performed an RNA sequencing study on colon biopsy samples from patients with IBD stratified according to clinical severity and modalities of treatment. We generated the VIVA mouse transgenic model, which specifically overexpresses Vnn1 on intestinal epithelial cells and explored its susceptibility to colitis. We developed a pharmacological mimicry of Vnn1 overexpression by administration of Vnn1 derivatives. RESULTS: VNN1 overexpression on colonocytes correlates with IBD severity. VIVA mice are resistant to experimentally induced colitis. The pantetheinase activity of Vnn1 is cytoprotective in colon: it enhances CoA regeneration and metabolic adaptation of colonocytes; it favours microbiota-dependent production of short chain fatty acids and mostly butyrate, shown to regulate mucosal energetics and to be reduced in patients with IBD. This prohealing phenotype is recapitulated by treating control mice with the substrate (pantethine) or the products of pantetheinase activity prior to induction of colitis. In severe IBD, the protection conferred by the high induction of VNN1 might be compromised because its enzymatic activity may be limited by lack of available substrates. In addition, we identify the elevation of indoxyl sulfate in urine as a biomarker of Vnn1 overexpression, also detected in patients with IBD. CONCLUSION: The induction of Vnn1/VNN1 during colitis in mouse and human is a compensatory mechanism to reinforce the mucosal barrier. Therefore, enhancement of vitamin B5-driven metabolism should improve mucosal healing and might increase the efficacy of anti-inflammatory therapy.


Assuntos
Colite , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Colite/metabolismo , Colo/patologia , Mucosa Intestinal/metabolismo , Doenças Inflamatórias Intestinais/genética , Ácidos Graxos Voláteis/metabolismo , Vitaminas , Sulfato de Dextrana , Modelos Animais de Doenças
17.
J Crohns Colitis ; 17(1): 111-122, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-35917251

RESUMO

BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.


Assuntos
Colite , Oxazolona , Camundongos , Animais , Oxazolona/efeitos adversos , Acetilmuramil-Alanil-Isoglutamina/efeitos adversos , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Colite/metabolismo , Inflamação , Transdução de Sinais , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo
18.
Biol Open ; 11(12)2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36350252

RESUMO

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. We set out to systematically compare the effect of two ECM classes, the interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In contrast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture resembled gut development in vivo. The dramatic ECM remodelling was accompanied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse resulted in a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.


Assuntos
Laminina , Células-Tronco , Animais , Camundongos , Colágeno/metabolismo , Matriz Extracelular , Laminina/metabolismo , Laminina/farmacologia , Celulas de Paneth/metabolismo , Intestinos
19.
Sci Rep ; 12(1): 17591, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266398

RESUMO

Live biotherapeutic products constitute an emerging therapeutic approach to prevent or treat inflammatory bowel diseases. Lactobacillus acidophilus is a constituent of the human microbiota with probiotic potential, that is illustrated by improvement of intestinal inflammation and antimicrobial activity against several pathogens. In this study, we evaluated the immunomodulatory properties of the L. acidophilus strain BIO5768 at steady state and upon acute inflammation. Supplementation of naïve mice with BIO5768 heightened the transcript level of some IL-17 target genes encoding for protein with microbicidal activity independently of NOD2 signaling. Of these, the BIO5768-induced expression of Angiogenin-4 was blunted in monocolonized mice that are deficient for the receptor of IL-17 (but not for NOD2). Interestingly, priming of bone marrow derived dendritic cells by BIO5768 enhanced their ability to support the secretion of IL-17 by CD4+ T cells. Equally of importance, the production of IL-22 by type 3 innate lymphoid cells is concomitantly heightened in response to BIO5768. When administered alone or in combination with Bifidobacterium animalis spp. lactis BIO5764 and Limosilactobacillus reuteri, BIO5768 was able to alleviate at least partially intestinal inflammation induced by Citrobacter rodentium infection. Furthermore, BIO5768 was also able to improve colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). In conclusion, we identify a new potential probiotic strain for the management of inflammatory bowel diseases, and provide some insights into its IL-17-dependent and independent mode of action.


Assuntos
Colite , Imunidade Inata , Doenças Inflamatórias Intestinais , Lactobacillus acidophilus , Probióticos , Animais , Camundongos , Bifidobacterium animalis , Colite/induzido quimicamente , Colite/terapia , Colite/microbiologia , Infecções por Enterobacteriaceae/terapia , Inflamação , Doenças Inflamatórias Intestinais/terapia , Interleucina-17 , Linfócitos , Probióticos/farmacologia , Probióticos/uso terapêutico , Ácido Trinitrobenzenossulfônico/efeitos adversos
20.
Front Immunol ; 13: 928886, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36189256

RESUMO

Asthma is an extremely prevalent chronic inflammatory disease of the airway where innate and adaptive immune systems participate collectively with epithelial and other structural cells to cause airway hyperresponsiveness, mucus overproduction, airway narrowing, and remodeling. The nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular innate immune sensors that detect microbe-associated molecular patterns and damage-associated molecular patterns, well-recognized for their central roles in the maintenance of tissue homeostasis and host defense against bacteria, viruses and fungi. In recent times, NLRs have been increasingly acknowledged as much more than innate sensors and have emerged also as relevant players in diseases classically defined by their adaptive immune responses such as asthma. In this review article, we discuss the current knowledge and recent developments about NLR expression, activation and function in relation to asthma and examine the potential interventions in NLR signaling as asthma immunomodulatory therapies.


Assuntos
Asma , Proteína Adaptadora de Sinalização NOD2 , Proteínas de Transporte , Humanos , Imunidade Inata , Nucleotídeos/metabolismo
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