RESUMO
Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems.
Assuntos
Imunidade Inata , Lipídeos/imunologia , Animais , Membrana Celular/química , Fibroblastos/metabolismo , Doença de Gaucher/imunologia , Humanos , Interleucina-6/imunologia , Leucodistrofia de Células Globoides/imunologia , Redes e Vias Metabólicas , Camundongos , Esfingolipídeos/metabolismo , Receptores Toll-Like/imunologiaRESUMO
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 SinaisRESUMO
Fundamental insight gained over the last decades led to the discovery of cytokines as pivotal drivers of inflammatory diseases such as rheumatoid arthritis, psoriasis/psoriasis arthritis, inflammatory bowel diseases, atopic dermatitis and spondylarthritis. A deeper understanding of the pro-inflammatory and anti-inflammatory effects of various cytokines has prompted new cytokine-targeting therapies, which revolutionised the treatment options in the last years for patients with inflammatory disorders. Disease-associated immune responses typically involve a complex interplay of multiple cytokines. Therefore, blockade of one single cytokine does not necessarily lead to a persistent remission in all patients with inflammatory disorders and fostered new therapeutic strategies targeting intracellular pathways shared by multiple cytokines. By inhibiting JAK-STAT signalling pathways common to families of cytokines, JAK-inhibitors (JAKinibs) have created a new paradigm for the treatment of inflammatory diseases. Multiple agents have been approved for various disorders and more are being investigated for several new indications. Second-generation selective JAKinibs have been devised with the aim to achieve an increased selectivity and a possible reduced risk of side effects. In the current review, we will summarise the current body of evidence of pan versus selective JAKinibs and the most recent insights on new side effects and indications, including COVID-19.
Assuntos
Artrite Psoriásica , Artrite Reumatoide , Doenças Autoimunes , Inibidores de Janus Quinases , Humanos , Inibidores de Janus Quinases/efeitos adversos , Artrite Reumatoide/tratamento farmacológico , Citocinas/metabolismo , Artrite Psoriásica/tratamento farmacológico , Janus Quinases/metabolismoRESUMO
BACKGROUND: The significance of muscle biopsy as a diagnostic tool in idiopathic inflammatory myopathies (IIM) remains elusive. We aimed to determine the diagnostic weight that has been given to muscle biopsy in patients with suspected IIM, particularly in terms of clinical diagnosis and therapeutic decisions. MATERIAL AND METHODS: In this retrospective multicentric study, we analyzed muscle biopsy results of adult patients with suspected IIM referred to a tertiary center between January 1, 2007, and October 31, 2021. Information regarding referral department, suspected diagnosis, biopsy site, demographic, clinical, laboratory data, and imaging results were extracted. Statistical analyses included the level of agreement between suspected and histological diagnosis and calculation of diagnostic performance (positive and negative predictive values, positive and negative likelihood ratios, sensitivity, and specificity of muscle biopsy in relation to clinical diagnosis and/or treatment initiation). Performance was tested in different strata based on clinical pre-test probability. RESULTS: Among 758 muscle biopsies, IIM was histologically compatible in 357/758 (47.1%) cases. Proportion of IIM was higher if there was a solid clinical pre-test probability (64.3% vs. 42.4% vs. 48% for high, medium and low pre-test probability). Sensitivity and specificity of muscle biopsy were highest (82%) when the diagnosis by the clinician was used as outcome scenario. Negative predictive value was only moderate (between 63% and 80%) and lowest if autoantibodies were positive (35%). CONCLUSION: In patients with clinically suspected IIM, approximately 50% of biopsies revealed features indicative of IIM. Diagnostic performance of muscle biopsy was moderate to high depending on clinical pre-test probability.
Assuntos
Miosite , Adulto , Humanos , Estudos Retrospectivos , Miosite/diagnóstico , Miosite/patologia , Biópsia , Tomada de Decisão Clínica , Autoanticorpos , MúsculosRESUMO
BACKGROUND: An understanding vaccine-dependent effects on protective and sustained humoral immune response is crucial to planning future vaccination strategies against coronavirus disease 2019 (COVID-19). METHODS: In this multicenter, population-based, cohort study including 4601 individuals after primary vaccination against COVID-19 ≥ 4 months earlier we compared factors associated with residual antibody levels against severe acute respiratory syndrome coronavirus-2 receptor-binding domain (RBD) across different vaccination strategies (BNT162b2, mRNA-1273, or ChAdOx1). RESULTS: Our main model including 3787 individuals (2 × BNT162b2, n = 2271; 2 × mRNA-1273, n = 251; 2 × ChAdOx1, n = 1265), predicted significantly lower levels of anti-RBD antibodies after 6 months in individuals vaccinated with ChAdOx1 (392.7 binding antibody units per milliliter [BAU/mL]) compared with those vaccinated with BNT162b2 (1179.5 BAU/mL) or mRNA-1273 (2098.2 BAU/mL). Vaccine-dependent association of antibody levels was found for age with a significant predicted difference in BAU/ml per year for BNT162b2 (-21.5; 95% confidence interval [CI], -24.7 to -18.3) and no significant association for mRNA-1273 (-4.0; 95% CI, -20.0 to 12.1) or ChAdOx1 (1.7; 95% CI, .2 to 3.1). The predicted decrease over time since full immunization was highest in mRNA-1273 (-23.4; 95% CI, -31.4 to -15.4) compared with BNT162b2 (-5.9; 95% CI, -7 to -4.8). CONCLUSIONS: Our study revealed population-based evidence of vaccine-dependent effects of age and time since full immunization on humoral immune response. Findings underline the importance of individualized vaccine selection, especially in elderly individuals.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Idoso , Humanos , Vacina BNT162 , Vacina de mRNA-1273 contra 2019-nCoV , Estudos de Coortes , COVID-19/prevenção & controleRESUMO
INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.
Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Citocinas , Fator de Necrose Tumoral alfa/farmacologia , Membrana Sinovial/patologia , Sinoviócitos/patologia , Fibroblastos/patologia , Células CultivadasRESUMO
OBJECTIVES: A third COVID-19 vaccination is recommended for immunosuppressed patients. However, data on immunogenicity and safety of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMIDs) are sparse and therefore addressed within this clinical trial. METHODS: 60 immunosuppressed patients and 48 healthy controls (HCs) received a third vaccination with an mRNA vaccine. The primary endpoint was defined as the presence of antibody levels against the receptor-binding domain (RBD)>1500 BAU/mL in patients with IMIDs versus HCs. Further endpoints included differences in neutralising antibodies and cellular immune responses after the third vaccination. Reactogenicity was recorded for 7 days, and safety was evaluated until week 4. RESULTS: Rate of individuals with anti-RBD antibodies>1500 BAU/mL was not significantly different after the third vaccination between patients with IMIDs and HCs (91% vs 100% p=0.101). Anti-RBD and neutralising antibody levels were significantly lower in patients with IMIDs after the third vaccination than in HCs (p=0.002 and p=0.016, respectively). In contrast, fold increase in antibody levels between week 0 and 4 was higher in patients with IMIDs. Treatment with biological (b) disease-modifying anti-rheumatic drugs (DMARD) or combination of bDMARDs and conventional synthetic DMARDs was associated with reduced antibody levels. Enhanced cellular immune response to wild type and Omicron peptide stimulation was observed after the third vaccination. No serious adverse event was attributed to the third vaccination. CONCLUSION: Our clinical trial data support the immunogenicity and safety of a third COVID-19 vaccination in patients with IMIDs. However, effects of DMARD therapy on immunogenicity should be considered. TRIAL REGISTRATION NUMBER: EudraCT No: 2021-002693-10.
Assuntos
Vacinas contra COVID-19 , Humanos , Anticorpos Antivirais , Antirreumáticos , COVID-19 , Vacinas contra COVID-19/efeitos adversos , Imunogenicidade da Vacina , Agentes de Imunomodulação , VacinaçãoRESUMO
BACKGROUND: A 3rd COVID-19 vaccination is currently recommended for patients under immunosuppression. However, a fast decline of antibodies against the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein has been observed. Currently it remains unclear whether immunosuppressive therapy affects kinetics of humoral and cellular immune responses. METHODS: 50 patients under immunosuppression and 42 healthy controls (HCs) received a 3rd dose of an mRNA-based vaccine and were monitored over a 12-weeks period. Humoral immune response was assessed 4 and 12 weeks after 3rd dose. Antibodies were quantified using the Elecsys Anti-SARS-CoV-2 Spike immunoassay against the receptor-binding domain (RBD) of the spike protein. SARS-CoV-2-specific T cell responses were quantified by IFN-γ ELISpot assays. Adverse events, including SARS-CoV-2 infections, were monitored over a 12-week period. RESULTS: At week 12, reduced anti-RBD antibody levels were observed in IMID patients as compared to HCs (median antibody level 5345 BAU/ml [1781-10,208] versus 9650 BAU/ml [6633-16,050], p < 0.001). Reduction in relative antibody levels was significantly higher in IMID patients as compared to HCs at week 12 (p < 0.001). Lowest anti-RBD antibody levels were detected in IMID patients who received biological disease-modifying anti-rheumatic drugs (DMARDs) or a combination therapy with conventional synthetic and biological DMARDs. Number of SARS-CoV-2-specific T cells against wildtype and Omicron variants remained stable over 12 weeks in IMID patients. No serious adverse events were reported. CONCLUSION: Due to a fast decline in anti-RBD antibodies in IMID patients an early 4th vaccination should be considered in this vulnerable group of patients.
Assuntos
Antirreumáticos , COVID-19 , Humanos , Vacinas contra COVID-19 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2 , Anticorpos , Imunidade Humoral , Anticorpos Antivirais , VacinaçãoRESUMO
OBJECTIVES: SARS-CoV-2-induced COVID-19 has led to exponentially rising mortality, particularly in immunosuppressed patients, who inadequately respond to conventional COVID-19 vaccination. METHODS: In this blinded randomised clinical trial, we compare the efficacy and safety of an additional booster vaccination with a vector versus mRNA vaccine in non-seroconverted patients. We assigned 60 patients under rituximab treatment, who did not seroconvert after their primary mRNA vaccination with either BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna), to receive a third dose, either using the same mRNA or the vector vaccine ChAdOx1 nCoV-19 (Oxford-AstraZeneca). Patients were stratified according to the presence of peripheral B cells. The primary efficacy endpoint was the difference in the SARS-CoV-2 antibody seroconversion rate between vector (heterologous) and mRNA (homologous) vaccinated patients by week 4. Key secondary endpoints included the overall seroconversion and cellular immune response; safety was assessed at week 1 and week 4. RESULTS: Seroconversion rates at week 4 were comparable between vector (6/27 patients, 22%) and mRNA (9/28, 32%) vaccines (p=0.6). Overall, 27% of patients seroconverted; specific T cell responses were observed in 20/20 (100%) vector versus 13/16 (81%) mRNA vaccinated patients. Newly induced humoral and/or cellular responses occurred in 9/11 (82%) patients. 3/37 (8%) of patients without and 12/18 (67%) of the patients with detectable peripheral B cells seroconverted. No serious adverse events, related to immunisation, were observed. CONCLUSIONS: This enhanced humoral and/or cellular immune response supports an additional booster vaccination in non-seroconverted patients irrespective of a heterologous or homologous vaccination regimen.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , ChAdOx1 nCoV-19 , Humanos , RNA Mensageiro , Soroconversão , Vacinação , Vacinas Sintéticas , Vacinas de mRNARESUMO
OBJECTIVES: Patients under rituximab therapy are at high risk for a severe COVID-19 disease course. Humoral immune responses to SARS-CoV-2 vaccination are vastly diminished in B-cell-depleted patients, even after a third vaccine dose. However, it remains unclear whether these patients benefit from a fourth vaccination and whether continued rituximab therapy affects antibody development. METHODS: In this open-label extension trial, 37 rituximab-treated patients who received a third dose with either a vector or mRNA-based vaccine were vaccinated a fourth time with an mRNA-based vaccine (mRNA-1273 or BNT162b2). Key endpoints included the humoral and cellular immune response as well as safety after a fourth vaccination. RESULTS: The number of patients who seroconverted increased from 12/36 (33%) to 21/36 (58%) following the fourth COVID-19 vaccination. In patients with detectable antibodies to the spike protein's receptor-binding domain (median: 8.0 binding antibody units (BAU)/mL (quartiles: 0.4; 13.8)), elevated levels were observed after the fourth vaccination (134.0 BAU/mL (quartiles: 25.5; 1026.0)). Seroconversion and antibody increase were strongly diminished in patients who received rituximab treatment between the third and the fourth vaccination. The cellular immune response declined 12 weeks after the third vaccination, but could only be slightly enhanced by a fourth vaccination. No unexpected safety signals were detected, one serious adverse event not related to vaccination occurred. CONCLUSIONS: A fourth vaccine dose is immunogenic in a fraction of rituximab-treated patients. Continuation of rituximab treatment reduced humoral immune response, suggesting that rituximab affects a second booster vaccination. It might therefore be considered to postpone rituximab treatment in clinically stable patients. TRIAL REGISTRATION NUMBER: 2021-002348-57.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Rituximab/efeitos adversos , Anticorpos Antivirais , SARS-CoV-2 , Vacina BNT162 , Vacinação , RNA Mensageiro , Imunogenicidade da VacinaRESUMO
OBJECTIVES: TNF-induced activation of fibroblast-like synoviocytes (FLS) is a critical determinant for synovial inflammation and joint destruction in RA. The detrimental role of TNF-receptor 1 (TNFR1) has thoroughly been characterized. The contributions of TNFR2, however, are largely unknown. This study was performed to delineate the role of TNFR2 in human FLS activation. METHODS: TNFR2 expression in synovial tissue samples was determined by immunohistochemistry. Expression of TNFR2 was silenced using RNAi or CRISPR/Cas9 technologies. Global transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to validate RNA-seq results and to uncover pathways operating downstream of TNFR2 in FLS. RESULTS: TNFR2 expression was increased in RA when compared with OA synovial tissues. In particular, RA-FLS demonstrated higher levels of TNFR2 when compared with OA-FLS. TNFR2 expression in RA-FLS correlated with RA disease activity, synovial T- and B-cell infiltration. TNF and IL1ß were identified as inflammatory mediators that upregulate TNFR2 in RA-FLS. Silencing of TNFR2 in RA-FLS markedly diminished the TNF-induced expression of inflammatory cytokines and chemokines, including CXCR3-binding chemokines and the B-cell activating factor TNFSF13B. Immunobiochemical analyses revealed that TNFR2-mediated expression of inflammatory mediators critically depends on STAT1. CONCLUSION: Our results define a critical role for TNFR2 in FLS-driven inflammation and unfold its participation in the unresolved course of synovial inflammation in RA.
Assuntos
Artrite Reumatoide , Receptores Tipo II do Fator de Necrose Tumoral , Sinoviócitos , Humanos , Artrite Reumatoide/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Membrana Sinovial/metabolismo , Sinoviócitos/metabolismoRESUMO
OBJECTIVES: Evidence suggests that B cell-depleting therapy with rituximab (RTX) affects humoral immune response after vaccination. It remains unclear whether RTX-treated patients can develop a humoral and T-cell-mediated immune response against SARS-CoV-2 after immunisation. METHODS: Patients under RTX treatment (n=74) were vaccinated twice with either mRNA-1273 or BNT162b2. Antibodies were quantified using the Elecsys Anti-SARS-CoV-2 S immunoassay against the receptor-binding domain (RBD) of the spike protein and neutralisation tests. SARS-CoV-2-specific T-cell responses were quantified by IFN-γ enzyme-linked immunosorbent spot assays. Prepandemic healthy individuals (n=5), as well as healthy individuals (n=10) vaccinated with BNT162b2, served as controls. RESULTS: All healthy controls developed antibodies against the SARS-CoV-2 RBD of the spike protein, but only 39% of the patients under RTX treatment seroconverted. Antibodies against SARS-CoV-2 RBD significantly correlated with neutralising antibodies (τ=0.74, p<0.001). Patients without detectable CD19+ peripheral B cells (n=36) did not develop specific antibodies, except for one patient. Circulating B cells correlated with the levels of antibodies (τ=0.4, p<0.001). However, even patients with a low number of B cells (<1%) mounted detectable SARS-CoV-2-specific antibody responses. SARS-CoV-2-specific T cells were detected in 58% of the patients, independent of a humoral immune response. CONCLUSIONS: The data suggest that vaccination can induce SARS-CoV-2-specific antibodies in RTX-treated patients, once peripheral B cells at least partially repopulate. Moreover, SARS-CoV-2-specific T cells that evolved in more than half of the vaccinated patients may exert protective effects independent of humoral immune responses.
Assuntos
Antirreumáticos/uso terapêutico , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Hospedeiro Imunocomprometido/imunologia , Imunogenicidade da Vacina/imunologia , Rituximab/uso terapêutico , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Feminino , Humanos , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunogenicidade da Vacina/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2 , Linfócitos T/imunologiaRESUMO
Through their capacity to sense danger signals and to generate active interleukin-1ß (IL-1ß), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1ß, little is known about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1ß was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.
Assuntos
Caspase 1/metabolismo , Inflamassomos/imunologia , Interleucina-1alfa/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/imunologia , Proteínas de Ligação a DNA , Feminino , Humanos , Inflamassomos/metabolismo , Interleucina-1alfa/antagonistas & inibidores , Interleucina-1alfa/metabolismo , Interleucina-1beta/biossíntese , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Peritonite/imunologia , Processamento de Proteína Pós-Traducional , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/imunologiaRESUMO
Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismoRESUMO
Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.
Assuntos
Proteínas de Transporte/fisiologia , Movimento Celular/genética , Células Dendríticas/imunologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Mutação PuntualRESUMO
The enzyme acid sphingomyelinase-like phosphodiesterase 3B (SMPDL3B) was shown to act as a negative regulator of innate immune signaling, affecting cellular lipid composition and membrane fluidity. Furthermore, several reports identified this enzyme as an off target of the therapeutic antibody rituximab, with implications in kidney disorders. However, structural information for this protein is lacking. Here we present the high resolution crystal structure of murine SMPDL3B, which reveals a substrate binding site strikingly different from its paralogs. The active site is located in a narrow boot-shaped cavity. We identify a unique loop near the active site that appears to impose size constraints on incoming substrates. A structure in complex with phosphocholine indicates that the protein recognizes this head group via an aromatic box, a typical choline-binding motif. Although a potential substrate for SMPDL3B is sphingomyelin, we identify other possible substrates such as CDP-choline, ATP, and ADP. Functional experiments employing structure-guided mutagenesis in macrophages highlight amino acid residues potentially involved in recognition of endogenous substrates. Our study is an important step toward elucidating the specific function of this poorly characterized enzyme.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Difosfato de Adenosina/química , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Colina/química , Colina/genética , Colina/metabolismo , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Camundongos , Domínios Proteicos , Estrutura Secundária de Proteína , Células Sf9 , Esfingomielinas/química , Esfingomielinas/genética , Esfingomielinas/metabolismo , Spodoptera , Especificidade por SubstratoRESUMO
Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication, and account for more than two-thirds of known drug targets.1-8 In the past years, several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach.9-11 We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation, and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54% (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90% of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. Glycosylphosphatidylinositol (GPI)-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications.
Assuntos
Proteínas de Membrana/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Antibacterianos/farmacologia , Biotinilação , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatografia Líquida/métodos , Humanos , Espectrometria de Massas/métodos , Proteínas de Membrana/genética , Proteoma/genética , Reprodutibilidade dos Testes , Tunicamicina/farmacologiaRESUMO
Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular proteins involved in innate-driven inflammatory responses. The function of the family member NLR caspase recruitment domain containing protein 5 (NLRC5) remains a matter of debate, particularly with respect to NF-κB activation, type I IFN, and MHC I expression. To address the role of NLRC5, we generated Nlrc5-deficient mice (Nlrc5(Δ/Δ)). In this article we show that these animals exhibit slightly decreased CD8(+) T cell percentages, a phenotype compatible with deregulated MHC I expression. Of interest, NLRC5 ablation only mildly affected MHC I expression on APCs and, accordingly, Nlrc5(Δ/Δ) macrophages efficiently primed CD8(+) T cells. In contrast, NLRC5 deficiency dramatically impaired basal expression of MHC I in T, NKT, and NK lymphocytes. NLRC5 was sufficient to induce MHC I expression in a human lymphoid cell line, requiring both caspase recruitment and LRR domains. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Consistent with downregulated MHC I expression, the elimination of Nlrc5(Δ/Δ) lymphocytes by cytotoxic T cells was markedly reduced and, in addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Hence, loss of NLRC5 expression represents an advantage for evading CD8(+) T cell-mediated elimination by downmodulation of MHC I levels-a mechanism that may be exploited by transformed cells. Our data show that NLRC5 acts as a key transcriptional regulator of MHC I in lymphocytes and support an essential role for NLRs in directing not only innate but also adaptive immune responses.
Assuntos
Genes MHC Classe I , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Linfócitos T Citotóxicos/imunologia , Imunidade Adaptativa , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Medula Óssea/imunologia , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/imunologia , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Linfócitos T Citotóxicos/citologiaRESUMO
Impaired immune response to COVID-19 (coronavirus disease 2019) vaccination has been reported in patients with inborn errors of immunity (IEI). Repetitive vaccinations are recommended for this vulnerable group. Due to the high diversity within IEI patients, additional safety and immunogenicity data are needed to better understand these aspects especially in less common immunodeficiency syndromes. In this prospective open-label clinical trial, we assessed the humoral immune response and the Tcell response in patients with IEI or severe MBL (mannose-binding lectin) deficiency (IEI/MBLdef) after three vaccinations. A total of 16 patients and 16 matched healthy controls (HC) with suboptimal humoral response defined by anti-SARS-CoV2 RBD (severe acute respiratory syndrome coronavirus type 2 receptor binding domain) antibodies below 1500 BAU/ml (binding antibody units per ml) after the second COVID-19 vaccination were enrolled in this study and qualified for a third mRNA vaccine dose. After 4 weeks following vaccination, 100% of HC and 75% of IEI/MBLdef patients exhibited anti-SARS-CoV2 RBD antibodies >â¯1500 BAU/ml, although the difference was not statistically significant (75% vs. 100%; pâ¯= 0.109). Although post-vaccination IEI/MBLdef patients demonstrated significantly increased anti-SARS-CoV2 RBD antibodies and neutralizing antibodies compared to baseline, these responses were significantly lower in IEI/MBLdef patients compared to HCs. Notably, the third vaccination augmented the cellular immune response to both wild-type and omicron peptide stimulation. No serious adverse events were reported within the 4week follow-up period and, importantly, vaccination had little to no effect on the long-term disease activity and fatigue. This trial strongly supports the recommendation of repeated COVID-19 vaccinations for patients suffering from immunodeficiencies, especially when they exhibit an initially limited response to the vaccine.
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.