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1.
Cells ; 10(10)2021 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-34685525

RESUMO

The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages' supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.


Assuntos
Autoimunidade , COVID-19/sangue , COVID-19/imunologia , Armadilhas Extracelulares/imunologia , Imunidade Humoral , Inflamação , Neutrófilos/imunologia , Anticorpos Antinucleares , Peptídeos Catiônicos Antimicrobianos/sangue , Autoanticorpos/metabolismo , Estudos Transversais , Citocinas/metabolismo , Citocinas/farmacologia , Citometria de Fluxo , Granulócitos/metabolismo , Proteína HMGB1/sangue , Voluntários Saudáveis , Humanos , Microscopia Confocal , Monócitos/citologia , Neutrófilos/citologia , SARS-CoV-2 , Ubiquitinas/farmacologia
2.
Carbohydr Polym ; 273: 118567, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34560978

RESUMO

Diffuse alveolar injury and pulmonary fibrosis (PF) are the main causes of death of Covid-19 cases. In this study a low molecular weight fucoidan (LMWF) with unique structural was obtained from Laminaria japonica, and its anti- PF and anti-epithelial-mesenchymal transition (EMT) bioactivity were investigated both in vivo and in vitro. After LWMF treatment the fibrosis and inflammatory factors stimulated by Bleomycin (BLM) were in lung tissue. Immunohistochemical and Western-blot results found the expression of COL2A1, ß-catenin, TGF-ß, TNF-α and IL-6 were declined in mice lung tissue. Besides, the phosphorylation of PI3K and Akt were inhibited by LMWF. In addition, the progression of EMT induced by TGF-ß1 was inhibited by LMWF through down-regulated both TGF-ß/Smad and PI3K/AKT signaling pathways. These data indicate that unique LMWF can protect the lung from fibrosis by weakening the process of inflammation and EMT, and it is a promising therapeutic option for the treatment of PF.


Assuntos
COVID-19/complicações , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Polissacarídeos/química , Fibrose Pulmonar/complicações , Fibrose Pulmonar/tratamento farmacológico , SARS-CoV-2 , Células A549 , Animais , Bleomicina/efeitos adversos , COVID-19/virologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Citocinas/farmacologia , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/mortalidade , Transdução de Sinais/efeitos dos fármacos
3.
J Neurochem ; 159(3): 574-589, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34482548

RESUMO

A contributing factor to the development of obesity is the consumption of a diet high in saturated fatty acids, such as palmitate. These fats induce hypothalamic neuroinflammation, which dysregulates neuronal function and induces orexigenic neuropeptide Y (Npy) to promote food intake. An inflammatory cytokine array identified multiple candidates that could mediate palmitate-induced up-regulation of Npy mRNA levels. Of these, visfatin or nicotinamide phosphoribosyltransferase (NAMPT), macrophage migratory inhibitory factor (MIF), and IL-17F were chosen for further study. Direct treatment of the neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing mHypoE-46 neuronal cell line with the aforementioned cytokines demonstrated that visfatin could directly induce Npy mRNA expression. Preventing the intracellular metabolism of palmitate through long-chain acyl-CoA synthetase (ACSL) inhibition was sufficient to block the palmitate-mediated increase in Npy gene expression. Furthermore, thin-layer chromatography revealed that in neurons, palmitate is readily incorporated into ceramides and defined species of phospholipids. Exogenous C16 ceramide, dipalmitoyl-phosphatidylcholine, and dipalmitoyl-phosphatidylethanolamine were sufficient to significantly induce Npy expression. This study suggests that the intracellular metabolism of palmitate and elevation of metabolites, including ceramide and phospholipids, are responsible for the palmitate-mediated induction of the potent orexigen Npy. Furthermore, this suggests that the regulation of Npy expression is less reliant on inflammatory cytokines per se than palmitate metabolites in a model of NPY/AgRP neurons. These lipid species likely induce detrimental downstream cellular signaling events ultimately causing an increase in feeding, resulting in an overweight phenotype and/or obesity.


Assuntos
Citocinas/farmacologia , Neuropeptídeo Y/biossíntese , Palmitatos/farmacologia , Acil Coenzima A/metabolismo , Animais , Linhagem Celular , Ceramidas/metabolismo , Meios de Cultivo Condicionados , Dieta Hiperlipídica , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
4.
Front Immunol ; 12: 668307, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489927

RESUMO

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Citocinas/farmacologia , Gangliosídeos/antagonistas & inibidores , Efeito Enxerto vs Tumor , Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/transplante , Ativação Linfocitária/efeitos dos fármacos , Neuroblastoma/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia
5.
Cells ; 10(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34440780

RESUMO

Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine highly expressed by epithelial cells and several innate and adaptive immune cells. TSLP exerts its biological effects by binding to a heterodimeric complex composed of TSLP receptor (TSLPR) and IL-7Rα. In humans, there are two TSLP isoforms: the short form (sfTSLP), constitutively expressed, and the long form (lfTSLP), which is upregulated in inflammation. TSLP has been implicated in the induction and progression of several experimental and human cancers. Primary human lung macrophages (HLMs), monocyte-derived macrophages (MDMs), and peripheral blood monocytes consitutively expressed sfTSLP mRNA. Incubation of HLMs, MDMs, and monocytes with lipopolysaccharide (LPS) or IL-4, but not with IL-13, induced TSLP release from HLMs. LPS, but not IL-4 or IL-13, induced CXCL8 release from HLMs. LPS, IL-4 alone or in combination with IL-13, induced the expression of lfTSLP, but not of sfTSLP from HLMs. Preincubation of HLMs with IL-4, alone or in combination with IL-13, but not IL-13 alone, synergistically enhanced TSLP release from LPS-activated macrophages. By contrast, IL-4, alone or in combination with IL-13, inhibited LPS-induced CXCL8 release from HLMs. Immunoreactive TSLP was detected in lysates of HLMs, MDMs, and monocytes. Incubation of HLMs with TSLP induced the release of proinflammatory (TNF-α), angiogenic (VEGF-A, angiopoietin 2), and lymphangiogenic (VEGF-C) factors. TSLP, TSLPR, and IL-7Rα were expressed in intratumoral and peritumoral areas of human lung cancer. sfTSLP and lfTSLP mRNAs were differentially expressed in peritumoral and intratumoral lung cancer tissues. The TSLP system, expressed in HLMs, MDMs, and monocytes, could play a role in chronic inflammatory disorders including lung cancer.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Citocinas/metabolismo , Neoplasias Pulmonares/metabolismo , Ativação de Macrófagos , Macrófagos Alveolares/metabolismo , Microambiente Tumoral , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Idoso , Proteínas Angiogênicas/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/farmacologia , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Lipopolissacarídeos/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Linfangiogênese , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Transdução de Sinais
6.
Mol Immunol ; 137: 221-227, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34284214

RESUMO

Natural Killer (NK) cells are considered the first line of defense against viral infections and tumors. Several factors affect NK cytotoxic activity rendering it dysfunctional and thereby impeding the ability to scavenge abnormal cells as a part of immune escaping mechanisms induced by different types of cancers. NK cells play a crucial role augmenting the activity of various types of anticancer mAb since dysfunctional NK cells are the main reason for the low response to these therapies. To this light, we examined the phenotypic characters of the circulating NK cells isolated from HCC patients compared to healthy controls. Then, dysfunctional NK cells, from HCC patients, were reactivated with cytokines cocktail and their cytotoxic activity with the anti-EGFR mAb "cetuximab" was investigated. This showed a downregulation of patients NK cells activating receptors (NKP30, NKP46, NKG2D and CD16) as well as CD56 and up-regulation of NKG2A inhibitory receptor. We also reported an increase in aberrant CD56- NK cells subset in peripheral blood of HCC patients compared to healthy controls. Thus, confirming the dysfunctionality of peripheral NK cells isolated from HCC patients. Cytokines re-activation of those NK cells lead to upregulation of NK activating receptors and downregulation of inhibitory receptor. Moreover, the percentage of aberrant CD56- NK cells subset was reduced. Here, we proved that advanced HCC patients have an increased percentage of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for the low cytotoxicity noticed in those patients. A significant improvement in the cytotoxicity against HCC was noticed upon using reactivated NK cells combined with cetuximab. Therefore, this study highlights the potential recruitment of NK immune cells along with cetuximab to enhance cytotoxicity against HCC.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Hepatocelular/terapia , Cetuximab/uso terapêutico , Citocinas/farmacologia , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/terapia , Antígeno CD56/metabolismo , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo
7.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299012

RESUMO

Atopic dermatitis (AD) represents a severe global burden on physical, physiological and mental health. Innate immune cell basophils are essential for provoking allergic inflammation in AD. However, the roles of novel immunoregulatory cytokine IL-37 in basophils remain elusive. We employed in vitro co-culture of human basophils and human keratinocyte HaCaT cells and an in vivo MC903-induced AD murine model to investigate the anti-inflammatory mechanism of IL-37. In the in vitro model, IL-37b significantly decreased Der p1-induced thymic stromal lymphopoietin (TSLP) overexpression in HaCaT cells and decreased the expression of TSLP receptor as well as basophil activation marker CD203c on basophils. IL-37 could also reduce Th2 cytokine IL-4 release from TSLP-primed basophils ex vivo. In the in vivo model, alternative depletion of basophils ameliorated AD symptoms and significantly lowered the Th2 cell and eosinophil populations in the ear and spleen of the mice. Blocking TSLP alleviated the AD-like symptoms and reduced the infiltration of basophils in the spleen. In CRISPR/Cas9 human IL-37b knock-in mice or mice with direct treatment by human IL-37b antibody, AD symptoms including ear swelling and itching were significantly alleviated upon MC903 challenge. Notably, IL-37b presence significantly reduced the basophil infiltration in ear lesions. In summary, IL-37b could regulate the TSLP-mediated activation of basophils and reduce the release of IL-4. The results, therefore, suggest that IL-37 may target TSLP-primed basophils to alleviate AD.


Assuntos
Basófilos/imunologia , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Interleucina-1/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Basófilos/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Citocinas/antagonistas & inibidores , Citocinas/farmacologia , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Regulação para Baixo , Orelha/patologia , Eosinófilos/metabolismo , Técnicas de Introdução de Genes , Humanos , Interleucina-1/genética , Interleucina-1/farmacologia , Interleucina-1/uso terapêutico , Interleucina-4/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Baço/imunologia , Baço/metabolismo , Células Th2/imunologia , Regulação para Cima
8.
Nat Commun ; 12(1): 4560, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315876

RESUMO

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment.


Assuntos
Quimiocinas/biossíntese , Citocinas/farmacologia , Elementos Facilitadores Genéticos , Hepatite Alcoólica/genética , Animais , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Regiões Promotoras Genéticas/genética , RNA-Seq , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
9.
Anticancer Res ; 41(7): 3247-3252, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230118

RESUMO

Cancer immunotherapy is an evolving field of research. Cytokines have been conceptualized as an anticancer therapy for longer than most other cancer immunotherapy modalities. Yet, to date, only two cytokines are FDA-approved: IFN-α and IL-2. Despite the initial breakthrough, both agents have been superseded by other, more efficacious agents such as immune checkpoint inhibitors. Several issues persist with cytokine-based cancer therapies; these are broadly categorised into a) high toxicity and b) low efficacy. Despite the only moderate benefits with early cytokine-based cancer therapies, advances in molecular engineering, genomics, and molecular analysis hold promise to optimise and reinstate cytokine-based therapies in future clinical practice. This review considers five important concepts for the successful clinical application of cytokine-based cancer therapies including: (i) improving pharmacokinetics and pharmacodynamics, (ii) improving local administration strategies, (iii) understanding context-dependent interactions in the tumour-microenvironment, (iv) elucidating the role of genetic polymorphisms, and (v) optimising combination therapies. IL-10 has been the focus of attention in recent years and is discussed herein as an example.


Assuntos
Citocinas/farmacologia , Citocinas/uso terapêutico , Interleucina-10/farmacologia , Interleucina-10/uso terapêutico , Neoplasias/imunologia , Neoplasias/terapia , Animais , Humanos , Imunoterapia/métodos , Polimorfismo Genético/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
10.
J Virol ; 95(19): e0022721, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287050

RESUMO

Latent HIV reservoirs persist in people living with HIV despite effective antiretroviral therapy and contribute to rebound viremia upon treatment interruption. Macrophages are an important reservoir cell type, but analysis of agents that modulate latency in macrophages is limited by lack of appropriate in vitro models. We therefore generated an experimental system to investigate this by purifying nonproductively infected human monocyte-derived macrophages (MDM) following in vitro infection with an M-tropic enhanced green fluorescent protein reporter HIV clone and quantified activation of HIV transcription using live-cell fluorescence microscopy. The proportion of HIV-infected MDM was quantified by qPCR detection of HIV DNA, and GFP expression was validated as a marker of productive HIV infection by colabeling of HIV Gag protein. HIV transcription spontaneously reactivated in latently infected MDM at a rate of 0.22% ± 0.04% cells per day (mean ± the standard error of the mean, n = 10 independent donors), producing infectious virions able to infect heterologous T cells in coculture experiments, and both T cells and TZM-bl cells in a cell-free infection system using MDM culture supernatants. Polarization to an M1 phenotype with gamma interferon plus tumor necrosis factor resulted in a 2.3-fold decrease in initial HIV infection of MDM (P < 0.001, n = 8) and a 1.4-fold decrease in spontaneous reactivation (P = 0.025, n = 6), whereas M2 polarization using interleukin-4 prior to infection led to a 1.6-fold decrease in HIV infectivity (P = 0.028, n = 8) but a 2.0-fold increase in the rate of HIV reactivation in latently infected MDM (P = 0.023, n = 6). The latency reversing agents bryostatin and vorinostat, but not panobinostat, significantly induced HIV reactivation in latently infected MDM (P = 0.031 and P = 0.038, respectively, n = 6). IMPORTANCE Agents which modulate latent HIV reservoirs in infected cells are of considerable interest to HIV cure strategies. The present study characterizes a robust, reproducible model enabling quantification of HIV reactivation in primary HIV-infected human MDM which is relatively insensitive to the monocyte donor source and hence suitable for evaluating latency modifiers in MDM. The rate of initial viral infection was greater than the rate of HIV reactivation, suggesting that different mechanisms regulate these processes. HIV reactivation was sensitive to macrophage polarization, suggesting that cellular and tissue environments influence HIV reactivation in different macrophage populations. Importantly, latently infected MDM showed different susceptibilities to certain latency-reversing agents known to be effective in T cells, indicating that dedicated strategies may be required to target latently infected macrophage populations in vivo.


Assuntos
HIV-1/genética , HIV-1/fisiologia , Macrófagos/virologia , Transcrição Genética , Ativação Viral , Briostatinas/farmacologia , Citocinas/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Panobinostat/farmacologia , Linfócitos T/virologia , Latência Viral , Replicação Viral , Vorinostat/farmacologia
11.
Front Immunol ; 12: 678036, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305908

RESUMO

The epithelium-associated cytokine thymic stromal lymphopoietin (TSLP) can induce OX40L and CCL17 expression by myeloid dendritic cells (mDCs), which contributes to aberrant Th2-type immune responses. Herein, we report that such TSLP-induced Th2-type immune response can be effectively controlled by Dectin-1, a C-type lectin receptor expressed by mDCs. Dectin-1 stimulation induced STAT3 activation and decreased the transcriptional activity of p50-RelB, both of which resulted in reduced OX40L expression on TSLP-activated mDCs. Dectin-1 stimulation also suppressed TSLP-induced STAT6 activation, resulting in decreased expression of the Th2 chemoattractant CCL17. We further demonstrated that Dectin-1 activation was capable of suppressing ragweed allergen (Amb a 1)-specific Th2-type T cell response in allergy patients ex vivo and house dust mite allergen (Der p 1)-specific IgE response in non-human primates in vivo. Collectively, this study provides a molecular explanation of Dectin-1-mediated suppression of Th2-type inflammatory responses and suggests Dectin-1 as a target for controlling Th2-type inflammation.


Assuntos
Citocinas/farmacologia , Células Dendríticas/imunologia , Hipersensibilidade/imunologia , Imunidade/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th2/imunologia , Fator de Transcrição RelB/metabolismo , Adulto , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/administração & dosagem , Antígenos de Dermatophagoides/imunologia , Antígenos de Plantas/farmacologia , Estudos de Casos e Controles , Dermatophagoides farinae/imunologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hipersensibilidade/sangue , Lectinas Tipo C/agonistas , Macaca mulatta , Masculino , Pessoa de Meia-Idade , Ligante OX40/metabolismo , Proteínas de Plantas/farmacologia , Células Th2/efeitos dos fármacos , beta-Glucanas/farmacologia
12.
Curr Top Med Chem ; 21(14): 1251-1267, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34238160

RESUMO

BACKGROUND AND OBJECTIVE: Mesenchymal stem cells (MSCs), particularly bone MSCs (BMSCs) offer great potentials for targeted therapeutic applications owing to their migratory and differentiation capacities. Significant advances have been achieved in the differentiation of hepatocyte or hepatocyte-like cells both in vitro and in vivo. However, there is limited knowledge on the differentiation of BMSCs into bipotential hepatic progenitor cells or cholangiocyte. This study reviews the potentials and advances in using MSCs as vehicles for targeted drug delivery and proposes a new method for the induction of differentiation in rat BMSCs into hepatic progenitor cells in vitro and assesses the differential and migratory capacities. METHODS: The BMSCs of Sprague Dawley (SD) rats were harvested from the femur and the tibiae of the rats. After isolation and culturing, BMSCs from Passage 1 were used for the study. The in vitro differentiation of the hepatic progenitor cells was performed using a 2-step induction approach after 5-day serum deprivation from the BMSCs and culturing in Dulbecco's modified eagle medium. Spontaneous in vitro differentiation of BMSCs was examined in the absence of growth factors for 15 days as control treatment. Hepatocytes differentiation was achieved by exposing the culture to collagen type I-coated plates. Cholangiocytes differentiation was achieved with replating the BMC-HepPCs on a layer of Matrigel. Immunofluorescence was conducted on twelve-well plates to determine cell differentiations. Real-Time Quantitative Reverse Transcription PCR (qRTPCR) was used to determine the total RNA extracted using the Trizol LS reagent. In the hepatocyte differentiation group, after periodic acid-schiff (PAS) staining for glycogen, inverted microscope was used to determine differentiations and undifferentiated BMC-HepPCs served as controls. The amount of low-density lipoprotein (LDL) uptake by the BMSCs-derived hepatocytes was assessed using fluorescence microscopy. The secretion of rat albumin was quantified using a quantitative ELISA kit. RESULTS: Differentiation induction is indicative of the sequential supplementation of sodium butyrate and cytokines, which are involved in the embryonic development of the mammalian liver. Hepatic progenitor cells, derived from bone marrow, can be differentiated bidirectionally in vitro into both hepatocyte and cholangiocyte cell-lines. The differentiated cells, including hepatic progenitor cells, hepatocytes, and bile duct-like cells, were identified and analyzed at mRNA and protein levels. CONCLUSION: Our findings show that BMSCs can be utilized as novel bipotential hepatic progenitor cells and thereby for hepatobiliary disease treatment or hepatobiliary tissue-engineering.


Assuntos
Ácido Butírico/farmacologia , Citocinas/farmacologia , Sistemas de Liberação de Medicamentos , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Hepatócitos , Ratos , Ratos Sprague-Dawley
13.
Sci Rep ; 11(1): 13131, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162938

RESUMO

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, ß4galNT2, SLA-I α chain, and ß2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.


Assuntos
Animais Geneticamente Modificados/imunologia , Antígenos Heterófilos/imunologia , Células Endoteliais/imunologia , Suínos/imunologia , Transplante Heterólogo/métodos , Animais , Anticorpos Heterófilos/imunologia , Reações Antígeno-Anticorpo , Antígenos Heterófilos/genética , Sistemas CRISPR-Cas , Degranulação Celular , Linhagem Celular Transformada , Citocinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Técnicas de Inativação de Genes , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/imunologia , Fígado/citologia , Ativação Linfocitária , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/imunologia , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/imunologia , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia
14.
Am J Physiol Renal Physiol ; 320(6): F1174-F1190, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33998295

RESUMO

Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention. However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of the present study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS + transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat [autotransplantation (ATx)]. Complement proteins (C3 and C5b-9) were evaluated with Western blot analysis (reducing and nonreducing conditions) and immunostaining. Western blot analysis of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS + transplant compared with ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS + transplant but was absent in sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS + transplant. Real-time PCR (SYBR green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS mediates aberrant activation of the complement system in renal grafts following transplantation.NEW & NOTEWORTHY This study highlights cold storage-mediated aberrant activation of complement components in renal allografts following transplantation. Specifically, the results demonstrate, for the first time, that cold storage functions in exacerbation of C5b-9, a terminal cytolytic membrane attack complex, in renal grafts following transplantation. In addition, the results indicated that cold storage induces local C3 biogenesis in renal proximal cells/tubules and that TNF-α promotes C3 biogenesis and activation in renal proximal tubular cells.


Assuntos
Proteínas do Sistema Complemento/metabolismo , Rim/metabolismo , Animais , Linhagem Celular , Temperatura Baixa , Ativação do Complemento , Proteínas do Sistema Complemento/genética , Citocinas/administração & dosagem , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transplante de Rim , Túbulos Renais Proximais/citologia , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew
15.
Life Sci ; 278: 119544, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33945827

RESUMO

Drug-induced liver injury (DILI) is one of the most frequent sources of liver failure and the leading cause of liver transplant. Common non-prescription medications such as non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, and other prescription drugs when taken at more than the recommended doses may lead to DILI. The severity of DILI is affected by factors such as age, ethnicity, race, gender, nutritional status, on-going liver diseases, renal function, pregnancy, alcohol consumption, and drug-drug interactions. Characteristics of DILI-associated inflammation include apoptosis and necrosis of hepatocytes and hepatic infiltration of pro-inflammatory immune cells. If untreated or if the inflammation continues, DILI and associated hepatic inflammation may lead to development of hepatocarcinoma. The therapeutic approach for DILI-associated hepatic inflammation depends on whether the inflammation is acute or chronic. Discontinuing the causative medication, vaccination, and special dietary supplementation are some of the conventional approaches to treat DILI. In this review, we discuss a concise overview of DILI-associated liver complications, and current therapeutic options with special emphasis on biologics including the scope of cytokine therapy in hepatic repair and resolution of inflammation caused by over- the-counter (OTC) or prescription drugs.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Citocinas/uso terapêutico , Interleucina-2/uso terapêutico , Fígado/efeitos dos fármacos , Analgésicos não Narcóticos/efeitos adversos , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citocinas/farmacologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Interleucina-2/farmacologia , Fígado/patologia
16.
Front Immunol ; 12: 631353, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34017325

RESUMO

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/fisiologia , Sangue Fetal/citologia , Doença Enxerto-Hospedeiro/prevenção & controle , Células-Tronco Mesenquimais/metabolismo , Animais , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/imunologia , Citocinas/farmacologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos NOD , Controle de Qualidade
17.
Nat Commun ; 12(1): 2538, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953160

RESUMO

Group 2 innate lymphoid cells (ILC2s) play a critical role in protection against helminths and in diverse inflammatory diseases by responding to soluble factors such as the alarmin IL-33, that is often overexpressed in cancer. Nonetheless, regulatory factors that dictate ILC2 functions remain poorly studied. Here, we show that peroxisome proliferator-activated receptor gamma (PPARγ) is selectively expressed in ILC2s in humans and in mice, acting as a central functional regulator. Pharmacologic inhibition or genetic deletion of PPARγ in ILC2s significantly impair IL-33-induced Type-2 cytokine production and mitochondrial fitness. Further, PPARγ blockade in ILC2s disrupts their pro-tumoral effect induced by IL-33-secreting cancer cells. Lastly, genetic ablation of PPARγ in ILC2s significantly suppresses tumor growth in vivo. Our findings highlight a crucial role for PPARγ in supporting the IL-33 dependent pro-tumorigenic role of ILC2s and suggest that PPARγ can be considered as a druggable pathway in ILC2s to inhibit their effector functions. Hence, PPARγ targeting might be exploited in cancer immunotherapy and in other ILC2-driven mediated disorders, such as asthma and allergy.


Assuntos
Imunidade Inata/imunologia , Interleucina-33/metabolismo , Linfócitos/metabolismo , Neoplasias/terapia , PPAR gama/metabolismo , Animais , Asma , Citocinas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Hipersensibilidade , Imunoterapia , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias , Neoplasias/patologia , PPAR gama/genética
18.
Front Immunol ; 12: 645850, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815404

RESUMO

Cytokines are soluble and membrane-bound factors that dictate immune responses. Dogmatically, cytokines are divided into families that promote type 1 cell-mediated immune responses (e.g., IL-12) or type 2 humoral responses (e.g., IL-4), each capable of antagonizing the opposing family of cytokines. The discovery of additional families of cytokines (e.g., IL-17) has added complexity to this model, but it was the realization that immune responses frequently comprise mixtures of different types of cytokines that dismantled this black-and-white paradigm. In some cases, one type of response may dominate these mixed milieus in disease pathogenesis and thereby present a clear therapeutic target. Alternatively, synergistic or blended cytokine responses may obfuscate the origins of disease and perplex clinical decision making. Most immune cells express receptors for many types of cytokines and can mediate a myriad of functions important for tolerance, immunity, tissue damage, and repair. In this review, we will describe the unconventional effects of a variety of cytokines on the activity of a prototypical type 1 effector, the natural killer (NK) cell, and discuss how this may impact the contributions of these cells to health and disease.


Assuntos
Citocinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Viroses/imunologia , Humanos , Interleucinas/farmacologia , Células Matadoras Naturais/imunologia , Fator de Crescimento Transformador beta/fisiologia
19.
Methods Mol Biol ; 2285: 65-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928543

RESUMO

CD4+ T helper (TH) cells are key mediators of immunity, and according to their effector functions, they can be divided into different subsets, namely, TH1, TH2, TH17, and TH22. In order to maintain systemic homeostasis and peripheral tolerance, CD4+ TH cells are counterbalanced by CD4+ T cells with regulatory properties, namely, Foxp3+ regulatory T cells (Foxp3+TREG) and TR1 cells. Here, we describe how to in vitro differentiate murine naïve CD4+ T cells toward helper (TH1, TH2, TH17, and TH22) and regulatory (Foxp3+TREG and TR1) cells.


Assuntos
Diferenciação Celular , Plasticidade Celular , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Plasticidade Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Citocinas/farmacologia , Citometria de Fluxo , Separação Imunomagnética , Camundongos Endogâmicos C57BL , Fenótipo , Projetos de Pesquisa , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fluxo de Trabalho
20.
Front Immunol ; 12: 659996, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912188

RESUMO

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-ß cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-ß, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-ß and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.


Assuntos
Neutrófilos/metabolismo , Receptores de Interleucina-8B/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Citocinas/farmacologia , Feminino , Humanos , Ligantes , Células MCF-7 , Infiltração de Neutrófilos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Neoplasias de Mama Triplo Negativas/patologia
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