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Impaired chronic viral and tumor clearance has been attributed to CD8+ T cell exhaustion, a differentiation state in which T cells have reduced and altered effector function that can be partially reversed upon blockade of inhibitory receptors. The role of the exhaustion program and transcriptional networks that control CD8+ T cell function and fate in autoimmunity is not clear. Here we show that intra-islet CD8+ T cells phenotypically, transcriptionally, epigenetically and metabolically possess features of canonically exhausted T cells, yet maintain important differences. This 'restrained' phenotype can be perturbed and disease accelerated by CD8+ T cell-restricted deletion of the inhibitory receptor lymphocyte activating gene 3 (LAG3). Mechanistically, LAG3-deficient CD8+ T cells have enhanced effector-like functions, trafficking to the islets, and have a diminished exhausted phenotype, highlighting a physiological role for an exhaustion program in limiting autoimmunity and implicating LAG3 as a target for autoimmune therapy.
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Linfócitos T CD8-Positivos , Neoplasias , Autoimunidade , Humanos , Neoplasias/patologia , FenótipoRESUMO
Regulatory T cells (Treg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. Treg cells subvert beneficial anti-tumor immunity by modulating inhibitory receptor expression on tumor-infiltrating lymphocytes (TILs); however, the underlying mediators and mechanisms have remained elusive. Here, we found that the cytokines IL-10 and IL-35 (Ebi3-IL-12α heterodimer) were divergently expressed by Treg cell subpopulations in the tumor microenvironment (TME) and cooperatively promoted intratumoral T cell exhaustion by modulating several inhibitory receptor expression and exhaustion-associated transcriptomic signature of CD8+ TILs. While expression of BLIMP1 (encoded by Prdm1) was a common target, IL-10 and IL-35 differentially affected effector T cell versus memory T cell fates, respectively, highlighting their differential, partially overlapping but non-redundant regulation of anti-tumor immunity. Our results reveal previously unappreciated cooperative roles for Treg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8+ TILs that limits effective anti-tumor immunity.
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Imunidade Celular , Interleucina-10/metabolismo , Interleucinas/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Melanoma Experimental , Camundongos , Neoplasias/patologia , Transdução de Sinais , TranscriptomaRESUMO
Approaches to reverse or limit regulatory T cell (Treg) insufficiency are of great interest for development of immunotherapeutic treatments for autoimmune patients, including type 1 diabetes. Treg insufficiency is heavily implicated in the progression of autoimmune diabetes in the NOD mouse model and is characterized by defects in Treg numbers, development, and/or function. Utilizing a Treg-centric screen, we show that intraislet Tregs have a uniquely dysfunctional phenotype, hallmarked by an almost complete lack of neuropilin-1 (Nrp1), a cell surface receptor required to maintain Treg stability. Intraislet Nrp1- Tregs exhibit hallmark features of fragility, including reduced suppressive capacity, decreased CD73 and Helios, and increased Rorγt and Tbet. Intraislet Nrp1- Tregs also exhibit decreased Foxp3 expression on a per cell basis, suggesting that Nrp1 may also be required for long-term Treg stability. Mechanistically, Treg-restricted augmentation of Nrp1 expression limited the onset of autoimmune diabetes in NOD mice suggesting that Nrp1 critically impacts intraislet Treg function. Transcriptional analysis showed that Nrp1 restoration led to an increase in markers and pathways of TCR signaling, survival, and suppression, and when Nrp1 protein expression is examined by cellular indexing of transcriptomes and epitopes by sequencing, significant differences were observed between Nrp1+ and Nrp1- Tregs in all tissues, particularly in markers of Treg fragility. This translated into substantive differences between Nrp1+ and Nrp1- Tregs that afforded the former with a competitive advantage in the islets. Taken together, these data suggest that maintenance of Nrp1 expression and signaling on Tregs limits diabetes onset and may serve as a strategy to combat Treg insufficiency in autoimmune disease.
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The immune system is guided by a series of checks and balances, a major component of which is a large array of co-stimulatory and co-inhibitory pathways that modulate the host response. Although co-stimulation is essential for boosting and shaping the initial response following signaling through the antigen receptor, inhibitory pathways are also critical for modulating the immune response. Excessive co-stimulation and/or insufficient co-inhibition can lead to a breakdown of self-tolerance and thus to autoimmunity. In this review, we will focus on the role of co-stimulatory and co-inhibitory pathways in two systemic (systemic lupus erythematosus and rheumatoid arthritis) and two organ-specific (multiple sclerosis and type 1 diabetes) emblematic autoimmune diseases. We will also discuss how mechanistic analysis of these pathways has led to the identification of potential therapeutic targets and initiation of clinical trials for autoimmune diseases, as well as outline some of the challenges that lie ahead.
Assuntos
Artrite Reumatoide/imunologia , Receptores Coestimuladores e Inibidores de Linfócitos T/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Imunoterapia/métodos , Lúpus Eritematoso Sistêmico/imunologia , Esclerose Múltipla/imunologia , Receptor Cross-Talk , Linfócitos T/imunologia , Animais , Artrite Reumatoide/terapia , Autoimunidade , Diabetes Mellitus Tipo 1/terapia , Humanos , Imunoterapia/tendências , Lúpus Eritematoso Sistêmico/terapia , Ativação Linfocitária , Terapia de Alvo Molecular , Esclerose Múltipla/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
AIM: To investigate the synergetic regulatory effect of miR-22 on HIF-1α and NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1ß and IL-18 in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis. METHODOLOGY: Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to determine the localization of miR-22-3p, NLRP3 and HIF-1α in human dental pulp tissues (HDPTs). The miR-22 mimics and inhibitor or plasmid of NLRP3 or HIF-1α were used to upregulate or downregulate miR-22 or NLRP3 or HIF-1α in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay were conducted to confirm target association. The mRNA and protein expression of HIF-1α, NLRP3, caspase-1, IL-1ß and IL-18 were determined by qRT-PCR and western blotting, respectively. The release of IL-1ß and IL-18 was analysed by ELISA. The significance of the differences between the experimental and control groups was determined by one-way analysis of variance, p < .05 indicated statistical significance. RESULTS: A decrease in miR-22 and an increase in HIF-1α and NLRP3 in HDPTs occurred during the transformation of reversible pulpitis into irreversible pulpitis compared with that in the healthy pulp tissues (p < .05). In the normal HDPTs, miR-22-3p was extensively expressed in dental pulp cells. HIF-1α and NLRP3 were mainly expressed in the odontoblasts and vascular endothelial cells. Whereas in the inflamed HDPTs, the odontoblast layers were disrupted. HDPFs were positive for miR-22-3p, HIF-1α and NLRP3. Computational prediction via TargetScan 5.1 and luciferase reporter assays confirmed that both NLRP3 and HIF-1α were direct targets of miR-22 in HDPFs. The miR-22 inhibitor further promoted the activation of NLRP3/CASP1 inflammasome pathway induced by ATP plus LPS and hypoxia (p < .05). In contrast, the miR-22 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS and hypoxia (p < .05). CONCLUSION: MiR-22, as a synergetic negative regulator, is involved in controlling the secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3 and HIF-1α. These results provide a novel function and mechanism of miR-22-HIF-1α-NLRP3 signalling in the control of proinflammatory cytokine secretion, thus indicating a potential therapeutic strategy for future endodontic treatment.
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MicroRNAs , Pulpite , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Caspase 1/metabolismo , Citocinas/metabolismo , Polpa Dentária , Células Endoteliais/metabolismo , Fibroblastos , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hibridização in Situ Fluorescente , Inflamassomos/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-18/farmacologia , Lipopolissacarídeos/farmacologia , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pulpite/metabolismo , RNA Mensageiro/metabolismoRESUMO
Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.
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Clostridium cellulovorans/genética , Expressão Gênica , Engenharia Metabólica/métodos , Plasmídeos/genética , Transformação Bacteriana , Biocombustíveis , Biomassa , Butanóis/metabolismo , Celulose/metabolismo , Clostridium cellulovorans/metabolismo , Eletroporação , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Células-TroncoRESUMO
Autoreactive T cells infiltrating the target organ can possess a broad TCR affinity range. However, the extent to which such biophysical parameters contribute to T cell pathogenic potential remains unclear. In this study, we selected eight InsB9-23-specific TCRs cloned from CD4(+) islet-infiltrating T cells that possessed a relatively broad range of TCR affinity to generate NOD TCR retrogenic mice. These TCRs exhibited a range of two-dimensional affinities (â¼ 10(-4)-10(-3) µm(4)) that correlated with functional readouts and responsiveness to activation in vivo. Surprisingly, both higher and lower affinity TCRs could mediate potent insulitis and autoimmune diabetes, suggesting that TCR affinity does not exclusively dictate or correlate with diabetogenic potential. Both central and peripheral tolerance mechanisms selectively impinge on the diabetogenic potential of high-affinity TCRs, mitigating their pathogenicity. Thus, TCR affinity and multiple tolerance mechanisms converge to shape and broaden the diabetogenic T cell repertoire, potentially complicating efforts to induce broad, long-term tolerance.
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Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Ligação Competitiva/imunologia , Transplante de Medula Óssea/métodos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Citometria de Fluxo , Insulina/deficiência , Insulina/genética , Insulina/imunologia , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
BACKGROUND: Fused teeth usually involve several complications, such as the development of caries in the groove between fused crowns, tooth impaction, diastemas, aesthetic and periodontal problems, and pulpal pathosis, due to the complex anatomical structure of fused teeth. A thorough diagnosis is paramount to forming an accurate treatment plan and obtaining a favourable prognosis. With the advent of cone-beam computed tomography (CBCT), accurate 3-dimensional images of teeth and their surrounding dentoalveolar structures can now be readily obtained, and the technology can accurately provide a minimally invasive approach to acquire detailed diagnostic information. Therefore, we utilize CBCT data herein to generate a digital model for the infected region in a patient, and this model enables us to better plan the management of his case. CASE SUMMARY: This report details the diagnosis and endodontic treatment of a rare case involving a fused maxillary second molar and two paramolars with apical periodontitis. The patient experienced pain upon biting and cold sensitivity in the area of the maxillary left molar. No caries or other defects were identified in these teeth, and a normal response to a pulp electric viability test was observed. With the aid of CBCT and digital model technology, we initially suspected that the infection originated from the isthmus between the maxillary second molar and two paramolars. Therefore, we only treated the isthmus by an endodontic approach and did not destroy the original tooth structure; furthermore, the vital pulp was retained, and good treatment outcomes were observed at the 24-month follow-up. CONCLUSION: This finding may provide new insights and perspectives on the diagnosis and treatment of fused teeth.
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Background: The caries-preventive effect of topical fluoride application has been corroborated by a number of clinical studies. However, the effect of fluoride on oral microecology remains unclear. Objective: To monitor the effect of fluoride on dental plaque microecology and demineralization/remineralization balance of enamel initial caries. Methods: Three-year-old children were enrolled and treated with fluoride at baseline and 6 months. International Caries Detection and Assessment System II indices of 52 subjects were measured at baseline, 3, 6, and 12 months. Supragingival plaque samples of 12 subjects were collected at baseline, 3 and 14 days for 16S rRNA sequencing. Results: Changes in microbial community structure were observed at 3 days after fluoridation. Significant changes in the relative abundance of microorganisms were observed after fluoride application, especially Capnocytophaga, unidentified Prevotellaceae and Rothia. Functional prediction revealed that cell movement, carbohydrate and energy metabolism were affected significantly after fluoride application. Fluoride significantly inhibited enamel demineralization and promoted remineralization of early demineralized caries enamel at 3 months. Conclusion: Fluoride application significantly inhibited the progression of enamel initial caries and reversed the demineralization process, possibly by disturbing dental plaque microecology and modulating the physicochemical action of demineralization/remineralization. This deepened our understanding of caries-preventive effects and mechanisms of fluoride.
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TRAIL (Tnfsf10/TRAIL/CD253/Apo2L) is an important immune molecule that mediates apoptosis. TRAIL can play key roles in regulating cell death in the tumor and autoimmune microenvironments. However, dissecting TRAIL function remains difficult because of the lack of optimal models. We have now generated a conditional knockout (Tnfsf10 L/L) for cell type-specific analysis of TRAIL function on C57BL/6, BALB/c, and NOD backgrounds. Previous studies have suggested a role for TRAIL in regulatory T cell (Treg)-mediated suppression. We generated mice with a Treg-restricted Tnfsf10 deletion and surprisingly found no impact on tumor growth in C57BL/6 and BALB/c tumor models. Furthermore, we found no difference in the suppressive capacity of Tnfsf10-deficient Tregs and no change in function or proliferation of T cells in tumors. We also assessed the role of TRAIL on Tregs in two autoimmune mouse models: the NOD mouse model of autoimmune diabetes and the myelin oligodendrocyte glycoprotein (MOG) C57BL/6 model of experimental autoimmune encephalomyelitis. We found that deletion of Tnfsf10 on Tregs had no effect on disease progression in either model. We conclude that Tregs do not appear to be dependent on TRAIL exclusively as a mechanism of suppression in both the tumor and autoimmune microenvironments, although it remains possible that TRAIL may contribute in combination with other mechanisms and/or in different disease settings. Our Tnfsf10 conditional knockout mouse should prove to be a useful tool for the dissection of TRAIL function on different cell populations in multiple mouse models of human disease.
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Tolerância Periférica , Linfócitos T Reguladores/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Animais , Apoptose , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Citometria de Fluxo , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/imunologia , Neoplasias/imunologia , Linfócitos T Reguladores/citologiaRESUMO
Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1-deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1-deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K-AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1-deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity.
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Diabetes Mellitus Experimental/imunologia , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Diabetes Mellitus Experimental/genética , Encefalomielite Autoimune Experimental/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes Neurológicos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Receptor de Morte Celular Programada 1/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/genéticaRESUMO
Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in TregEbi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3+ and Foxp3neg (iTr35) T cells. Furthermore, IL-35 producers, although rare, secrete Ebi3 and p35 on extracellular vesicles (EVs) targeting a 25- to 100-fold higher number of T and B lymphocytes, causing them to acquire surface IL-35. This surface IL-35 is absent when EV production is inhibited or if Ebi3 is genetically deleted in Treg cells. The unique ability of EVs to coat bystander lymphocytes with IL-35, promoting exhaustion in, and secondary suppression by, non-Treg cells identifies a novel mechanism of infectious tolerance.
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Vesículas Extracelulares/metabolismo , Tolerância Imunológica , Subunidade p35 da Interleucina-12/metabolismo , Interleucinas/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Receptores de Citocinas/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Técnicas de Cocultura , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/ultraestrutura , Feminino , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Inativação de Genes , Transplante de Coração , Terapia de Imunossupressão , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Linfócitos T Reguladores/metabolismoRESUMO
Inhibitory receptors (IRs) are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (Tconv) cell proliferation, viability, and function. However, the role of IRs on regulatory T cells (Tregs) remains obscure because they could be required for suppressive activity and/or limit Treg function. We evaluated the role of lymphocyte activation gene 3 (LAG3; CD223) on Tregs by generating mice in which LAG3 is absent on the cell surface of Tregs in a murine model of type 1 diabetes. Unexpectedly, mice that lacked LAG3 expression on Tregs exhibited reduced autoimmune diabetes, consistent with enhanced Treg proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3-deficient Tregs was largely comparable, substantial differences between intra-islet Tregs were evident and involved a subset of genes and pathways that promote Treg maintenance and function. Consistent with these observations, Lag3-deficient Tregs outcompeted WT Tregs in the islets but not in the periphery in cotransfer experiments because of enhanced interleukin-2-signal transducer and activator of transcription 5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits Treg proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment, and may contribute to Treg insufficiency in autoimmune disease.
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Beclomethasone dipropionate (BDP), which is a member in the inhaled glucocorticosteroid class, is commonly used in the treatment of asthma by pulmonary delivery. The purpose of this study is to prepare ultrafine BDP particles for dry powder inhalation (DPI) administration by combining microfluidic antisolvent precipitation without surfactant, high-pressure homogenization (HPH) and spray drying. T-junction microchannel was adopted for the preparation of needle-like BDP particles. The needle-like particles could be easily broken down into smaller particles during HPH, which were assembled into uniform low-density spherical BDP aggregates by spray drying. The effects of the operation parameters, such as the flow rates of BDP methanol solution and antisolvent, the overall flow rate, the BDP concentration, and the change of the injection phase on BDP particle size were explored. The results indicated that the BDP particle size greatly decreased with the reduction of BDP solution flow rate and the increase of antisolvent flow rate. However, the BDP particle size firstly decreased and then increased with the increase of the overall flow rate and the increase of BDP concentration. Also, BDP solution as the injection phase could form the smaller BDP particles. 10 HPH cycles are enough to forming short rod-like particles. After spray drying, the BDP spherical aggregates with a 2-3 µm size could be achieved. They have an excellent aerosol performance, 2.8 and 1.4 times as many as raw BDP and vacuum-dried BDP particles, respectively.
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Antiasmáticos/química , Beclometasona/química , Química Farmacêutica/métodos , Inaladores de Pó Seco , Administração por Inalação , Antiasmáticos/administração & dosagem , Beclometasona/administração & dosagem , Composição de Medicamentos , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
This article presents the preparation of nanoparticles of amorphous cefuroxime axetil (CFA) in a microporous tube-in-tube microchannel reactor (MTMCR). The experimental results indicated that CFA particle with a tunable size of 400-1400 nm could be achieved under a high throughput in the range of 1.5-6L/min. The average particle size decreased with increasing overall volumetric flow rate and decreasing CFA concentration, micropore size, and annular channel width. The produced CFA nanoparticles were characterized by SEM, XRD, FT-IR, DSC and a dissolution test, which indicated that the nanosized CFA was amorphous and exhibited higher dissolution rate compared to the raw CFA. The MTMCR might offer a general and facile pathway for mass production of the nanoparticles of hydrophobic pharmaceuticals thanks to its high throughput capacity and excellent micromixing performance.