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1.
Nat Immunol ; 25(6): 969-980, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38831104

ABSTRACT

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.


Subject(s)
Genetic Predisposition to Disease , Lupus Erythematosus, Systemic , Toll-Like Receptor 7 , Lupus Erythematosus, Systemic/genetics , Humans , Animals , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Mice , Child , Female , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Male , Age of Onset , Genetic Variation , NF-kappa B/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Adolescent , THP-1 Cells , Interferon Type I/metabolism
2.
Sci Adv ; 10(18): eadn6537, 2024 May 03.
Article in English | MEDLINE | ID: mdl-38701219

ABSTRACT

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.


Subject(s)
Autoimmunity , Macrophages , Toll-Like Receptor 7 , X Chromosome Inactivation , Animals , Female , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Autoimmunity/genetics , Mice , Male , Macrophages/metabolism , Macrophages/immunology , RNA, Long Noncoding/genetics , Signal Transduction , Dendritic Cells/immunology , Dendritic Cells/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology
3.
Int J Mol Sci ; 25(10)2024 May 14.
Article in English | MEDLINE | ID: mdl-38791389

ABSTRACT

The pathogenesis of systemic lupus erythematosus (SLE) is linked to the differential roles of toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. TLR7 overexpression or gene duplication, as seen with the Y-linked autoimmune accelerator (Yaa) locus or TLR7 agonist imiquimod, correlates with increased SLE severity, and specific TLR7 polymorphisms and gain-of-function variants are associated with enhanced SLE susceptibility and severity. In addition, the X-chromosome location of TLR7 and its escape from X-chromosome inactivation provide a genetic basis for female predominance in SLE. The absence of TLR8 and TLR9 have been shown to exacerbate the detrimental effects of TLR7, leading to upregulated TLR7 activity and increased disease severity in mouse models of SLE. The regulatory functions of TLR8 and TLR9 have been proposed to involve competition for the endosomal trafficking chaperone UNC93B1. However, recent evidence implies more direct, regulatory functions of TLR9 on TLR7 activity. The association between age-associated B cells (ABCs) and autoantibody production positions these cells as potential targets for treatment in SLE, but the lack of specific markers necessitates further research for precise therapeutic intervention. Therapeutically, targeting TLRs is a promising strategy for SLE treatment, with drugs like hydroxychloroquine already in clinical use.


Subject(s)
Lupus Erythematosus, Systemic , Toll-Like Receptor 7 , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/metabolism , Animals , Humans , Mice , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Toll-Like Receptors/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 8/genetics , Disease Models, Animal , Genetic Predisposition to Disease
4.
J Exp Med ; 221(8)2024 Aug 05.
Article in English | MEDLINE | ID: mdl-38780621

ABSTRACT

Nucleic acid-sensing Toll-like receptors (TLR) 3, 7/8, and 9 are key innate immune sensors whose activities must be tightly regulated to prevent systemic autoimmune or autoinflammatory disease or virus-associated immunopathology. Here, we report a systematic scanning-alanine mutagenesis screen of all cytosolic and luminal residues of the TLR chaperone protein UNC93B1, which identified both negative and positive regulatory regions affecting TLR3, TLR7, and TLR9 responses. We subsequently identified two families harboring heterozygous coding mutations in UNC93B1, UNC93B1+/T93I and UNC93B1+/R336C, both in key negative regulatory regions identified in our screen. These patients presented with cutaneous tumid lupus and juvenile idiopathic arthritis plus neuroinflammatory disease, respectively. Disruption of UNC93B1-mediated regulation by these mutations led to enhanced TLR7/8 responses, and both variants resulted in systemic autoimmune or inflammatory disease when introduced into mice via genome editing. Altogether, our results implicate the UNC93B1-TLR7/8 axis in human monogenic autoimmune diseases and provide a functional resource to assess the impact of yet-to-be-reported UNC93B1 mutations.


Subject(s)
Autoimmunity , Animals , Humans , Mice , Autoimmunity/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , DNA Mutational Analysis , Toll-Like Receptors/metabolism , Toll-Like Receptors/genetics , Mutation , Female , Male , Mice, Inbred C57BL , HEK293 Cells , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology
5.
Dev Comp Immunol ; 157: 105197, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38763479

ABSTRACT

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.


Subject(s)
Fish Proteins , Ictaluridae , Imidazoles , Imiquimod , Immunity, Innate , Lysosomes , Toll-Like Receptor 7 , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/genetics , Imidazoles/pharmacology , Ictaluridae/immunology , Lysosomes/metabolism , Fish Proteins/genetics , Fish Proteins/metabolism , Signal Transduction , Humans , Aminoquinolines/pharmacology , Poly I-C/immunology
6.
Cell Mol Life Sci ; 81(1): 199, 2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38683377

ABSTRACT

Tyrosine kinase 2 (TYK2) is involved in type I interferon (IFN-I) signaling through IFN receptor 1 (IFNAR1). This signaling pathway is crucial in the early antiviral response and remains incompletely understood on B cells. Therefore, to understand the role of TYK2 in B cells, we studied these cells under homeostatic conditions and following in vitro activation using Tyk2-deficient (Tyk2-/-) mice. Splenic B cell subpopulations were altered in Tyk2-/- compared to wild type (WT) mice. Marginal zone (MZ) cells were decreased and aged B cells (ABC) were increased, whereas follicular (FO) cells remained unchanged. Likewise, there was an imbalance in transitional B cells in juvenile Tyk2-/- mice. RNA sequencing analysis of adult MZ and FO cells isolated from Tyk2-/- and WT mice in homeostasis revealed altered expression of IFN-I and Toll-like receptor 7 (TLR7) signaling pathway genes. Flow cytometry assays corroborated a lower expression of TLR7 in MZ B cells from Tyk2-/- mice. Splenic B cell cultures showed reduced proliferation and differentiation responses after activation with TLR7 ligands in Tyk2-/- compared to WT mice, with a similar response to lipopolysaccharide (LPS) or anti-CD40 + IL-4. IgM, IgG, IL-10 and IL-6 secretion was also decreased in Tyk2-/- B cell cultures. This reduced response of the TLR7 pathway in Tyk2-/- mice was partially restored by IFNα addition. In conclusion, there is a crosstalk between TYK2 and TLR7 mediated by an IFN-I feedback loop, which contributes to the establishment of MZ B cells and to B cell proliferation and differentiation.


Subject(s)
B-Lymphocytes , Interferon Type I , Signal Transduction , Spleen , TYK2 Kinase , Toll-Like Receptor 7 , Animals , Mice , B-Lymphocytes/metabolism , B-Lymphocytes/immunology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Interferon Type I/metabolism , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Spleen/cytology , Spleen/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , TYK2 Kinase/metabolism , TYK2 Kinase/genetics
7.
Proc Natl Acad Sci U S A ; 121(19): e2319569121, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38683985

ABSTRACT

Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5'-half molecules of tRNAHisGUG (the 5'-tRNAHisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5'-tRNA half molecules and identified the 5'-tRNAValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5'-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5'-tRNAValCAC/AAC half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with Mycobacterium tuberculosis. The 5'-tRNAValCAC/AAC half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5'-tRNAValCAC/AAC half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.


Subject(s)
Macrophages , Toll-Like Receptor 7 , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Humans , Macrophages/metabolism , Macrophages/immunology , Ligands , Mycobacterium tuberculosis/immunology , RNA, Transfer, His/metabolism , RNA, Transfer, His/genetics , Lipopolysaccharides
8.
Viruses ; 16(4)2024 04 17.
Article in English | MEDLINE | ID: mdl-38675965

ABSTRACT

Epstein-Barr virus (EBV), a Herpesviridae family member, is associated with an increased risk of autoimmune disease development in the host. We previously demonstrated that EBV DNA elevates levels of the pro-inflammatory cytokine IL-17A and that inhibiting Toll-like receptor (TLR) 3, 7, or 9 reduces its levels. Moreover, this DNA exacerbated colitis in a mouse model of inflammatory bowel disease (IBD). In the study at hand, we examined whether inhibition of TLR3, 7, or 9 alleviates this exacerbation. Mice were fed 1.5% dextran sulfate sodium (DSS) water and administered EBV DNA. Then, they were treated with a TLR3, 7, or 9 inhibitor or left untreated. We also assessed the additive impact of combined inhibition of all three receptors. Mice that received DSS, EBV DNA, and each inhibitor alone, or a combination of inhibitors, showed significant improvement. They also had a decrease in the numbers of the pathogenic colonic IL-17A+IFN-γ+ foci. Inhibition of all three endosomal TLR receptors offered no additive benefit over administering a single inhibitor. Therefore, inhibition of endosomal TLRs reduces EBV DNA exacerbation of mouse colitis, offering a potential approach for managing IBD patients infected with EBV.


Subject(s)
DNA, Viral , Herpesvirus 4, Human , Inflammatory Bowel Diseases , Toll-Like Receptors , Animals , Female , Mice , Colitis/chemically induced , Colitis/drug therapy , Colitis/virology , Dextran Sulfate , Disease Models, Animal , DNA, Viral/adverse effects , DNA, Viral/pharmacology , Endosomes/drug effects , Endosomes/metabolism , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/drug therapy , Inflammatory Bowel Diseases/chemically induced , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/virology , Interleukin-17/metabolism , Mice, Inbred C57BL , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/antagonists & inhibitors , Toll-Like Receptor 9/metabolism , Toll-Like Receptors/antagonists & inhibitors , Toll-Like Receptors/metabolism
9.
J Immunol ; 212(11): 1680-1692, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38607278

ABSTRACT

Plasmacytoid dendritic cells (pDCs) are strongly implicated as a major source of IFN-I in systemic lupus erythematosus (SLE), triggered through TLR-mediated recognition of nucleic acids released from dying cells. However, relatively little is known about how TLR signaling and IFN-I production are regulated in pDCs. In this article, we describe a role for integrin αvß3 in regulating TLR responses and IFN-I production by pDCs in mouse models. We show that αv and ß3-knockout pDCs produce more IFN-I and inflammatory cytokines than controls when stimulated through TLR7 and TLR9 in vitro and in vivo. Increased cytokine production was associated with delayed acidification of endosomes containing TLR ligands, reduced LC3 conjugation, and increased TLR signaling. This dysregulated TLR signaling results in activation of B cells and promotes germinal center (GC) B cell and plasma cell expansion. Furthermore, in a mouse model of TLR7-driven lupus-like disease, deletion of αvß3 from pDCs causes accelerated autoantibody production and pathology. We therefore identify a pDC-intrinsic role for αvß3 in regulating TLR signaling and preventing activation of autoreactive B cells. Because αvß3 serves as a receptor for apoptotic cells and cell debris, we hypothesize that this regulatory mechanism provides important contextual cues to pDCs and functions to limit responses to self-derived nucleic acids.


Subject(s)
Autoimmunity , Dendritic Cells , Integrin alphaVbeta3 , Lupus Erythematosus, Systemic , Mice, Knockout , Signal Transduction , Toll-Like Receptor 7 , Animals , Mice , Dendritic Cells/immunology , Integrin alphaVbeta3/immunology , Integrin alphaVbeta3/metabolism , Autoimmunity/immunology , Toll-Like Receptor 7/immunology , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Lupus Erythematosus, Systemic/immunology , Signal Transduction/immunology , Mice, Inbred C57BL , Cytokines/metabolism , Cytokines/immunology , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolism , B-Lymphocytes/immunology , Autoantibodies/immunology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Lymphocyte Activation/immunology , Disease Models, Animal
10.
Cell Commun Signal ; 22(1): 220, 2024 Apr 08.
Article in English | MEDLINE | ID: mdl-38589923

ABSTRACT

Endosomal single-stranded RNA-sensing Toll-like receptor-7/8 (TLR7/8) plays a pivotal role in inflammation and immune responses and autoimmune diseases. However, the mechanisms underlying the initiation of the TLR7/8-mediated autoimmune signaling remain to be fully elucidated. Here, we demonstrate that miR-574-5p is aberrantly upregulated in tissues of lupus prone mice and in the plasma of lupus patients, with its expression levels correlating with the disease activity. miR-574-5p binds to and activates human hTLR8 or its murine ortholog mTlr7 to elicit a series of MyD88-dependent immune and inflammatory responses. These responses include the overproduction of cytokines and interferons, the activation of STAT1 signaling and B lymphocytes, and the production of autoantigens. In a transgenic mouse model, the induction of miR-574-5p overexpression is associated with increased secretion of antinuclear and anti-dsDNA antibodies, increased IgG and C3 deposit in the kidney, elevated expression of inflammatory genes in the spleen. In lupus-prone mice, lentivirus-mediated silencing of miR-574-5p significantly ameliorates major symptoms associated with lupus and lupus nephritis. Collectively, these results suggest that the miR-574-5p-hTLR8/mTlr7 signaling is an important axis of immune and inflammatory responses, contributing significantly to the development of lupus and lupus nephritis.


Subject(s)
Lupus Nephritis , MicroRNAs , Humans , Mice , Animals , Lupus Nephritis/genetics , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/genetics , Toll-Like Receptor 8/metabolism , Kidney/metabolism , Mice, Transgenic , MicroRNAs/genetics
11.
Exp Mol Med ; 56(5): 1164-1177, 2024 May.
Article in English | MEDLINE | ID: mdl-38689088

ABSTRACT

Recent evidence of gut microbiota dysbiosis in the context of psoriasis and the increased cooccurrence of inflammatory bowel disease and psoriasis suggest a close relationship between skin and gut immune responses. Using a mouse model of psoriasis induced by the Toll-like receptor (TLR) 7 ligand imiquimod, we found that psoriatic dermatitis was accompanied by inflammatory changes in the small intestine associated with eosinophil degranulation, which impaired intestinal barrier integrity. Inflammatory responses in the skin and small intestine were increased in mice prone to eosinophil degranulation. Caco-2 human intestinal epithelial cells were treated with media containing eosinophil granule proteins and exhibited signs of inflammation and damage. Imiquimod-induced skin and intestinal changes were attenuated in eosinophil-deficient mice, and this attenuation was counteracted by the transfer of eosinophils. Imiquimod levels and the distribution of eosinophils were positively correlated in the intestine. TLR7-deficient mice did not exhibit intestinal eosinophil degranulation but did exhibit attenuated inflammation in the skin and small intestine following imiquimod administration. These results suggest that TLR7-dependent bidirectional skin-to-gut communication occurs in psoriatic inflammation and that inflammatory changes in the intestine can accelerate psoriasis.


Subject(s)
Cell Degranulation , Disease Models, Animal , Eosinophils , Imiquimod , Intestine, Small , Psoriasis , Toll-Like Receptor 7 , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Psoriasis/pathology , Psoriasis/metabolism , Mice , Eosinophils/metabolism , Eosinophils/immunology , Humans , Intestine, Small/pathology , Intestine, Small/metabolism , Skin/pathology , Skin/metabolism , Inflammation/pathology , Inflammation/metabolism , Mice, Knockout , Caco-2 Cells , Membrane Glycoproteins
12.
Org Biomol Chem ; 22(14): 2764-2773, 2024 04 03.
Article in English | MEDLINE | ID: mdl-38497199

ABSTRACT

Pattern recognition receptors (PRRs) play a critical role in the innate immune response, and toll-like receptor 7 (TLR7) is an important member of PRRs. Although several TLR7 agonists are available, most of them are being tested clinically, with only one available on the market. Thus, it is imperative to develop new TLR7 agonists. In this study, we designed and synthesized three kinds of quinazoline derivatives and five kinds of pyrrolo[3,2-d]pyrimidine derivatives targeting TLR7. The antiviral efficacy of these compounds was evaluated in vitro and in vivo. Our findings indicated that four kinds of compounds showed exceptional antiviral activity. Furthermore, molecular docking studies confirmed that compound 11 successfully positioned itself in the pocket of the TLR7 guanosine loading site with a binding energy of -4.45 kcal mol-1. These results suggested that these compounds might be potential antiviral agents.


Subject(s)
Quinazolines , Toll-Like Receptor 7 , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/metabolism , Quinazolines/chemistry , Molecular Docking Simulation , Adjuvants, Immunologic , Antiviral Agents/pharmacology , Pyrimidines/chemistry
13.
Front Immunol ; 15: 1329805, 2024.
Article in English | MEDLINE | ID: mdl-38481993

ABSTRACT

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity.


Subject(s)
Influenza Vaccines , Interferon Type I , Humans , Male , Female , Adolescent , Interferon-alpha , Influenza Vaccines/metabolism , Toll-Like Receptor 7/metabolism , Androgens/metabolism , BNT162 Vaccine , mRNA Vaccines , Interferon Type I/metabolism , Vaccination , Dendritic Cells , Immunoglobulin G/metabolism
14.
J Innate Immun ; 16(1): 216-225, 2024.
Article in English | MEDLINE | ID: mdl-38461810

ABSTRACT

INTRODUCTION: Toll-like receptors play crucial roles in the sepsis-induced systemic inflammatory response. Septic shock mortality correlates with overexpression of neutrophilic TLR2 and TLR9, while the role of TLR4 overexpression remains a debate. In addition, TLRs are involved in the pathogenesis of viral infections such as COVID-19, where the single-stranded RNA of SARS-CoV-2 is recognized by TLR7 and TLR8, and the spike protein activates TLR4. METHODS: In this study, we conducted a comprehensive analysis of TLRs 1-10 expressions in white blood cells from 71 patients with bacterial and viral infections. Patients were divided into 4 groups based on disease type and severity (sepsis, septic shock, moderate, and severe COVID-19) and compared to 7 healthy volunteers. RESULTS: We observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in septic shock neutrophils compared to the control group (p < 0.001). Severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils compared to controls (p < 0.05). Septic shock patients also showed a similar increase in TLR7 in neutrophils along with elevated intermediate monocytes (CD14+CD16+) compared to the control group (p < 0.005 and p < 0.001, respectively). However, TLR expression remained unchanged in lymphocytes. CONCLUSION: This study provides further insights into the mechanisms of TLR activation in various infectious conditions. Additional analysis is needed to assess their correlation with patient outcome and to evaluate the impact of TLR-pathway modulation during septic shock and severe COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Toll-Like Receptor 10 , Aged , Female , Humans , Male , Middle Aged , Bacterial Infections/immunology , COVID-19/immunology , COVID-19/blood , Leukocytes/immunology , Leukocytes/metabolism , Lipopolysaccharide Receptors/metabolism , Neutrophils/immunology , SARS-CoV-2/immunology , Sepsis/immunology , Shock, Septic/immunology , Shock, Septic/blood , Toll-Like Receptor 1/metabolism , Toll-Like Receptor 1/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/genetics , Toll-Like Receptors/metabolism , Aged, 80 and over
15.
J Autoimmun ; 145: 103189, 2024 May.
Article in English | MEDLINE | ID: mdl-38442677

ABSTRACT

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.


Subject(s)
Arthritis, Rheumatoid , Cell Differentiation , Dendritic Cells , MicroRNAs , Osteoclasts , Toll-Like Receptor 7 , Toll-Like Receptor 8 , Humans , Dendritic Cells/immunology , Dendritic Cells/metabolism , MicroRNAs/genetics , Toll-Like Receptor 8/metabolism , Osteoclasts/metabolism , Osteoclasts/immunology , Animals , Toll-Like Receptor 7/metabolism , Mice , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Synovial Fluid/immunology , Synovial Fluid/metabolism , Cells, Cultured , Female , Male
16.
Cells ; 13(5)2024 Feb 26.
Article in English | MEDLINE | ID: mdl-38474371

ABSTRACT

Toll-like receptors (TLRs) are a collection of pattern recognition sensors that form a first line of defence by detecting pathogen- or damage-associated molecular patterns and initiating an inflammatory response. TLR activation in microglia, the major immune cells in the brain, can trigger the release of inflammatory molecules, which may contribute to various CNS diseases including Alzheimer's disease. Recently, some microRNAs were shown to serve as signalling molecules for TLRs. Here, we present miR-154-5p as a novel TLR7 ligand. Exposing microglia to miR-154-5p results in cytokine release and alters expression of the TLR signalling pathway dependent on TLR7. Additionally, miR-154-5p causes neuronal injury in enriched cortical neuron cultures and additive toxicity in the presence of microglia. Finally, intrathecal injection of miR-154-5p into mice leads to neuronal injury and accumulation of microglia in the cerebral cortex dependent on TLR7 expression. In conclusion, this study establishes miR-154-5p as a direct activator of TLR7 that can cause neuroinflammation and neuronal injury, which may contribute to CNS disease.


Subject(s)
MicroRNAs , Microglia , Toll-Like Receptor 7 , Animals , Mice , Ligands , Microglia/metabolism , MicroRNAs/metabolism , Neurons/metabolism , Toll-Like Receptor 7/metabolism , Humans
17.
Cell Mol Life Sci ; 81(1): 110, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38429401

ABSTRACT

Toll-like receptors (TLRs), especially TLR7, play an important role in systemic lupus erythematosus (SLE) pathogenesis. However, the regulatory mechanism underlying the abnormal activation of TLR pathways in patients with SLE has not been elucidated. Notably, accumulating evidence indicates that myeloid-derived suppressor cells (MDSCs) are important regulators of inflammation and autoimmune diseases. Compared with healthy control subjects, patients with SLE have a greater proportion of MDSCs among peripheral blood mononuclear cells (PBMCs); however, the effect of MDSCs on TLR7 pathway activation has not been determined. In the present study, lupus MDSCs significantly promoted TLR7 pathway activation in macrophages and dendritic cells (DCs), exacerbating the imiquimod-induced lupus model. RNA-sequencing analysis revealed significant overexpression of S100 calcium-binding protein A8 (S100A8) and S100A9 in MDSCs from diseased MRL/lpr mice. In vitro and in vivo studies demonstrated that S100A8/9 effectively promoted TLR7 pathway activation and that S100A8/9 deficiency reversed the promoting effect of MDSCs on TLR7 pathway activation in lupus. Mechanistically, MDSC-derived S100A8/9 upregulated interferon gamma (IFN-γ) secretion by macrophages and IFN-γ subsequently promoted TLR7 pathway activation in an autocrine manner. Taken together, these findings suggest that lupus MDSCs promote TLR7 pathway activation and lupus pathogenesis through the S100A8/9-IFN-γ axis. Our study identified an important target for SLE therapy.


Subject(s)
Calgranulin A , Calgranulin B , Lupus Erythematosus, Systemic , Myeloid-Derived Suppressor Cells , Animals , Mice , Dendritic Cells/metabolism , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/pathology , Macrophages/metabolism , Mice, Inbred MRL lpr , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Calgranulin A/metabolism , Calgranulin B/metabolism
18.
Physiol Rep ; 12(3): e15949, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38346802

ABSTRACT

Toll-like receptor-7 (TLR7) activation promotes autoimmunity, and metabolic syndrome (MetS) is a common comorbidity in patients with autoimmune disease. We previously demonstrated hyperinsulinemia in TLR7 agonist imiquimod (IMQ)-treated, high-fat diet (HFD)-fed female C57BL/6 mice. Since mouse strains differ in susceptibility to MetS and target organ damage, this study investigated whether 12 weeks of exposure to HFD and IMQ promoted MetS, autoimmunity, and target organ damage in female FVB/N mice. Supporting early-stage autoimmunity, spleen-to-tibia ratio, and anti-nuclear antibodies (ANA) were significantly increased by IMQ. No significant effect of IMQ on urinary albumin excretion or left ventricular hypertrophy was observed. HFD increased liver-to-tibia ratio, which was further exacerbated by IMQ. HFD increased fasting blood glucose levels at the end of 12 weeks, but there was no significant effect of IMQ treatment on fasting blood glucose levels at 6 or 12 weeks of treatment. However, oral glucose tolerance testing at 12 weeks revealed impaired glucose tolerance in HFD-fed mice compared to control diet mice together with IMQ treatment exacerbating the impairment. Accordingly, these data suggest TLR7 activation also exacerbates HFD-induced dysregulation of glucose handling FVB/N mice, supporting the possibility that endogenous TLR7 activation may contribute to dysglycemia in patients with autoimmune disease.


Subject(s)
Autoimmune Diseases , Metabolic Syndrome , Humans , Female , Mice , Animals , Imiquimod/pharmacology , Diet, High-Fat/adverse effects , Blood Glucose/metabolism , Toll-Like Receptor 7/metabolism , Glycemic Control , Mice, Inbred C57BL , Mice, Inbred Strains
19.
J Med Chem ; 67(5): 3321-3338, 2024 Mar 14.
Article in English | MEDLINE | ID: mdl-38363069

ABSTRACT

Immunotherapy targeting the toll-like receptor 7 (TLR7) is a promising strategy for cancer treatment. Herein, we describe the design and synthesis of a series of imidazoquinoline-based TLR7 agonists and assess NF-κB pathway activation using HEK-Blue hTLR7 cells to identify the most potent small-molecule TLR7 agonist, SMU-L11 (EC50 = 0.024 ± 0.002 µM). In vitro experiments demonstrated that SMU-L11 specifically activated TLR7, resulting in recruitment of the MyD88 adaptor protein and activation of the NF-κB and MAPK signaling pathways. Moreover, SMU-L11 was found to exert immune-enhancing effects by significantly inducing the secretion of proinflammatory cytokines in murine dendritic cells, macrophages, and human peripheral blood mononuclear cells while promoting M1 macrophage polarization. In vivo studies using a B16-F10 mouse tumor model showed that SMU-L11 significantly enhanced immune cell activation and augmented CD4+ T and CD8+ T-cell proliferation, directly killing tumor cells and inhibiting tumor growth.


Subject(s)
Melanoma , Humans , Animals , Mice , Melanoma/drug therapy , Melanoma/metabolism , NF-kappa B/metabolism , Toll-Like Receptor 7/metabolism , Tumor Microenvironment , Leukocytes, Mononuclear/metabolism , Adjuvants, Immunologic/metabolism
20.
Adv Healthc Mater ; 13(11): e2303910, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38180445

ABSTRACT

Self-assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two-component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self-assembly in vitro. Single-stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length-dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small-molecule TLR7/8 agonist show that co-delivery of antigen and adjuvant results in a more than 20-fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo-loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.


Subject(s)
Nanoparticles , Nanoparticles/chemistry , Animals , Mice , Proteins/chemistry , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 8/agonists , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/agonists
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