Aberrant RNA sensing in regulatory T cells causes systemic autoimmunity.

Chronic and aberrant nucleic acid sensing causes type I IFN-driven autoimmune diseases, designated type I interferonopathies. We found a significant reduction of regulatory T cells (Tregs) in patients with type I interferonopathies caused by mutations in ADAR1 or IFIH1 (encoding MDA5). We analyzed the underlying mechanisms using murine models and found that Treg-specific deletion of Adar1 caused peripheral Treg loss and scurfy-like lethal autoimmune disorders. Similarly, knock-in mice with Treg-specific expression of an MDA5 gain-of-function mutant caused apoptosis of peripheral Tregs and severe autoimmunity. Moreover, the impact of ADAR1 deficiency on Tregs is multifaceted, involving both MDA5 and PKR sensing. Together, our results highlight the dysregulation of Treg homeostasis by intrinsic aberrant RNA sensing as a potential determinant for type I interferonopathies.

Notch2 with retinoic acid license IL-23 expression by intestinal EpCAM+ DCIR2+ cDC2s in mice.

Despite the importance of IL-23 in mucosal host defense and disease pathogenesis, the mechanisms regulating the development of IL-23-producing mononuclear phagocytes remain poorly understood. Here, we employed an Il23aVenus reporter strain to investigate the developmental identity and functional regulation of IL-23-producing cells. We showed that flagellin stimulation or Citrobacter rodentium infection led to robust induction of IL-23-producing EpCAM+ DCIR2+ CD103- cDC2s, termed cDCIL23, which was confined to gut-associated lymphoid tissues, including the mesenteric lymph nodes, cryptopatches, and isolated lymphoid follicles. Furthermore, we demonstrated that Notch2 signaling was crucial for the development of EpCAM+ DCIR2+ cDC2s, and the combination of Notch2 signaling with retinoic acid signaling controlled their terminal differentiation into cDCIL23, supporting a two-step model for the development of gut cDCIL23. Our findings provide fundamental insights into the developmental pathways and cellular dynamics of IL-23-producing cDC2s at steady state and during pathogen infection.

Targeting abatacept-resistant T-helper-17 cells by aldehyde dehydrogenase inhibition.

IL-17-producing helper T (Th17) cells are long-lived and serve as central effector cells in chronic autoimmune diseases. The underlying mechanisms of Th17 persistence remain unclear. We demonstrated that abatacept, a CD28 antagonist, effectively prevented the development of skin disease in a Th17-dependent experimental autoimmune dermatitis model. Abatacept selectively inhibited the emergence of IL-7R-negative effector-phenotype T cells while allowing the survival and proliferation of IL-7R+ memory-phenotype cells. The surviving IL-7R+ Th17 cells expressed genes associated with alcohol/aldehyde detoxification and showed potential to transdifferentiate into IL-7R-negative effector cells. Inhibiting aldehyde dehydrogenase reduced IL-7R+ Th17 cells in vivo, independently of CD28, and exhibited additive effects when combined with abatacept. Our findings suggest that CD28 blockade prevents inflammation without eliminating persistent memory cells. These remaining memory cells can be targeted by other drugs, such as aldehyde dehydrogenase inhibitors, to limit their survival, thereby facilitating the treatment of chronic autoimmune diseases.

Identification of Surface Markers and Functional Characterization of Myeloid Derived Suppressor Cell-Like Adherent Cells.

Myeloid-derived suppressor cell (MDSC)-like adherent cells (MLACs) are a recently identified CD11b+ F4/80- myeloid cell subset that can infiltrate tumors early in development and promote their growth. Because of these functions, MLACs play an important role in establishing an immunosuppressive tumor microenvironment (TME). However, the lack of MLAC-specific markers has hampered further characterization of this cell type. This study identifies the gene signature of MLACs by analyzing RNA-sequencing (RNA-seq) and public single-cell RNA-seq data, revealing that MLACs are an independent cell population that are distinct from other intratumoral myeloid cells. After combining proteome analysis of membrane proteins with RNA-seq data, H2-Ab1 and CD11c are indicated as marker proteins that can support the isolation of MLAC subsets from CD11b+ F4/80- myeloid cells by fluorescence-activated cell sorting. The CD11b+ F4/80- H2-Ab1+ and CD11b+ F4/80- CD11c+ MLAC subsets represent approximately half of the MLAC population that is isolated based on their adhesion properties and possess gene signatures and functional properties similar to those of the MLAC population. Additionally, membrane proteome analysis suggests that MLACs express highly heterogeneous surface proteins. This study facilitates an integrated understanding of heterogeneous intratumoral myeloid cells, as well as the molecular and cellular details of the development of an immunosuppressive TME.

Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease.

Thymic selection and peripheral activation of conventional T (Tconv) and regulatory T (Treg) cells depend on TCR signaling, whose anomalies are causative of autoimmunity. Here, we expressed in normal mice mutated ZAP-70 molecules with different affinities for the CD3 chains, or wild type ZAP-70 at graded expression levels under tetracycline-inducible control. Both manipulations reduced TCR signaling intensity to various extents and thereby rendered those normally deleted self-reactive thymocytes to become positively selected and form a highly autoimmune TCR repertoire. The signal reduction more profoundly affected Treg development and function because their TCR signaling was further attenuated by Foxp3 that physiologically repressed the expression of TCR-proximal signaling molecules, including ZAP-70, upon TCR stimulation. Consequently, the TCR signaling intensity reduced to a critical range generated pathogenic autoimmune Tconv cells and concurrently impaired Treg development/function, leading to spontaneous occurrence of autoimmune/inflammatory diseases, such as autoimmune arthritis and inflammatory bowel disease. These results provide a general model of how altered TCR signaling evokes autoimmune disease.

Attenuation of regulatory T cell function by type I IFN signaling in an MDA5 gain-of-function mutant mouse model.

Melanoma differentiation-associated gene 5 (MDA5) is an essential viral double-stranded RNA sensor to trigger antiviral immune responses, including type I interferon (IFN) induction. Aberrant activation of this viral sensor is known to cause autoimmune diseases designated as type I interferonopathies. However, the cell types responsible for these diseases and the molecular mechanisms behind their onset and development are still largely unknown. In this study, we revealed the attenuation of regulatory T cell (Treg) function by type I IFN signaling in a mouse model expressing a gain-of-function MDA5 G821S mutant. We found that experimental colitis induced by adoptive transfer of naïve T cells in Rag2-/- mice was rescued by simultaneous transfer of Tregs from wild-type but not from the MDA5 mutant mice. Type I IFN receptor deficiency in the MDA5 mutant mice recovered the suppressive function of MDA5 mutant Tregs. These results suggest that constitutive MDA5 and type I IFN signaling in Tregs decreases the suppressive function of Tregs, potentially contributing to the onset and exacerbation of autoimmune disorders in interferonopathies.

Uniformity and Efficacy of Dry Powders Delivered to the Lungs of a Mycobacterial-Surrogate Rat Model of Tuberculosis.

PURPOSE: Pulmonary administration of dry drug powder is a considered promising strategy in the treatment of various lung diseases such as tuberculosis and is more effective than systemic medication. However, in the pre-clinical study phase, there is a lack of devices for effective delivery of dry powders to the lungs of small rodents. In this study, an administration device which utilizes Venturi effect to deliver dry powders to the lungs homogeneously was developed. METHODS: A Venturi-effect administration device which synchronizes with breathes by use of a ventilator and aerosolizes the dry powders was created. Pulmonary distribution of inhalable dry powders prepared by spray-drying poly(lactic-co-glycolic) acid and an antituberculosis agent rifampicin and anti-tuberculosis effect of the powders on mycobacteria infected rats by administration with the Venturi-effect administration device and a conventional insufflation device were evaluated. RESULTS: Homogeneous distribution of the dry powders in the lung was achieved by the Venturi-effect administration device due to efficient and recurring aerosolization of loaded dry powders while synchronizing with breathes. Amount of rifampicin delivered to the lungs by the Venturi-effect administration device was three times higher than that by a conventional insufflation device, demonstrating three times greater antimycobacterial activity. CONCLUSIONS: The Venturi-effect administration device aerosolized inhalable antituberculosis dry powders efficiently, achieved uniform pulmonary distribution, and aided the dry powders to exert antituberculosis activity on lung-residing mycobacteria.

Effect of Impaired T Cell Receptor Signaling on the Gut Microbiota in a Mouse Model of Systemic Autoimmunity.

OBJECTIVE: T cell receptor (TCR) signaling abnormalities and gut dysbiosis are thought to be involved in the development of systemic lupus erythematosus (SLE). However, it is not known whether these mechanisms are interrelated. This study was undertaken to explore the impact of defective TCR signaling on microbiota-driven immune responses and the consequent triggering of systemic autoimmunity. METHODS: The responses of B6SKG mice harboring a mutation in ZAP-70 leading to spontaneous development of SLE were evaluated under specific pathogen-free (SPF) and germ-free (GF) conditions. The gut microbiome was analyzed using 16S ribosomal RNA sequencing. Secretory IgA production in the gut and follicular helper T (Tfh) cell development in the spleen and Peyer's patches were analyzed. Interleukin-17 (IL-17)-deficient mice and segmented filamentous bacteria (SFB)-specific TCR-transgenic mice were used to examine the role of IL-17 and thymic selection. RESULTS: SLE development in B6SKG mice was significantly more attenuated under GF conditions than under SPF conditions. The gut microbiota in B6SKG mice was altered, which was associated with the expansion of SFB and consequent development of SLE by driving Th17 cell differentiation, which was in turn blunted by IL-17 deficiency. Notably, although systemic Tfh development and autoantibody IgG response were enhanced, local gut Tfh and IgA responses were impaired. Moreover, experiments in SFB-specific TCR-transgenic mice revealed that this differential response was caused by altered thymic selection of self- and microbiota-reactive TCR because of defective TCR signaling. CONCLUSION: Our findings indicate that defective TCR signaling alters the gut microbiota and promotes systemic autoimmunity by driving Th17 cell differentiation.

Dispensable roles of Gsdmd and Ripk3 in sustaining IL-1ß production and chronic inflammation in Th17-mediated autoimmune arthritis.

Programmed necrosis, such as necroptosis and pyroptosis, is a highly pro-inflammatory cellular event that is associated with chronic inflammation. Although there are various triggers of pyroptosis and necroptosis in autoimmune tissue inflammation and subsequent lytic forms of cell death release abundant inflammatory mediators, including damage-associated molecular patterns and IL-1ß, capable of amplifying autoimmune Th17 effector functions, it remains largely unclear whether the programs play a crucial role in the pathogenesis of autoimmune arthritis. We herein report that Gasdermin D (Gsdmd) and receptor interacting serine/threonine kinase 3 (Ripk3)-key molecules of pyroptosis and necroptosis, respectively-are upregulated in inflamed synovial tissues, but dispensable for IL-1ß production and the development of IL-17-producing T helper (Th17) cell-mediated autoimmune arthritis in SKG mice. Gsdmd-/-, Ripk3-/-, or Gsdmd-/- Ripk3-/- SKG mice showed severe arthritis with expansion of arthritogenic Th17 cells in the draining LNs and inflamed joints, which was comparable to that in wild-type SKG mice. Despite the marked reduction of IL-1ß secretion from Gsdmd-/- or Ripk3-/- bone marrow-derived DCs by canonical stimuli, IL-1ß levels in the inflamed synovium were not affected in the absence of Gsdmd or Ripk3. Our results revealed that T cell-mediated autoimmune arthritis proceeds independently of the pyroptosis and necroptosis pathways.

Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells.

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.

Foxp3+ Regulatory T Cells Inhibit CCl4-Induced Liver Inflammation and Fibrosis by Regulating Tissue Cellular Immunity.

Foxp3+ regulatory T (Treg) cells are pivotal in maintaining immunological self-tolerance and tissue homeostasis; however, it remains unclear how tissue Treg cells respond to liver injury and regulate chronic inflammation, which can cause liver fibrosis. We report here that hepatic Treg cells play a critical role in preventing liver pathology by suppressing inflammatory cellular immunity that can promote liver damage and fibrosis. Chronic liver inflammation induced by injections of carbon tetrachloride (CCl4) led to preferential expansion of hepatic Treg cells that prevented liver fibrosis. In contrast, depletion of Treg cells in the CCl4-induced liver fibrosis model exacerbated the severity of liver pathology. Treg depletion unleashed tissue cellular immunity and drove the activation and expansion of the pro-fibrotic IL-4-producing T helper 2 cells, as well as CCR2high Ly-6Chigh inflammatory monocytes/macrophages in the inflamed liver. Although Treg expression of amphiregulin plays a key role in tissue remodeling and repair in various inflammation models, amphiregulin from hepatic Treg cells, the largest producer among liver immune cells, was dispensable for maintaining liver homeostasis and preventing liver fibrosis during CCl4-induced chronic inflammation. Our results indicate that Treg cells control chronic liver inflammation and fibrosis by regulating the aberrant activation and functions of immune effector cells. Harnessing Treg functions, which effectively regulate tissue cellular immunity, may be a therapeutic strategy for preventing and treating liver fibrosis.

Impaired T cell receptor signaling and development of T cell-mediated autoimmune arthritis.

Mutations of the genes encoding T-cell receptor (TCR)-proximal signaling molecules, such as ZAP-70, can be causative of immunological diseases ranging from T-cell immunodeficiency to T-cell-mediated autoimmune disease. For example, SKG mice, which carry a hypomorphic point mutation of the Zap-70 gene, spontaneously develop T-cell-mediated autoimmune arthritis immunopathologically similar to human rheumatoid arthritis (RA). The Zap-70 mutation alters the sensitivity of developing T cells to thymic positive/negative selection by self-peptides/MHC complexes, shifting self-reactive TCR repertoire to include a dominant arthritogenic specificity and also affecting thymic development and function of autoimmune suppressive regulatory T (Treg) cells. Polyclonal self-reactive T cells, including potentially arthritogenic T cells, thus produced by the thymus recognize self-peptide/MHC complexes on antigen-presenting cells (APCs) in the periphery and stimulate them to produce cytokines including IL-6 to drive the arthritogenic T cells to differentiate into arthritogenic T-helper 17 (Th17) cells. Insufficient Treg suppression or activation of APCs via microbial and other environmental stimuli evokes arthritis by activating granulocyte-macrophage colony-stimulating factor-secreting effector Th17 cells, mediating chronic bone-destructive joint inflammation by activating myeloid cells, innate lymphoid cells, and synoviocytes in the joint. These findings obtained from the study of SKG mouse arthritis are instrumental in understanding how arthritogenic T cells are produced, become activated, and differentiate into effector T cells mediating arthritis, and may help devising therapeutic measures targeting autoimmune pathogenic Th17 cells or autoimmune-suppressing Treg cells to treat and prevent RA.

Conversion of antigen-specific effector/memory T cells into Foxp3-expressing Treg cells by inhibition of CDK8/19.

A promising way to restrain hazardous immune responses, such as autoimmune disease and allergy, is to convert disease-mediating T cells into immunosuppressive regulatory T (Treg) cells. Here, we show that chemical inhibition of the cyclin-dependent kinase 8 (CDK8) and CDK19, or knockdown/knockout of the CDK8 or CDK19 gene, is able to induce Foxp3, a key transcription factor controlling Treg cell function, in antigen-stimulated effector/memory as well as naïve CD4+ and CD8+ T cells. The induction was associated with STAT5 activation, independent of TGF-ß action, and not affected by inflammatory cytokines. Furthermore, in vivo administration of a newly developed CDK8/19 inhibitor along with antigen immunization generated functionally stable antigen-specific Foxp3+ Treg cells, which effectively suppressed skin contact hypersensitivity and autoimmune disease in animal models. The results indicate that CDK8/19 is physiologically repressing Foxp3 expression in activated conventional T cells and that its pharmacological inhibition enables conversion of antigen-specific effector/memory T cells into Foxp3+ Treg cells for the treatment of various immunological diseases.

Synovial Tissue Inflammation Mediated by Autoimmune T Cells.

In rheumatoid arthritis (RA), various hematopoietic and non-hematopoietic cells present in the synovial tissue secrete numerous inflammatory mediators including pro-inflammatory cytokines critical for the induction of chronic joint inflammation and bone destruction. Fibroblast-like synoviocytes (FLSs) in the non-hematopoietic cell compartment are key inflammatory cells activated in inflamed joints and driving the disease; yet how synovial tissue inflammation is modulated by autoimmune T cells is not fully understood. In this review, mainly based on recent findings with a mouse model of spontaneous autoimmune arthritis, we discuss the mechanism of Th17-mediated synovial tissue inflammation; that is, what environmental stimuli and arthritogenic self-antigens trigger arthritis, how arthritogenic T cells initiate joint inflammation by stimulating FLSs, and how the cellular sources of GM-CSF from lymphoid and tissue stromal cells in the synovium contribute to the development of arthritis. We also highlight possible plasticity of Th17 cells toward pathogenic GM-CSF producers, and the functional instability of regulatory T cells under inflammatory conditions in RA joints.

Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection.

During thymic negative selection, autoreactive thymocytes carrying T cell receptor (TCR) with overtly strong affinity to self-MHC/self-peptide are removed by Bim-dependent apoptosis, but how Bim is specifically regulated to link TCR activation and apoptosis induction is unclear. Here we identify a murine T cell-specific genomic enhancer EBAB (Bub1-Acoxl-Bim), whose deletion leads to accumulation of thymocytes expressing high affinity TCRs. Consistently, EBAB knockout mice have defective negative selection and fail to delete autoreactive thymocytes in various settings, with this defect accompanied by reduced Bim expression and apoptosis induction. By contrast, EBAB is dispensable for maintaining peripheral T cell homeostasis via Bim-dependent pathways. Our data thus implicate EBAB as an important, developmental stage-specific regulator of Bim expression and apoptosis induction to enforce thymic negative selection and suppress autoimmunity. Our study unravels a part of genomic enhancer codes that underlie complex and context-dependent gene regulation in TCR signaling.

Correction to: The pathogenicity of Th17 cells in autoimmune diseases.

Unfortunately, an error occurred in the following passus of the article. The word "receptor" was missing in the sentence "Because T cells do not express GM-CSF receptor [41], GM-CSF affects non-T cells."

The pathogenicity of Th17 cells in autoimmune diseases.

IL-17-producing T helper (Th17) cells have been implicated in the pathogenesis of many inflammatory and autoimmune diseases. Targeting the effector cytokines IL-17 and GM-CSF secreted by autoimmune Th17 cells has been shown to be effective for the treatment of the diseases. Understanding a molecular basis of Th17 differentiation and effector functions is therefore critical for the regulation of the pathogenicity of tissue Th17 cells in chronic inflammation. Here, we discuss the roles of proinflammatory cytokines and environmental stimuli in the control of Th17 differentiation and chronic tissue inflammation by pathogenic Th17 cells in humans and in mouse models of autoimmune diseases. We also highlight recent advances in the regulation of pathogenic Th17 cells by gut microbiota and immunometabolism in autoimmune arthritis.

Satb1 regulates the effector program of encephalitogenic tissue Th17 cells in chronic inflammation.

The genome organizer, special AT-rich sequence-binding protein-1 (Satb1), plays a pivotal role in the regulation of global gene networks in a cell type-dependent manner and is indispensable for the development of multiple cell types, including mature CD4+ T, CD8+ T, and Foxp3+ regulatory T cells in the thymus. However, it remains unknown how the differentiation and effector program of the Th subsets in the periphery are regulated by Satb1. Here, we demonstrate that Satb1 differentially regulates gene expression profiles in non-pathogenic and pathogenic Th17 cells and promotes the pathogenic effector program of encephalitogenic Th17 cells by regulating GM-CSF via Bhlhe40 and inhibiting PD-1 expression. However, Satb1 is dispensable for the differentiation and non-pathogenic functions of Th17 cells. These results indicate that Satb1 regulates the specific gene expression and function of effector Th17 cells in tissue inflammation.

Reverse Engineering the 1-Month Lupron Depot®.

The 1-month Lupron Depot® (LD) encapsulating water-soluble leuprolide in poly(lactic-co-glycolic acid) (PLGA) microspheres is a benchmark product upon which modern long-acting release products are often compared. Despite expiration of patent coverage, no generic product for the LD has been approved in the USA, likely due to the complexity of components and manufacturing processes involved in the product. Here, we describe the reverse engineering of the LD composition and important product attributes. Specific attributes analyzed for microspheres were as follows: leuprolide content by three methods; gelatin content, type, and molecular weight distribution; PLGA content, lactic acid/glycolic acid ratio, and molecular weight distribution; mannitol content; in vitro drug release; residual solvent and moisture content; particle size distribution and morphology; and glass transition temperature. For the diluent, composition, viscosity, and specific gravity were analyzed. Analyzed contents of the formulation and the determined PLGA characteristics matched well with the official numbers stated in the package insert and those found in literature, respectively. The gelatin was identified as type B consistent with ~ 300 bloom. The 11-µm volume-median microspheres in the LD slowly released the drug in vitro in a zero-order manner after ~ 23% initial burst release. Very low content of residual moisture (< 0.5%) and methylene chloride (< 1 ppm) in the product indicates in-water drying is capable of removing solvents to extremely low levels during manufacturing. The rigorous approach of reverse engineering described here may be useful for development of generic leuprolide-PLGA microspheres as well as other new and generic PLGA microsphere formulations.

Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis.

Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention.