Ssu72 phosphatase directly binds to ZAP-70, thereby providing fine-tuning of TCR signaling and preventing spontaneous inflammation.

ZAP-70 is required for the initiation of T cell receptor (TCR) signaling, and Ssu72 is a phosphatase that regulates RNA polymerase II activity in the nucleus. However, the mechanism by which ZAP-70 regulates the fine-tuning of TCR signaling remains elusive. Here, we found that Ssu72 contributed to the fine-tuning of TCR signaling by acting as tyrosine phosphatase for ZAP-70. Affinity purification-mass spectrometry and an in vitro assay demonstrated specific interaction between Ssu72 and ZAP-70 in T cells. Upon TCR stimulation, Ssu72-deficient T cells increased the phosphorylation of ZAP-70 and downstream molecules and exhibited hyperresponsiveness, which was restored by reducing ZAP-70 phosphorylation. In vitro assay demonstrated that recombinant Ssu72 reduced tyrosine phosphorylation of ZAP-70 via phosphatase activity. Cd4-CreSsu72fl/fl mice showed a defect in the thymic development of invariant natural killer T cells and reductions in CD4+ and CD8+ T cell numbers in the periphery but more CD44hiCD62Llo memory T cells and fewer CD44loCD62Lhi naïve T cells, compared with wild-type mice. Furthermore, Cd4-CreSsu72fl/fl mice developed spontaneous inflammation at 6 mo. In conclusion, Ssu72 phosphatase regulates the fine-tuning of TCR signaling by binding to ZAP-70 and regulating its tyrosine phosphorylation, thereby preventing spontaneous inflammation.

Design and Optimization of an α-Helical Bundle Dimer Cell-Penetrating Peptide for In Vivo Drug Delivery.

To deliver membrane-impermeable drugs into eukaryotic cells, a lot of cell-penetrating peptides (CPPs) were discovered. Previously we designed an amphipathic α-helical peptide which dimerizes itself via its two C-residues. This bis-disulfide-linked dimeric bundle, LK-3, has remarkable cell-penetrating ability at nanomolar concentration, which is an essential prerequisite for CPP. In an effort to optimize the sequence of LK-3, we adjusted its length and evaluated changes in the dimerization rate. We found that a 10-amino-acid monomer has the fastest dimerization rate and subsequently modified its hydrophobic and hydrophilic residues to construct a small peptide library. The evaluation of cell permeability of these derivatives showed that their cell-penetrating ability is comparable to that of the LK-3, except V- or H-containing ones. In this library, diLR10 was found to display fast nanomolar cell membrane penetration, low toxicity, and ease of production. The methotrexate (MTX) conjugate of diLR10, MTX-diLR10, has a 19-fold increased efficacy over MTX in MDA-MB-231 cells and efficiently deflates lesions in a rheumatoid arthritis (RA) in vivo mouse model.

Innate Type-2 Cytokines: From Immune Regulation to Therapeutic Targets.

The intricate role of innate type-2 cytokines in immune responses is increasingly acknowledged for its dual nature, encompassing both protective and pathogenic dimensions. Ranging from defense against parasitic infections to contributing to inflammatory diseases like asthma, fibrosis, and obesity, these cytokines intricately engage with various innate immune cells. This review meticulously explores the cellular origins of innate type-2 cytokines and their intricate interactions, shedding light on factors that amplify the innate type-2 response, including TSLP, IL-25, and IL-33. Recent advancements in therapeutic strategies, specifically the utilization of biologics targeting pivotal cytokines (IL-4, IL-5, and IL-13), are discussed, offering insights into both challenges and opportunities. Acknowledging the pivotal role of innate type-2 cytokines in orchestrating immune responses positions them as promising therapeutic targets. The evolving landscape of research and development in this field not only propels immunological knowledge forward but also holds the promise of more effective treatments in the future.

Invariant natural killer T cells in lung diseases.

Invariant natural killer T (iNKT) cells are a subset of T cells that are characterized by a restricted T-cell receptor (TCR) repertoire and a unique ability to recognize glycolipid antigens. These cells are found in all tissues, and evidence to date suggests that they play many immunological roles in both homeostasis and inflammatory conditions. The latter include lung inflammatory diseases such as asthma and infections: the roles of lung-resident iNKT cells in these diseases have been extensively researched. Here, we provide insights into the biology of iNKT cells in health and disease, with a particular focus on the role of pulmonary iNKT cells in airway inflammation and other lung diseases.

Development and Functions of Alveolar Macrophages.

Macrophages residing in various tissue types are unique in terms of their anatomical locations, ontogenies, developmental pathways, gene expression patterns, and immunological functions. Alveolar macrophages (AMs) reside in the alveolar lumen of the lungs and serve as the first line of defense for the respiratory tract. The immunological functions of AMs are implicated in the pathogenesis of various pulmonary diseases such as allergic asthma, chronic obstructive pulmonary disorder (COPD), pulmonary alveolar proteinosis (PAP), viral infection, and bacterial infection. Thus, the molecular mechanisms driving the development and function of AMs have been extensively investigated. In this review article, we discuss the roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-ß in AM development, and provide an overview of the anti-inflammatory and proinflammatory functions of AMs in various contexts. Notably, we examine the relationships between the metabolic status of AMs and their development processes and functions. We hope that this review will provide new information and insight into AM development and function.

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2.

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas-/-)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.

Sodium chloride inhibits IFN-γ, but not IL-4, production by invariant NKT cells.

Invariant NKT (iNKT) cells are a distinct subset of T cells that exert Janus-like functions in vivo by producing IFN-γ and IL-4. Sodium chloride modulates the functions of various immune cells, including conventional CD4+ T cells and macrophages. However, it is not known whether sodium chloride affects iNKT cell function, so we addressed this issue. Sodium chloride inhibited IFN-γ, but not IL-4, production by iNKT cells upon TCR or TCR-independent (IL-12 and IL-18) stimulation in a dose-dependent manner. Consistently, sodium chloride reduced the expression level of tbx21, but not gata-3, in iNKT cells stimulated with TCR engagement or IL-12 + IL-18. Sodium chloride increased phosphorylated p38 expression in iNKT cells and inhibitors of p38, NFAT5, SGK1, and TCF-1 restored IFN-γ production by iNKT cells stimulated with sodium chloride and TCR engagement. Furthermore, adoptive transfer of iNKT cells pretreated with sodium chloride restored antibody-induced joint inflammation to a lesser extent than for untreated iNKT cells in Jα18 knockout mice. These findings suggest that sodium chloride inhibits IFN-γ production by iNKT cells in TCR-dependent and TCR-independent manners, which is dependent on p38, NFAT5, SGK1, and TCF-1. These findings highlight the functional role of sodium chloride in iNKT cell-mediated inflammatory diseases.

GM-CSF and IL-4 produced by NKT cells inversely regulate IL-1ß production by macrophages.

Natural Killer T (NKT) cells are distinct T cell subset that link innate and adaptive immune responses. IL-1ß, produced by various immune cells, plays a key role in the regulation of innate immunity in vivo. However, it is unclear whether NKT cells regulate IL-1ß production by macrophages. To address this, we co-cultured NKT cells and peritoneal macrophages in the presence of TCR stimulation and inflammasome activators. Among cytokines secreted from NKT cells, GM-CSF enhanced IL-1ß production by macrophages via regulating LPS-mediated pro-IL-1ß expression and NLRP3-dependent inflammasome activation, whereas IL-4 enhanced M2-differentiation of macrophages and decreased IL-1ß production. Together, our findings suggest the NKT cells have double-sided effects on IL-1ß-mediated innate immune responses by producing IL-4 and GM-CSF. These findings may be helpful for a comprehensive understanding of NKT cell-mediated regulatory mechanisms of the pro-inflammatory effects of IL-1ß in inflammatory diseases in vivo.

The roles of innate lymphoid cells in the development of asthma.

Asthma is a common pulmonary disease with several different forms. The most studied form of asthma is the allergic form, which is mainly related to the function of Th2 cells and their production of cytokines (IL-4, IL-5, and IL-13) in association with allergen sensitization and adaptive immunity. Recently, there have been many advances in understanding non-allergic asthma, which seems to be related to environmental factors such as air pollution, infection, or even obesity. Cells of the innate immune system, including macrophages, neutrophils, and natural killer T cells as well as the newly described innate lymphoid cells, are effective producers of a variety of cytokines and seem to play important roles in the development of non-allergic asthma. In this review, we focus on recent findings regarding innate lymphoid cells and their roles in asthma.