Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Macrophage-mediated extracellular matrix remodeling controls host Staphylococcus aureus susceptibility in the skin.

Hypodermis is the predominant site of Staphylococcus aureus infections that cause cellulitis. Given the importance of macrophages in tissue remodeling, we examined the hypodermal macrophages (HDMs) and their impact on host susceptibility to infection. Bulk and single-cell transcriptomics uncovered HDM subsets with CCR2-dichotomy. HDM homeostasis required the fibroblast-derived growth factor CSF1, ablation of which abrogated HDMs from the hypodermal adventitia. Loss of CCR2- HDMs resulted in accumulation of the extracellular matrix component, hyaluronic acid (HA). HDM-mediated HA clearance required sensing by the HA receptor, LYVE-1. Cell-autonomous IGF1 was required for accessibility of AP-1 transcription factor motifs that controlled LYVE-1 expression. Remarkably, loss of HDMs or IGF1 limited Staphylococcus aureus expansion via HA and conferred protection against cellulitis. Our findings reveal a function for macrophages in the regulation of HA with an impact on infection outcomes, which may be harnessed to limit the establishment of infection in the hypodermal niche.

Mouse Models for Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial disease with underlying barrier disruption and altered microbial flora, resulting in dry skin and eczematous inflammation with persistent pruritis. Mouse models have been heavily used to investigate AD pathophysiology. Among various AD mouse models, AD-like inflammation induced by topical calcipotriol, a vitamin D3 analog referred to as MC903 in experimental settings, is a versatile model that can be applied to any strain of mice, which can be used for immunologic and morphologic studies. Herein, we provide basic protocols for the topical application of MC903 and approaches to assess phenotypes. After inducing AD-like inflammation, the skin is harvested for flow cytometry analysis, as well as for histologic and immunofluorescence microscopy analyses. The combination of these approaches enables accurate characterization of the degree of inflammation, type of inflammatory infiltrate, and localization of immune infiltrates. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Application of MC903 and gross phenotype assessment Basic Protocol 2: Processing skin for flow cytometry analysis Support Protocol: Skin immune cell surface staining and flow cytometry analysis Basic Protocol 3: Harvesting skin for histologic analysis Basic Protocol 4: Immunofluorescence staining to identify immune cell infiltrates.

cis interaction of CD153 with TCR/CD3 is crucial for the pathogenic activation of senescence-associated T cells.

With age, senescence-associated (SA) CD4+ T cells that are refractory to T cell receptor (TCR) stimulation are increased along with spontaneous germinal center (Spt-GC) development prone to autoantibody production. We demonstrate that CD153 and its receptor CD30 are expressed in SA-T and Spt-GC B cells, respectively, and deficiency of either CD153 or CD30 results in the compromised increase of both cell types. CD153 engagement on SA-T cells upon TCR stimulation causes association of CD153 with the TCR/CD3 complex and restores TCR signaling, whereas CD30 engagement on GC B cells induces their expansion. Administration of an anti-CD153 antibody blocking the interaction with CD30 suppresses the increase in both SA-T and Spt-GC B cells with age and ameliorates lupus in lupus-prone mice. These results suggest that the molecular interaction of CD153 and CD30 plays a central role in the reciprocal activation of SA-T and Spt-GC B cells, leading to immunosenescent phenotypes and autoimmunity.

A case of methotrexate-associated Epstein-Barr virus-positive mucocutaneous ulcer.

Epstein-Barr virus-positive mucocutaneous ulcer (EBVMCU) is a B-cell proliferative disorder that has been designated as a provisional entity in the 2017 World Health Organization classification for lymphoid neoplasms. While EBVMCU may contain varying numbers of cells with Hodgkin and Reed-Sternberg cells-like morphology, the clinical course is benign and must be distinguished from lymphomas. Patients who develop EBVMCU are commonly immunocompromised, with methotrexate (MTX) as the leading cause. Most previously reported cases of EBVMCU describe mucosal ulcers with very little documentation on skin lesions and its course. Here, we report a case of MTX-associated EBVMCU of the lower leg that underwent spontaneous regression after MTX withdrawal, during which negative conversion of local Epstein-Barr virus activation was confirmed.

What is the "washout" of hepatocellular carcinoma as observed on the equilibrium phase CT?: consideration based on the concept of extracellular volume fraction.

PURPOSE: To verify the hypothesis that extracellular volume fraction (ECV) and precontrast CT density are the main determinants of washout of hepatocellular carcinoma (HCC) at the equilibrium phase CT. MATERIALS AND METHODS: Between 2018 and 2020, patients with surgically resected HCC were recruited who had undergone preoperative 4-phase CT. Those larger than 6 cm were excluded to minimize the possibility of intratumoral hemorrhage or degeneration. Two radiologists reviewed the whole images in consensus and divided cases into washout positive and negative groups. Washout positive group at the equilibrium phase was defined as "HCC showing relatively low density as compared to the surrounding background liver (BGL), irrespective of the presence of early enhancement or fibrous capsule". Several clinico-pathological and radiological features, including ECV and precontrast CT density, were correlated to the presence of washout, using uni- and multi-variable analyses. RESULTS: 27 HCC in 24 patients met the inclusion criteria. 22 (82%) and five HCC belonged to washout positive and negative groups, respectively. Univariable analysis revealed ECV of HCC and BGL, ECV difference between HCC and BGL, and presence of fibrous capsule on the equilibrium phase CT were the significant factors. Multivariable analysis showed ECV of HCC and BGL, and precontrast CT density of BGL, were the independently significant factors related to washout, suggesting washout is more likely observed with lower HCC ECV, higher BGL ECV, and higher BGL precontrast CT density. CONCLUSION: Major determinants of washout of HCC may be ECV of HCC and BGL, and precontrast CT density of BGL.

Flow cytometry analysis of the subpopulations of mouse keratinocytes and skin immune cells.

Skin is our body's outermost physical barrier and an immunological interface enriched with various immune and non-immune cells. However, efficient generation of single-cell suspensions for flow cytometry analysis can be challenging. Here, we provide protocols to obtain epidermal and whole skin cell suspensions as well as gating strategies to identify mouse keratinocytes and skin immune cell subsets via flow cytometry. For complete details on the use and execution of this protocol, please refer to Sakamoto et al. (2021).

Disruption of the endopeptidase ADAM10-Notch signaling axis leads to skin dysbiosis and innate lymphoid cell-mediated hair follicle destruction.

Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting ß-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.

Importance of extracellular volume fraction of the spleen as a predictive biomarker for high-risk esophago-gastric varices in patients with chronic liver diseases: A preliminary report.

PURPOSE: To clarify clinico-radiological factors for high-risk esophago-gastric varices (EGV), including extracellular volume fraction (ECV) of the liver, pancreas, and the spleen. METHODS: Between 2014 and 2018, 70 chronic liver disease patients who underwent 4-phase CT of the upper abdomen and either of upper gastrointestinal tract endoscopy, or actual treatment for bleeding EGV, within three months after CT, were retrospectively included. Patients were subdivided into high-risk EGV group (HRG), who had high-risk endoscopic findings or actual hemostatic treatments, and non-high-risk EGV group (NHRG). ECV of the liver, pancreas, and the spleen was measured on the ECV map generated from routine diagnostic CT data, and additional clinico-radiological factors including direct visualization of EGV on portal venous phase CT, were correlated to HRG, using both univariable and multivariable analyses. RESULTS: There were 8 and 62 patients in HRG, and NHRG, respectively. None had symptoms related to EGV at the time of CT examinations. Univariable analysis revealed splenic volume, liver and splenic ECVs, and EGV visualization on portal venous phase CT, as significant factors. Multivariable analysis suggested that EGV visualization, splenic ECV, and splenic volume were independently significant factors. Using these three factors, sensitivity/specificity/positive predictive value/negative predictive value/accuracy = 100/85/40/100/87% were obtained with partition model analysis. CONCLUSIONS: High-risk EGV can be predicted with acceptable accuracy using routine diagnostic CT data including splenic ECV.

Immediate effects of short period lower limb ergometer exercise in adolescent and young adult patients with cerebral palsy and spastic diplegia.

[Purpose] To determine the effects of lower limb ergometer exercise on the spasticity and joint range of motion of the lower extremity and gait function in patients with cerebral palsy and spastic paralysis. [Participants and Methods] This study included 8 participants with cerebral palsy and spastic paralysis (GMFCS levels I to IV) who received care at the outpatient clinic. After a 5-min rest, the lower limb ergometer exercises were performed for 10 min. We measured the participants' arterial blood pressure, pulse rate, passive range of knee joint extension, muscle tone using the Modified Ashworth scale (MAS) and Modified Tardieu scale (MTS), and 10-m walk test (10MWT). Measurements were collected three times (at baseline before exercise, immediately at the end of exercise, and 5 min after exercise during recovery). [Results] The 10-min lower limb ergometer exercise significantly improved the knee joint extension, MAS and MTS scores, and reduced lower extremity spasticity. Furthermore, it significantly increased the range of knee joint extension and decreased the 10MWT score. [Conclusion] The results showed that the 10-minute lower limb ergometer exercise is beneficial in reducing the spasticity of the lower limb muscles and in increasing the range of motion of knee extension in paraplegic patients with cerebral palsy, suggesting that its implementation in young children could prevent spasticity and enhance motor function.

Mucinous cystic neoplasm of the liver communicated with intrahepatic duct exhibiting peculiar chronological change in MR imaging appearances: a case report.

A 70-year-old woman has been followed up for chronic hepatitis C and hepatocellular carcinoma which had been successfully controlled by several sessions of radiofrequency ablation. A small cystic lesion in segment IV associated with adjacent intrahepatic duct dilatation was firstly noted 4 years before on MR imaging, which showed gradual increase in size and significant interval change in the MRI signal intensity of the cystic content on the follow-up examinations. The mass finally reached 4 cm in its largest dimension, associated with slightly enhancing thickened wall, suggesting its neoplastic nature. The mass was surgically resected and a final diagnosis of mucinous cystic neoplasm (MCN) of the liver was made. MCN is usually considered to have no communication with intrahepatic duct, but in this particular case, the communication with the biliary duct was suggested from its early stage of the lesion, which would be the cause of peculiar chronological change in MR appearance.

Targeted therapy guided by single-cell transcriptomic analysis in drug-induced hypersensitivity syndrome: a case report.

Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a potentially fatal multiorgan inflammatory disease associated with herpesvirus reactivation and subsequent onset of autoimmune diseases1-4. Pathophysiology remains elusive and therapeutic options are limited. Cases refractory to corticosteroid therapy pose a clinical challenge1,5 and approximately 30% of patients with DiHS/DRESS develop complications, including infections and inflammatory and autoimmune diseases1,2,5. Progress in single-cell RNA sequencing (scRNA-seq) provides an opportunity to dissect human disease pathophysiology at unprecedented resolutions6, particularly in diseases lacking animal models, such as DiHS/DRESS. We performed scRNA-seq on skin and blood from a patient with refractory DiHS/DRESS, identifying the JAK-STAT signaling pathway as a potential target. We further showed that central memory CD4+ T cells were enriched with DNA from human herpesvirus 6b. Intervention via tofacitinib enabled disease control and tapering of other immunosuppressive agents. Tofacitinib, as well as antiviral agents, suppressed culprit-induced T cell proliferation in vitro, further supporting the roles of the JAK-STAT pathway and herpesviruses in mediating the adverse drug reaction. Thus, scRNA-seq analyses guided successful therapeutic intervention in the patient with refractory DiHS/DRESS. scRNA-seq may improve our understanding of complicated human disease pathophysiology and provide an alternative approach in personalized medicine.

Usefulness of iodine-blood material density images in estimating degree of liver fibrosis by calculating extracellular volume fraction obtained from routine dual-energy liver CT protocol equilibrium phase data: preliminary experience.

PURPOSE: To assess whether extracellular volume fraction (ECV) calculated from iodine(-blood) density images (I-B) of dual-energy liver CT (DECT) equilibrium phase data (EqD) is useful in estimating the degree of liver fibrosis. MATERIALS AND METHODS: Consecutive 52 patients with chronic liver disease who underwent fast kV switching DECT and liver MR elastography (MRE) were retrospectively enrolled. Iodine(-water) density images (I-W) and I-B generated from EqD and ECV were calculated. As blood pools, abdominal aorta (Ao) and suprahepatic inferior vena cava (IVC) were chosen, and, therefore, 4 types of ECV (ECVI-W Ao, ECVI-W IVC, ECVI-B Ao, ECVI-B IVC) were obtained. ECV was also calculated using conventional method (ECVconv Ao). The correlation coefficients (R2 or rho) of these five ECVs versus liver stiffness (MRE) or pathologically proven fibrosis grades were compared. RESULTS: As for correlation with liver stiffness, R2 for ECVconv.Ao, ECVI-W Ao, ECVI-B Ao, ECVI-W IVC, and ECVI-B IVC, were 0.26, 0.34, 0.44, 0.39, and 0.52, respectively (all p < 0.0001). Histopathological correlation was available in 28 patients, and rho values were 0.61, 0.60, 0.71, 0.68, and 0.76, respectively (all p < 0.001). CONCLUSION: ECVI-B IVC calculated from EqD of DECT is useful in estimating the degree of liver fibrosis.

Undifferentiated carcinoma of the pancreas with osteoclast-like giant cells showing intraductal growth and intratumoral hemorrhage: MRI features.

We report a case of undifferentiated carcinoma of the pancreas with osteoclast-like giant cells ocalized within the main pancreatic duct (MPD). A 61-year-old woman was referred to our hospital for evaluation of dilatation of the MPD that was detected on screening sonogram. Preoperative MR imaging revealed a small hypervascular tumor within the dilated MPD, showing high signal on R2* map and signal reduction on in-phase as compared to out-of-phase. R2* hyperintensity and in-phase signal reduction may be a characteristic feature of undifferentiated carcinoma of the pancreas with osteoclast-like giant cells, which indicates intratumoral hemorrhage even if they are small.

Cell-autonomous FLT3L shedding via ADAM10 mediates conventional dendritic cell development in mouse spleen.

Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.

Torsion of right lung sequestration mimicking a posterior mediastinal mass presenting as acute abdomen: Usefulness of MR imaging.

A 15-year-old boy with extralobar sequestration torsion is presented, who presented as an acute abdomen. Chest X-ray and computed tomography on admission revealed an apparent posterior mediastinal mass on the right side at the lower thoracic vertebral level. MR imaging, however, clearly showed scanty fluid around the mass and the subpleural fat layer between the vertebral body and the mass, suggesting its extrapulmonary and intrapleural cavity location. Its hemorrhagic nature was also suggested by the reduced signal on the in-phase as compared to out-of-phase chemical shift images, which helped make correct preoperative diagnosis.