CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

The Prostaglandin D2 Receptor CRTH2 Contributes to Airway Hyperresponsiveness during Airway Inflammation Induced by Sensitization without an Adjuvant in Mice.

INTRODUCTION: Prostaglandin D2 (PGD2), which is produced mainly by Th2 cells and mast cells, promotes a type-2 immune response by activating Th2 cells, mast cells, eosinophils, and group 2 innate lymphoid cells (ILC2s) via its receptor, chemoattractant receptor-homologous molecules on Th2 cells (CRTH2). However, the role of CRTH2 in models of airway inflammation induced by sensitization without adjuvants, in which both IgE and mast cells may play major roles, remain unclear. METHODS: Wild-type (WT) and CRTH2-knockout (KO) mice were sensitized with ovalbumin (OVA) without an adjuvant and then challenged intranasally with OVA. Airway inflammation was assessed based on airway hyperresponsiveness (AHR), lung histology, number of leukocytes, and levels of type-2 cytokines in the bronchoalveolar lavage fluid (BALF). RESULTS: AHR was significantly reduced after OVA challenge in CRTH2 KO mice compared to WT mice. The number of eosinophils, levels of type-2 cytokines (IL-4, IL-5, and IL-13) in BALF, and IgE concentration in serum were decreased in CRTH2 KO mice compared to WT mice. However, lung histological changes were comparable between WT and CRTH2 KO mice. CONCLUSION: CRTH2 is responsible for the development of asthma responses in a mouse model of airway inflammation that features prominent involvement of both IgE and mast cells.

Hand Grip Strength Assessment Based on Sarcopenia Diagnostic Criteria Predicts Swallowing Function.

The purpose of this study is to clarify whether swallowing function can be inferred from grip strength. Based on the diagnostic criteria of sarcopenia, patients were divided into two groups according to grip strength, and it was analyzed whether there was a difference in the evaluation index for swallowing function between the two groups. Among the cases requesting evaluation of swallowing function from June 10, 2020 to October 28, 2020, 83 cases (mean age: 71.7 years, 59 males and 24 females) who received assessment tests and swallowing endoscopy were included. According to the diagnostic criteria for grip strength in the Asian working group in Sarcopenia, less than 28 kgf and 18 kgf were defined as the weak group for men and women, respectively. Hyodo scores, repeated salivary swallowing tests (RSST), maximum vocalization time (MPT), and dysphagia severity classification (DSS) were compared between the two groups. Of the 83 patients, 29 and 54 were in the normal group and weak group, respectively. In all indicators, the normal group showed significantly better results than the weak group: Hyodo score (2.4 vs. 4.0, p < 0.01), RSST (4.1 times vs. 2.4 times, p < 0.01), MPT (12.1 s vs. 5.9 s, p < 0.001), DSS (4.5 vs. 5.9, p < 0.001), respectively. In multiple regression analysis with DSS as the dependent variable, grip strength was a significant independent variable of DSS even after adjusting for age, gender, and body mass index. Grip strength assessment based on sarcopenia criteria can be a useful tool for estimating swallowing function.

Successful Treatment of a Patient with Drug-Refractory Rheumatoid Arthritis-Associated Interstitial Lung Disease with Upadacitinib: A Case Report.

Insufficient evidence exists regarding the efficacy of Janus kinase inhibitors (JAKis), a class of targeted synthetic disease-modifying anti-rheumatic drugs (tsDMARDs), in the treatment of rheumatoid arthritis (RA)-associated interstitial lung disease (ILD). Herein, we present a case of RA-ILD refractory to previous treatments that exhibited favorable response to upadacitinib. A 69-year-old man, former smoker, was diagnosed with RA-ILD based on persistent symmetric polyarthritis, elevated C-reactive protein levels and erythrocyte sedimentation rate, reduced diffusing capacity for carbon monoxide/alveolar volume (DLCO 69.9%), and bilateral ground-glass attenuation with traction bronchiectasis, predominantly in the lower lung lobe. Initial treatment with oral prednisolone and methotrexate was started; however, the patient showed worsening dyspnea, chest high-resolution computed tomography abnormalities, and decreased pulmonary function. The dose of prednisolone was increased, and methotrexate was shifted to tacrolimus; however, tacrolimus was eventually discontinued because of renal dysfunction. Subsequent treatment changes included abatacept followed by intravenous cyclophosphamide, but ILD activity continued to worsen and met the criteria of progressive pulmonary fibrosis. Approximately 4.5 years after the RA diagnosis, dyspnea, radiological abnormalities, and DLCO improved following treatment switch to upadacitinib, one of JAKis. JAKi therapy may have potential as a treatment option for refractory RA-ILD.

PGD2 /CRTH2 signaling promotes acquired immunity against bee venom by enhancing IgE production.

IgE-dependent/independent activation of mast cell (MC) has been assumed to play a host defensive role against venom injection in skin. However, its detailed mechanisms remain unknown. We aimed to investigate the contribution of MC-derived prostaglandin D2 (PGD2 )-mediated signaling in host defense against bee venom (BV). To achieve this, we utilized gene-deficient mice of a PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). We first confirmed that subcutaneous injection of BV produced PGD2 equally in wild-type (WT) and CRTH2-deficient (Crth2-/- ) mice skins. The BV injection dropped body temperature and impaired kidney equally in both lines of mice. In WT mice, pre-injection of BV (3 weeks) significantly inhibited the hypothermia and kidney impairment caused by second BV injection. In contrast, this pre-injection was not effective for the second BV injection in Crth2-/- mice. We also found that BV injections increased serum BV-specific IgE levels in WT mice, and its serum transfused mice improved the BV-induced hypothermia in naïve WT mice. In contrast, serum BV-specific IgE level was significantly lower in Crth2-/- mice. FACS analysis showed the BV injection stimulate migration of dendritic cells (DCs) into regional lymph nodes in WT mice. In Crth2-/- mice, its number was significantly smaller than that of WT mice. In conclusion, PGD2 /CRTH2 signaling plays defensive role against second BV injection. This signaling promotes BV-specific IgE production at least partially by promoting DCs migration into regional lymph node.

Otorhinolaryngologists/head and neck surgeons' knowledge, attitudes, and practices regarding fertility preservation in young cancer patients treated with chemotherapy: an anonymous questionnaire survey.

BACKGROUND: It is well known that chemotherapy for adolescent and young adult (AYA) patients with cancer can reduce fertility regardless of the regimen. A decline in fertility greatly affects the quality of life of cancer survivors in the AYA age group; however, few patients are thought to be receiving fertility preservation measures. METHODS: A questionnaire survey was conducted to assess the current understanding and consideration of fertility among otorhinolaryngologists/head and neck surgeons who treat AYA patients with cancer, and to inform them of the guidelines for fertility preservation. A total of 275 otorhinolaryngologists/head and neck surgeons working at our hospital in Ehime, Japan, six neighboring universities, and their affiliated facilities were included in this study. The questionnaire was mailed and requested to be returned by fax. Twenty questions were asked about respondents' years of experience as physicians, specialties, experience in medical care and chemotherapy for AYA patients with cancer, and knowledge and experience in fertility reduction measures. RESULTS: Although 58.7% of the physicians were aware that cryopreservation of eggs and sperm prior to chemotherapy was recommended, only 7 out of 40 physicians (17.5%) had referred AYA patients with cancer to an appropriate medical facility (department) after obtaining informed consent for chemotherapy. CONCLUSIONS: Although fertility preservation has been discussed at professional conferences and seminars, consideration and actions in the field of otorhinolaryngology/head and neck surgery have not been sufficient. We hope that the results of this survey will help raise awareness of fertility preservation.

An autopsy case report of neuronal intermediate filament inclusion disease presenting with predominantly upper motor neuron features.

We describe an autopsy case of neuronal intermediate filament inclusion disease (NIFID), a subtype of frontotemporal lobar degeneration (FTLD) with the appearance of fused-in-sarcoma (FUS) inclusions (FTLD-FUS). A 57-year-old man developed dysarthria and dysphagia. One year and five months later, he was admitted to a hospital, and pseudobulbar palsy and right upper motor neuron signs were observed on examination. Needle electromyography revealed no active or chronic denervation. His neurological symptoms gradually deteriorated, and behavioral alterations occurred. He died of hemoperitoneum secondary to rupture of a ureteric tumor. The total duration of the disease was six years and 10 months. Neuropathologically, the frontal cortex, including the motor cortex, and the pyramidal tract were severely affected, whereas the lower motor neurons in the spinal cord and brainstem were mildly damaged. The striatum and substantia nigra were also severely damaged. Hyaline conglomerate inclusions, neuronal cytoplasmic inclusions with a distinct eosinophilic core (so-called cherry spot), Pick body-like inclusions, and eosinophilic round inclusions were observed in the remaining neurons. Immunohistochemical examination revealed that these inclusions were immunoreactive for FUS. HC inclusions were also immunoreactive for α-internexin and phosphorylated neurofilament protein. FUS-immunoreactive NCIs were abundant in the basal ganglia but not in the hippocampus, in contrast to previously reported NIFID cases. Furthermore, Bunina bodies identified by immunohistochemistry for cystatin C were also observed in the lower motor neurons. Bunina bodies may be present in NIFID. This case confirms the pathological heterogeneity of NIFID and supports the notion of the difference between amyotrophic lateral sclerosis and NIFID.

Classification of grazing-incidence small-angle X-ray scattering patterns by convolutional neural network.

Grazing-incidence small-angle X-ray scattering (GISAXS) patterns have multiple superimposed contributions from the shape of the nanoscale structure, the coupling between the particles, the partial pair correlation, and the layer geometry. Therefore, it is not easy to identify the model manually from the huge amounts of combinations. The convolutional neural network (CNN), which is one of the artificial neural networks, can find regularities to classify patterns from large amounts of combinations. CNN was applied to classify GISAXS patterns, focusing on the shape of the nanoparticles. The network found regularities from the GISAXS patterns and showed a success rate of about 90% for the classification. This method can efficiently classify a large amount of experimental GISAXS patterns according to a set of model shapes and their combinations.

Epithelial cell-derived prostaglandin D2 inhibits chronic allergic lung inflammation in mice.

The precise role of prostaglandin D (PGD)2 in allergic lung inflammation remains controversial. Here, we aimed to clarify the role of PGD2 in chronic allergic lung inflammation using hematopoietic PGD synthase (H-PGDS)-deficient mice. Repeated intranasal administration of ovalbumin (OVA) resulted in eosinophilic infiltration and mucin production in the lungs of wild type (WT) mice, leading to respiratory dysfunction. H-PGDS deficiency exacerbated these effects, which were accompanied by increased mRNA expression of TNF-α and eosinophil chemoattractants. The bronchial epithelium expressed both H-PGDS and TNF-α in the inflamed WT lung, and H-PGDS deficiency increased TNF-α expression further. In cultured bronchial tissue of WT mice, treatment with LPS elevated mRNA expression of TNF-α and eosinophil chemoattractants. H-PGDS deficiency promoted the expression of these factors, which was inhibited by treatment with PGD2 receptor, D prostanoid (DP) receptor agonist, or PGD2 metabolite 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2). Treatment with TNF-α receptor antibody inhibited eosinophil chemoattractant expression. In vivo, administration of DP agonist or 15d-PGJ2 inhibited OVA-induced allergic lung inflammation. Bronchial epithelial cell-derived PGD2 attenuated lung eosinophilic infiltration with chronic allergic inflammation; these phenomena are at least partly attributed to the inhibition of TNF-α production via DP activation or 15-deoxy-Δ12,14-PGJ2 signaling.-Maehara, T., Nakamura, T., Maeda, S., Aritake, K., Nakamura, M., Murata, T. Epithelial cell-derived prostaglandin D2 inhibits chronic allergic lung inflammation in mice.

Deficiency of CRTH2, a Prostaglandin D2 Receptor, Aggravates Bleomycin-induced Pulmonary Inflammation and Fibrosis.

Chemoattractant receptor homologous with T-helper cell type 2 cells (CRTH2), a receptor for prostaglandin D2, is preferentially expressed on T-helper cell type 2 lymphocytes, group 2 innate lymphoid cells, eosinophils, and basophils, and elicits the production of type 2 cytokines, including profibrotic IL-13. We hypothesized that lack of CRTH2 might protect against fibrotic lung disease, and we tested this hypothesis using a bleomycin-induced lung inflammation and fibrosis model in CRTH2-deficient (CRTH2-/-) or wild-type BALB/c mice. Compared with wild-type mice, CRTH2-/- mice treated with bleomycin exhibited significantly higher mortality, enhanced accumulation of inflammatory cells 14-21 days after bleomycin injection, reduced pulmonary compliance, and increased levels of collagen and total protein in the lungs. These phenotypes were associated with decreased levels of IFN-γ, IL-6, IL-10, and IL-17A in BAL fluid. Adoptive transfer of splenocytes from wild-type, but not CRTH2-/-, mice 2 days before injection of bleomycin resolved the sustained inflammation as well as the increased collagen and protein accumulation in the lungs of CRTH2-/- mice. We consider that the disease model is driven by γδT cells that express CRTH2; thus, the adoptive transfer of γδT cells could ameliorate bleomycin-induced alveolar inflammation and fibrosis.

[Radiation Exposure in Computed Tomography Localizer Radiograph].

This research measured the radiation exposure of the computed tomography(CT) localizer radiograph of the trunk of the body. The entrance surface dose for CT localizer radiograph was measured using radiophotoluminescent glass dosimeter(RPLD) on four points of measurement, including the center of the phantom, on the surface of a phantom placed in the center of a CT bed, assuming that the subject has a thickness of 20 cm. The entrance surface dose of the localizer radiograph under the chest CT protocol manufacturer's initial setting conditions of 120 kV 35 mA was 0.80 mGy at the center and 0.53 for the 4-location average for the upper X-ray tube (excluding the CT bed), and 0.74 mGy at the center and 0.48 mGy for the 4-location average for the lower X-ray tube (including the CT bed). Compared to the Japan DRLs 2015 chest X-ray (P→A), the entrance surface dose was 2.67 times at the center and 1.77 times for the 4-location average for the upper X-ray tube and 2.47 times at the center and 1.60 times for the 4-location average for the lower X-ray tube. The CT radiation dose also cannot be ignored for the localizer radiograph entrance surface dose.

Loss of Tbx1 induces bone phenotypes similar to cleidocranial dysplasia.

T-box transcription factor, TBX1, is the major candidate gene for 22q11.2 deletion syndrome (DiGeorge/ Velo-cardio-facial syndrome) characterized by facial defects, thymus hypoplasia, cardiovascular anomalies and cleft palates. Here, we report that the loss of Tbx1 in mouse (Tbx1(-/-)) results in skeletal abnormalities similar to those of cleidocranial dysplasia (CCD) in humans, which is an autosomal-dominant skeletal disease caused by mutations in RUNX2. Tbx1(-/-) mice display short stature, absence of hyoid bone, failed closure of fontanelle, bifid xiphoid process and hypoplasia of clavicle and zygomatic arch. A cell-type-specific deletion of Tbx1 in osteochondro-progenitor (Tbx1(OPKO)) or mesodermal (Tbx1(MKO)) lineage partially recapitulates the Tbx1(-/-) bone phenotypes. Although Tbx1 expression has not been previously reported in neural crest, inactivation of Tbx1 in the neural crest lineage (Tbx1(NCKO)) leads to an absence of the body of hyoid bone and postnatal lethality, indicating an unanticipated role of Tbx1 in neural crest development. Indeed, Tbx1 is expressed in the neural crest-derived hyoid bone primordium, in addition to mesoderm-derived osteochondral progenitors. Ablation of Tbx1 affected Runx2 expression in calvarial bones and overexpression of Tbx1 induced Runx2 expression in vitro. Taken together, our current studies reveal that Tbx1 is required for mesoderm- and neural crest-derived osteoblast differentiation and normal skeletal development. TBX1 mutation could lead to CCD-like bone phenotypes in human.

Prostaglandin D2 acts through the Dp2 receptor to influence male germ cell differentiation in the foetal mouse testis.

Through intercellular signalling, the somatic compartment of the foetal testis is able to program primordial germ cells to undergo spermatogenesis. Fibroblast growth factor 9 and several members of the transforming growth factor ß superfamily are involved in this process in the foetal testis, counteracting the induction of meiosis by retinoic acid and activating germinal mitotic arrest. Here, using in vitro and in vivo approaches, we show that prostaglandin D2 (PGD2), which is produced through both L-Pgds and H-Pgds enzymatic activities in the somatic and germ cell compartments of the foetal testis, plays a role in mitotic arrest in male germ cells by activating the expression and nuclear localization of the CDK inhibitor p21(Cip1) and by repressing pluripotency markers. We show that PGD2 acts through its Dp2 receptor, at least in part through direct effects in germ cells, and contributes to the proper differentiation of male germ cells through the upregulation of the master gene Nanos2. Our data identify PGD2 signalling as an early pathway that acts in both paracrine and autocrine manners, and contributes to the differentiation of germ cells in the foetal testis.

Rapidly progressive psychotic symptoms triggered by infection in a patient with methylenetetrahydrofolate reductase deficiency: a case report.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn error of metabolism inherited in autosomal recessive pattern and is associated with a wide spectrum of neurological abnormalities. CASE PRESENTATION: We herein describe a 15-year-old boy with MTHFR deficiency who presented with a slowly progressive decline of school performance and a spastic gait. Rapidly deteriorating psychosis and repetitive seizures triggered by a febrile infection prompted neurological investigation. He had significantly elevated total plasma homocysteine and urinary homocystine levels, as well as a decreased plasma methionine level. Brain magnetic resonance imaging (MRI) revealed leukoencephalopathy. DNA gene sequencing showed c.446_447 del GC ins TT and c.137G > A, and c.665C > T heterozygous mutations in the MTHFR gene of the patient. Oral administration of betaine drastically improved his clinical symptoms within a few months. After 8 months of treatment, his total plasma homocysteine level moderately decreased; and the plasma methionine concentration became normalized. Furthermore, the white matter lesions on MRI had disappeared. CONCLUSION: This patient demonstrates the possibility that MTHFR deficiency should be considered in mentally retarded adolescents who display an abnormally elevated plasma level of homocysteine in association with progressive neurological dysfunction and leukoencephalopathy. Febrile infections may be an aggravating factor in patients with MTHFR deficiency.

A genome-wide analysis identifies a notch-RBP-Jκ-IL-7Rα axis that controls IL-17-producing γδ T cell homeostasis in mice.

Notch signaling is an important regulator for the development and function of both αß and γδ T cells, whereas roles of Notch signaling in T cell maintenance remain unclear. We reported previously that the Notch-Hes1 pathway was involved in the intrathymic development of naturally occurring IL-17-producing (IL-17(+)) γδ T cells. To gain insight into additional roles for the Notch axis in the homeostasis of γδ T cells, we performed a genome-wide analysis of Notch target genes and identified the novel promoter site of IL-7Rα driven by the Notch-RBP-Jκ pathway. Constitutive Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro, whereas conditional deletion of RBP-Jκ abrogated IL-7Rα expression, but not Hes1 expression, by γδ T cells and selectively reduced the pool size of IL-7Rα(high) IL-17(+) γδ T cells in the periphery. In the absence of IL-7Rα-mediated signaling, IL-17(+) γδ T cells were barely maintained in adult mice. Addition of exogenous IL-7 in vitro selectively expanded IL-17(+) γδ T cells. Thus, our results revealed a novel role for the Notch-RBP-Jκ-IL-7Rα axis that is independent of Hes1 for homeostasis of IL-17(+) γδ T cells.

Krüppel-like factor 2 represses transcription of the telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) in human T cells.

In normal human T cells, telomerase activity is strictly regulated. T cells are thought to express telomerase to avoid replicative senescence, unlike most normal somatic cells with definite replicative lifespan. T cells in blood and tissues are usually in a state of quiescence without expression of the limiting catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). In contrast to activation, repression of hTERT transcription has not been studied well. Our previous studies have found an hTERT promoter element with repressive function. Here we identified KLF2, which represses hTERT transcription by binding to the putative promoter element. KLF2 and hTERT exhibited reciprocal mRNA expression patterns in primary human T cells. In activated T cells, KLF2 binding to the hTERT promoter was eliminated, relieving the repression of hTERT transcription found in resting T cells. Our results suggest that KLF2 is involved in strict repression of hTERT expression through binding to the promoter in primary human T cells.

A deficiency in the prostaglandin D2 receptor CRTH2 exacerbates adjuvant-induced joint inflammation.

Although the cyclooxygenase metabolites PGs are known to be involved in the progression of arthritis, the role of PGD2 remains unclear. In this study, we evaluated the contribution of signaling mediated through a PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), in the progression of adjuvant-induced joint inflammation. Injection of CFA into the ankle joint stimulated PGD2 production and induced paw swelling in both CRTH2-naive (WT) and CRTH2(-/-) mice. CRTH2(-/-) mice presented more severe arthritic manifestations than did WT mice. Through bone marrow transplantation experiments between WT and CRTH2(-/-) mice, we showed that CRTH2 deficiency in bone marrow-derived immune cells is involved in disease progression. Morphological studies showed that CRTH2 deficiency accelerated the infiltration of macrophages into the inflamed paw. Consistent with this finding, we observed that treatment with the macrophage inactivator GdCl3 or the macrophage-depleting agent liposomal clodronate improved arthritis symptoms in CRTH2(-/-) mice. Adoptive transfer of CRTH2(-/-) macrophages exacerbated joint inflammation in WT mice. In addition, CRTH2 deficiency accelerated, whereas CRTH2 agonism inhibited, the expression of a macrophage-activating cytokine (GM-CSF) and a chemokine receptor (CXCR2) in CFA-treated peritoneal macrophages. Together, these observations demonstrate that PGD2-CRTH2 signaling plays a protective role in joint inflammation by attenuating the infiltration of macrophages.

IFN-γ-producing and IL-17-producing γδ T cells differentiate at distinct developmental stages in murine fetal thymus.

γδ T cells develop at the double-negative (DN) 2 and DN3 stages and acquire functions to produce IL-17 and IFN-γ in fetal thymus. However, the relationship between differentiation stages and their functions was unclear. In this study, we found that, although IFN-γ-producing and IL-17-producing γδ T cells developed from DN2 cells, only IFN-γ-producing γδ T cells developed from DN3 cells, indicating the direct generation of IL-17-producing γδ T cells from the DN2 stage, not through the DN3 stage. Single-cell analysis revealed that DN2 cells contained heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Inactivation of B cell leukemia/lymphoma 11b, a zinc finger transcription factor responsible for transition from early to late stages of DN2 cells, completely abrogated the development of IL-17-producing γδ T cells, although a unique subset of IFN-γ-producing γδ T cells expressing a high level of promyelocytic leukemia zinc finger was able to develop. Thus, our results reveal that γδ T cells are functionally differentiated to IFN-γ and IL-17 producers at different developmental stages in fetal thymus.

Opposing immunomodulatory roles of prostaglandin D2 during the progression of skin inflammation.

The effects of PGD2 are extremely context dependent. It can have pro- or anti-inflammatory effects in clinically important pathological conditions. A greater mechanistic insight into the determinants of PGD2 activity during inflammation is thus required. In this study, we investigated the role of PGD2 in croton oil-induced dermatitis using transgenic (TG) mice overexpressing hematopoietic PGD synthase. Administration of croton oil caused tissue swelling and vascular leakage in the mouse ear. Compared with wild-type animals, TG mice produced more PGD2 and showed decreased inflammation in the early phase, but more severe manifestations during the late phase. Data obtained from bone marrow transplantation between wild-type and TG mice indicated that PGD2 produced by tissue resident cells in the TG mice attenuated early-phase inflammation, whereas PGD2 produced from hematopoietic lineage cells exacerbated late-phase inflammation. There are two distinct PGD2 receptors: D-prostanoid receptor (DP) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). In TG mice, treatment with a DP antagonist exacerbated inflammation in the early phase, whereas treatment with a CRTH2 antagonist attenuated inflammation during the late phase. In vitro experiments showed that DP agonism enhanced vascular endothelial barrier formation, whereas CRTH2 agonism stimulated neutrophil migration. Collectively, these results show that when hematopoietic PGD synthase is overexpressed, tissue resident cell-derived PGD2 suppresses skin inflammation via DP in the early phase, but hematopoietic lineage cell-derived PGD2 stimulates CRTH2 and promotes inflammation during the late phase. DP-mediated vascular barrier enhancement or CRTH2-mediated neutrophil activation may be responsible for these effects. Thus, PGD2 represents opposite roles in inflammation, depending on the disease phase in vivo.