A role of Achaete-scute complex homolog 2 in T follicular regulatory cell development.

T follicular regulatory (Tfr) cells, a subset of CD4+ Foxp3+ regulatory T (Treg) cells, locate to the lymphoid follicle and germinal center (GC) and regulate antibody responses. Tfr cells express the functional molecules of follicular helper T (Tfh) cells, including CXCR5 and Bcl6. CD25- mature Tfr cells differentiate from CD25+ Treg cells through CD25+ immature Tfr cells. Others and we have shown that Achaete-scute complex homolog 2 (Ascl2) plays a role in Tfh cell development; however, the role of Ascl2 in the development of Tfr cells remains unclear. Here, we found that Ascl2 was highly and preferentially expressed in CD25+ Tfr cells and CD25- Tfr cells, and that the differentiation from CD25+ Tfr cells to CD25- Tfr cells was impaired by the absence of Ascl2. Furthermore, the forced Ascl2 expression in Treg cells downregulated CD25 expression and suppressed IL-2-induced phosphorylation of STAT5, which is known to suppress CD25- Tfr cell development. Finally, we found that the downregulation of CD25 by Ascl2 in Treg cells is independent of Bach2, which also regulates CD25 downregulation in CD25+ Tfr cells. These results suggest that Ascl2 plays a vital role in developing Tfr cells, possibly by downregulating CD25 expression in a Bach2-independent mechanism.

Visceral disseminated varicella zoster virus infection during non-intensive maintenance therapy in a patient with systemic lupus erythematosus.

Visceral disseminated varicella zoster virus infection (VD-VZV) is a rare complication in immunocompromised patients. Although systemic lupus erythematosus (SLE) patients have a higher risk of VZV infection, only a few reports describe VD-VZV in SLE. Here, we report a 48-year-old woman with SLE who had received maintenance therapy. She was transferred to the hospital because of severe epigastric pain. There were no significant abnormalities in abdominal computed tomography and upper gastrointestinal endoscopy. On hospital day 4, she developed vesicular eruption on her face and abdomen. VZV antigen was detected in specimens obtained from skin lesions, and treatment with acyclovir was started. VZV DNA in blood turned out to be positive, and the epigastric pain was thought to be caused by VD-VZV. There is a risk of VD-VZV in patients with SLE, even in those receiving non-intensive maintenance therapy.

NF-κB1 Contributes to Imiquimod-Induced Psoriasis-Like Skin Inflammation by Inducing Vγ4+Vδ4+γδT17 Cells.

Recent studies have identified NF-κB1 as a new disease susceptibility gene for psoriasis. Although accumulating evidence has shown the importance of NF-κB signaling in various cell types in the pathogenesis of psoriasis, it remains unclear how NF-κB1 contributes to the pathogenesis of psoriasis. In this study, we examined psoriasis-like skin diseases induced by topical administration of imiquimod in Nf-κb1‒deficient (Nf-κb1-/-) mice and littermate wild-type (WT) mice. Compared with WT mice, Nf-κb1-/- mice exhibited attenuated skin inflammation. The numbers of Vγ4+Vδ4+γδT17 cells, which cause skin inflammation in this model, were significantly reduced in the skin and draining lymph nodes in imiquimod-treated Nf-κb1-/- mice. Nf-κb1 is preferentially phosphorylated in Vγ4+Vδ4+γδT17 cells in WT mice. In vitro proliferation of Vγ4+Vδ4+γδT17 cells but not conventional CD4+ T cells was significantly impaired in Nf-κb1-/- mice compared with that in WT mice. RNA-sequencing analyses revealed that the expression of E2 factor target genes was decreased in Vγ4+Vδ4+γδT cells by the absence of NF-κB1. Consistently, the cell cycle progression of Vγ4+Vδ4+γδT cells was reduced in Nf-κb1-/- mice compared with that in WT mice. These results suggest that Nf-κb1 plays a crucial role in the pathogenesis of imiquimod-induced psoriasis-like skin inflammation by promoting the proliferation of Vγ4+Vδ4+γδT17 cells.

Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model.

OBJECTIVE: Recently, podocytes have been recognised not only as a physical barrier to prevent urinary protein loss but also as producers of proinflammatory cytokines. However, the roles of podocytes in the pathogenesis of lupus nephritis (LN) remain largely unknown. This study aims to determine the roles of suppressor of cytokine signalling (SOCS) family members expressed in glomeruli in the regulation of LN. METHODS: We investigated the expression of SOCS family members in glomeruli in murine lupus model induced by repeated epicutaneous administration of the TLR7/8 agonist imiquimod. We also investigated the roles of SOCS3 expressed in podocytes in the imiquimod-induced glomerulonephritis and systemic autoimmunity by using podocyte-specific SOCS3-deficient mice (podocin-Cre x SOCS3fl/fl mice (SOCS3-cKO mice)). Finally, we investigated the expression of proinflammatory cytokines and chemokines in SOCS3-deficient podocyte cell lines. RESULTS: qPCR analysis revealed that among SOCS family members, SOCS3 was preferentially induced in glomeruli on epicutaneous administration of imiquimod and that interleukin 6 (IL-6) induced SOCS3 expression in podocyte cell lines. SOCS3-cKO mice exhibited severe glomerulonephritis, high levels of serum creatinine and urine albumin and decreased survival rate compared with control SOCS3-WT mice. Levels of anti-double-strand DNA antibody, SOCS (GC) formation and the numbers of follicular helper T (Tfh) cells and GC B cells in the spleen were higher in SOCS3-cKO mice than those in SOCS3-WT mice. Serum IL-6 levels and expression of IL-6 mRNA in glomeruli were also elevated in SOCS3-cKO mice. IL-6-induced IL-6 expression was enhanced in SOCS3-deficient podocyte cell lines compared with that in SOCS3-sufficient podocyte cell lines. CONCLUSION: SOCS3 expressed in podocytes plays protective roles for the development of glomerulonephritis and inhibits autoantibody production in the imiquimod-induced lupus model presumably by suppressing IL-6 production of podocytes.

Dual optical recordings for action potentials and calcium handling in induced pluripotent stem cell models of cardiac arrhythmias using genetically encoded fluorescent indicators.

Reprogramming of human somatic cells to pluripotency has been used to investigate disease mechanisms and to identify potential therapeutics. However, the methods used for reprogramming, in vitro differentiation, and phenotyping are still complicated, expensive, and time-consuming. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free induced pluripotent stem cells (iPSCs) from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from patients with Timothy syndrome into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared with the controls. The results are consistent with previous reports using a retroviral method for reprogramming and an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording the action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and that these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to investigate mechanisms underlying cardiac arrhythmias and to test potential therapeutics.

Chronic Stimulation of Alpha-2A-Adrenoceptors With Guanfacine Protects Rodent Prefrontal Cortex Dendritic Spines and Cognition From the Effects of Chronic Stress.

The prefrontal cortex (PFC) provides top-down regulation of behavior, cognition, and emotion, including spatial working memory. However, these PFC abilities are greatly impaired by exposure to acute or chronic stress. Chronic stress exposure in rats induces atrophy of PFC dendrites and spines that correlates with working memory impairment. As similar PFC grey matter loss appears to occur in mental illness, the mechanisms underlying these changes need to be better understood. Acute stress exposure impairs PFC cognition by activating feedforward cAMP-calcium-K+ channel signaling, which weakens synaptic inputs and reduces PFC neuronal firing. Spine loss with chronic stress has been shown to involve calcium-protein kinase C signaling, but it is not known if inhibiting cAMP signaling would similarly prevent the atrophy induced by repeated stress. The current study examined whether inhibiting cAMP signaling through alpha-2A-adrenoceptor stimulation with chronic guanfacine treatment would protect PFC spines and working memory performance during chronic stress exposure. Guanfacine was selected due to 1) its established effects on cAMP signaling at post-synaptic alpha-2A receptors on spines in PFC, and 2) its increasing clinical use for the treatment of pediatric stress disorders. Daily guanfacine treatment compared to vehicle control was found to prevent dendritic spine loss in layer II/III pyramidal neurons of prelimbic PFC in rats exposed to chronic restraint stress. Guanfacine also protected working memory performance; cognitive performance correlated with dendritic spine density. These findings suggest that chronic guanfacine use may have clinical utility by protecting PFC gray matter from the detrimental effects of stress.

eIF2alpha Phosphorylation-dependent translation in CA1 pyramidal cells impairs hippocampal memory consolidation without affecting general translation.

Protein synthesis inhibitor antibiotics are widely used to produce amnesia, and have been recognized to inhibit general or global mRNA translation in the basic translational machinery. For instance, anisomycin interferes with protein synthesis by inhibiting peptidyl transferase or the 80S ribosomal function. Therefore, de novo general or global protein synthesis has been thought to be necessary for long-term memory formation. However, it is unclear which mode of translation-gene-specific translation or general/global translation-is actually crucial for the memory consolidation process in mammalian brains. Here, we generated a conditional transgenic mouse strain in which double-strand RNA-dependent protein kinase (PKR)-mediated phosphorylation of eIF2alpha, a key translation initiation protein, was specifically increased in hippocampal CA1 pyramidal cells by the chemical inducer AP20187. Administration of AP20187 significantly increased activating transcription factor 4 (ATF4) translation and concomitantly suppressed CREB-dependent pathways in CA1 cells; this led to impaired hippocampal late-phase LTP and memory consolidation, with no obvious reduction in general translation. Conversely, inhibition of general translation by low-dose anisomycin failed to block hippocampal-dependent memory consolidation. Together, these results indicated that CA1-restricted genetic manipulation of particular mRNA translations is sufficient to impair the consolidation and that consolidation of memories in CA1 pyramidal cells through eIF2alpha dephosphorylation depends more on transcription/translation of particular genes than on overall levels of general translation. The present study sheds light on the critical importance of gene-specific translations for hippocampal memory consolidation.