Neutrophils impaired by anti-galectin-3 antibodies elicit inflammation of endothelial cells to aggregate the development of lupus cutaneous vasculitis.

OBJECTIVES: To investigate whether the interplay of anti-galectin-3 antibodies (anti-Gal3 Abs) with neutrophils contributes to the development of lupus cutaneous vasculitis. METHODS: Enzyme-linked immunosorbent assay was used to determine the serum level of anti-Gal3 Abs in lupus patients. Flow cytometry, quantitative PCR and western blot were performed to investigate the expression of cell surface receptors, proinflammatory cytokines and signalling molecules in neutrophils stimulated by serum from lupus patients or healthy controls (HCs) or anti-Gal3 Ab, respectively. Immunofluorescence was performed to visualise the formation of neutrophil extracellular traps (NETs). Human umbilical vein endothelial cells were co-cultured with the supernatants from neutrophils stimulated by anti-Gal3 Ab, and cytokine production was measured at mRNA and protein levels. Immunohistochemistry was adopted to reveal the distribution of Gal3, cytokines and myeloperoxidase within lupus skin lesions. REULTS: Serum levels of anti-Gal3 Abs were negatively correlated with peripheral counts of neutrophils. Anti-Gal3 Abs positive sera from SLE patients accelerated neutrophil death, altered cell phenotype and promoted formation of NETs with the involvement of p38 MAPK pathway. Supernatants collected from neutrophils co-cultured with anti-Gal3 Ab provoked endothelial cells to produce cytokines such as IL-1, ICAM-1, SELE and particularly IL-6. Consistently, IL-6 was higher in SLE patients with anti-Gal3 Ab positive sera and enriched in the area of vascular inflammation together with enhanced expression of Gal3 protein and infiltration of neutrophils. CONCLUSIONS: Overall, these findings suggested that neutrophils were crucial mediators in anti-Gal3 Ab induced lupus cutaneous vasculitis.

TNF-α promotes CXCL-1/8 production in keratinocytes by downregulating galectin-3 through NF-κB and hsa-miR-27a-3p pathway to contribute psoriasis development.

OBJECTIVE: Treatment with TNF-α inhibitors improve psoriasis with minimize/minor neutrophils infiltration and CXCL-1/8 expression in psoriatic lesions. However, the fine mechanism of TNF-α initiating psoriatic inflammation by tuning keratinocytes is unclear. Our previous research identified the deficiency of intracellular galectin-3 was sufficient to promote psoriasis inflammation characterized by neutrophil accumulation. This study aims to investigate whether TNF-α participated in psoriasis development through dysregulating galectin-3 expression. METHODS: mRNA levels were assessed through quantitative real-time PCR. Flow cytometry was used to detect cell cycle/apoptosis. Western blot was used to evaluate the activation of the NF-κB signaling pathway. HE staining and immunochemistry were used to detect epidermal thickness and MPO expression, respectively. Specific small interfering RNA (siRNA) was used to knock down hsa-miR-27a-3p while plasmids transfection was used to overexpress galectin-3. Further, the multiMiR R package was utilized to predict microRNA-target interaction. RESULTS AND DISCUSSION: We found that TNF-α stimulation altered cell proliferation and differentiation and promoted the production of psoriasis-related inflammatory mediators along with the inhibition of galectin-3 expression in keratinocytes. Supplement of galectin-3 could counteract the rise of CXCL-1/8 but not the other phenotypes of keratinocytes induced by TNF-α. Mechanistically, inhibition of the NF-κB signaling pathway could counteract the decrease of galectin-3 and the increase of hsa-miR-27a-3p expression whereas silence of hsa-miR-27a-3p could counteract the decrease of galectin-3 expression induced by TNF-α treatment in keratinocytes. Intradermal injection of murine anti-CXCL-2 antibody greatly alleviated imiquimod-induced psoriasis-like dermatitis. CONCLUSION: TNF-α initiates psoriatic inflammation by increasing CXCL-1/8 in keratinocytes mediated by the axis of NF-κB-hsa-miR-27a-3p-galectin-3 pathway.

16p11.2 CNV gene Doc2α functions in neurodevelopment and social behaviors through interaction with Secretagogin.

Copy-number variations (CNVs) of the human 16p11.2 genetic locus are associated with neurodevelopmental disorders, including autism spectrum disorders (ASDs) and schizophrenia. However, it remains largely unclear how this locus is involved in the disease pathogenesis. Doc2α is localized within this locus. Here, using in vivo and ex vivo electrophysiological and morphological approaches, we show that Doc2α-deficient mice have neuronal morphological abnormalities and defects in neural activity. Moreover, the Doc2α-deficient mice exhibit social and repetitive behavioral deficits. Furthermore, we demonstrate that Doc2α functions in behavioral and neural phenotypes through interaction with Secretagogin (SCGN). Finally, we demonstrate that SCGN functions in social/repetitive behaviors, glutamate release, and neuronal morphology of the mice through its Doc2α-interacting activity. Therefore, Doc2α likely contributes to neurodevelopmental disorders through its interaction with SCGN.

Topical histone deacetylase 1 inhibitor Entinostat ameliorates psoriasiform dermatitis through suppression of IL-17A response.

BACKGROUND: Biologics against IL-17A, IL-23 and TNF-α achieve a great success in treating psoriasis. However, the majority of patients still have some residual lesions left and require combination therapy to reach complete clearance. Topical medicine is an optional choice but only has limited categories. Besides, drug resistance is very often. Thus, topical medicine targeting new signaling pathway is still in an urgent need in the biologics era. OBJECTIVE: To investigate the role of topical Entinostat, a selective inhibitor of histone deacetylases 1 (HDAC1) that has been tested in clinic trials to treat solid tumors and hematological malignancies, in psoriasis therapy. METHODS: Efficacious Entinostat were tested in a mouse imiquimod (IMQ)-induced psoriasiform dermatitis (PsD) model. An in vitro model consisting of human CD4 + T cell, murine T cells and NHEKs were used to screen Entinostat for inhibition of cutaneous inflammatory genes. RESULTS: Topical application of Entinostat significantly improved psoriasiform inflammation in imiquimod-induced mice model with great reduction of IL-17A+ Î³Î´T cell infiltration in skin. Entinostat is powerful agent in inhibition of Th17 cell generation and the expression of psoriasis-related inflammatory mediators by primary keratinocytes upon CD4+ T cells stimulation. CONCLUSION: Our findings suggest Entinostat is a promising topical medicine for psoriasis treatment.

Are centenarians successful agers? Evidence from China.

BACKGROUND: It is unclear to what degree centenarians are successful agers. We assess successful aging (SA) and its subtypes in a large Chinese sample. METHODS: Based on a large national sample of 18,311 Chinese centenarians, we first estimated the prevalence of SA among centenarians, and then used the Latent Class Analysis to classify centenarians into different types based on the five dimensions of SA. Multinomial regression analysis was used to examine how demographic, socioeconomic, and lifestyle covariates are associated with these identified types. RESULTS: 5.7% of centenarians fulfilled all five criteria of SA, and 1.3% failed all five criteria. The remainder could be classified into six types of SA. The regression analysis further revealed that these SA types were related to various social factors. For example, with timely access to medical care, centenarians were three times more likely to be successful agers. CONCLUSIONS: Centenarians demonstrate substantial heterogeneity in terms of achieving five SA criteria. Social factors are found to be significantly associated with centenarians' aging performances. Specific subtypes of SA among centenarians may be associated with and help explore different underlying biological-environmental mechanisms of exceptional longevity.

5-ALA mediated photodynamic therapy with combined treatment improves anti-tumor efficacy of immunotherapy through boosting immunogenic cell death.

Photodynamic therapy (PDT) is clinically promising in destructing primary tumors and immunotherapy awakes host immunity to control distant metastases. 5-aminolevulinic acid (5-ALA), a smart photosensitizer, converts into a physiological PDT agent with no dark toxicity in vivo. In this study, we found for the first time 5-ALA-PDT induced colorectal cancer (CRC) cells death by immunogenic cell death (ICD) upon AKT inhibition. Dying cancer cells induced by 5-ALA-PDT efficiently activated bone-marrow derived dendritic cells (BMDCs). Simultaneously, autophagy was observed after AKT inhibition by 5-ALA-PDT. Besides, we found cells died more remarkable by ICD under a circumstance of low occurrence of autophagy. To evaluate the effects of 5-ALA-PDT in vivo, we applied subcutaneous tumor mouse model and delightedly found 5-ALA-PDT induced a systemic antitumor immune response to control both primary tumors and distant metastases. Meanwhile, 5-ALA-PDT enhanced Th1 immunity, leading cytotoxic T lymphocyte response, and raised tumor-specific T cells. Combining with Chloroquine (CQ), 5-ALA-PDT further augmented tumor-specific immunity effects indicating protective role of autophagy. Together, the combination therapy of 5-ALA-PDT and autophagy inhibitor synergistically led to a novel clinical approach and potential ICD-based tumor vaccine for CRC patients.

Generation of an induced pluripotent stem cell line (IPCASi001-A) from an autism spectrum disorder individual without intellectual disability.

Autism spectrum disorder (ASD) is a highly inheritable neurodevelopmental disorder that causes diverse deficits in social communication and restricted repetitive sensorimotor behaviors. Here, we studied a human-induced pluripotent cell line from an autistic patient with impaired social function and a normal intelligence quotient (IQ > 70). The cell line was validated by its morphology, gene expression, and potential to differentiate into three germ layers. This model can be used to explore the pathophysiological and molecular mechanisms in patients with ASD, compared those of with patients with normal cognitive abilities.

Scalable Preparation of Complete Stereo-Complexation Polylactic Acid Fiber and Its Hydrolysis Resistance.

Due to their high sensitivity to temperature and humidity, the applications of polylactic acid (PLA) products are limited. The stereo-complexation (SC) formed by poly(L-lactic acid) (PLLA) and its enantiomer poly(D-lactic acid) (PDLA) can effectively improve the heat resistance and hydrolysis resistance of PLA products. In this work, the blended melt-spinning process of PLLA/PDLA was carried out using a polyester fiber production line to obtain PLA fiber with a complete SC structure. The effects of high-temperature tension heat-setting on the crystalline structure, thermal properties, mechanical properties, and hydrolysis resistance were discussed. The results indicated that when the tension heat-setting temperature reached 190 °C, the fiber achieved an almost complete SC structure, and its melting point was 222.5 °C. An accelerated hydrolysis experiment in a 95 °C water bath proved that the SC crystallites had better hydrolysis resistance than homocrystallization (HC). The monofilament strength retention rate of SC-190 fiber reached as high as 78.5% after hydrolysis for 24 h, which was significantly improved compared with PLLA/PDLA drawn fiber.

Profiling Serum Cytokines and Anticytokine Antibodies in Psoriasis Patients.

Cytokines like IL-17A have been consistently found to be elevated in psoriatic lesional skin, and therapeutic antibodies to IL-17 have demonstrated efficacy in treating psoriatic skin and joint disease. However, results about the circulating cytokines in psoriasis patients remained controversial. Anticytokine autoantibodies (ACAAs) were detected in various autoimmune diseases but remained largely unknown in psoriasis. We aimed to investigate the serum levels of cytokines and ACAAs in psoriasis patients. The study included 44 biologics-naive psoriasis patients and 40 healthy controls. Serum cytokines and the corresponding autoantibodies were measured by multiplex bead-based technology. The bioactivity of serum IL-17A was determined by IL-8 production in primary keratinocytes. Herein, we found serum levels of IL-12B (median: 6.16 vs. 9.03, p = 0.0194) and Th17 cytokines (IL-17A: median: 0.32 vs. 1.05, p = 0.0026; IL-22: median: 4.41 vs. 4.41, p = 0.0120) were increased in psoriasis patients. More interestingly, bioactive IL-17A was identified in a proportion of patients and positively correlated with disease severity. A few of cytokines were closely associated with each other and formed into a distinct panel in psoriasis. Of 13 anticytokine antibodies, anti-IL-22 was moderately lower (median: 262.8 vs.190.5, p = 0.0418), and anti-IL-15 was slightly higher (median: 25.5 vs. 30.5, p = 0.0069) in psoriasis than controls. None of ACAAs was related to disease severity. Consequently, the ratios of antibodies to cytokines varied with the pattern of cytokines. In summary, our finding suggested that the levels of circulating bioactive IL-17A were associated with disease activity in psoriasis patients. In contrast, the titers of ACAAs were not significantly altered nor correlated with disease severity. However, the functionality of ACAAs remains to be further demonstrated in vitro in future studies.

Enhanced Migratory Ability of Neutrophils Toward Epidermis Contributes to the Development of Psoriasis via Crosstalk With Keratinocytes by Releasing IL-17A.

Microabscess of neutrophils in epidermis is one of the histological hallmarks of psoriasis. The axis of neutrophil-keratinocyte has been thought to play a critical role in the pathogenesis of psoriasis. However, the features and mechanism of interaction between the two cell types remain largely unknown. Herein, we found that blood neutrophils were increased in psoriasis patients, positively correlated with disease severity and highly expressed CD66b, but not CD11b and CD62L compared to healthy controls. Keratinocytes expressed high levels of psoriasis-related inflammatory mediators by direct and indirect interaction with neutrophils isolated from psoriasis patients and healthy controls. The capacity of neutrophils in provoking keratinocytes inflammatory response was comparable between the two groups and is dependent on IL-17A produced by itself. Neutrophils isolated from psoriasis patients displayed more transcriptome changes related to integrin and increased migration capacity toward keratinocytes with high CD11b expression on cell surface. Of interest, neutrophils were more susceptible to keratinocyte stimulation than to fibroblasts and human umbilical vein endothelial cells (HUVECs) in terms of CD11b expression and the production of ROS and NETs. In conclusion, neutrophils from psoriasis patients gain a strong capacity of IL-17A production and integrins expression that possibly facilitates their abilities to promote production of psoriasis-related inflammatory mediators and migration, a phenomenon likely induced by their interaction with keratinocytes but not with fibroblasts. These findings provide a proof-of-concept that development of new drugs targeting migration of neutrophils could be a more specific and safe solution to treat psoriasis.

Development and validation of a nomogram risk prediction model for malignancy in dermatomyositis patients: a retrospective study.

BACKGROUND: Dermatomyositis accompanied with malignancy is a common poor prognostic factor of dermatomyositis. Thus, the early prediction of the risk of malignancy in patients with dermatomyositis can significantly improve the prognosis of patients. However, the identification of antibodies related to malignancy in dermatomyositis patients has not been widely implemented in clinical practice. Herein, we established a predictive nomogram model for the diagnosis of dermatomyositis associated with malignancy. METHODS: We retrospectively analyzed 240 cases of dermatomyositis patients admitted to Sun Yat-sen Memorial Hospital, Sun Yat-sen University from January 2002 to December 2019. According to the year of admission, the first 70% of the patients were used to establish a training cohort, and the remaining 30% were assigned to the validation cohort. Univariate analysis was performed on all variables, and statistically relevant variables were further included in a multivariate logistic regression analysis to screen for independent predictors. Finally, a nomogram was constructed based on these independent predictors. Bootstrap repeated sampling calculation C-index was used to evaluate the model's calibration, and area under the curve (AUC) was used to evaluate the model discrimination ability. RESULTS: Multivariate logistic analysis showed that patients older than 50-year-old, dysphagia, refractory itching, and elevated creatine kinase were independent risk factors for dermatomyositis associated with malignancy, while interstitial lung disease was a protective factor. Based on this, we constructed a nomogram using the above-mentioned five factors. The C-index was 0.780 (95% CI [0.690-0.870]) in the training cohort and 0.756 (95% CI [0.618-0.893]) in the validation cohort, while the AUC value was 0.756 (95% CI [0.600-0.833]). Taken together, our nomogram showed good calibration and was effective in predicting which dermatomyositis patients were at a higher risk of developing malignant tumors.

Synaptotagmin-7-mediated activation of spontaneous NMDAR currents is disrupted in bipolar disorder susceptibility variants.

Synaptotagmin-7 (Syt7) plays direct or redundant Ca2+ sensor roles in multiple forms of vesicle exocytosis in synapses. Here, we show that Syt7 is a redundant Ca2+ sensor with Syt1/Doc2 to drive spontaneous glutamate release, which functions uniquely to activate the postsynaptic GluN2B-containing NMDARs that significantly contribute to mental illness. In mouse hippocampal neurons lacking Syt1/Doc2, Syt7 inactivation largely diminishes spontaneous release. Using 2 approaches, including measuring Ca2+ dose response and substituting extracellular Ca2+ with Sr2+, we detect that Syt7 directly triggers spontaneous release via its Ca2+ binding motif to activate GluN2B-NMDARs. Furthermore, modifying the localization of Syt7 in the active zone still allows Syt7 to drive spontaneous release, but the GluN2B-NMDAR activity is abolished. Finally, Syt7 SNPs identified in bipolar disorder patients destroy the function of Syt7 in spontaneous release in patient iPSC-derived and mouse hippocampal neurons. Therefore, Syt7 could contribute to neuropsychiatric disorders through driving spontaneous glutamate release.

miR-218-2 regulates cognitive functions in the hippocampus through complement component 3-dependent modulation of synaptic vesicle release.

microRNA-218 (miR-218) has been linked to several cognition related neurodegenerative and neuropsychiatric disorders. However, whether miR-218 plays a direct role in cognitive functions remains unknown. Here, using the miR-218 knockout (KO) mouse model and the sponge/overexpression approaches, we showed that miR-218-2 but not miR-218-1 could bidirectionally regulate the contextual and spatial memory in the mice. Furthermore, miR-218-2 deficiency induced deficits in the morphology and presynaptic neurotransmitter release in the hippocampus to impair the long term potentiation. Combining the RNA sequencing analysis and luciferase reporter assay, we identified complement component 3 (C3) as a main target gene of miR-218 in the hippocampus to regulate the presynaptic functions. Finally, we showed that restoring the C3 activity in the miR-218-2 KO mice could rescue the synaptic and learning deficits. Therefore, miR-218-2 played an important role in the cognitive functions of mice through C3, which can be a mechanism for the defective cognition of miR-218 related neuronal disorders.

Synaptotagmin-7 deficiency induces mania-like behavioral abnormalities through attenuating GluN2B activity.

Synaptotagmin-7 (Syt7) probably plays an important role in bipolar-like behavioral abnormalities in mice; however, the underlying mechanisms for this have remained elusive. Unlike antidepressants that cause mood overcorrection in bipolar depression, N-methyl-d-aspartate receptor (NMDAR)-targeted drugs show moderate clinical efficacy, for unexplained reasons. Here we identified Syt7 single nucleotide polymorphisms (SNPs) in patients with bipolar disorder and demonstrated that mice lacking Syt7 or expressing the SNPs showed GluN2B-NMDAR dysfunction, leading to antidepressant behavioral consequences and avoidance of overcorrection by NMDAR antagonists. In human induced pluripotent stem cell (iPSC)-derived and mouse hippocampal neurons, Syt7 and GluN2B-NMDARs were localized to the peripheral synaptic region, and Syt7 triggered multiple forms of glutamate release to efficiently activate the juxtaposed GluN2B-NMDARs. Thus, while Syt7 deficiency and SNPs induced GluN2B-NMDAR dysfunction in mice, patient iPSC-derived neurons showed Syt7 deficit-induced GluN2B-NMDAR hypoactivity that was rescued by Syt7 overexpression. Therefore, Syt7 deficits induced mania-like behaviors in mice by attenuating GluN2B activity, which enabled NMDAR antagonists to avoid mood overcorrection.

Synaptotagmin-7 is a key factor for bipolar-like behavioral abnormalities in mice.

The pathogenesis of bipolar disorder (BD) has remained enigmatic, largely because genetic animal models based on identified susceptible genes have often failed to show core symptoms of spontaneous mood cycling. However, pedigree and induced pluripotent stem cell (iPSC)-based analyses have implicated that dysfunction in some key signaling cascades might be crucial for the disease pathogenesis in a subpopulation of BD patients. We hypothesized that the behavioral abnormalities of patients and the comorbid metabolic abnormalities might share some identical molecular mechanism. Hence, we investigated the expression of insulin/synapse dually functioning genes in neurons derived from the iPSCs of BD patients and the behavioral phenotype of mice with these genes silenced in the hippocampus. By these means, we identified synaptotagmin-7 (Syt7) as a candidate risk factor for behavioral abnormalities. We then investigated Syt7 knockout (KO) mice and observed nocturnal manic-like and diurnal depressive-like behavioral fluctuations in a majority of these animals, analogous to the mood cycling symptoms of BD. We treated the Syt7 KO mice with clinical BD drugs including olanzapine and lithium, and found that the drug treatments could efficiently regulate the behavioral abnormalities of the Syt7 KO mice. To further verify whether Syt7 deficits existed in BD patients, we investigated the plasma samples of 20 BD patients and found that the Syt7 mRNA level was significantly attenuated in the patient plasma compared to the healthy controls. We therefore concluded that Syt7 is likely a key factor for the bipolar-like behavioral abnormalities.

Mapping cis-regulatory chromatin contacts in neural cells links neuropsychiatric disorder risk variants to target genes.

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes. We identify hundreds of thousands of long-range cis-interactions between promoters and distal promoter-interacting regions, enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several promoter-interacting regions by using clustered regularly interspaced short palindromic repeats (CRISPR) techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP and DRD2 are transcriptionally regulated by physically linked enhancers.

Author Correction: CRISPR interference-based specific and efficient gene inactivation in the brain.

In the version of this article initially published, the affiliation for Jian Zhang and Shuangli Mi was incomplete. In addition to the Key Laboratory of Genomics and Precision Medicine, they are also affiliated with the University of Chinese Academy of Sciences, Beijing, China. In Supplementary Fig. 1h,l, the molecular mass marker accompanying Snap25 was labeled 58 kDa; the correct value is 25 kDa. In Supplementary Fig. 9b,c, the top panel was labeled Syt1, with molecular mass markers ranging from 46 to 100 kDa; it is actually Snap25, with molecular mass markers ranging from 17 to 46 kDa. The errors have been corrected in the HTML and PDF versions of the article.

CRISPR interference-based specific and efficient gene inactivation in the brain.

CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency. By developing conditional CRISPRi tools targeting synaptotagmin I (Syt1), we modified the excitatory to inhibitory balance in the dentate gyrus of the mouse hippocampus and found that the dentate gyrus has distinct regulatory roles in learning and affective processes in mice. We therefore recommend CRISPRi as a useful tool for more rapid investigation of gene function in the mammalian brain.

Application of induced pluripotent stem cells to understand neurobiological basis of bipolar disorder and schizophrenia.

The etiology of neuropsychiatric disorders, such as schizophrenia and bipolar disorder, usually involves complex combinations of genetic defects/variations and environmental impacts, which hindered, for a long time, research efforts based on animal models and patients' non-neuronal cells or post-mortem tissues. However, the development of human induced pluripotent stem cell (iPSC) technology by the Yamanaka group was immediately applied to establish cell research models for neuronal disorders. Since then, techniques to achieve highly efficient differentiation of different types of neural cells following iPSC modeling have made much progress. The fast-growing iPSC and neural differentiation techniques have brought valuable insights into the pathology and neurobiology of neuropsychiatric disorders. In this article, we first review the application of iPSC technology in modeling neuronal disorders and discuss the progress in the accompanying neural differentiation. Then, we summarize the progress in iPSC-based research that has been accomplished so far regarding schizophrenia and bipolar disorder.