Expression Of C3 Research Articles

Complement C3a receptor inactivation attenuates retinal degeneration induced by oxidative damage

Retinal degeneration causes vision loss and threatens the health of elderly individuals worldwide. Evidence indicates that the activation of the complement system is associated with retinal degeneration. However, the mechanism of complement signaling in retinal degeneration needs to be further studied. In this study, we show that the expression of C3 and C3a receptor (C3ar1) is positively associated with the inflammatory response and retinal degeneration. Genetic deletion of C3 and pharmacological inhibition of C3ar1 resulted in the alleviation of neuroinflammation, prevention of photoreceptor cell apoptosis and restoration of visual function. RNA sequencing (RNA-seq) identified a C3ar1-dependent network shown to regulate microglial activation and astrocyte gliosis formation. Mechanistically, we found that STAT3 functioned downstream of the C3-C3ar1 pathway and that the C3ar1-STAT3 pathway functionally mediated the immune response and photoreceptor cell degeneration in response to oxidative stress. These findings reveal an important role of C3ar1 in oxidative-induced retinal degeneration and suggest that intervention of the C3ar1 pathway may alleviate retinal degeneration.

Psoriatic Dermal-Derived Mesenchymal Stem Cells Induced C3 Expression in Keratinocytes.

PurposeOur recent studies found a splice region mutation in C3 accompanied by a significantly increased C3 in psoriatic peripheral blood. Mesenchymal stem cells (MSCs) are a key immunological suppression cell. We further investigate the regulation of MSCs on C3 in psoriasis.Patients and MethodsWe analyzed the C3 and its upstream S100A9, S100A8 and downstream MCP1 in psoriatic and control skin, and in normal human epidermal keratinocytes (NHEKs) co-cultured with psoriatic versus control dermal-derived mesenchymal stem cells (DMSCs) by mRNA, iTRAQ (isobaric tags for relative and absolute quantitative) and simple Western analysis.ResultsThe mRNA and Simple Western analysis showed that the expression of C3, S100A8 and S100A9 are upregulated in psoriatic lesion (C3: mRNA, 9.23-fold, p = 0.0092; protein, 3.56-fold, p = 0.0244. S100A8: mRNA, 28.35-fold, p = 0.0015; protein, 4.68-fold, p = 0.0215. S100A9: mRNA, 79.45-fold, p = 0.0066; protein, 12.42-fold, p > 0.05). Moreover, the iTRAQ showed that C3 and S100A9 were significantly increased in NHEKs after co-cultured with psoriatic DMSCs compared to that of control DMSCs (C3: 3.40-fold, p = 0, FDR = 0; S100A9: 2.30-fold, p = 9.86E-241, FDR = 6.50E-239), verified by Simple Western. However, the expression of S100A8 and MCP1 was slightly different between the two groups.ConclusionOur results suggest that psoriatic DMSCs contribute to the increased C3 expression in psoriatic lesion via upregulating S100A9, providing the theoretical basis for the role of C3 and DMSCs in the pathogenesis of psoriasis.

Divergent complement system activation in two clinically distinct murine models of multiple sclerosis.

Multiple sclerosis (MS) is a neurological disease featuring neuroinflammation and neurodegeneration in young adults. So far, most research has focused on the peripheral immune system, which appears to be the driver of acute relapses. Concurrently, the mechanisms underlying neurodegeneration in the progressive forms of the disease remain unclear. The complement system, a molecular component of the innate immunity, has been recently implicated in several neurological disorders, including MS. However, it is still unknown if the complement proteins detected in the central nervous system (CNS) are actively involved in perpetuating chronic inflammation and neurodegeneration. To address this knowledge gap, we compared two clinically distinct mouse models of MS: 1) proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (rEAE) resembling a relapsing-remitting disease course, and 2) Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) resembling a progressive disease. Real-time PCR was performed in the spinal cord of rEAE mice, TMEV-IDD mice and age-matched sham controls to quantify gene expression for a broad range of complement components. In both experimental models, we found significantly increased expression of complement factors, such as C1q, C3, CfB, and C3aR. We showed that the complement system, specifically the classical complement pathway, was associated with TMEV-IDD pathogenesis, as the expression of C1q, C3 and C3aR1 were all significantly correlated to a worse disease outcome (all P≤0.0168). In line with this finding, C1q and C3 deposition was observed in the spinal cord of TMEV-IDD mice. Furthermore, C1q deposition was detected in spinal cord regions characterized by inflammation, demyelination, and axonal damage. Conversely, activation of the classical complement cascade seemed to result in protection from rEAE (C1q: P=0.0307). Interestingly, the alternative pathway related to a worse disease outcome in rEAE (CFb: P=0.0006). Overall, these results indicate potential divergent roles for the complement system in MS. The chronic-progressive disease form is more reliant on the activation of the classic complement pathway, while protecting from acute relapses. Conversely, relapsing MS appears more likely affected by the alternative pathway. Understanding the functions of the complement system in MS is critical and can lead to better, more targeted therapies in the future.

Complement factor B inhibitor LNP023 improves lupus nephritis in MRL/lpr mice

BackgroundLupus nephritis (LN) is the most common complication of systemic lupus erythematosus (SLE), and the abnormal activation of the alternative complement pathway is associated with the pathogenesis of LN. As an inhibitor of complement factor B (CFB) in the alternative pathway, LNP023 has been used in the treatment of a variety of renal diseases with abnormal complement system involvement, such as paroxysmal nocturnal hemoglobinuria, IgA nephropathy, and membranous nephropathy. The aim of our study was to explore whether LNP023 improved LN in MRL/lpr mice by inhibiting the activation of the alternative complement pathway. MethodsThe mice were divided into a normal control group (Normal group) (n = 6), MRL/lpr model group (n = 6), and LNP023 group (n = 6). The LNP023 group was administered LNP023 for 2 weeks by gavage; the MRL/lpr model group was administered saline for 2 weeks by gavage; and the Normal group was administered saline for 2 weeks by gavage. External signs, renal pathology, renal function, renal immune complex and complement deposition, serum anti-dsDNA, serum ANA concentration, and the expression of core complement factors in the alternative complement pathway were analyzed in the 3 groups of animals. The core targets of LNP023 in the treatment of LN were screened using network pharmacology. The pathogenicity of the core targets in LN was verified by analyzing the mRNA expression of the core targets in the peripheral blood mononuclear cells (PBMCs) of normal individuals, SLE patients, and LN patients. The mRNA and protein expression of core targets in the Normal group, MRL/lpr group, and LNP023 group were analyzed to verify whether LNP023 exerted it LN therapeutic effect through the regulation of core targets. ResultsCompared with the MRL/lpr group, the LNP023 group had reduced lupus-like signs, improved renal function, decreased serum anti-dsDNA and ANA concentrations, and reduced renal IgM, IgG, IgG1, C1q, C3, and C4 deposition. Renal pathology showed that LNP023 attenuated pathological damage in the kidneys of MRL/lpr mice. Compared with the MRL/lpr model group, the treatment group had no crescent formation, less immune deposition, no nuclear fragmentation, and less inflammatory cell infiltration. The expression of complement proteins C3, C3b, CR1, CFB, and C5b-9 in kidney tissues and liver was decreased, and the expression of C5 was increased. Network pharmacology screening indicated that AKT, TNF-α, MDM2, UBC, STST3, ESR1, and TP53 were core targets of LNP023 in the treatment of LN. Compared with that in the Normal group, the mRNA expression of the core target in the SLE and LN groups was different; compared with the MRL/lpr group, the LNP023 treatment group showed different mRNA and protein expression levels of AKT, TNF-α, and STST3. ConclusionLNP023 improves LN in MRL/lpr mice. The mechanism is as follows: LNP023 binds to CFB to inhibit the activation of the alternative complement pathway. LNP023 treatment for LN may also play a role in regulating the protein expression of AKT, TNF-α, and STST3.

Effects of dietary chrysophyte (Poterioochromonas malhamensis) rich in beta-glucan on the growth performance, intestinal health, lipid metabolism, immune gene expression, and disease resistance against Aeromonas salmonicida in juvenile rainbow trout (Oncorhynchus mykiss)

An eight-week growth trial was conducted to investigate the effects of different dietary levels (0%, 2%, 4%, and 8%) of chrysophyte (Poterioochromonas malhamensis) rich in water-soluble beta-glucan on the growth performance, intestinal health, lipid metabolism, immune responses, and disease resistance of juvenile rainbow trout (Oncorhynchus mykiss). At the end of the feeding trial, no significant difference in weight gain was observed between the non-supplemented control and moderate doses (2% and 4%), but decreased weight gain was found at the highest dose (8%) of P. malhamensis compared with the control group. The survival rate in each group was above 97%, with no significant differences between groups. P. malhamensis supplementation in diets changed bacterial diversity and altered the composition of intestinal microbes. In particular, the abundance of the beneficial bacteria Lactobacillus was increased with moderate doses, and the abundance of the pathogenic bacteria Acinetobacter was enhanced with the highest dose (8%) of P. malhamensis. The mRNA expression levels of tight junction protein genes (e.g., claudin-1, occludin, and tricellulin) were also increased after P. malhamensis supplementation. Meanwhile, P. malhamensis supplementation altered lipid metabolism, including reduced crude lipid content in whole fish and decreased CHO, TG, HDL, and LDL in the serum of the highest dose group. Several free fatty acids, including C14:0, C16:1, and C18:3n3, were also reduced in the liver in the highest dose group. In contrast, P. malhamensis induced mRNA expression of the immune-related genes c3, c4, and c5 and the upregulation of mRNA expression in pro-inflammatory cytokines (IL1β and IL8) in the highest dose group. After the 8-week feeding trial, fish were intraperitoneally injected with a bacterial pathogen, Aeromonas salmonicida. The survival rate increased by 14% in the moderate dose groups and decreased by 11% in the highest dose group compared with the control. In addition, the mRNA expression levels of cytokines and complement system genes were differentially influenced by the challenge test, with an increase of c7 in the moderate dose groups, a decrease of c3 in the highest dose group, a reduction of IL1β in the moderate dose groups, and an upregulation of the anti-inflammatory cytokine IL10 in the moderate dose groups. In conclusion, the dietary inclusion of P. malhamensis at optimum levels (2–4%) efficiently regulated lipid metabolism, improved intestinal health, heightened immune responses, and increased resistance to A. salmonicida in juvenile rainbow trout. P. malhamensis could be a good source of beta-glucan for fish.

Abstract 1320: Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer

Abstract Background: The use of immune PD-1/-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced non-small cell lung cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified certain genomic subsets of NSCLC as drivers of primary resistance to ICI treatment. Methods: We evaluated NSCLC patients treated at Roswell Park Comprehensive Cancer Center from 2017-2020 with successful genomic and immune profiling using a CLIA-certified laboratory developed test that included targeted next-generation genomic sequencing, PD-L1 assessment by immunohistochemistry (IHC), and targeted RNA-seq of 394 immune transcripts. Results: A total of 379 treatment-naive non-squamous NSCLC subjects were identified with 113 subjects treated with an FDA approved ICI regimen and evaluable for response. Using a gene expression signature based on differential gene expression analysis in the 113 subjects with and without disease control with ICI treatment, we performed unsupervised clustering of the larger 379 subject cohort and identified a responder (R) cluster and non-responder (NR) cluster. The R cluster was associated with high PD-L1 expression, T-cell infiltration, and KRAS mutations. The NR cluster was associated with negative PD-L1 expression, lack of T-cell infiltration, and mutations in STK11, APC/CTNNB1, and NFE2L2. Of the mutation subsets, STK11 was most associated with NR with a trend towards lower PD-L1 expression by clinical IHC, but much more significantly associated by gene expression signatures with low PD-L1 and T-cell inflammation. In our gene panel, we identified elevated TRIM29 expression in STK11 mutant NSCLC tumors and confirmed TRIM29 expression by IHC on a separate 64 subject cohort with known STK11 mutation status and archival formalin fixed paraffin embedded (FFPE) tissue. A subset of STK11 mutant NSCLC cell lines also demonstrated elevated TRIM29 expression and we generated knockouts of TRIM29 with CRISPR-Cas9 in these cell lines which resulted in decreased tumor growth in xenograft models. Using Nanostring nCounter multiplex inflammation gene expression panel on both the 64 patient FFPE cohort and TRIM29 knockout cell lines, we identified the innate immune complement pathway as a target of therapeutic interest and specifically the high expression of complement component C3 by TRIM29 high tumor cells. We then generated a syngeneic mouse model where loss of Stk11 resulted in elevated expression of Trim29 and C3 for preclinical studies to evaluate the therapeutic modulation of the complement pathway on response to ICI. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immune environment of mutational subsets of NSCLC and identified the complement pathway as a potential therapeutic target in STK11 mutant NSCLC. Citation Format: Edwin H. Yau, Bojidar Kandar, Lei Deng, Te-An Chen, Wiam Bshara, Sean Glenn, Sarabjot Pabla, Antonios Papanicolau-Sengos, Mary Nesline, Hongbin Chen, Amy Early, Carl Morrison, Grace Dy. Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1320.

Effect of rottlerin on astrocyte phenotype polarization after trimethyltin insult in the dentate gyrus of mice

BackgroundIt has been demonstrated that reactive astrocytes can be polarized into pro-inflammatory A1 phenotype or anti-inflammatory A2 phenotype under neurotoxic and neurodegenerative conditions. Microglia have been suggested to play a critical role in astrocyte phenotype polarization by releasing pro- and anti-inflammatory mediators. In this study, we examined whether trimethyltin (TMT) insult can induce astrocyte polarization in the dentate gyrus of mice, and whether protein kinase Cδ (PKCδ) plays a role in TMT-induced astrocyte phenotype polarization.MethodsMale C57BL/6 N mice received TMT (2.6 mg/kg, i.p.), and temporal changes in the mRNA expression of A1 and A2 phenotype markers were evaluated in the hippocampus. In addition, temporal and spatial changes in the protein expression of C3, S100A10, Iba-1, and p-PKCδ were examined in the dentate gyrus. Rottlerin (5 mg/kg, i.p. × 5 at 12-h intervals) was administered 3–5 days after TMT treatment, and the expression of A1 and A2 transcripts, p-PKCδ, Iba-1, C3, S100A10, and C1q was evaluated 6 days after TMT treatment.ResultsTMT treatment significantly increased the mRNA expression of A1 and A2 phenotype markers, and the increased expression of A1 markers remained longer than that of A2 markers. The immunoreactivity of the representative A1 phenotype marker, C3 and A2 phenotype marker, S100A10 peaked 6 days after TMT insult in the dentate gyrus. While C3 was expressed evenly throughout the dentate gyrus, S100A10 was highly expressed in the hilus and inner molecular layer. In addition, TMT insult induced microglial p-PKCδ expression. Treatment with rottlerin, a PKCδ inhibitor, decreased Iba-1 and C3 expression, but did not affect S100A10 expression, suggesting that PKCδ inhibition attenuates microglial activation and A1 astrocyte phenotype polarization. Consistently, rottlerin significantly reduced the expression of C1q and tumor necrosis factor-α (TNFα), which has been suggested to be released by activated microglia and induce A1 astrocyte polarization.ConclusionWe demonstrated the temporal and spatial profiles of astrocyte polarization after TMT insult in the dentate gyrus of mice. Taken together, our results suggest that PKCδ plays a role in inducing A1 astrocyte polarization by promoting microglial activation and consequently increasing the expression of pro-inflammatory mediators after TMT insult.

Gypenoside XVII, an Active Ingredient from Gynostemma Pentaphyllum, Inhibits C3aR-Associated Synaptic Pruning in Stressed Mice.

Gynostemma pentaphyllum is a herbal medicine widely used in Asian countries, and its saponin extracts have been shown to possess potent anti-inflammatory effects. Gypenoside XVII, an active ingredient isolated from Gynostemma pentaphyllum, has been found to alleviate the inflammation induced by LPS in the BV2 microglia, according to our preliminary study. This study aims to evaluate whether Gypenoside XVII could attenuate depression-like symptoms in vivo and tries to demonstrate the involvement of the complement regulation in its antidepressant-like effect. The results showed that Gypenoside XVII significantly attenuated depression-like behaviors in the forced swimming test, tail suspension test and sucrose preference test. It also alleviated the acute stress-induced hyperactivity of serum corticosterone levels. Additionally, Gypenoside XVII significantly inhibited the activation of microglia and the expression of C3 in mice exposed to chronic unpredictable mild stress (CUMS). Meanwhile, the activation of C3aR/STAT3 signaling and the expression of proinflammatory cytokines was reversed by Gypenoside XVII. Moreover, CUMS induced excessive synaptic pruning by activating microglia, while Gypenoside XVII restored it in the prefrontal cortex. Our data demonstrated that Gypenoside XVII, the active ingredient of Gynostemma pentaphyllum, produced the antidepressant-like effects in mice, which was mediated by the inhibition of complement C3/C3aR/STAT3/cytokine signaling in the prefrontal cortex.

The SNPs of three immunity genes in striped catfish, Pangasianodon hypophthalmus are associated with resistance against infection of Aeromonas hydrophila

Three important immunity-related genes, NLRC3 (nucleotide-oligomerization domain (NOD)-like receptor subfamily C), IL4R (interleukin 4 receptor), and C4 (complement component 4), can respond to infection by Aeromonas hydrophila in the spleen of striped catfish, Pangasianodon hypophthalmus. However, their association with resistance against infection by A. hydrophila are unknown. In this study, the expression of NLRC3, IL4R, and C4 in heart, liver, spleen, head kidney, trunk kidney, and gill of striped catfish after A. hydrophila infection was determined using quantitative real-time PCR. Single nucleotide polymorphism (SNP) identification and genotyping were performed by Sanger sequencing. We then analyzed the correlation between these polymorphisms and resistance to A. hydrophila infection. NLRC3, IL4R, and C4 were strongly induced by A. hydrophila infection in the liver, spleen, trunk kidney, and gill that peaked in the liver at 12 h post-infection. Three SNPs from NLRC3, two SNPs from IL4R, and two SNPs from C4 were identified by direct sequencing, in which NLRC3-CM018541.1-359661A/T, NLRC3-CM018541.1-359672G/T, NLRC3-CM018541.1-359815G/C, IL4R-CM018563.1-17094599G > A, and C4-CM018554.1-20401796A > T were associated with the incidence of resistant and susceptible phenotypes in striped catfish. Additionally, three SNP loci (NLRC3-CM018541.1-359661A/T, NLRC3-CM018541.1-359672G/T, and NLRC3-CM018541.1-359815G/C) showed strong pairwise linkage disequilibrium, and the haplotype AGC generated with these three SNPs was associated with resistance to A. hydrophila in striped catfish. These findings suggest strongly a relationship between the NLRC3, IL4R, C4 polymorphisms and A. hydrophila resistance in striped catfish, and that the NLRC3 gene could be used as a candidate marker for genetic selection of A. hydrophila-resistant striped catfish.

Altered Complement System Activity in Schizophrenia: Overexpression of C4 and/or Abnormal Expression of Complement Control Proteins in the DLPFC, Parietal Cortex, Temporal Cortex, Associative Striatum, Hippocampus, Cerebellum and Whole Blood

IntroductionIn schizophrenia, abnormal synaptic pruning during adolescence may be due to an altered Complement system activity. While this hypothesis is supported by C4 overexpression in various brain regions of individuals with schizophrenia, such alterations should be replicated and extended to other brain regions relevant to schizophrenia. Moreover, transcriptional studies of genes coding for proteins regulating the Complement system activity are lacking. Furthermore, it remains unknown whether cerebral and peripheral expression of C4 and Complement control proteins (CCP) are related.ObjectivesTo identify altered expression of C4 and CCP (CSMD1, CSMD2, CD46) coding genes at the cerebral and peripheral levels in schizophrenic individuals.MethodsWe explored C4 and CCP coding genes expression at the cerebral and peripheral levels. Using shinyGEO application we analyzed gene expression from eight Gene Expression Omnibus datasets obtained from 196 schizophrenic individuals and 182 control subjects. First, we compared gene expression between schizophrenic patients and controls in postmortem cerebral samples from 7 different brain regions. Then, we compared gene expression between schizophrenic patients and controls in 4 peripheral tissues.ResultsWe observed C4 overexpression in the DLPFC, parietal, temporal cortex and associative striatum of schizophrenic individuals. We report altered transcriptional patterns of CCP genes in the DLPFC, hippocampus and cerebellum of schizophrenic individuals. CD46 expression was altered in opposite directions between brain and blood of schizophrenic individuals. No significant alteration of C4 expression was observed in peripheral tissues.ConclusionsOur results support the hypothesis of an altered Complement system activity in various brain regions of schizophrenic individuals which may disrupt the synaptic pruning process during adolescence.DisclosureNo significant relationships.

Human Melanoma-Associated Mast Cells Display a Distinct Transcriptional Signature Characterized by an Upregulation of the Complement Component 3 That Correlates With Poor Prognosis.

Cutaneous melanoma is one of the most aggressive human malignancies and shows increasing incidence. Mast cells (MCs), long-lived tissue-resident cells that are particularly abundant in human skin where they regulate both innate and adaptive immunity, are associated with melanoma stroma (MAMCs). Thus, MAMCs could impact melanoma development, progression, and metastasis by secreting proteases, pro-angiogenic factors, and both pro-inflammatory and immuno-inhibitory mediators. To interrogate the as-yet poorly characterized role of human MAMCs, we have purified MCs from melanoma skin biopsies and performed RNA-seq analysis. Here, we demonstrate that MAMCs display a unique transcriptome signature defined by the downregulation of the FcεRI signaling pathway, a distinct expression pattern of proteases and pro-angiogenic factors, and a profound upregulation of complement component C3. Furthermore, in melanoma tissue, we observe a significantly increased number of C3+ MCs in stage IV melanoma. Moreover, in patients, C3 expression significantly correlates with the MC-specific marker TPSAB1, and the high expression of both markers is linked with poorer melanoma survival. In vitro, we show that melanoma cell supernatants and tumor microenvironment (TME) mediators such as TGF-β, IL-33, and IL-1β induce some of the changes found in MAMCs and significantly modulate C3 expression and activity in MCs. Taken together, these data suggest that melanoma-secreted cytokines such as TGF-β and IL-1β contribute to the melanoma microenvironment by upregulating C3 expression in MAMCs, thus inducing an MC phenotype switch that negatively impacts melanoma prognosis.

Characterization of the Estrogen Response Helps to Predict Prognosis and Identify Potential Therapeutic Targets in Cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive malignancy originating from the epithelium of the bile duct. The prognosis of patients is poor regardless of radical resection and chemoradiotherapy. The current classification and prognostic model of CCA are unable to satisfy the requirements for predicting the clinical outcome and exploring therapeutic targets. Estrogen signaling is involved in diverse cancer types, and it has long been established that CCA could be regulated by estrogen. In our study, estrogen response was identified to be significantly and stably correlated with poor prognosis in CCA. Employing several algorithms, CCA was classified into ES cluster A and B. ES cluster B was mainly composed of patients with fluke infection and overlapped with CCA cluster 1/2, and ES cluster A was mainly composed of patients without fluke infection and overlapped with CCA cluster 3/4. COMT and HSD17B1 were identified to be responsible for the differential estrogen response between ES clusters A and B, and the estrogen response may be correlated with the differentiation and cancer stemness of CCA at the single-cell level. Complement activation and the expression of C3 and C5, which are mainly expressed by CCA cells, were significantly downregulated in ES cluster B. An estrogen response risk score (ESRS) model was constructed to predict the prognosis of CCA, followed by a nomogram integrating ESRS and clinical features. Finally, altered pathways, applicable drugs and sensitivity to chemical drugs were analyzed specific to the estrogen response. In summary, our results provide insights into the role of the estrogen response in CCA progression as well as applicable drugs and potential therapeutic targets in estrogen metabolism, the complement system and ESRS-related pathways.

Molecular signature of immunological mechanism behind impaired endometrial receptivity in polycystic ovarian syndrome.

Despite the treatment of anovulation, infertility is still one of the main complications in PCOS women during reproductive age, which appears to be mainly due to impaired uterine receptivity. This study investigated the transcriptome profiles of endometrium in PCOS patients and healthy fertile individuals as the control group. Total mRNA was extracted from endometrial tissues of PCOS patients (n = 12) and healthy fertile individuals (n = 10) during the luteal phase. After cDNA synthesis, PCR array was performed using Human Female Infertility RT2 Profiler PCR Array kit (Qiagen, Cat.No: PAHS-164Z) for evaluating expression of 84 genes contributing to the female infertility. PCR Array data analysis identified significantly greater expression of CSF, IL11, IL15, IL1r1, IL1b, TNF, LIF, TNFRSF10B, TGFβ, C3, ITGA4 (Cd49d), SPP1, and Calca in PCOS women than in controls (P < 0.05). However, the expression of LIFR, C2, CD55, CFD, CALCA, LAM1, LAMC2, MMP2, MMP7, MMP9, ESR, SELL, ITGB3, and VCAM1 was significantly lower in PCOS group than in controls (P < 0.05). The results revealed dysregulation of immune-inflammatory molecules, complement activation and downregulation of IGF-I as well as adhesion molecules in PCOS group. The findings of this study indicated some potential causes of reduced receptivity of endometrium thus compromising the fertility in PCOS patients.

Integrated Pharmacology Reveals the Molecular Mechanism of Gegen Qinlian Decoction Against Lipopolysaccharide-induced Acute Lung Injury.

ALI is a severe inflammatory disease of the lungs. In previous studies, we found that GQD was effective against ALI, but specific molecular mechanism is still unclear. Therefore, this study was to examine effect of GQD on LPS-induced ALI rats and underlying mechanisms using multi-omics and molecular methods. The results showed that GQD significantly improved lung tissue damage, reduced pulmonary edema, inhibited MPO activity, and improved respiratory function in ALI rat. Additionally, GQD significantly reduced the levels of TNF-α, IL-1β, and IL-6 in serum and BALF. Furthermore, metabolomic analysis showed that GQD reduced pulmonary inflammation by improving metabolic remodeling. Moreover, transcriptomic analysis showed that GQD inhibited the activation of complement pathway and regulated Th17 and Treg cells balance. Additionally, GQD inhibited the expression of C3, C5a, and IL-17, and promoted the expression of TGF-β and CYP1A1 at the mRNA and protein levels. Gut microbial assay showed that GQD treatment increased the relative abundance of Firmicutes and their genera in intestinal microbiota, and increased short-chain fatty acids concentration. Overall, GQD treated ALI by improving metabolic remodeling, affecting immune-related pathways and regulating intestinal microbiota. This study provides a solid scientific basis for promoting the clinical use of GQD in treating ALI.

A Neuron, Microglia, and Astrocyte Triple Co-culture Model to Study Alzheimer's Disease.

Glial cells are essential to understand Alzheimer’s disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-β1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aβ-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.

Single-Cell Transcriptome Analysis Reveals the Role of Pancreatic Secretome in COVID-19 Associated Multi-organ Dysfunctions.

The SARS-CoV-2 infection affects the lungs, heart, kidney, intestine, olfactory epithelia, liver, and pancreas and brings forward multi-organ dysfunctions (MODs). However, mechanistic details of SARS-CoV-2-induced MODs are unclear. Here, we have investigated the role of pancreatic secretory proteins to mechanistically link COVID-19 with MODs using single-cell transcriptome analysis. Secretory proteins were identified using the Human Protein Atlas. Gene ontology, pathway, and disease enrichment analyses were used to highlight the role of upregulated pancreatic secretory proteins (secretome). We show that SARS-CoV-2 infection shifts the expression profile of pancreatic endocrine cells to acinar and ductal cell-specific profiles, resulting in increased expression of acinar and ductal cell-specific genes. Among all the secretory proteins, the upregulated expression of IL1B, AGT, ALB, SPP1, CRP, SERPINA1, C3, TFRC, TNFSF10, and MIF was mainly associated with disease of diverse organs. Extensive literature and experimental evidence are used to validate the association of the upregulated pancreatic secretome with the coagulation cascade, complement activation, renin-angiotensinogen system dysregulation, endothelial cell injury and thrombosis, immune system dysregulation, and fibrosis. Our finding suggests the influence of an upregulated secretome on multi-organ systems such as nervous, cardiovascular, immune, digestive, and urogenital systems. Our study provides evidence that an upregulated pancreatic secretome is a possible cause of SARS-CoV-2-induced MODs. This finding may have a significant impact on the clinical setting regarding the prevention of SARS-CoV-2-induced MODs.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s12539-022-00513-3.

Effects of electroacupuncture combined with hydrogel on the formation and changes in the glial scar in rats with spinal cord injury

To observe the effect of electroacupuncture (EA) combined with oriented conductive bioprotein hydrogel (OCBH) on the recovery of nerve function in rats with complete spinal cord injury (SCI) and to explore its effect and mechanism on the formation and changes of glial scars. A total of 72 female Sprague–Dawley rats were randomly divided into groups according to the treatment received. A rat model of complete SCI was constructed using a spinal cord transection. Behavioral assessments, hematoxylin-eosin (H&E) staining, immunofluorescence staining, and Western blotting were performed at a fixed period after the operation. The material group and the material + EA group obtained better results in the behavioral assessments (all P < .05) and the H&E staining. In the immunofluorescence staining and Western blotting, the GFAP protein was expressed more and denser in the material group and the material + EA group than in the model group, and the density of the GFAP expression in the material + EA group was lower at week 12 than in the material group (all P < .05). The expression of complement C3 in the model, material, and material + EA groups decreased in turn. Some inflammatory factors and the NF-κB signaling pathway showed similar results in the Western blotting (all P < .05). The expression of the GDNF protein in the material + EA group was significantly higher than that in the model group and the material group (both P < .01). EA combined with OCBH can promote the recovery of motor functions after SCI by facilitating the formation of glial scars in the early stage, preventing the further spread of an inflammatory response that would affect the activation of A1/A2 astrocytes and change the morphology of glial scars at the spinal cord–material interface in its late stage.

Therapeutic Small Interfering RNA Targeting Complement C3 in a Mouse Model of C3 Glomerulopathy.

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.

Early Immune Modulation in European Seabass (Dicentrarchus labrax) Juveniles in Response to Betanodavirus Infection

The early host–pathogen interaction between European seabass (Dicentrarchus labrax) and Betanodavirus was examined by using juvenile fish infected intramuscularly with RGNNV (red-spotted grouper nervous necrosis virus). The time course selected for sampling (0–144 h post-infection (hpi)) covered the early stages of infection, with hematological, antioxidant and immunological responses examined. Early activation of the host’s immune system was seen in the first few hours post-infection (6 to 9 hpi), as evidenced by an increase in tnfα, cd28 and c3 expression in the head kidney of infected fish. Most hematological parameters that were examined showed significant differences between sampling times, including differences in the number of thrombocytes and various leukocyte populations. The plasma lysozyme concentration decreased significantly over the course of the trial, and most antioxidant parameters examined in the liver showed significant differences over the infection period. At 144 hpi, peak expression of tnfα and il-1β coincided with the appearance of disease symptoms, peak levels of virus in the brain and high levels of fish mortality. The results of the study show the importance of analyzing the early interactions between European seabass and Betanodavirus to establish early indicators of infection to prevent more severe outcomes of the infection from occurring.

Three Different Types of β-Glucans Enhance Cognition: The Role of the Gut-Brain Axis.

BackgroundDietary fiber is fermented in the lower gastrointestinal tract, potentially impacting the microbial ecosystem and thus may improve elements of cognition and brain function via the gut-brain axis. β-glucans, soluble dietary fiber, have different macrostructures and may exhibit different effects on the gut-brain axis. This study aimed to compare the effects of β-glucans from mushroom, curdlan and oats bran, representing β-(1,3)/(1,6)-glucan, β-(1,3)-glucan or β-(1,3)/(1,4)-glucan, on cognition and the gut-brain axis.MethodsC57BL/6J mice were fed with either control diet or diets supplemented with β-glucans from mushroom, curdlan and oats bran for 15 weeks. The cognitive functions were evaluated by using the temporal order memory and Y-maze tests. The parameters of the gut-brain axis were examined, including the synaptic proteins and ultrastructure and microglia status in the hippocampus and prefrontal cortex (PFC), as well as colonic immune response and mucus thickness and gut microbiota profiles.ResultsAll three supplementations with β-glucans enhanced the temporal order recognition memory. Brain-derived neurotrophic factor (BDNF) and the post-synaptic protein 95 (PSD95) increased in the PFC. Furthermore, mushroom β-glucan significantly increased the post-synaptic thickness of synaptic ultrastructure in the PFC whilst the other two β-glucans had no significant effect. Three β-glucan supplementations decreased the microglia number in the PFC and hippocampus, and affected complement C3 and cytokines expression differentially. In the colon, every β-glucan supplementation increased the number of CD206 positive cells and promoted the expression of IL-10 and reduced IL-6 and TNF-α expression. The correlation analysis highlights that degree of cognitive behavior improved by β-glucan supplementations was significantly associated with microglia status in the hippocampus and PFC and the number of colonic M2 macrophages. In addition, only β-glucan from oat bran altered gut microbiota and enhanced intestinal mucus.ConclusionsWe firstly demonstrated long-term supplementation of β-glucans enhanced recognition memory. Comparing the effects of β-glucans on the gut-brain axis, we found that β-glucans with different molecular structures exhibit differentia actions on synapses, inflammation in the brain and gut, and gut microbiota. This study may shed light on how to select appropriate β-glucans as supplementation for the prevention of cognitive deficit or improving immune function clinically.