Infliximab inhibits TNF-α-dependent activation of the NLRP3/IL-1ß pathway in acne inversa.

Background: Acne inversa (AI) is a refractory inflammatory skin disease, and TNF-α plays an important role in the pathogenesis of AI. By blocking TNF-α, infliximab (IFX) has been proven to be a promising method. Objectives: To explore the underlying mechanisms of IFX treatment in AI patients. Methods: In this research, we integrated transcriptome sequencing data from the samples of our patients with AI and the GEO database. Ex vivo skin culture of AI patients was conducted to evaluate the efficacy of IFX treatment. Animal studies and cell experiments were used to explore the therapeutic effect and mechanism of IFX treatment. Results: Both TNF-α and NLRP3 inflammasome-related pathways were enriched in skin lesions of AI patients and murine AI models. After IFX treatment, the NLRP3 inflammasome-related pathway was effectively blocked, and the IL-1ß level was normalized in ex vivo AI skin explants and murine AI models. Mechanistically, IFX suppressed the NF-κB signaling pathway to lower the expression of NLRP3 and IL-1ß in keratinocytes. Conclusions: IFX treatment alleviated skin lesions in murine AI models and downregulated NLRP3 and IL-1ß expression levels by inhibiting the NF-κB signaling pathway, which was helpful for understanding the mechanism of IFX therapy.

Metallofullerenol alleviates alcoholic liver damage via ROS clearance under static magnetic and electric fields.

Alcoholic liver disease (ALD) is a common chronic redox disease caused by increased alcohol consumption. Abstinence is a major challenge for people with alcohol dependence, and approved drugs have limited efficacy. Therefore, this study aimed to explore a new treatment strategy for ALD using ferroferric oxide endohedral fullerenol (Fe3O4@C60(OH)n) in combination with static magnetic and electric fields (sBE). The primary hepatocytes of 8-9-week-old female BALB/c mice were used to evaluate the efficacy of the proposed combination treatment. A mouse chronic binge ethanol feeding model was established to determine the alleviatory effect of Fe3O4@C60(OH)n on liver injury under sBE exposure. Furthermore, the ability of Fe3O4@C60(OH)n to eliminate •OH was evaluated. Alcohol-induced hepatocyte and mitochondrial damage were reversed in vitro. Additionally, the combination therapy reduced liver damage, alleviated oxidative stress by improving antioxidant levels, and effectively inhibited liver lipid accumulation in animal experiments. Here, we used a combination of magnetic derivatives of fullerenol and sBE to further improve the ROS clearance rate, thereby alleviating ALD. The developed combination treatment may effectively improve alcohol-induced liver damage and maintain redox balance without apparent toxicity, thereby enhancing therapy aimed at ALD and other redox diseases.

Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy.

The CRISPR-Cas9 system offers substantial potential for cancer therapy by enabling precise manipulation of key genes involved in tumorigenesis and immune response. Despite its promise, the system faces critical challenges, including the preservation of cell viability post-editing and ensuring safe in vivo delivery. To address these issues, this study develops an in vivo CRISPR-Cas9 system targeting tumor-associated macrophages (TAMs). We employ bacterial protoplast-derived nanovesicles (NVs) modified with pH-responsive PEG-conjugated phospholipid derivatives and galactosamine-conjugated phospholipid derivatives tailored for TAM targeting. Utilizing plasmid-transformed E. coli protoplasts as production platforms, we successfully load NVs with two key components: a Cas9-sgRNA ribonucleoprotein targeting Pik3cg, a pivotal molecular switch of macrophage polarization, and bacterial CpG-rich DNA fragments, acting as potent TLR9 ligands. This NV-based, self-assembly approach shows promise for scalable clinical production. Our strategy remodels the tumor microenvironment by stabilizing an M1-like phenotype in TAMs, thus inhibiting tumor growth in female mice. This in vivo CRISPR-Cas9 technology opens avenues for cancer immunotherapy, overcoming challenges related to cell viability and safe, precise in vivo delivery.

Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.

The sustained release of profibrotic cytokines, mainly transforming growth factor-ß (TGF-ß), leads to the occurrence of kidney fibrosis and chronic kidney disease (CKD). Connective tissue growth factor (CTGF) appears to be an alternative target to TGF-ß for antifibrotic therapy in CKD. In this study, we found that long noncoding RNA AI662270 was significantly increased in various renal fibrosis models. In vivo, ectopic expression of AI662270 alone was sufficient to activate interstitial fibroblasts and drive kidney fibrosis, whereas inhibition of AI662270 blocked the activation of interstitial fibroblasts and ameliorated kidney fibrosis in various murine models. Mechanistic studies revealed that overexpression of AI662270 significantly increased CTGF product, which was required for the role of AI662270 in driving kidney fibrosis. Furthermore, AI662270 binds to the CTGF promoter and directly interacts with METTL3, the methyltransferase of RNA N6 -methyladenosine (m6 A) modification. Functionally, AI662270-mediated recruitment of METTL3 increased the m6 A methylation of CTGF mRNA and consequently enhanced CTGF mRNA stability. In conclusion, our results support that AI662270 promotes CTGF expression at the posttranscriptional stage by recruiting METTL3 to the CTGF promoter and depositing m6 A modifications on the nascent mRNA, thereby, uncovering a novel regulatory mechanism of CTGF in the pathogenesis of kidney fibrosis.

3,3'-Diindolylmethane improves antitumor immune responses of PD-1 blockade via inhibiting myeloid-derived suppressor cells.

BACKGROUND: Immune checkpoint inhibitors that target programmed cell death protein 1 (PD-1) have obtained encouraging results, but a fraction of tumor patients failed to respond to anti-PD-1 treatment due to the existence of multiple immune suppressive elements such as myeloid-derived suppressor cells (MDSCs). Traditional Chinese medicine or natural products from medicinal plants could enhance immunity and may be helpful for cancer immunotherapy. As a digestive metabolite from cruciferous plants, 3,3'-diindolylmethane (DIM) has been widely used in chemotherapy, but its influence on cancer immunotherapy remains unclear. Here we investigate the function of DIM on MDSCs and examine the therapeutic effects of DIM in conjunction with PD-1 antibody against mouse tumors. METHODS: Flow cytometry analysis, Western blot analysis and qRT-PCR assay were used to examine the inhibitory effects and mechanisms of DIM on MDSCs in vitro and in vivo. The therapeutic effects of DIM on cancer immunotherapy by PD-1 antibody were evaluated in mouse models of breast cancer and melanoma tumor. RESULTS: DIM exerted the inhibitory effect on MDSCs via downregulating miR-21 level and subsequently activating PTEN/PIAS3-STAT3 pathways. Adoptive transfer of MDSCs impaired the therapeutic effects of DIM, indicating that the antitumor activity of DIM might be due to the suppression of MDSCs. Furthermore, in mouse models of breast cancer and melanoma tumor, the addition of DIM can enhance the therapeutic effect of PD-1 antibody through promoting T cells responses, and thereby inhibiting tumor growth. CONCLUSIONS: Overall, the strategy based on the combination treatment of anti-PD-1 antibody and DIM may provide a new approach for cancer immunotherapy. Cruciferae plants-rich diet which contains high amount of DIM precursor may be beneficial for cancer patients that undergo the anti-PD-1 treatment.

In Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System.

Introduction: Tumor extracellular vesicles (EVs) and their relevance to various processes of tumor growth have been vigorously investigated over the past decade. However, obtaining direct evidence of spontaneous EV transfer in vivo remains challenging. In our previous study, a single-guide RNA (sgRNA): Cas9 ribonucleoprotein complex, which can efficiently delete target genes, was delivered into recipient cells using an engineered EV. Aim: Applying this newly discovered exosomal bio-cargo to track the uptake and distribution of tumor EVs. Methods: Tumor cells of interest were engineered to express and release the sgRNA:Cas9 complex, and a reporter cell/system containing STOP-fluorescent protein (FP) elements was also generated. EV-delivered Cas9 proteins from donor cells were programmed by a pair of sgRNAs to completely delete a blockade sequence and, in turn, recuperated the expression of FP in recipient reporter cells. Thus, fluorescently illuminated cells indicate the uptake of EVs. To improve the efficiency and sensitivity of this tracking system in vivo, we optimized the sgRNA design, which could more efficiently trigger the expression of reporter proteins. Results: We demonstrated the EV-mediated crosstalk between tumor cells, and between tumor cells and normal cells in vitro. In vivo, we showed that intravenously administered EVs can be taken up by the liver. Moreover, we showed that EVs derived from melanoma xenografts in vivo preferentially target the brain and liver. This distribution resembles the manifestation of organotrophic metastasis of melanoma. Conclusion: This study provides an alternative tool to study the distribution and uptake of tumor EVs.

Correlation of the detection rate of upper GI cancer with artificial intelligence score: results from a multicenter trial (with video).

BACKGROUND AND AIMS: The quality of EGD is a prerequisite for a high detection rate of upper GI lesions, especially early gastric cancer. Our previous study showed that an artificial intelligence system, named intelligent detection endoscopic assistant (IDEA), could help to monitor blind spots and provide an operation score during EGD. Here, we verified the effectiveness of IDEA to help evaluate the quality of EGD in a large-scale multicenter trial. METHODS: Patients undergoing EGD in 12 hospitals were consecutively enrolled. All hospitals were equipped with IDEA developed using deep convolutional neural networks and long short-term memory. Patients were examined by EGD, and the results were recorded by IDEA. The primary outcome was the detection rate of upper GI cancer. Secondary outcomes were part scores, total scores, and endoscopic procedure time, which were analyzed by IDEA. RESULTS: A total of 17,787 patients were recruited. The total detection rate of cancer-positive cases was 1.50%, ranging from .60% to 3.94% in each hospital. The total detection rate of early cancer-positive cases was .36%, ranging from .00% to 1.58% in each hospital. The average total score analyzed by IDEA ranged from 64.87 ± 16.87 to 83.50 ± 9.57 in each hospital. The cancer detection rate in each hospital was positively correlated with total score (r = .775, P = .003). Similarly, the early cancer detection rate was positively correlated with total score (r = .756, P = .004). CONCLUSIONS: This multicenter trial confirmed that the quality of the EGD result is positively correlated with the detection rate of cancer, which can be monitored by IDEA. (Clinical trial registration number: ChiCTR2000029001.).

Tubule-derived INHBB promotes interstitial fibroblast activation and renal fibrosis.

Upstream stimuli for myofibroblast activation are of considerable interest for understanding the mechanisms underlying renal fibrosis. Activin B, a member of the TGF-ß family, exists as a homodimer of inhibin subunit beta B (INHBB), but its role in renal fibrosis remains unknown. We found that INHBB expression was significantly increased in various renal fibrosis models and human chronic kidney disease specimens with renal fibrosis. Notably, the increase of INHBB occurred mainly in the tubular epithelial cells (TECs). In vivo, inhibiting INHBB blocked the activation of interstitial fibroblasts and ameliorated the renal fibrosis induced by unilateral ureteral obstruction or ischemia-reperfusion injury, while ectopic expression of INHBB in the TECs was able to activate interstitial fibroblasts and initiate interstitial fibrosis. In vitro, overexpression of INHBB in TECs led to the secretion of activin B, thereby promoting the proliferation and activation of interstitial fibroblasts through activin B/Smad signaling. Furthermore, inhibition of activin B/Smad signaling attenuated the fibrotic response caused by tubular INHBB. Mechanistically, the upregulation of INHBB depended on the transcription factor Sox9 in the injured TECs. Clinical analyses also identified a positive correlation between Sox9 and INHBB expression in human specimens, suggesting the Sox9/INHBB axis as a positive regulator of renal fibrosis. In conclusion, tubule-derived INHBB is implicated in the pathogenesis of renal fibrosis by activating the surrounding fibroblasts in a paracrine manner, thereby exhibiting as a potential therapeutic target. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CBFß promotes colorectal cancer progression through transcriptionally activating OPN, FAM129A, and UPP1 in a RUNX2-dependent manner.

Colorectal cancer (CRC) is commonly associated with aberrant transcription regulation, but characteristics of the dysregulated transcription factors in CRC pathogenesis remain to be elucidated. In the present study, core-binding factor ß (CBFß) is found to be significantly upregulated in human CRC tissues and correlates with poor survival rate of CRC patients. Mechanistically, CBFß is found to promote CRC cell proliferation, migration, invasion, and inhibit cell apoptosis in a RUNX2-dependent way. Transcriptome studies reveal that CBFß and RUNX2 form a transcriptional complex that activates gene expression of OPN, FAM129A, and UPP1. Furthermore, CBFß significantly promotes CRC tumor growth and live metastasis in a mouse xenograft model and a mouse liver metastasis model. In addition, tumor-suppressive miR-143/145 are found to inhibit CBFß expression by specifically targeting its 3'-UTR region. Consistently, an inverse correlation between miR-143/miR-145 and CBFß expression levels is present in CRC patients. Taken together, this study uncovers a novel regulatory role of CBFß-RUNX2 complex in the transcriptional activation of OPN, FAM129A, and UPP1 during CRC development, and may provide important insights into CRC pathogenesis.

HG-9-91-01 Attenuates Murine Experimental Colitis by Promoting Interleukin-10 Production in Colonic Macrophages Through the SIK/CRTC3 Pathway.

BACKGROUND: Interleukin-10 (IL-10) is a potent immunoregulatory cytokine that plays a pivotal role in maintaining mucosal immune homeostasis. As a novel synthetic inhibitor of salt-inducible kinases (SIKs), HG-9-91-01 can effectively enhance IL-10 secretion at the cellular level, but its in vivo immunoregulatory effects remain unclear. In this study, we investigated the effects and underlying mechanism of HG-9-91-01 in murine colitis models. METHODS: The anti-inflammatory effects of HG-9-91-01 were evaluated on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-, dextran sulfate sodium-induced colitis mice, and IL-10 knockout chronic colitis mice. The in vivo effector cell of HG-9-91-01 was identified by fluorescence-activated cell sorting and quantitative real-time polymerase chain reaction. The underlying mechanism of HG-9-91-01 was investigated via overexpressing SIKs in ANA-1 macrophages and TNBS colitis mice. RESULTS: Treatment with HG-9-91-01 showed favorable anticolitis effects in both TNBS- and DSS-treated mice through significantly promoting IL-10 expression in colonic macrophages but failed to protect against IL-10 KO murine colitis. Further study indicated that HG-9-91-01 markedly enhanced the nuclear level of cAMP response element-binding protein (CREB)-regulated transcription coactivator 3 (CRTC3), whereas treatment with lentiviruses encoding SIK protein markedly decreased the nuclear CRTC3 level in HG-9-91-01-treated ANA-1 macrophages. In addition, intracolonic administration with lentiviruses encoding SIK protein significantly decreased the nuclear CRTC3 level in the lamina propria mononuclear cells and ended the anti-inflammatory activities of HG-9-91-01. CONCLUSIONS: We found that HG-9-91-01 promoted the IL-10 expression of colonic macrophages and exhibited its anticolitis activity through the SIK/CRTC3 axis, and thus it may represent a promising strategy for inflammatory bowel disease therapy.

3,3'-Diindolylmethane alleviates acute atopic dermatitis by regulating T cell differentiation in a mouse model.

Atopic dermatitis is a severe, chronic relapsing inflammatory disease of the skin with family clustering. It is characterized into acute phase, which is dominated by T helper 2-type immune responses, and chronic phase, which is dominated by T helper 1-type immune responses. Studies have shown that 3,3'-diindolylmethane not only has antitumor effects but also can relieve symptoms of inflammatory diseases by inhibiting the nuclear factor-κB signaling pathway and regulating T cell differentiation. To study the effect of 3,3'-diindolylmethane on atopic dermatitis and the underlying mechanism, a mouse model of acute atopic dermatitis was established using 2,4-dinitrofluorobenzene. After intraperitoneal injection of 3,3'-diindolylmethane, skin erythema and edema in mice were significantly alleviated. Furthermore, 3,3'-diindolylmethane reduced immune activation, probably by inhibiting the secretion of thymic stromal lymphopoietin by keratinocytes. 3,3'-Diindolylmethane also promoted the differentiation of regulatory T cells and inhibited the activation of T helper 2 and T helper 17 cells to reduce atopic dermatitis-related immune responses. However, it showed no significant effect on the differentiation of T helper 1 cells. These results indicate that 3,3'-diindolylmethane has a significant inhibitory effect on T helper 2 cells in the acute phase of atopic dermatitis. Our findings may provide not only more insights into the pathological mechanism of AD, but also a new candidate medicine for it.

Sox9/INHBB axis-mediated crosstalk between the hepatoma and hepatic stellate cells promotes the metastasis of hepatocellular carcinoma.

The activation of hepatic stellate cells (HSCs) and liver fibrosis in the peri-tumoral tissue contributes to the progression of hepatocellular carcinoma (HCC). However, the mechanisms underlying the crosstalk between hepatoma and peri-tumoral HSCs remain elusive. We found that the Sox9/INHBB axis is upregulated in HCC and is associated with tumor metastasis. Using gain- and loss-of-function approaches, we revealed that the Sox9/INHBB axis promotes the growth and metastasis of an orthotopic HCC tumor by activating the peri-tumoral HSCs. Mechanistically, Sox9 induces INHBB expression by directly binding to its enhancer, thus aiding in the secretion of activin B from hepatoma cells, and in turn, promoting the activation of the surrounding HSCs through activin B/Smad signaling. Furthermore, inhibition of activin B/Smad singaling attenuates the fibrotic response in the peri-tumoral tissue and decreases the incidence of metastasis. Finally, clinical analyses indicated a positive correlation between Sox9 and INHBB expression in HCC specimens and identified the Sox9/INHBB axis as a positive regulator of liver fibrosis. In conclusion, Sox9/INHBB axis-mediated crosstalk between hepatoma cells and HSCs induces a fertile environment favoring HCC metastasis, thereby exhibiting as a potential therapeutic target.

A Novel Sox9/lncRNA H19 Axis Contributes to Hepatocyte Death and Liver Fibrosis.

Sox9 has been previously characterized as a transcription factor responsible for the extracellular matrix production during liver fibrosis. However, the deregulation and functional role of hepatocyte Sox9 in the progression of liver fibrosis remains elusive. Here, we found a significant increase of Sox9 in the hepatocytes isolated from CCl4-induced fibrotic liver and showed that antisense oligoribonucleotides depletion of Sox9 was sufficient to attenuate CCl4-induced liver fibrosis. Notably, the increase of Sox9 in hepatocyte was associated with the upregulation of long noncoding RNA H19 in both in vitro and in vivo systems. Mechanistic studies revealed that Sox9 induced H19 by binding to a conserved promoter region of H19. In vitro, hepatocyte injury triggered the increase of Sox9/H19 axis, whereas silence of H19 greatly alleviated the H2O2-induced hepatocyte apoptosis, suggesting that H19 functions as a downstream effector of Sox9 signaling and is involved in hepatocyte apoptosis. In animal experiments, inhibition of H19 alleviated the activation of hepatic stellate cells and reduced the extent of liver fibrosis, whereas ectopic expression of H19 abolished the inhibitory effects of Sox9 depletion on liver fibrosis, suggesting that the profibrotic effect of hepatocyte Sox9 depends on H19. Finally, we investigated the clinical relevance of Sox9/H19 axis to liver fibrosis and identified the increase of Sox9/H19 axis in liver cirrhosis patients. In conclusion, our findings link Sox9/H19 axis to the intrinsic mechanisms of hepatocyte apoptosis and may represent a hitherto unknown paradigm in hepatocyte injury associated with the progression of liver fibrosis.

An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells.

CRISPR-Cas9 is a versatile genome-editing technology that is a promising gene therapy tactic. However, the delivery of CRISPR-Cas9 is still a major obstacle to its broader clinical application. Here, we confirm that the components of CRISPR-Cas9-sgRNA and Cas9 protein-can be packaged into exosomes, where sgRNA and Cas9 protein exist as a sgRNA:Cas9 ribonucleoprotein complex. Although exosomal CRISPR-Cas9 components can be delivered into recipient cells, they are not adequate to abrogate the target gene in recipient cells. To solve this, we engineered a functionalized exosome (M-CRISPR-Cas9 exosome) that could encapsulate CRISPR-Cas9 components more efficiently. To improve the loading efficiency of Cas9 proteins into exosomes, we artificially engineered exosomes by fusing GFP and GFP nanobody with exosomal membrane protein CD63 and Cas9 protein, respectively. Therefore, Cas9 proteins could be captured selectively and efficiently loaded into exosomes due to the affinity of GFP-GFP nanobody rather than random loading. sgRNA and Cas9 protein exist as a complex in functionalized exosomes and can be delivered into recipient cells. To show the function of modified exosomes-delivered CRISPR-Cas9 components in recipient cells visually, we generated a reporter cell line (A549stop-DsRed) that produced a red fluorescent signal when the stop element was deleted by the sgRNA-guided endonuclease. Using A549stop-DsRed reporter cells, we showed that modified exosomes loaded with CRISPR-Cas9 components abrogated the target gene more efficiently in recipient cells. Our study reports an alternative tactic for CRISPR-Cas9 delivery.

Dual-Functionalized MSCs that Express CX3CR1 and IL-25 Exhibit Enhanced Therapeutic Effects on Inflammatory Bowel Disease.

Mesenchymal stem cells (MSCs) have shown great promise in inflammatory bowel disease (IBD) treatment, owing to their immunosuppressive capabilities, but their therapeutic effectiveness is sometimes thwarted by their low efficiency in entering the inflamed colon and variable immunomodulatory ability in vivo. Here, we demonstrated a new methodology to manipulate MSCs to express CX3C chemokine receptor 1 (CX3CR1) and interleukin-25 (IL-25) to promote their delivery to the inflamed colon and enhance their immunosuppressive capability. Compared to MSCs without treatment, MSCs infected with a lentivirus (LV) encoding CX3CR1 and IL-25 (CX3CR1&IL-25-LV-MSCs) exhibited enhanced targeting to the inflamed colon and could further move into extravascular space of the colon tissues via trans-endothelial migration in dextran sodium sulfate (DSS)-challenged mice after MSC intravenous injection. The administration of the CX3CR1&IL-25-LV-MSCs achieved a better therapeutic effect than that of the untreated MSCs, as indicated by pathological indices and inflammatory markers. Antibody-blocking studies indicated that the enhanced therapeutic effects of dual-functionalized MSCs were dependent on CX3CR1 and IL-25 function. Overall, this strategy, which is based on enhancing the homing and immunosuppressive abilities of MSCs, represents a promising therapeutic approach that may be valuable in IBD therapy.

Cyanidin-3-O-Glucoside and Cyanidin Protect Against Intestinal Barrier Damage and 2,4,6-Trinitrobenzenesulfonic Acid-Induced Colitis.

Anthocyanin-rich extracts have shown anti-inflammation activity in mouse colitis models. Cyanidin-3-glucoside (C3G) is one of the widespread anthocyanins in plants, and cyanidin (Cy) is the aglycone of C3G that can be generated in intestine under gut microorganism metabolism. To explore the anti-inflammatory activity of single anthocyanins compound and show the potential mechanism, the protective effects of C3G and its aglycone Cy on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice and lipopolysaccharide (LPS)-stimulated Caco-2 cellular monolayer inflammation were studied. The results showed that both C3G and Cy significantly improved the clinical symptoms and relieved the histological damage in TNBS-challenged mice. The activity of myeloperoxidase and the excretion of inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interferon-γ were also significantly inhibited at the administration dosage of 200 µmol/kg. In vitro studies showed that when LPS-stimulated Caco-2 cells were pretreated with C3G and Cy, the destruction of the intestinal epithelial barrier was ameliorated due to the improvement of the transepithelial electrical resistance and Lucifer yellow flux values, while there were no significant difference between C3G and Cy groups at the same dosage. Similarly, both C3G and Cy suppressed nitric oxide production and inflammatory cytokines secretion of LPS-induced Caco-2 cells. C3G and its aglycone Cy had similar anti-inflammatory activity in both colitis mice and Caco-2 cells. The results suggest that C3G and Cy may exert anti-inflammatory effects by protecting the intestinal barrier as well as by suppressing inflammatory cytokine secretion. Thus, C3G or Cy could be potential preventive agents or supplementary medicines for inflammatory bowel disease.

Outer-Frame-Degradable Nanovehicles Featuring Near-Infrared Dual Luminescence for in Vivo Tracking of Protein Delivery in Cancer Therapy.

In vivo monitoring of cargo protein delivery is critical for understanding the pharmacological efficacies and mechanisms during cancer therapy, but it still remains a formidable challenge because of the difficulty in observing nonfluorescent proteins at high resolution and sensitivity. Here we report an outer-frame-degradable nanovehicle featuring near-infrared (NIR) dual luminescence for real-time tracking of protein delivery in vivo. Upconversion nanoparticles (UCNPs) and fluorophore-doped degradable macroporous silica (DS) with spectral overlap were coupled to form a core-shell nanostructure as a therapeutic protein nanocarrier, which was eventually enveloped with a hyaluronic acid (HA) shell to prevent protein leakage and for recognizing tumor sites. The DS layer served as both a container to accommodate the therapeutic proteins and a filter to attenuate upconversion luminescence (UCL) of the inner UCNPs. After the nanovehicles selectively accumulated at tumor sites and entered cancer cells, intracellular hyaluronidase (HAase) digested the outermost HA protective shell and initiated the outer frame degradation-induced protein release and UCL restoration of UCNPs in the intracellular environment. Significantly, the biodistribution of the nanovehicles can be traced at the 710 nm NIR fluorescence channel of DS, whereas the protein release can be monitored at the 660 nm NIR fluorescence channel of UCNPs. Real-time tracking of protein delivery and release was achieved in vitro and in vivo by NIR fluorescence imaging. Moreover, in vitro and in vivo studies manifest that the protein cytochrome c-loaded nanovehicles exhibited excellent cancer therapeutic efficacy. This nanoplatform assembled by the outer-frame-degradable nanovehicles featuring NIR dual luminescence not only advances our understanding of where, when, and how therapeutic proteins take effect in vivo but also provides a universal route for visualizing the translocation of other bioactive macromolecules in cancer treatment and intervention.

Intestinal epithelial PKM2 serves as a safeguard against experimental colitis via activating ß-catenin signaling.

The pyruvate kinase M2 (PKM2)-mediated aerobic glycolysis has been shown to play a critical role in promoting cell survival and proliferation. However, little is known about the function of intestinal epithelial PKM2 in intestine homeostasis. Here we investigate whether and how intestinal epithelial PKM2 modulates the morphology and function of the adult intestine in experimental colitis. Analyzing colonoscopic biopsies from Crohn's disease and ulcerative colitis patients, we found significantly decreased level of intestinal epithelial PKM2 in patients compared to that in non-inflamed tissues. Similar reduction of intestinal epithelial PKM2 was observed in mice with dextran sulfate sodium-induced colitis. Moreover, intestinal epithelial-specific PKM2-knockout (Pkm2-/-) mice displayed more severe intestinal inflammation, as evidenced by a shortened colon, disruption of epithelial tight junctions, an increase in inflammatory cytokine levels, and immune cell infiltration, when compared to wild-type mice. Gene profiling, western blot, and function analyses indicated that cell survival signals, particularly the Wnt/ß-catenin pathways, were associated with PKM2 activity. Increasing mouse intestinal epithelial PKM2 expression via delivery of a PKM2-expressing plasmid attenuated experimental colitis. In conclusion, our studies demonstrate that intestinal epithelial PKM2 increases cell survival and wound healing under the colitic condition via activating the Wnt/ß-catenin signaling.

Upregulation of HER2 in tubular epithelial cell drives fibroblast activation and renal fibrosis.

Tubular epithelial cell-derived profibrotic factors are known as the driving force in renal fibrosis for their roles in activating the surrounding fibroblast. However, the mechanisms driving their expressions remain undefined. Here, we find that kidney human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, significantly increased in unilateral ureteral obstruction-induced renal fibrosis, in type 1 and type 2 diabetic nephropathy, and in kidney biopsies from patients with renal fibrosis. Notably, the upregulation of HER2 mainly occurred in proximal tubular epithelial cells (PTECs). In vivo, the ectopic expression of HER2 in these cells was sufficient to activate the interstitial fibroblast and initiate interstitial fibrosis, whereas inhibiting HER2 reduced the accumulation of myofibroblasts and the extent of renal fibrosis in the mouse obstruction model and in streptozotocin (STZ)-induced diabetic mice. We also generated a tubular epithelial cell subline stably expressing HER2 and performed transcriptome RNA sequence analysis. This showed that sustained HER2 expression significantly induced the expression of profibrotic connective tissue growth factor (CTGF). Mechanistically, the induction of CTGF depended on the HER2-mediated activation of Stat3 in the tubular epithelium. In vitro, the incubation of kidney fibroblasts with culture medium from HER2-overexpressed tubular epithelial cells promoted fibroblast proliferation and activation, whereas silencing CTGF impeded the profibrotic effects of the tubular epithelial cell preconditioned media. Thus, our results highlight the significance of HER2 in tubular injury and characterize its role in promoting surrounding fibroblast activation and renal fibrosis in a paracrine manner.

Phosphorothioated antisense oligodeoxynucleotide suppressing interleukin-10 is a safe and potent vaccine adjuvant.

While vaccination is highly effective for the prevention of many infectious diseases, the number of adjuvants licensed for human use is currently very limited. The aim of this study was to evaluate the safety, efficacy, and to clarify the mechanism of a phosphorothioated interleukin (IL)-10-targeted antisense oligonucleotide (ASO) as an immune adjuvant in intradermal vaccination. The cytotoxicity of IL-10 ASO and its ability to promote T cell proliferation were assessed by Cell Counting Kit-8 (CCK-8) assay. The contents of IL-6, IL-8, TNF-α, IL-1ß, and IL-10 in inoculated local tissue and the antigen-specific antibody titers in mouse serum samples were determined by ELISA. The target cells of IL-10 ASO were observed using immunofluorescent staining. The results showed that the specific antibody titer of ovalbumin (OVA), a model antigen, was increased 100-fold upon addition of IL-10 ASO as an adjuvant compared to that of OVA alone. IL-10 ASO showed an immunopotentiation efficacy similar to that of Freund's incomplete adjuvant, with no detectable cell or tissue toxicity. In vitro and in vivo experiments confirmed that IL-10 ASO enhances immune responses by temporarily suppressing IL-10 expression from local dendritic cells and consequently promoting T cell proliferation. In conclusion, IL-10 ASO significantly enhances immune responses against co-delivered vaccine antigens with high efficacy and low toxicity. It has the potential to be developed into a safe and efficient immune adjuvant.