Intraepithelial mast cells drive gasdermin C-mediated type 2 immunity.

A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEß7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Human adipose and synovial mesenchymal stem cells improve osteoarthritis in rats by reducing chondrocyte reactive oxygen species and inhibiting inflammatory response.

BACKGROUND: We explored the therapeutic effects of Adipose-derived mesenchymal stem cells (ADMSCs) and Synovial-derived mesenchymal stem cells (SDMSCs) on osteoarthritis (OA). METHODS: SDMSCs and ADMSCs were co-cultured with chondrocytes and stimulated with interleukin (IL)-1ß. An OA model was established on rats by intra-articular injection with ADMSCs and SDMSCs. After 8 weeks, the joint diameter difference was detected, and histological staining was used to observe the pathological changes in cartilage tissue. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of IL-6, tumor necrosis factor (TNF)-α and IL-1ß in joint fluid. The expressions of COL2A1, Aggrecan, Matrix metalloproteinase (MMP)-13, SOX9, IL-6, TNF-α and IL-1ß were detected by qRT-PCR and Western blotting in cartilage tissue. Reactive oxygen species (ROS) content in cells and cartilage tissues was detected by ROS kit. RESULTS: SDMSCs and ADMSCs co-cultured with chondrocytes could reduce MMP-13 expression, increase the expressions of COL2A1, Aggrecan and SOX9, as well as reverse the effects of IL-1ß on promoting ROS content and inflammatory factors levels. After the OA model was established, the injection of ADMSCs and SDMSCs reduced the differences in joint diameter and tissue lesions in OA rats. The OA model led to increased levels of IL-6, TNF-α and IL-1ß in joint fluid and cartilage tissue, while the injection of ADMSCs and SDMSCs inhibited the inflammatory factor levels in OA rats, and increased the expressions of COL2A1, Aggrecan and SOX9 in OA rats. CONCLUSION: ADMSCs and SDMSCs improve osteoarthritis in rats by reducing chondrocyte ROS and inhibiting inflammatory response.

MicroRNA-1253 Suppresses Cell Proliferation Migration and Invasion of Osteosarcoma by Targeting MMP9.

PURPOSE: MicroRNAs play an important role in osteosarcoma (OS) development and progress. Although miR-1253 was considered as a tumor-inhibitor in some cancers, it's function in the OS is not clear. METHODS: In our study, we examined the expression of miR-1253 in OS cells and osteoblast cells using quantitative real-time PCR. The proliferation of OS cells was measured by BrdU assay, and we performed transwell to detect migration and invasion of OS cells. Meanwhile, EMT proteins were tested by western blot. We used Bioinformatics to predict the target genes of miR-1253 and found out Matrix metalloproteinases9 (MMP9) was one of that. The direct combination between miR-1253 and MMP9 was verified by double luciferase reporting experiment. Quantitative real-time PCR and western blot were used to detect the expression of MMP9. RESULTS: We found that the expression level of miR-1253 in OS cells was significantly lower than that in osteoblast cells. Overexpression of miR-1253 could significantly inhibit OS cell proliferation, migration, invasion and EMT. And then, MMP9 was predicted as a downstream target of miR-1253 by Bioinformatics analysis. Further experiments showed that miR-1253 could reduce the protein level of MMP9 by directly binding to the 3'-UTR of MMP9. Afterward, we performed a rescue experiment, in which both MMP9 and miR-1253 were overexpressed. Compared with the groups overexpressed miR-1253 alone, cell proliferation, migration and invasion in co-overexpression groups were improved. CONCLUSIONS: In summary, these results suggested that miR-1253 down-regulated in OS cells, and could suppress the proliferation, migration and invasion of OS cells. Its molecular regulatory mechanism was that inhibits the expression of the downstream target gene MMP9 by directly binding, thus affect OS cell functions. Therefore, miR-1253 has the potential to become a biomarker and therapeutic target for OS therapy.

[Oxidative stress preconditioning alleviates oxidative stress-induced damage of bone marrow mesenchymal stem cells].

Objective To investigate the effect of oxidative stress preconditioning on the oxidative stress-induced damage of bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated and cultured by density gradient centrifugation combined with adherence method. The cells were divided into three groups: control group (control medium), oxidative damage group (treated with 1000 µmol/L H2O2 for 24 hours), and preconditioning group (preincubated with 50 µmol/L H2O2 for 8 hours before treatment with 1000 µmol/L H2O2 for 24 hours). DCFH-DA staining was used to analyze the level of reactive oxygen species (ROS). Mitochondrial membrane potential was measured by JC-1 staining. DNA damage was detected by TUNEL. Malondialdehyde (MDA) content was detected by thiobarbituric acid (TBA) method, and superoxide dismutase (SOD) activity was detected by water soluble tetrazolium-1 (WST-1) assay. CCK-8 assay was used to detect cell viability and flow cytometry to detect cell apoptosis. Results Compared with the oxidative damage group, the preconditioning group had reduced ROS level, reduced DNA damage, higher mitochondrial membrane potential, significantly decreased MDA content, increased SOD activity, increased cell viability, and significantly decreased apoptosis. Conclusion Oxidative stress preconditioning can enhance the anti-oxidative stress ability of BMSCs and promote its survival under oxidative stress.

Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.

Cytotoxic lymphocyte-mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)-positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon-γ (IFN-γ) up-regulates GSDMB expression and promotes pyroptosis. GSDMB is highly expressed in certain tissues, particularly digestive tract epithelia, including derived tumors. Introducing GZMA-cleavable GSDMB into mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte-killing mechanism, which may enhance antitumor immunity.

Repair of soft tissue defects of the fingers with medial plantar venous flap.

OBJECTIVE: To report the surgical procedures and efficacy of using medial plantar venous flap for the repair of soft tissue defects of the fingers. METHODS: From March 2010 to April 2012, medial plantar venous flaps were harvested to repair the wounds of 31 fingers in 29 cases. Among them, there were 13 middle fingers with defects at the tips in 11 cases, 7 fingers with defects in the dorsal part in 7 cases, and 11 fingers with defects in the finger pulp in 11 cases. The size of the defects ranged from 1.2cm×1.5 cm to 2.5cm × 3.5cm. Medial plantar venous flaps of 1.5cm × 2cm - 3×4 cm were harvested. Full-thickness skin grafts were adopted for the donor areas. RESULTS: All 31 flaps survived, except for 1 flap with arterial crisis and 2 cases with venous crisis. These conditions were timely corrected by secondary anastomosis of artery and vein and the flaps survived. The wounds and the donor areas achieved healing by the first intention. All grafted skins survived. Postoperative follow-up was conducted for 26 fingers in 24 cases for 4-12 months, excluding 5 cases with lost follow-up. The dorsal part of the damaged fingers had normal morphology, and the skin color and texture were similar to those of the normal skin. After the repair of defects in the fingertip and pulp, fingerprints appeared, and the protective sensation was restored. CONCLUSION: The soft tissue defects of the fingers can be satisfactorily repaired with medial plantar venous flap, and little damage is caused to the donor area. This method is proven effective for the repair of soft tissue defects of the fingers.

Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-ß-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR-Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.

Repair of soft tissue defects of the fingers with medial plantar venous flap

SUMMARY OBJECTIVE To report the surgical procedures and efficacy of using medial plantar venous flap for the repair of soft tissue defects of the fingers. Methods From March 2010 to April 2012, medial plantar venous flaps were harvested to repair the wounds of 31 fingers in 29 cases. Among them, there were 13 middle fingers with defects at the tips in 11 cases, 7 fingers with defects in the dorsal part in 7 cases, and 11 fingers with defects in the finger pulp in 11 cases. The size of the defects ranged from 1.2cm×1.5 cm to 2.5cm × 3.5cm. Medial plantar venous flaps of 1.5cm × 2cm - 3×4 cm were harvested. Full-thickness skin grafts were adopted for the donor areas. RESULTS All 31 flaps survived, except for 1 flap with arterial crisis and 2 cases with venous crisis. These conditions were timely corrected by secondary anastomosis of artery and vein and the flaps survived. The wounds and the donor areas achieved healing by the first intention. All grafted skins survived. Postoperative follow-up was conducted for 26 fingers in 24 cases for 4-12 months, excluding 5 cases with lost follow-up. The dorsal part of the damaged fingers had normal morphology, and the skin color and texture were similar to those of the normal skin. After the repair of defects in the fingertip and pulp, fingerprints appeared, and the protective sensation was restored. CONCLUSION The soft tissue defects of the fingers can be satisfactorily repaired with medial plantar venous flap, and little damage is caused to the donor area. This method is proven effective for the repair of soft tissue defects of the fingers.

RESUMO OBJETIVO Relatar os procedimentos cirúrgicos e a eficácia do uso de retalhos plantares mediais venosos para reparo de defeitos de tecidos moles dos dedos. METODOLOGIA De março de 2010 a abril de 2012, foram colhidos retalhos plantares mediais venosos para reparar ferimentos de 31 dedos em 29 casos. Entre eles, 13 dedos médios com defeitos nas pontas em 11 casos, 7 dedos com defeitos na parte dorsal em 7 casos e 11 dedos com defeitos na polpa digital em 11 casos. O tamanho dos defeitos variava de 1,2 cm × 1,5 cm a 2,5 cm × 3,5 cm. Foram colhidos retalhos plantares mediais venosos de 1,5 cm × 2 cm a 3 cm × 4 cm. Foram adotados enxertos de pele de espessura total na área doadora. RESULTADOS Todos os 31 retalhos sobreviveram, com exceção de 1 retalho com crise arterial e 2 casos com crise venosa. Esses problemas foram corrigidos a tempo com anastomoses secundárias das artérias e veias e os retalhos sobreviveram. Os ferimentos e áreas doadoras atingiram cicatrização por primeira intenção. Todos os enxertos de pele sobreviveram. Foi realizado acompanhamento pós-operatório em de 26 dedos em 24 casos por 4 a 12 meses, sendo que dos casos tratados 5 não tiveram acompanhamento. As partes dorsais dos dedos lesionados apresentaram morfologia normal, com cor e textura da pele muito similares a da pele normal. Após o reparo dos defeitos nas pontas e polpas digitais, impressões digitais apareceram e a sensação protetora foi restaurada. CONCLUSÃO Os defeitos de tecido mole dos dedos podem ser reparados de forma satisfatória com retalhos plantares mediais venosos, com poucos danos à área doadora. Este método mostrou se eficaz para o reparo de defeitos de tecido mole dos dedos.

[miR-125b suppresses the aerobic glycolysis of osteosarcoma HOS cells by downregulating the expression of hexokinase-2].

Objective To investigate the effect of miR-125b on the aerobic glycolysis of osteosarcoma HOS cells and its underlying mechanism. Methods Real-time quantitative PCR was performed to detect the expression of miR-125b in HOSB normal human osteoblast cells and HOS osteosarcoma cells. The glucose uptake rate was assessed with 3H-2 deoxyglucose (3H-2DG) and lactate production was tested with the kits to observe the effect of miR-125b-mimics on the aerobic glycolysis of osteosarcoma HOS cells. Hexokinase-2 (HK2) protein was detected by Western blot analysis. Dual luciferase reporter gene assay was used to determine whether HK2 was the direct target of miR-125b. Results Compared with HOSB normal human osteoblast cells, the expression of miR-125b was significantly lower in HOS cells. The glucose uptake and lactate production were downregulated in HOS cells transfected with miR-125b-mimics. Aerobic glycolysis of HOS cells was markedly inhibited. Protein level of HK2 was significantly inhibited in HOS cells transfected with miR-125b-mimics. Luciferase assay indicated that HK2 was the direct target of miR-125b. Conclusion miR-125b is down-regulated in HOS cells, and it inhibits the aerobic glycolysis of HOS cells by directly regulating the expression of HK2.

Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.

Pyroptosis is a form of cell death that is critical for immunity. It can be induced by the canonical caspase-1 inflammasomes or by activation of caspase-4, -5 and -11 by cytosolic lipopolysaccharide. The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity. GSDMD belongs to a gasdermin family that shares the pore-forming domain. The functions and mechanisms of activation of other gasdermins are unknown. Here we show that GSDME, which was originally identified as DFNA5 (deafness, autosomal dominant 5), can switch caspase-3-mediated apoptosis induced by TNF or chemotherapy drugs to pyroptosis. GSDME was specifically cleaved by caspase-3 in its linker, generating a GSDME-N fragment that perforates membranes and thereby induces pyroptosis. After chemotherapy, cleavage of GSDME by caspase-3 induced pyroptosis in certain GSDME-expressing cancer cells. GSDME was silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibited GSDME-dependent pyroptosis upon activation of caspase-3 by chemotherapy drugs. Gsdme-/- (also known as Dfna5-/-) mice were protected from chemotherapy-induced tissue damage and weight loss. These findings suggest that caspase-3 activation can trigger necrosis by cleaving GSDME and offer new insights into cancer chemotherapy.

Association between the c.3751G>a genetic variant of MDR1 and hepatocellular carcinoma risk in a Chinese Han population.

The objective of this study was to evaluate the influence of a genetic variant in the multidrug resistance 1 gene (MDR1) on hepatocellular carcinoma (HCC) risk. This case-control study was conducted in a Chinese population of 645 HCC cases and 658 cancer-free controls. The genotype of the c.3751G>A genetic variant in the MDR1 gene was investigated by created restriction site-polymerase chain reaction (CRS-PCR) and DNA sequencing methods. Our data demonstrated significantly differences detected in the allelic and genotypic frequencies between HCC cases and those of cancer-free controls. Association analyses indicated that there were statistically increased risk of HCC in the homozygote comparison (AA versus (vs.) GG: OR = 2.22, 95% CI 1.51-3.27, χ(2) = 16.90, P < 0.001), dominant model (AA/GA vs. GG: OR = 1.25, 95% CI 1.00-1.55, χ(2) = 3.98, P = 0.046), recessive model (AA vs. GA/GG: OR = 2.14, 95% CI 1.47-3.09, χ(2) = 16.68, P < 0.001) and allele comparison (A vs. G: OR = 1.33, 95% CI 1.13-1.57, χ(2) = 11.66, P = 0.001). The allele-A and genotype-AA may contribute to HCC susceptibility. These preliminary findings suggest that the c.3751G>A genetic variant in the MDR1 gene is potentially related to HCC susceptibility in a Chinese Han population, and might be used as a molecular marker for evaluating HCC susceptibility.

Synergistically killing activity of aspirin and histone deacetylase inhibitor valproic acid (VPA) on hepatocellular cancer cells.

Aspirin and valproic acid (VPA) have been extensively studied for inducing various malignancies growth inhibition respectively, despite their severe side effects. Here, we developed a novel combination by aspirin and VPA on hepatocellular cancer cells (HCCs). The viability of HCC lines were analyzed by MTT assay, apoptotic analysis of HepG2 and SMMC-7721 cell was performed. Real time-PCR and Western blotting were performed to determine the expression of apoptosis related genes and proteins such as Survivin, Bcl-2/Bax, Cyclin D1 and p15. Moreover, orthotopic xenograft tumors were challenged in nude mice to establish murine model, and then therapeutic effect was analyzed after drug combination therapy. The viability of HCC lines' significantly decreased after drug combination treatment, and cancer cell apoptosis in combination group increasingly induced compared with single drug use. Therapeutic effect was significantly enhanced by combination therapy in tumor volume and tumor weight decrease. From the data shown here, aspirin and VPA combination have a synergistic killing effect on hepatocellular cancers cells proliferation and apoptosis.

Pseudorabies virus infected porcine epithelial cell line generates a diverse set of host microRNAs and a special cluster of viral microRNAs.

Pseudorabies virus (PRV) belongs to Alphaherpesvirinae subfamily that causes huge economic loss in pig industry worldwide. It has been recently demonstrated that many herpesviruses encode microRNAs (miRNAs), which play crucial roles in viral life cycle. However, the knowledge about PRV-encoded miRNAs is still limited. Here, we report a comprehensive analysis of both viral and host miRNA expression profiles in PRV-infected porcine epithelial cell line (PK-15). Deep sequencing data showed that the ∼4.6 kb intron of the large latency transcript (LLT) functions as a primary microRNA precursor (pri-miRNA) that encodes a cluster of 11 distinct miRNAs in the PRV genome, and 209 known and 39 novel porcine miRNAs were detected. Viral miRNAs were further confirmed by stem-loop RT-PCR and northern blot analysis. Intriguingly, all of these viral miRNAs exhibited terminal heterogeneity both at the 5' and 3' ends. Seven miRNA genes produced mature miRNAs from both arms and two of the viral miRNA genes showed partially overlapped in their precursor regions. Unexpectedly, a terminal loop-derived small RNA with high abundance and one special miRNA offset RNA (moRNA) were processed from a same viral miRNA precursor. The polymorphisms of viral miRNAs shed light on the complexity of host miRNA-processing machinery and viral miRNA-regulatory mechanism. The swine genes and PRV genes were collected for target prediction of the viral miRNAs, revealing a complex network formed by both host and viral genes. GO enrichment analysis of host target genes suggests that PRV miRNAs are involved in complex cellular pathways including cell death, immune system process, metabolic pathway, indicating that these miRNAs play significant roles in virus-cells interaction of PRV and its hosts. Collectively, these data suggest that PRV infected epithelial cell line generates a diverse set of host miRNAs and a special cluster of viral miRNAs, which might facilitate PRV replication in cells.