One-Stage Arthroscopic Multiple Ligament Reconstruction for Schenck IV Knee Dislocation.

PURPOSE: Schenck IV knee dislocation patients have dissatisfactory knee function and return-to-sport rate with the existing treatment methods. The purpose of this study was to illustrate a one-stage arthroscopic multiple ligament reconstruction method for treating Schenck IV knee dislocations. METHODS: A retrospective case series study was performed. All patients with a history of Schenck IV knee dislocation who underwent one-stage arthroscopic multi-ligament reconstruction from 2010 to 2018 were followed for 24 months. The outcomes, including general patient data, Lysholm scores, International Knee Documentation Committee (IKDC) scores, visual analog scale (VAS) pain scores, knee active range of motion, and complications, were reviewed. The data was analyzed with paired-samples t-test. RESULTS: A total of 12 patients, comprising nine males and three females, were followed up and reviewed. The mean age at the time of the surgical procedure was 40.3 ± 9.0 (22-57) years. The mean body mass index (BMI) was 24.6 ± 4.9 (15.2-32.5) kg/m2 . The mean IKDC score and Lysholm score before surgery were 30.4 ± 6.1 (21-42) and 28.2 ± 6.2 (22-39), respectively. The average operation time was 121.8 minutes. The mean IKDC score and Lysholm score at the 24-month follow-up were 80.6 ± 6.5 (68-92) and 82.0 ± 7.5 (72-95), respectively. There were significant differences in the IKDC and Lysholm scores between the preoperative and 24-month postoperative time points (p < 0.01). The mean knee range of motion was 124.6° ± 6.6° (115°-135°) at the 24-month follow-up. No major complications occurred. CONCLUSIONS: The results of this retrospective study suggest that the new arthroscopic one-stage multi-ligament reconstruction technique is an effective way to treat Schenck IV knee dislocation with satisfactory postoperative knee function.

Transcriptomic analysis reveals proinflammatory signatures associated with acute myeloid leukemia progression.

Numerous studies have been performed over the last decade to exploit the complexity of genomic and transcriptomic lesions driving the initiation of acute myeloid leukemia (AML). These studies have helped improve risk classification and treatment options. Detailed molecular characterization of longitudinal AML samples is sparse, however; meanwhile, relapse and therapy resistance represent the main challenges in AML care. To this end, we performed transcriptome-wide RNA sequencing of longitudinal diagnosis, relapse, and/or primary resistant samples from 47 adult and 23 pediatric AML patients with known mutational background. Gene expression analysis revealed the association of short event-free survival with overexpression of GLI2 and IL1R1, as well as downregulation of ST18. Moreover, CR1 downregulation and DPEP1 upregulation were associated with AML relapse both in adults and children. Finally, machine learning-based and network-based analysis identified overexpressed CD6 and downregulated INSR as highly copredictive genes depicting important relapse-associated characteristics among adult patients with AML. Our findings highlight the importance of a tumor-promoting inflammatory environment in leukemia progression, as indicated by several of the herein identified differentially expressed genes. Together, this knowledge provides the foundation for novel personalized drug targets and has the potential to maximize the benefit of current treatments to improve cure rates in AML.

Genomic characterization of relapsed acute myeloid leukemia reveals novel putative therapeutic targets.

Relapse is the leading cause of death of adult and pediatric patients with acute myeloid leukemia (AML). Numerous studies have helped to elucidate the complex mutational landscape at diagnosis of AML, leading to improved risk stratification and new therapeutic options. However, multi-whole-genome studies of adult and pediatric AML at relapse are necessary for further advances. To this end, we performed whole-genome and whole-exome sequencing analyses of longitudinal diagnosis, relapse, and/or primary resistant specimens from 48 adult and 25 pediatric patients with AML. We identified mutations recurrently gained at relapse in ARID1A and CSF1R, both of which represent potentially actionable therapeutic alternatives. Further, we report specific differences in the mutational spectrum between adult vs pediatric relapsed AML, with MGA and H3F3A p.Lys28Met mutations recurrently found at relapse in adults, whereas internal tandem duplications in UBTF were identified solely in children. Finally, our study revealed recurrent mutations in IKZF1, KANSL1, and NIPBL at relapse. All of the mentioned genes have either never been reported at diagnosis in de novo AML or have been reported at low frequency, suggesting important roles for these alterations predominantly in disease progression and/or resistance to therapy. Our findings shed further light on the complexity of relapsed AML and identified previously unappreciated alterations that may lead to improved outcomes through personalized medicine.

Exposure of normal and chronic bronchitis-like mucosa models to aerosolized carbon nanoparticles: comparison of pro-inflammatory oxidative stress and tissue injury/repair responses.

Carbon nanoparticles (CNP) are generated by incomplete combustion of diesel engines. Several epidemiological studies associated higher susceptibility to particulate matter related adverse respiratory outcomes with preexisting conditions like chronic bronchitis (CB). Therefore, we compared the effect of CNP exposure on primary bronchial epithelial cells (PBEC) developed in air-liquid interface (ALI) models of normal versus CB-like-mucosa.PBEC cultured at ALI represented normal mucosa (PBEC-ALI). To develop CB-like-mucosa (PBEC-ALI/CB), 1 ng/ml interleukin-13 was added to the basal media of PBEC-ALI culturing. PBEC-ALI and PBEC-ALI/CB were exposed to sham or to aerosolized CNP using XposeALI® system. Protein levels of CXCL-8 and MMP-9 were measured in the basal media using ELISA. Transcript expression of pro-inflammatory (CXCL8, IL6, TNF, NFKB), oxidative stress (HMOX1, SOD3, GSTA1, GPx), tissue injury/repair (MMP9/TIMP1) and bronchial cell type markers (MUC5AC, CC10) were assessed using qRT-PCR.Increased secretion of CXCL-8 and MMP-9 markers was detected 24 h post-exposure in both PBEC-ALI and PBEC-ALI/CB with more pronounced effect in the later. Pro-inflammatory and tissue injury markers were increased at both 6 h and 24 h post-exposure in PBEC-ALI/CB. Oxidative stress markers exhibited similar responses at 6 h and 24 h post-exposure in PBEC-ALI/CB. The club cell specific marker CC10 was increased by 300 fold in PBEC-ALI/CB and 20 fold in PBEC-ALI following CNP exposure.Our data indicates an earlier and stronger reaction of pro-inflammatory, oxidative stress and tissue injury markers in PBEC-ALI/CB models compared to PBEC-ALI models following CNP exposure. The findings may provide insight into the plausible mechanisms of higher susceptibility among predisposed individuals to nanoparticle exposure.

MiR-92a inhibits fibroblast-like synoviocyte proliferation and migration in rheumatoid arthritis by targeting AKT2.

Growing data have indicated that the miR-17-92 cluster is implicated in inflammatory response and rheumatoid arthritis (RA). This study was aimed to investigate the effects of miR-92a on the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). Our results showed that miR-92a was significantly down-regulated in RA synovial tissue and RA-FLSs, whereas the protein level of AKT2 is increased. Restoration of miR-92a suppressed the proliferation and migration of RA-FLSs. Down-regulation of miR-92a promotes proliferation and migration of normal human FLSs. Dual luciferase reporter gene assay showed that miR-92a could specifically bind with the 30UTR of AKT2 and significantly repressed the luciferase activity. Down-regulation or up-regulation of miR-92a significantly increased or decreased the protein and phosphorylation levels of AKT2. siRNA-mediated down-regulation of AKT2 significantly prevented cell proliferation and migration of RA-FLSs, which were similar to the effects induced by overexpression of miR-92a. Moreover, AKT2 overexpression rescued miR-92a-mediated suppressive effect on proliferation and migration of RA-FLS. Thus, miR-92a could inhibit the proliferation and migration of RA-FLSs through regulation of AKT2 expression.

TNF­α increases inflammatory factor expression in synovial fibroblasts through the toll­like receptor­3­mediated ERK/AKT signaling pathway in a mouse model of rheumatoid arthritis.

Osteoarthritis is a type of joint disease that may lead to other joint diseases. Previous research has demonstrated that tumor necrosis factor (TNF)­α is associated with osteoarthritis activity and pathology. The possible mechanisms of the TNF­α­mediated signaling pathway have not been clearly elaborated in synovial fibroblasts. The present study aimed to investigate the potential mechanisms of TNF­α in a mouse model of iodoacetate­induced osteoarthritis. Reverse transcription­quantitative polymerase chain reaction, ELISA, western blotting and immunohistochemistry were performed to evaluate the role of TNF­α in the progression of osteoarthritis. The results revealed that the serum levels of TNF­α, interleukin (IL)­1ß, IL­4 and IL­6 were significantly upregulated in a mouse model of iodoacetate­induced osteoarthritis compared with healthy mice (P<0.01). TNF­α, IL­1ß, IL­4 and IL­6 mRNA and protein levels were also significantly upregulated in synovial fibroblasts in the experimental mice (P<0.01). It was demonstrated that TNF­α increased pro­inflammation factors matrix metalloproteinase (MMP)­3, MMP­9, nuclear factor (NF)­κB and receptor activator of NF­κB ligand (RANKL) in synovial fibroblasts. It was also observed that the toll­like receptor (TLR)­3 was significantly upregulated and extracellular signal­regulated kinase (ERK) and protein kinase B (AKT) were significantly downregulated in synovial fibroblasts in osteoarthritis mice (P<0.01). An in vitro assay demonstrated that TNF­α inhibitor decreased mRNA and protein levels of IL­1ß, IL­4 and IL­6 in synovial fibroblasts. The knockdown of TLR­3 abolished the TNF­α upregulated mRNA and protein levels of IL­1ß, IL­4 and IL­6 in synovial fibroblasts. In addition, the knockdown of TLR­3 also reversed TNF­α­upregulated ERK and AKT expression in synovial fibroblasts. In vivo assays demonstrated that TNF­α inhibitor significantly decreased the deposition of IL­1ß, IL­4 and IL­6 as well as bone destruction and significantly increased the body weight and osteoarthritis score for osteoarthritic mice (P<0.01). TNF­α inhibitor decreased TLR­3 and significantly increased the expression and phosphorylation of ERK and AKT in articular cartilage (P<0.01). In conclusion the results of the present study indicate that TNF­α serves an essential role in synovial fibroblasts in osteoarthritis, suggesting that inhibition of TNF­α may decrease inflammation via the TLR­3­mediated ERK/AKT signaling pathway in a mouse model of monosodium iodoacetate­induced osteoarthritis.

A genome-wide association study of IgM antibody against phosphorylcholine: shared genetics and phenotypic relationship to chronic lymphocytic leukemia.

Phosphorylcholine (PC) is an epitope on oxidized low-density lipoprotein (oxLDL), apoptotic cells and several pathogens like Streptococcus pneumoniae. Immunoglobulin M against PC (IgM anti-PC) has the ability to inhibit uptake of oxLDL by macrophages and increase clearance of apoptotic cells. From our genome-wide association studies (GWASs) in four European-ancestry cohorts, six single nucleotide polymorphisms (SNPs) in 11q24.1 were discovered (in 3002 individuals) and replicated (in 646 individuals) to be associated with serum level of IgM anti-PC (the leading SNP rs35923643-G, combined ß = 0.19, 95% confidence interval 0.13-0.24, P = 4.3 × 10-11). The haplotype tagged by rs35923643-G (or its proxy SNP rs735665-A) is also known as the top risk allele for chronic lymphocytic leukemia (CLL), and a main increasing allele for general IgM. By using summary GWAS results of IgM anti-PC and CLL in the polygenic risk score (PRS) analysis, PRS on the basis of IgM anti-PC risk alleles positively associated with CLL risk (explained 0.6% of CLL variance, P = 1.2 × 10-15). Functional prediction suggested that rs35923643-G might impede the binding of Runt-related transcription factor 3, a tumor suppressor playing a central role in the immune regulation of cancers. Contrary to the expectations from the shared genetics between IgM anti-PC and CLL, an inverse relationship at the phenotypic level was found in a nested case-control study (30 CLL cases with 90 age- and sex-matched controls), potentially reflecting reverse causation. The suggested function of the top variant as well as the phenotypic association between IgM anti-PC and CLL risk needs replication and motivates further studies.

Overexpressed miR-145 inhibits osteoclastogenesis in RANKL-induced bone marrow-derived macrophages and ovariectomized mice by regulation of Smad3.

BACKGROUND: MicroRNAs (miRs) play an important role in osteoclastogenesis. However, no study has investigated the underlying molecular mechanisms of miR-145 in this process. The purpose of the present study was to investigate the role of miR-145 and its post-transcriptional mechanism in the progression of osteoclast differentiation. METHODS: Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) were used to induce osteoclastogenesis originated from bone marrow-derived macrophages (BMMs). Female C57BL/6J mice were divided into sham, OVX, OVX + NC-agomir and OVX + miR-145-agomir groups. Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify osteoclasts in-vitro and in-vivo. The mRNA and protein levels in osteoclast and tibia were assayed by qRT-PCR and western blotting, respectively. RESULTS: miR-145 expression was inhibited in RANKL-induced osteoclastogenesis, whereas overexpression of miR-145 attenuated it. We further found that Smad3 is a direct target gene of miR-145 by binding with its 3'-UTR. Overexpression of miR-145 significantly suppressed Smad3 mRNA and protein expression. In-vivo, miR-145 agomir treatment inhibited osteoclast activity in OVX mice by inhibiting Smad3 expression. CONCLUSION: We provide the evidence that over-expression of miR-145 could inhibit osteoclast differentiation, at least partially, by decreasing Smad3 expression.

IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.

BACKGROUND AND AIMS: IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. METHODS: Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age- and sex-matched controls, and symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells was determined by flow cytometry in CD4+T cells after 6 days of culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. RESULTS: IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and a higher proportion of Th17 cells as compared to matched controls. IgM anti-PC, but not control antibodies, significantly reduced the production of IL-17 and TNF-α in cell cultures from SLE patients and atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage, potentially being tolerogenic. We observed differences in the IgM peptide expression levels in anti-PC compared to control antibodies. CONCLUSIONS: IgM anti-PC promote polarization of Tregs, which could represent a novel protective mechanism in atherosclerosis and autoimmune conditions as SLE.

Interleukin-26 Production in Human Primary Bronchial Epithelial Cells in Response to Viral Stimulation: Modulation by Th17 cytokines.

Interleukin (IL)-26 is abundant in human airways and this cytokine is involved in the local immune response to a bacterial stimulus in vivo. Specifically, local exposure to the toll-like receptor (TLR) 4 agonist endotoxin does increase IL-26 in human airways and this cytokine potentiates chemotactic responses in human neutrophils. In addition to T-helper (Th) 17 cells, alveolar macrophages can produce IL-26, but it remains unknown whether this cytokine can also be produced in the airway mucosa per se in response to a viral stimulus. Here, we evaluated whether this is the case using primary bronchial epithelial cells from the airway epithelium in vitro, and exploring the signaling mechanisms involved, including the modulatory effects of additional Th17 cytokines. Finally, we assessed IL-26 and its archetype signaling responses in healthy human airways in vivo. We found increased transcription and release of IL-26 protein after stimulation with the viral-related double stranded (ds) RNA polyinosinic-polycytidylic acid (poly-IC) and showed that this IL-26 release involved mitogen-activated protein (MAP) kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The release of IL-26 in response to a viral stimulus was modulated by additional Th17 cytokines. Moreover, there was transcription of IL26 mRNA and expression of the protein in epithelial cells of bronchial brush and tissue biopsies respectively after harvest in vivo. In addition, the extracellular IL-26 protein concentrations in bronchoalveolar lavage (BAL) samples did correlate with increased epithelial cell transcription of an archetype intracellular signaling molecule downstream of the IL-26-receptor complex, STAT1, in the bronchial brush biopsies. Thus, our study suggests that viral stimulation causes the production of IL-26 in lining epithelial cells of human airway structural cells that constitute a critical immune barrier and that this production is modulated by Th17 cytokines.

Oxidized Low-Density Lipoprotein (OxLDL)-Treated Dendritic Cells Promote Activation of T Cells in Human Atherosclerotic Plaque and Blood, Which Is Repressed by Statins: microRNA let-7c Is Integral to the Effect.

BACKGROUND: Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low-density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T-cell activation. METHODS AND RESULTS: Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T-cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL-treated DCs produced interferon-γ and interleukin (IL)-17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor-α, IL-1ß and IL-6, and increased transforming growth factor-ß and IL-10 secretion. Statin-treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T-bet and RORγt expression, and induced T regulatory cells with IL-10 production. OxLDL-induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let-7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results. CONCLUSIONS: Statins repress human DC maturation induced by oxLDL, limit T-cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let-7c is integral to the effects.

Autoimmune regulator­overexpressing dendritic cells induce T helper 1 and T helper 17 cells by upregulating cytokine expression.

The autoimmune regulator (Aire) protein is a transcriptional activator that is essential in central immune tolerance, as it regulates the ectopic expression of many tissue­restricted antigens in medullary thymic epithelial cells. Aire expression has also been described in hematopoietic cells, such as monocytes/macrophages and dendritic cells (DCs), in the peripheral immune system. However, the role of Aire expression in peripheral immune system cells, including DCs, remains to be elucidated. In the present study, the effects of secreted cytokines from Aire­overexpressing DCs on cluster of differentiation (CD)4+ T cell subsets were investigated. The dendritic cell line, DC2.4, which overexpresses Aire, was co­cultured with CD4+ T cells from splenocytes using Transwell inserts. The results indicate that Aire­overexpressing cells induce T helper (Th)1 subsets by increasing interleukin (IL)­12 expression, and induce Th17 subsets by upregulating IL­6 and transforming growth factor (TGF)­ß production. In addition, it was observed that increased levels of phosphorylated extracellular signal­regulated kinases and p38 upregulated the expression of cytokines in Aire­overexpressing cells. These data suggest that Aire may have a role in inducing Th1 and Th17 differentiation by upregulating cytokine expression in DCs.

The BH3 mimetic S1 induces endoplasmic reticulum stress-associated apoptosis in cisplatin-resistant human ovarian cancer cells although it activates autophagy.

SKOV3/DDP human ovarian cancer cells have been shown to be resistant to cisplatin. Although the BH3 mimetic S1 induces cell death in several types of tumor cells, it is unclear whether it induces death in drug-resistant cells. Herein, we found that S1 induced endoplasmic reticulum (ER) stress-associated apoptosis in both SKOV3 and SKOV3/DDP cells. S1 activated autophagy at early time points in SKOV3/DDP cells, and inhibition of autophagy increased ER stress-associated apoptosis. Collectively, our data indicate that autophagy plays a protective role, but it cannot protect against S1-induced cell death in cisplatin-resistant SKOV3/DDP cells.

[Case-control studies on double bundle posterior cruciate ligament reconstruction with remnant fiber preservation].

OBJECTIVE: To study the clinical efficacy of double bundle posterior cruciate ligament (PCL) reconstruction with remnant preservation. METHODS: From January 2007 to November 2011, 50 patients with PCL rupture met the inclusion criteria were divided into two groups: remnant preservation group (RP group) and remnant resection group (RR group). There were 19 males and 7 females in the RP group, ranging in age from 18 to 55 years, with a mean of (32.250 +/- 11.085) years old. The duration from injury to operation ranged from 2 to 66 months, with an average of (17.481 +/- 3.568) months. Among the RR group, 17 patients were male and 7 patients were female, ranging in age from 20 to 54 years old, with an average of (31.458 +/- 9.569) years. The duration from injury to operation ranged from 3 to 72 months, with a mean of (19.354 +/- 3.950) months. The patients in both groups suffered from instability of knee joint, got a positive result of posterior drawer test. In the RP group, the intercondylar notch remnant fiber, scar tissue and synovial were preserved in operation, only the free ligament in the intercondylar notch was resected. In the RR group, the remnant fiber, scar tissue and synovial tissue of adhesive parts were resected. In both groups, autologous semitendinosus and gracilis tendon double-bundle PCL reconstruction were carried out, the tibia was fixed with an absorbable interference screw with post-tie fixation, and the femur side was compositely fixed with absorbable interference screws and suspending fixation. Each patient received both subjective assessment (IKDC subjective evaluation, Lysholm scoring and Cincinnati rating) and objective clinical assessment (IKDC objective evaluation and Kneelax 3 tibia backward measurement) before operation and two years after operation. RESULTS: IKDC subjective evaluation: 92.167 +/- 4.177 in the RP group,which was higher than 87.542 +/- 5.687 in the RR group (P = 0.010). Lysholm scores: 90.917 +/- 4.413 in the RP group, which was higher than 87.083 +/- 5.149 in the RR group (P = 0.027). Cincinnati knee scores: 92.125 +/- 4.003 in the RP group, which was higher than 87.791 +/- 6.665 in the RR group (P = 0.027). IKDC objective evaluation:no significant statistical differences between RP group and RR group. Kneelax 3 assessment : tibia backward test with Kneelax 3 under 132 N showed no significant statistical difference between RP group and RR group, which were (3.958 +/- 0.693) mm and (4.029 +/- 0.846) mm respectively (P = 0.795). CONCLUSION: The study shows a significant advantage of remnant fiber preservation than remnant fiber resection in double-bundle PCL construction in terms of subjective knee function recovery after operation. There is no significant difference in postoperative knee stability.

Macrophages overexpressing Aire induce CD4+Foxp3+ T cells.

Aire plays an important role in central immune tolerance by regulating the transcription of thousands of genes. However, the role of Aire in the peripheral immune system is poorly understood. Regulatory T (Treg) cells are considered essential for the maintenance of peripheral tolerance, but the effect of Aire on Treg cells in the peripheral immune system is currently unknown. In this study, we investigated the effects of macrophages overexpressing Aire on CD4+Foxp3+ Treg cells by co-culturing Aire-overexpressing RAW264.7 cells or their supernatant with splenocytes. The results show that macrophages overexpressing Aire enhanced the expression of Foxp3 mRNA and induced different subsets of Treg cells in splenocytes through cell-cell contact or a co-culture supernatants. TGF-ß is a key molecule in the increases of CD4+CD45RA+Foxp3hi T cell and activating Treg (aTreg) levels observed following cell­supernatant co-culturing. Subsets of Treg cells were induced by Aire-overexpressing macrophages, and the manipulation of Treg cells by the targeting of Aire may provide a method for the treatment of inflammatory or autoimmune diseases.

Foxp3 expression in A549 cells is regulated by Toll-like receptor 4 through nuclear factor-κB.

Foxp3 is a master regulator of the development and function of CD4+CD25+ regulatory T cells (Tregs). Previous studies have reported that Foxp3 is also expressed in tumour cells and promotes tumour immune evasion. However, the regulation of the expression of Foxp3 in tumour cells remains unclear. Toll-like receptor 4 (TLR4), a member of the pattern recognition receptor family, is also expressed in tumour cells. Previous studies have found that the TLR4 signaling pathway is involved in tumour immune evasion in lung cancer cells, and that the transcription factor, nuclear factor-κB (NF-κB), plays a key role in the TLR4 signaling pathway. Moreover, recent studies found that NF-κB promotes the transcription of Foxp3. We hypothesised that TLR4 may also be involved in the regulation of Foxp3 in A549 cells through NF-κB. Therefore, we examined the effect of TLR4 and NF-κB on the expression of Foxp3 in the A549 lung cancer cell line. The results showed that Foxp3 and TLR4 are expressed in A549 cells; the expression of Foxp3 increased after lipopolysaccharide (LPS) stimulation and decreased after blocking the TLR4 signaling pathway. In addition, the expression of NF-κB (p65) increased after LPS stimulation and the expression of Foxp3 decreased after blocking NF-κB. These results suggest that TLR4 is involved in the regulation of Foxp3 in A549 cells through NF-κB.

Overexpressing autoimmune regulator regulates the expression of toll-like receptors by interacting with their promoters in RAW264.7 cells.

Autoimmune regulator (Aire) is a transcriptional activator that regulates the ectopic expression of many tissue-restricted antigens in medullary thymic epithelial cells, and that has an important role in the negative selection of autoreactive T cells. However, the roles of Aire expression in peripheral lymphoid tissues and hematopoietic cells, especially monocytes/macrophages, remain poorly understood. In this study, we found that the mRNA and protein expression levels of toll-like receptor (TLR)1, TLR3, and TLR8 were notably up-regulated in a mouse macrophage-like cell line (RAW264.7) stably expressing Aire, while the expression of TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9 were not significantly changed. In addition, the mRNA expression of TLR3 and TLR8 were significantly increased in primary peritoneal macrophages transiently transfected with Aire. Using chromatin immunoprecipitation and a luciferase activity assay, we also found that Aire interacted with the TLR1, TLR3, and TLR8 promoters and increased the luciferase transcriptional activity of these promoters in RAW264.7 cells. Moreover, after stimulation by Pam(3)CSK(4), a TLR1 ligand, and poly(I:C), a TLR3 ligand, we found that the mRNA expression levels of IL-1α, TNFα, iNOS, and IFNα were increased in RAW264.7 cells stably expressing Aire. Together, these data suggest that Aire has a crucial role in the recognition of pathogenic microorganisms and peripheral immune tolerance in antigen-presenting cells (APCs) by regulating the expression of TLRs.