Peripheral-derived regulatory T cells contribute to tumor-mediated immune suppression in a nonredundant manner.

Identifying tumor-mediated mechanisms that impair immunity is instrumental for the design of new cancer therapies. Regulatory T cells (Tregs) are a key component of cancer-derived immune suppression; however, these lymphocytes are necessary to prevent systemic autoimmunity in mice and humans, and thus, direct targeting of Tregs is not a clinical option for cancer patients. We have previously demonstrated that excising transcription factor Kruppel-like factor 2 (Klf2) within the T cell lineage blocks the generation of peripheral-derived Tregs (pTregs) without impairing production of thymic-derived Tregs. Using this mouse model, we have now demonstrated that eliminating pTregs is sufficient to delay/prevent tumor malignancy without causing autoimmunity. Cancer-bearing mice that expressed KLF2 converted tumor-specific CD4+ T cells into pTregs, which accumulated in secondary lymphoid organs and impaired further T cell effector activity. In contrast, pTreg-deficient mice retained cancer-specific immunity, including improved T cell infiltration into "cold" tumors, reduced T cell exhaustion in tumor beds, restricted generation of tumor-associated myeloid-derived suppressor cells, and the continued production of circulating effector T cells that arose in a cancer-dependent manner. Results indicate that tumor-specific pTregs are critical for early stages of cancer progression and blocking the generation of these inhibitory lymphocytes safely delays/prevents malignancy in preclinical models of melanoma and prostate cancer.

Wnt9 directs zebrafish heart tube assembly via a combination of canonical and non-canonical pathway signaling.

During zebrafish heart formation, cardiac progenitor cells converge at the embryonic midline where they form the cardiac cone. Subsequently, this structure transforms into a heart tube. Little is known about the molecular mechanisms that control these morphogenetic processes. Here, we use light-sheet microscopy and combine genetic, molecular biological and pharmacological tools to show that the paralogous genes wnt9a/b are required for the assembly of the nascent heart tube. In wnt9a/b double mutants, cardiomyocyte progenitor cells are delayed in their convergence towards the embryonic midline, the formation of the heart cone is impaired and the transformation into an elongated heart tube fails. The same cardiac phenotype occurs when both canonical and non-canonical Wnt signaling pathways are simultaneously blocked by pharmacological inhibition. This demonstrates that Wnt9a/b and canonical and non-canonical Wnt signaling regulate the migration of cardiomyocyte progenitor cells and control the formation of the cardiac tube. This can be partly attributed to their regulation of the timing of cardiac progenitor cell differentiation. Our study demonstrates how these morphogens activate a combination of downstream pathways to direct cardiac morphogenesis.

Tissue-Resident Macrophages Are Locally Programmed for Silent Clearance of Apoptotic Cells.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Med1 controls thymic T-cell migration into lymph node through enhancer-based Foxo1-Klf2 transcription program.

The migration is the key step for thymic T cells to enter circulation and then lymph nodes (LNs), essential for future immune surveillance. Although promoter-based transcriptional regulation through Foxo1, Klf2, Ccr7, and Sell regulates T-cell migration, it remains largely unexplored whether and how enhancers are involved in this process. Here we found that the conditional deletion of Med1, a component of the mediator complex and a mediator between enhancers and RNA polymerase II, caused a reduction of both CD4+ and CD8+ T cells in LNs, as well as a decrease of CD8+ T cells in the spleen. Importantly, Med1 deletion hindered the migration of thymic αßT cells into the circulation and then into LNs, accompanied by the downregulation of KLF2, CCR7, and CD62L. Mechanistically, Med1 promotes Klf2 transcription by facilitating Foxo1 binding to the Klf2 enhancer. Furthermore, forced expression of Klf2 rescued Ccr7 and Sell expression, as well as αßT-cell migration into LNs. Collectively, our study unveils a crucial role for Med1 in regulating the enhancer-based Foxo1-Klf2 transcriptional program and the migration of αßT cells into LNs, providing valuable insights into the molecular mechanisms underlying T-cell migration.

Krüppel-like factor 2 is an endoprotective transcription factor in diabetic kidney disease.

Diabetic kidney disease (DKD) is a microvascular complication of diabetes, and glomerular endothelial cell (GEC) dysfunction is a key driver of DKD pathogenesis. Krüppel-like factor 2 (KLF2), a shear stress-induced transcription factor, is among the highly regulated genes in early DKD. In the kidney, KLF2 expression is mostly restricted to endothelial cells, but its expression is also found in immune cell subsets. KLF2 expression is upregulated in response to increased shear stress by the activation of mechanosensory receptors but suppressed by inflammatory cytokines, both of which characterize the early diabetic kidney milieu. KLF2 expression is reduced in progressive DKD and hypertensive nephropathy in humans and mice, likely due to high glucose and inflammatory cytokines such as TNF-α. However, KLF2 expression is increased in glomerular hyperfiltration-induced shear stress without metabolic dysregulation, such as in settings of unilateral nephrectomy. Lower KLF2 expression is associated with CKD progression in patients with unilateral nephrectomy, consistent with its endoprotective role. KLF2 confers endoprotection by inhibition of inflammation, thrombotic activation, and angiogenesis, and thus KLF2 is considered a protective factor for cardiovascular disease (CVD). Based on similar mechanisms, KLF2 also exhibits renoprotection, and its reduced expression in endothelial cells worsens glomerular injury and albuminuria in settings of diabetes or unilateral nephrectomy. Thus KLF2 confers endoprotective effects in both CVD and DKD, and its activators could potentially be developed as a novel class of drugs for cardiorenal protection in diabetic patients.

The role of KLF2 in regulating hepatic lipogenesis and blood cholesterol homeostasis via the SCAP/SREBP pathway.

Liver steatosis is a common metabolic disorder resulting from imbalanced lipid metabolism, which involves various processes such as de novo lipogenesis, fatty acid uptake, fatty acid oxidation, and VLDL secretion. In this study, we discovered that KLF2, a transcription factor, plays a crucial role in regulating lipid metabolism in the liver. Overexpression of KLF2 in the liver of db/db mice, C57BL/6J mice, and Cd36-/- mice fed on a normal diet resulted in increased lipid content in the liver. Additionally, transgenic mice (ALB-Klf2) that overexpressed Klf2 in the liver developed liver steatosis after being fed a normal diet. We found that KLF2 promotes lipogenesis by increasing the expression of SCAP, a chaperone that facilitates the activation of SREBP, the master transcription factor for lipogenic gene expression. Our mechanism studies revealed that KLF2 enhances lipogenesis in the liver by binding to the promoter of SCAP and increasing the expression of genes involved in fatty acid synthesis. Reduction of KLF2 expression led to a decrease in SCAP expression and a reduction in the expression of SREBP1 target genes involved in lipogenesis. Overexpression of KLF2 also increased the activation of SREBP2 and the mRNA levels of its downstream target SOAT1. In C57BL/6J mice fed a high-fat diet, overexpression of Klf2 increased blood VLDL secretion, while reducing its expression decreased blood cholesterol levels. Our study emphasizes the novelty that hepatic KLF2 plays a critical role in regulating lipid metabolism through the KLF2/SCAP/SREBPs pathway, which is essential for hepatic lipogenesis and maintaining blood cholesterol homeostasis.

The effects of simvastatin-loaded nanoliposomes on human multilineage liver fibrosis microtissue.

In this in vitro study, for the first time, we evaluate the effects of simvastatin-loaded liposome nanoparticles (SIM-LipoNPs) treatment on fibrosis-induced liver microtissues, as simvastatin (SIM) has shown potential benefits in the non-alcoholic fatty liver disease process. We developed multicellular liver microtissues composed of hepatic stellate cells, hepatoblastoma cells and human umbilical vein endothelial cells. The microtissues were supplemented with a combination of palmitic acid and oleic acid to develop fibrosis models. Subsequently, various groups of microtissues were exposed to SIM and SIM-LipoNPs at doses of 5 and 10 mg/mL. The effectiveness of the treatments was evaluated by analysing cell viability, production of reactive oxygen species (ROS) and nitric oxide (NO), the expression of Kruppel-like factor (KLF) 2, and pro-inflammatory cytokines (interleukin(IL)-1 α, IL-1 ß, IL-6 and tumour necrosis factor-α), and the expression of collagen I. Our results indicated that SIM-LipoNPs application showed promising results. SIM-LipoNPs effectively amplified the SIM-klf2-NO pathway at a lower dosage compatible with a high dosage of free SIM, which also led to reduced oxidative stress by decreasing ROS levels. SIM-LipoNPs administration also resulted in a significant reduction in pro-inflammatory cytokines and Collagen I mRNA levels, as a marker of fibrosis. In conclusion, our study highlights the considerable therapeutic potential of using SIM-LipoNPs to prevent liver fibrosis progress, underscoring the remarkable properties of SIM-LipoNPs in activating the KLF2-NO pathway and anti-oxidative and anti-inflammatory response.

UM171 suppresses breast cancer progression by inducing KLF2.

PURPOSE: Breast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action. METHODS: The inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells. RESULTS: UM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture. CONCLUSION: These results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

Sustainability of shear stress conditioning in endothelial colony-forming cells compared to human aortic endothelial cells to underline suitability for tissue-engineered vascular grafts.

The endothelialization of cardiovascular implants is supposed to improve the long-term patency of these implants. In addition, in previous studies, it has been shown, that the conditioning of endothelial cells by dynamic cultivation leads to the expression of an anti-thrombogenic phenotype. For the creation of a tissue-engineered vascular graft (TEVG), these two strategies were combined to achieve optimal hemocompatibility. In a clinical setup, this would require the transfer of the already endothelialized construct from the conditioning bioreactor to the patient. Therefore, the reversibility of the dynamic conditioning of the endothelial cells with arterial-like high shear stress (20 dyn/cm2) was investigated to define the timeframe (tested in a range of up to 24 h) for the perseverance of dynamically induced phenotypical changes. Two types of endothelial cells were compared: endothelial colony-forming cells (ECFCs) and human aortic endothelial cells (HAECs). The results showed that ECFCs respond far more sensitively and rapidly to flow than HAECs. The resulting cell alignment and increased protein expression of KLF-2, Notch-4, Thrombomodulin, Tie2 and eNOS monomer was paralleled by increased eNOS and unaltered KLF-2 mRNA levels even under stopped-flow conditions. VCAM-1 mRNA and protein expression was downregulated under flow and did not recover under stopped flow. From these time kinetic results, we concluded, that the maximum time gap between the TEVG cultivated with autologous ECFCs in future reactor cultivations and the transfer to the potential TEVG recipient should be limited to ∼6 h.

TET1 inhibits liver fibrosis by blocking hepatic stellate cell activation.

Hepatic stellate cells (HSCs) are critical regulator contributing to the onset and progression of liver fibrosis. Chronic liver injury triggers HSCs to undergo vast changes and trans-differentiation into a myofibroblast HSCs, the mechanism remains to be elucidated. This study investigated that the involvement of hydroxymethylase TET1 (ten-eleven translocation 1) in HSC activation and liver fibrosis. It is revealed that TET1 levels were downregulated in the livers in mouse models of liver fibrosis and patients with cirrhosis, as well as activated HSCs in comparison to quiescent HSCs. In vitro data showed that the inhibition of TET1 promoted the activation HSC, whereas TET1 overexpression inhibited HSC activation. Moreover, TET1 could regulate KLF2 (Kruppel-like transcription factors) transcription by promoting hydroxymethylation of its promoter, which in turn suppressed the activation of HSCs. In vivo, it is confirmed that liver fibrosis was aggravated in Tet1 knockout mice after CCl4 injection, accompanied by excessive activation of primary stellate cells, in contrast to wild-type mice. In conclusion, we suggested that TET1 plays a significant role in HSC activation and liver fibrosis, which provides a promising target for anti-fibrotic therapies.

Netrin-1 protects blood-brain barrier (BBB) integrity after cerebral ischemia-reperfusion by activating the Kruppel-like factor 2 (KLF2)/occludin pathway.

Ischemia/reperfusion (I/R)-induced neural damage and neuroinflammation have been associated with pathological progression during stroke. Netrin-1 is an important member of the family of laminin-related secreted proteins, which plays an important role in governing axon elongation. However, it is unknown whether Netrin-1 possesses a beneficial role in stroke. Here, we employed the middle cerebral artery occlusion (MCAO) model to study the function of Netrin-1 in alleviating brain injuries. Our results demonstrate that Netrin-1 rescued poststroke neurological deficits and inhibited production of the inflammatory cytokines such as interleukin 6 (IL-6) and endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1). Importantly, Netrin-1 protected against MCAO-induced dysfunction of the blood-brain barrier (BBB) in mice and a reduction in the expression of the tight junction (TJ) protein occludin. Additionally, we report that Netrin-1 could ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury and prevent aggravation in endothelial monolayer permeability in bEnd.3 human brain microvascular endothelial cells (HBMVECs). Mechanistically, Netrin-1 ameliorated OGD/R-induced decrease in occludin and Kruppel-like factor 2 (KLF2) in HBMVECs. Notably, silencing of KLF2 abolished the beneficial effects of Netrin-1 in protecting endothelial permeability and occludin expression, suggesting that these effects are mediated by KLF2. In conclusion, our findings suggest that Netrin-1 could constitute a novel therapeutic strategy for ischemic stroke.

Hemodynamics During Development and Postnatal Life.

A well-developed heart is essential for embryonic survival. There are constant interactions between cardiac tissue motion and blood flow, which determine the heart shape itself. Hemodynamic forces are a powerful stimulus for cardiac growth and differentiation. Therefore, it is particularly interesting to investigate how the blood flows through the heart and how hemodynamics is linked to a particular species and its development, including human. The appropriate patterns and magnitude of hemodynamic stresses are necessary for the proper formation of cardiac structures, and hemodynamic perturbations have been found to cause malformations via identifiable mechanobiological molecular pathways. There are significant differences in cardiac hemodynamics among vertebrate species, which go hand in hand with the presence of specific anatomical structures. However, strong similarities during development suggest a common pattern for cardiac hemodynamics in human adults. In the human fetal heart, hemodynamic abnormalities during gestation are known to progress to congenital heart malformations by birth. In this chapter, we discuss the current state of the knowledge of the prenatal cardiac hemodynamics, as discovered through small and large animal models, as well as from clinical investigations, with parallels gathered from the poikilotherm vertebrates that emulate some hemodynamically significant human congenital heart diseases.

Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling.

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.

Comprehensive analysis of KLF2 as a prognostic biomarker associated with fibrosis and immune infiltration in advanced hepatocellular carcinoma.

PURPOSE: Most Hepatocellular carcinoma (HCC) patients are in advanced or metastatic stage at the time of diagnosis. Prognosis for advanced HCC patients is dismal. This study was based on our previous microarray results, and aimed to explore the promising diagnostic and prognostic markers for advanced HCC by focusing on the important function of KLF2. METHODS: The Cancer Genome Atlas (TCGA), Cancer Genome Consortium database (ICGC), and the Gene Expression Comprehensive Database (GEO) provided the raw data of this study research. The cBioPortal platform, CeDR Atlas platform, and the Human Protein Atlas (HPA) website were applied to analyze the mutational landscape and single-cell sequencing data of KLF2. Basing on the results of single-cell sequencing analyses, we further explored the molecular mechanism of KLF2 regulation in the fibrosis and immune infiltration of HCC. RESULTS: Decreased KLF2 expression was discovered to be mainly regulated by hypermethylation, and indicated a poor prognosis of HCC. Single-cell level expression analyses revealed KLF2 was highly expressed in immune cells and fibroblasts. The function enrichment analysis of KLF2 targets indicated the crucial association between KLF2 and tumor matrix. 33-genes related with cancer associated fibroblasts (CAFs) were collected to identify the significant association of KLF2 with fibrosis. And SPP1 was validated as a promising prognostic and diagnostic marker for advanced HCC patients. CXCR6 CD8+ T cells were noted as a predominant proportion in the immune microenvironment, and T cell receptor CD3D was discovered to be a potential therapeutic biomarker for HCC immunotherapy. CONCLUSION: This study identified that KLF2 is an important factor promoting HCC progression by affecting the fibrosis and immune infiltration, highlighting its great potential as a novel prognostic biomarker for advanced HCC.

Mechanism of tumor-derived extracellular vesicles in prostatic cancer progression through the circFMN2/KLF2/RNF128 axis.

Circular RNAs (circRNAs) are a major type of cargos encapsulated in extracellular vesicles (EVs) and regulate the progression of prostatic cancer (PC). This study was conducted to explore the role of tumor-derived EVs in PC cell proliferation, invasion, and migration via shuttle of circRNA formin 2 (circFMN2). RT-qPCR or Western blot assay showed that circFMN2 was upregulated while KLF2 and RNF128 were downregulated in PC tissues and cells. EVs were separated from PC cells and characterized and its internalization in PC cells was examined, which suggested that PC-EVs mediated the shuttle of circFMN2 to upregulate circFMN2 expression in PC cells. PC cell functions were determined by cell counting kit-8, colony formation and Transwell assays, which suggested that PC-EVs fueled the proliferation, invasion, and migration of PC cells. At cellular level, PC-EVs mediated the shuttle of circFMN2 to upregulate circFMN2 expression in PC cells, and circFMN2 binding to HuR decreased the HuR-KLF2 interaction and repressed KLF2 expression, which further reduced the KLF2-RNF128 promoter binding and repressed RNF128 transcription. Overexpression of KLF2/RNF128 ablated the effects of PC-EVs on the proliferation, invasion, and migration of PC cells. The xenograft tumor models and lung/liver metastasis models were established and revealed that PC-EVs accelerated tumorigenesis and metastasis in vivo via delivery of circFMN2 and repression of KLF2/RNF128.

Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling.

Kruppel-like factor 2 (KLF2) has been linked with fibrosis and neutrophil-associated thromboinflammation; however, its role in COVID-19 remains elusive. We investigated the effect of disease microenvironment on the fibrotic potential of human lung fibroblasts (LFs) and its association with KLF2 expression. LFs stimulated with plasma from severe COVID-19 patients down-regulated KLF2 expression at mRNA/protein and functional level acquiring a pre-fibrotic phenotype, as indicated by increased CCN2/collagen levels. Pre-incubation with the COMBI-treatment-agents (DNase I and JAKs/IL-6 inhibitors baricitinib/tocilizumab) restored KLF2 levels of LFs to normal abolishing their fibrotic activity. LFs stimulated with plasma from COMBI-treated patients at day-7 expressed lower CCN2 and higher KLF2 levels, compared to plasma prior-to-treatment, an effect not observed in standard-of-care treatment. In line with this, COMBI-treated patients had better outcome than standard-of-care group. These data link fibroblast KLF2 with NETosis and JAK/IL-6 signaling, suggesting the potential of combined therapeutic strategies in immunofibrotic diseases, such as COVID-19.

Prmt5 deficiency inhibits CD4+ T-cell Klf2/S1pr1 expression and ameliorates EAE disease.

BACKGROUND: Protein arginine methyltransferase 5 (Prmt5) is the main type II methyltransferase, catalyzes protein arginine residue symmetric dimethylation, and modulates normal cellular physiology and disease progression. Prmt5 inhibition or deletion in CD4+ T cells has been reported to ameliorate experimental autoimmune encephalomyelitis (EAE), but the detailed molecular mechanisms have not yet been elucidated. METHODS: EAE was induced by administration of myelin oligodendrocyte glycoprotein (MOG35-55) in T cells Prmt5 conditional knockout (CD4-cre-Prmt5fl/fl, Prmt5cko) and Prmt5fl/fl (WT) mice. Flow cytometry, single-cell RNA sequencing, ATAC sequencing and chromatin immunoprecipitation assay (ChIP) approaches were used to explore the detail mechanisms. RESULTS: We find that Prmt5cko mice are resistant to EAE; infiltrating inflammatory CD4+ T cells in the central nervous system (CNS) are greatly reduced. However, in Prmt5cko mice, T cells in the spleen show much more proliferation and activation properties, the total number of CD4+ T cells in the spleen is not reduced, and the percentage of Rora+ CD4+ T cells is elevated. Also, CD4+ T cells express lower levels of S1pr1 and Klf2 than WT mice, which may influence pathogenic CD4+ T-cell egress from the spleen and migration to the CNS. Moreover, the single-cell ATAC sequence and ChIP assay reveal that the transcription factor Klf2 is enriched at the S1pr1 promoter and that Klf2 motif activity is reduced in Prmt5cko mice. CONCLUSIONS: Our study delineates the undiscovered role of Prmt5 in T-cell biology in which Prmt5 may inhibit Klf2-S1pr1 pathway to ameliorate EAE disease. Controlling T-cell Prmt5 expression may be helpful for the treatment of autoimmune diseases.

PRC2 mediated KLF2 down regulation: a therapeutic and diagnostic axis during tumor progression.

Surgery and chemo-radiotherapy are used as the common first-line treatment options in many cancers. However, tumor relapse is observed in many cancer patients following such first-line treatments. Therefore, targeted therapy according to the molecular cancer biology can be very important in reducing tumor recurrence. In this regard, a wide range of monoclonal antibodies against the growth factors and their receptors can offer more targeted treatment in cancer patients. However, due to the importance of growth factors in the normal biology of body cells, side effects can also be observed following the application of growth factor inhibitors. Therefore, more specific factors should be introduced as therapeutic targets with less side effects. Krüppel-like factors 2 (KLF2) belongs to the KLF family of transcription factors that are involved in the regulation of many cellular processes. KLF2 deregulations have been also reported during the progression of many tumors. In the present review we discussed the molecular mechanisms of KLF2 during tumor growth and invasion. It has been shown that the KLF2 as a tumor suppressor is mainly inhibited by the non-coding RNAs (ncRNAs) through the polycomb repressive complex 2 (PRC2) recruitment. This review is an effective step towards introducing the KLF2 as a suitable diagnostic and therapeutic target in cancer patients.

Lovastatin inhibits erythroleukemia progression through KLF2-mediated suppression of MAPK/ERK signaling.

BACKGROUND: Lovastatin, an HMG-CoA inhibitor and an effective cholesterol lowering drug, exhibits anti-neoplastic activity towards several types of cancer, although the underlying mechanism is still not fully understood. Herein, we investigated mechanism of growth inhibition of leukemic cells by lovastatin. METHODS: RNAseq analysis was used to explore the effect of lovastatin on gene expression in leukemic cells. An animal model of leukemia was used to test the effect of this statin in vivo. FAM83A and DDIT4 expression was knocked-downed in leukemia cells via lentivirus-shRNA. Western blotting, RT-qPCR, cell cycle analysis and apoptosis assays were used to determine the effect of lovastatin-induced growth suppression in leukemic cells in vitro. RESULTS: Lovastatin treatment strongly inhibited cancer progression in a mouse model of erythroleukemia induced by Friend virus. In tissue culture, lovastatin inhibited cell proliferation through induction of G1 phase cell cycle arrest and apoptosis. Interestingly, lovastatin induced most known genes associated with cholesterol biosynthesis in leukemic cells. Moreover, it suppressed ERK1/2 phosphorylation by downregulating FAM83A and DDIT4, two mediators of MAP-Kinase signaling. RNAseq analysis of lovastatin treated leukemic cells revealed a strong induction of the tumor suppressor gene KLF2. Accordingly, lentivirus-mediated knockdown of KLF2 antagonized leukemia cell suppression induced by lovastatin, associated with higher ERK1/2 phosphorylation compared to control. We further show that KLF2 induction by lovastatin is responsible for lower expression of the FAM83A and DDIT4 oncogenes, involved in the activation of ERK1/2. KLF2 activation by lovastatin also activated a subset of cholesterol biosynthesis genes that may further contribute to leukemia suppression. CONCLUSIONS: These results implicate KLF2-mediated FAM83A/DDIT4/MAPK suppression and activation of cholesterol biosynthesis as the mechanism of leukemia cell growth inhibition by lovastatin.