Leukemia inhibitory factor drives transcriptional programs that promote lipid accumulation and M2 polarization in macrophages.

Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, plays a central role in homeostasis and disease. Interestingly, some of the pleiotropic effects of LIF have been attributed to the modulation of macrophage functions although the molecular underpinnings have not been explored at a genome-wide scale. Herein, we investigated LIF-driven transcriptional changes in murine bone marrow-derived macrophages (BMDM) by RNA-seq. In silico analyses revealed a selective and time-dependent remodelling of macrophage gene expression programs associated with lipid metabolism and cell activation. Accordingly, a subset of LIF-upregulated transcripts related to cholesterol metabolism and lipid internalization was validated by RT-qPCR. This was accompanied by a LIF-enhanced capacity for lipid accumulation in macrophages upon incubation with oxidated low-density lipoprotein (Ox-LDL). Mechanistically, LIF triggered the phosphorylation (Y705 and S727) and nuclear translocation of the transcription factor STAT3 in BMDM. Consistent with this, Ingenuity Pathway Analysis (IPA) identified STAT3 as an upstream regulator of a subset of transcripts, including Il4ra, in LIF-treated macrophages. Notably, LIF priming enhanced BMDM responses to IL-4-mediated M2 polarization (i.e., increased arginase activity and accumulation of transcripts encoding for M2 markers). Conversely, LIF stimulation had no significant effect in BMDM responses to M1 polarizing stimuli (IFNγ and LPS). Thus, our study provides insight into the transcriptional landscape of LIF-treated macrophages, shedding light on its role in lipid metabolism and M2 polarization responses. A better understanding of the regulatory mechanisms governing LIF-driven changes might help informing novel therapeutic approaches aiming to reprogram macrophage phenotypes in diseased states (e.g., cancer, atherosclerosis, infection, etc.).

Serum leukemia inhibitory factor (LIF) levels in patients with Takayasu's and Giant cell arteritis: A cross-sectional study.

INTRODUCTION AND OBJECTIVES: In this study, we aimed to evaluate LIF levels and its possible relationship with disease activity in patients with Takayasu's (TAK) and Giant cell arteritis (GCA) patients. MATERIALS AND METHODS: 23 Takayasu's arteritis, 9 Giant cell arteritis patients and 25 healthy volunteers were included in the study. Serum LIF levels were measured ELISA. RESULTS: The mean age of Giant cell arteritis patients was statistically significantly higher than the other groups (p<0.001). The rate of women was found to be higher in Takayasu's arteritis (p=0.021). When healthy control, patients with GCA and Takayasu arteritis were compared, there was a difference in LIF values (p=0.018). In subgroup analyzes, LIF values were found to be higher in GCA patients compared to healthy controls (p<0.05). There was no statistically significant correlation between LIF and CRP (Rho=-0.038, p=0.778), ESR (Rho=0.114, p=0.399) and ITAS (Rho=-0.357, p=0.094). While CRP was statistically significantly higher in patients with disease activity (p=0.003), there was no statistically significant difference between patients in terms of ESR and LIF values. While there was a statistically significant relationship between CRP (OR=1.19 [1.03-1.37], p=0.018) and disease activity in univariate analyses, no statistically significant variable was found in multivariable analyses. CONCLUSIONS: LIF values were significantly higher in patients with Giant cell arteritis compared to healthy controls.

Augmentative effects of leukemia inhibitory factor reveal a critical role for TYK2 signaling in vascular calcification.

Medial vascular calcification in chronic kidney disease (CKD) involves pro-inflammatory pathways induced by hyperphosphatemia. Several interleukin 6 family members have been associated with pro-calcific effects in vascular smooth muscle cells (VSMCs) and are considered as therapeutic targets. Therefore, we investigated the role of leukemia inhibitory factor (LIF) during VSMC calcification. LIF expression was found to be increased following phosphate exposure of VSMCs. LIF supplementation aggravated, while silencing of endogenous LIF or LIF receptor (LIFR) ameliorated the pro-calcific effects of phosphate in VSMCs. The soluble LIFR mediated antagonistic effects towards LIF and reduced VSMC calcification. Mechanistically, LIF induced phosphorylation of the non-receptor tyrosine-protein kinase 2 (TYK2) and signal transducer and activator of transcription-3 (STAT3) in VSMCs. TYK2 inhibition by deucravacitinib, a selective, allosteric oral immunosuppressant used in psoriasis treatment, not only blunted the effects of LIF, but also interfered with the pro-calcific effects induced by phosphate. Conversely, TYK2 overexpression aggravated VSMC calcification. Ex vivo calcification of mouse aortic rings was ameliorated by Tyk2 pharmacological inhibition and genetic deficiency. Cholecalciferol-induced vascular calcification in mice was improved by Tyk2 inhibition and in the Tyk2-deficient mice. Similarly, calcification was ameliorated in Abcc6/Tyk2-deficient mice after adenine/high phosphorus-induced CKD. Thus, our observations indicate a role for LIF in CKD-associated vascular calcification. Hence, the effects of LIF identify a central pro-calcific role of TYK2 signaling, which may be a future target to reduce the burden of vascular calcification in CKD.

The role of interleukin-6 family cytokines in cancer cachexia.

Cachexia is a wasting syndrome that manifests in more than half of all cancer patients. Cancer-associated cachexia negatively influences the survival of patients and their quality of life. It is characterized by a rapid loss of adipose and skeletal muscle tissues, which is partly mediated by inflammatory cytokines. Here, we explored the crucial roles of interleukin-6 (IL-6) family cytokines, including IL-6, leukemia inhibitory factor, and oncostatin M, in the development of cancer cachexia. These cytokines have been shown to exacerbate cachexia by promoting the wasting of adipose and muscle tissues, activating mechanisms that enhance lipolysis and proteolysis. Overlapping effects of the IL-6 family cytokines depend on janus kinase/signal transducer and activator of transcription 3 signaling. We argue that the blockade of these cytokine pathways individually may fail due to redundancy and future therapeutic approaches should target common downstream elements to yield effective clinical outcomes.

Altered Serum Proteins Suggest Inflammation, Fibrogenesis and Angiogenesis in Adult Patients with a Fontan Circulation.

Previous omics research in patients with complex congenital heart disease and single-ventricle circulation (irrespective of the stage of palliative repair) revealed alterations in cardiac and systemic metabolism, inter alia abnormalities in energy metabolism, and inflammation, oxidative stress or endothelial dysfunction. We employed an affinity-proteomics approach focused on cell surface markers, cytokines, and chemokines in the serum of 20 adult Fontan patients with a good functioning systemic left ventricle, and we 20 matched controls to reveal any specific processes on a cellular level. Analysis of 349 proteins revealed 4 altered protein levels related to chronic inflammation, with elevated levels of syndecan-1 and glycophorin-A, as well as decreased levels of leukemia inhibitory factor and nerve growth factor-ß in Fontan patients compared to controls. All in all, this means that Fontan circulation carries specific physiological and metabolic instabilities, including chronic inflammation, oxidative stress imbalance, and consequently, possible damage to cell structure and alterations in translational pathways. A combination of proteomics-based biomarkers and the traditional biomarkers (uric acid, γGT, and cholesterol) performed best in classification (patient vs. control). A metabolism- and signaling-based approach may be helpful for a better understanding of Fontan (patho-)physiology. Syndecan-1, glycophorin-A, leukemia inhibitory factor, and nerve growth factor-ß, especially in combination with uric acid, γGT, and cholesterol, might be interesting candidate parameters to complement traditional diagnostic imaging tools and the determination of traditional biomarkers, yielding a better understanding of the development of comorbidities in Fontan patients, and they may play a future role in the identification of targets to mitigate inflammation and comorbidities in Fontan patients.

Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation; however, the contribution of uterine gland structure to gland secretions, such as LIF, is not known. Here, we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma, and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a Müllerian duct Cre line, Pax2Cre, displays gland bud elongation but a failure in gland branching. Reduction of ESR1 in adult uterine epithelium using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of ESR1 and glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but mice deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium, even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

A novel termination site in a case of Stüve-Wiedemann syndrome: case report and review of literature.

Stüve-Wiedemann syndrome (SWS) is a rare autosomal recessive disorder that is characterized by bowing of long bones, dysautonomia, temperature dysregulation, swallowing and feeding difficulties, and frequent respiratory infections. Respiratory distress and hyperthermic events are the leading causes of early neonatal death, and most patients are not expected to survive past infancy. Here, we report on the survival of a 5-year-old male with SWS, discussing his case presentation, providing a brief clinical course, and discussing the outcome. This case adds to the literature surrounding rare instances of childhood survivors of SWS and raises awareness for this syndrome to facilitate an earlier recognition, intervention, and genetic counseling for the families, thereby improving understanding of this disease and the health outcomes for the children affected by this condition.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

BACKGROUND: Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). METHODS AND RESULTS: HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-ß. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). CONCLUSIONS: YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

Leukemia inhibitory factor protects against experimental periodontitis through immuno-modulations of both macrophages and periodontal ligament fibroblasts.

BACKGROUND: To explore the role of leukemia inhibitory factor (LIF) in periodontitis via in vivo and in vitro experiments. METHODS: The second upper molar of LIF knockout mice and their wild-type littermates were ligated for 8 days. Micro-computed tomography (micro-CT), histological analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. The expression levels of proinflammatory cytokines were examined in mouse bone marrow derived macrophages and human periodontal ligament fibroblasts (HPDLFs) after lipopolysaccharide (LPS) treatment. RESULTS: LIF deficiency promoted alveolar bone loss, inflammatory cells infiltration, osteoclasts formation and collagen fiber degradation in ligature-induced mouse, along with higher expressions of proinflammatory cytokines, including interleukin-6 (IL6), IL-1ß (IL1B), tumor necrosis factor-α (TNFA), matrix metalloproteinase 13 (MMP13), and RANKL/OPG ratio. Additionally, LIF deletion led to higher expression levels of these proinflammatory cytokines in mouse bone marrow-derived macrophages from both femur and alveolar bone and HPDLFs when treated with LPS. Administration of recombined LIF attenuated TNFA, IL1B, and RANKL/OPG ratio in HPDLFs. CONCLUSIONS: These findings indicate that LIF deficiency promotes the progress of periodontitis via modulating immuno-inflammatory responses of macrophages and periodontal ligament fibroblasts, and the application of LIF may be an adjunctive treatment for periodontitis to resolute inflammation.

LIF/JAK2/STAT1 Signaling Enhances Production of Galactose-Deficient IgA1 by IgA1-Producing Cell Lines Derived From Tonsils of Patients With IgA Nephropathy.

Introduction: Galactose-deficient IgA1 (Gd-IgA1) plays a key role in the pathogenesis of IgA nephropathy (IgAN). Tonsillectomy has been beneficial to some patients with IgAN, possibly due to the removal of tonsillar cytokine-activated cells producing Gd-IgA1. To test this hypothesis, we used immortalized IgA1-producing cell lines derived from tonsils of patients with IgAN or obstructive sleep apnea (OSA) and assessed the effect of leukemia inhibitory factor (LIF) or oncostatin M (OSM) on Gd-IgA1 production. Methods: Gd-IgA1 production was measured by lectin enzyme-linked immunosorbent assay; JAK-STAT signaling in cultured cells was assessed by immunoblotting of cell lysates; and validated by using small interfering RNA (siRNA) knock-down and small-molecule inhibitors. Results: IgAN-derived cells produced more Gd-IgA1 than the cells from patients with OSA, and exhibited elevated Gd-IgA1 production in response to LIF, but not OSM. This effect was associated with dysregulated STAT1 phosphorylation, as confirmed by STAT1 siRNA knock-down. JAK2 inhibitor, AZD1480 exhibited a dose-dependent inhibition of the LIF-induced Gd-IgA1 overproduction. Unexpectedly, high concentrations of AZD1480, but only in the presence of LIF, reduced Gd-IgA1 production in the cells derived from patients with IgAN to that of the control cells from patients with OSA. Based on modeling LIF-LIFR-gp130-JAK2 receptor complex, we postulate that LIF binding to LIFR may sequester gp130 and/or JAK2 from other pathways; and when combined with JAK2 inhibition, enables full blockade of the aberrant O-glycosylation pathways in IgAN. Conclusion: In summary, IgAN cells exhibit LIF-mediated overproduction of Gd-IgA1 due to abnormal signaling. JAK2 inhibitors can counter these LIF-induced effects and block Gd-IgA1 synthesis in IgAN.

Immunosuppressive role of BDNF in therapy-induced neuroendocrine prostate cancer.

Prostate stromal cells play a crucial role in the promotion of tumor growth and immune evasion in the tumor microenvironment (TME) through intricate molecular alterations in their interaction with prostate cancer (PCa) cells. While the impact of these cells on establishing an immunosuppressive response and influencing PCa aggressiveness remains incompletely understood. Our study shows that the activation of the leukemia inhibitory factor (LIF)/LIF receptor (LIFR) pathway in both prostate tumor and stromal cells, following androgen deprivation therapy (ADT), leads to the development of an immunosuppressive TME. Activation of LIF/LIFR signaling in PCa cells induces neuroendocrine differentiation (NED) and upregulates immune checkpoint expression. Inhibition of LIF/LIFR attenuates these effects, underscoring the crucial role of LIF/LIFR in linking NED to immunosuppression. Prostate stromal cells expressing LIFR contribute to NED and immunosuppressive marker abundance in PCa cells, while LIFR knockdown in prostate stromal cells reverses these effects. ADT-driven LIF/LIFR signaling induces brain-derived neurotrophic factor (BDNF) expression, which, in turn, promotes NED, aggressiveness, and immune evasion in PCa cells. Clinical analyses demonstrate elevated BDNF levels in metastatic castration-resistant PCa (CRPC) and a positive correlation with programmed death-ligand 1 (PDL1) and immunosuppressive signatures. This study shows that the crosstalk between PCa cells and prostate stromal cells enhances LIF/LIFR signaling, contributing to an immunosuppressive TME and NED in PCa cells through the upregulation of BDNF.

LncRNA H19 Participates in Leukemia Inhibitory Factor Mediated Stemness Promotion in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy and the third leading cause of cancer-related death worldwide. Cancer stem cells (CSCs) were considered to play important roles in the genesis and development of many tumors. In recent years, it has been observed that leukemia inhibitory factor (LIF) might be involved in the regulation of stemness in cancer cells. In this study, we observed that LIF could increase the spheroid formation and stemness marker expression (inculding Nanog and SOX2) in CRC cell lines, such as HCT116 and Caco2 cells. Meanwhile, we also observed that LIF could upregulate LncRNA H19 expression via PI3K/AKT pathway. Knockdown of the expression of LncRNA H19 could decrease the spheroid formation and SOX2 expression in LIF-treated HCT116 and Caco2 cells, and thereby LncRNA H19 knockdown could compensate for the stemness enhancement effects induced by LIF. Our results indicated that LncRNA H19 might participate in the stemness promotion of LIF in CRC cells.

LIF Aggravates Pulpitis by Promoting Inflammatory Response in Macrophages.

Leukemia inhibitory factor (LIF) has been recognized as a novel inflammatory modulator in inflammation-associated diseases. This study aimed to investigate the modulation of LIF in dental pulp inflammation. Experimental pulpitis was established in wild-type (WT) and Lif-deficient (Lif-/-) mice. Histological and immunostaining analyses were conducted to assess the role of LIF in the progression of pulpitis. Mouse macrophage cell line (RAW264.7) was treated with LPS to simulate an inflammatory environment. Exogenous LIF was added to this system to examine its modulation in macrophage inflammatory response in vitro. Primary bone marrow-derived macrophages (BMDMs) from WT and Lif-/- mice were isolated and stimulated with LPS to confirm the effect of Lif deletion on macrophage inflammatory response. Supernatants from LIF and LPS-treated human dental pulp cells (hDPCs) were collected and added to macrophages. Macrophage chemotaxis was assessed using transwell assays. The results showed an increased expression of LIF and LIFR with the progression of pulpitis, and LIFR was highly expressed in macrophages. Lif deficiency alleviated experimental pulpitis with the reduction of pro-inflammatory cytokines and macrophage infiltration. Exogenous LIF promoted inflammatory response of LPS-induced macrophages through a STAT3/p65-dependent pathway. Consistently, Lif deletion inhibited macrophage inflammatory response in vitro. Supernatants of LIF-treated hDPCs enhanced macrophage migration in LPS-induced inflammatory environment. Our findings demonstrated that LIF aggravates pulpitis by promoting macrophage inflammatory response through a STAT3/p65-dependent pathway. Furthermore, LIF plays a crucial role in driving the recruitment of macrophages to inflamed pulp tissue by promoting chemokine secretion in DPCs.

MDM2-p53 mediate a miR-181c-3p/LIF axis to regulate low dose-rate radiation-induced DNA damage in human B lymphocytes.

PURPOSE: Prolonged exposure to low dose-rate radiation (LDRR) is of growing concern to public health. Recent evidences indicates that LDRR causes deleterious health effects and is closely related to miRNAs. The aim of our study is to investigate the relationship between miRNAs and DNA damage caused by LDRR. MATERIALS AND METHODS: In this study, we irradiated C57BL/6J mice with 12.5µGy/h dose of γ ray emitted from uranium ore for 8 h a day for 120 days at a total dose of 12 mGy, and identified differentially expressed miRNAs from the mice long-term exposed to LDRR through isolating serum RNAs, constructing small RNA library, Illumina sequencing. To further investigate the role of differential miRNA under LDRR，we first built DNA damage model in Immortal B cells irradiated with 12.5µGy/h dose of γ ray for 28 days at a total dose of 9.4 mGy. Then, we chose the highly conserved miR-181c-3p among 12 miRNA and its mechanism in alleviating DNA damage induced by LDRR was studied by transfection, quantitative PCR, luciferase assay, and Western blot. RESULTS AND CONCLUSIONS: We have found that 12 differentially expressed miRNAs including miR-181c-3p in serum isolated from irradiated mice. Analysis of GO and KEGG indicated that target genes of theses 12 miRNA enriched in pathways related to membrane, protein binding and cancer. Long-term exposure to LDRR induced upregulation of gamma-H2A histone family member X (γ-H2AX) expression, a classical biomarker for DNA damage in B cells. miR-181c-3p inhibited Leukemia inhibitory factor (LIF) expression via combining its 3'UTR. LIF, MDM2, p53, and p-p53-s6 were upregulated after exposure to LDRR. In irradiated B cells, Transfection of miR-181c-3p reduced γ-H2AX expression and suppressed LIF and MDM2 protein levels, whereas p-p53-s6 expression was increased. As expected, the effect of LIF inhibition on irradiated B cells was similar to miR-181c-3p overexpression. Our results suggest that LDRR alters miRNA expression and induces DNA damage. Furthermore, miR-181c-3p can alleviate LDRR-induced DNA damage via the LIF/MDM2/p-p53-s6 pathway in human B lymphocytes. This could provide the basis for prevention and treatment of LDRR injury.

Asymmetric post-translational modifications regulate the nuclear translocation of STAT3 homodimers in response to leukemia inhibitory factor.

STAT3 is a pleiotropic transcription factor overactivated in 70% of solid tumours. We have recently reported that inactivating mutations on residues susceptible to post-translational modifications (PTMs) in only one of the monomers (i.e. asymmetric) caused changes in the cellular distribution of STAT3 homodimers. Here, we used more controlled experimental conditions, i.e. without the interference of endogenous STAT3 (STAT3-/- HeLa cells) and in the presence of a defined cytokine stimulus (Leukemia Inhibitory Factor, LIF), to provide further evidence that asymmetric PTMs affect the nuclear translocation of STAT3 homodimers. Time-lapse microscopy for 20 min after LIF stimulation showed that S727 dephosphorylation (S727A) and K685 inactivation (K685R) slightly enhanced the nuclear translocation of STAT3 homodimers, while K49 inactivation (K49R) delayed STAT3 nuclear translocation. Our findings suggest that asymmetrically modified STAT3 homodimers could be a new level of STAT3 regulation and, therefore, a potential target for cancer therapy.

Leukemia Inhibitory Factor Protects against Degeneration of Cone Photoreceptors Caused by RPE65 Deficiency.

BACKGROUND: Retinal pigment epithelium (RPE) 65 is a key enzyme in the visual cycle involved in the regeneration of 11-cis-retinal. Mutations in the human RPE65 gene cause Leber's congenital amaurosis (LCA), a severe form of an inherited retinal disorder. Animal models carrying Rpe65 mutations develop early-onset retinal degeneration. In particular, the cones degenerate faster than the rods. To date, gene therapy has been used successfully to treat RPE65-associated retinal disorders. However, gene therapy does not completely prevent progressive retinal degeneration in patients, possibly due to the vulnerability of cones in these patients. In the present study, we tested whether leukemia inhibitory factor (LIF), a trophic factor, protects cones in rd12 mice harboring a nonsense mutation in Rpe65. METHODS: LIF was administrated to rd12 mice by intravitreal microinjection. Apoptosis of retinal cells was analyzed by TUNEL assay. The degeneration of cone cells was evaluated by immunostaining of retinal sections and retinal flat-mounts. Signaling proteins regulated by LIF in the retinal and cultured cells were determined by immunoblotting. RESULTS: Intravitreal administration of LIF activated the STAT3 signaling pathway, thereby inhibiting photoreceptor apoptosis and preserving cones in rd12 mice. Niclosamide (NCL), an inhibitor of STAT3 signaling, effectively blocked STAT3 signaling and autophagy in cultured 661W cells treated with LIF. Co-administration of LIF with NCL to rd12 mice abolished the protective effect of LIF, suggesting that STAT3 signaling and autophagy mediate the protection. CONCLUSION: LIF is a potent factor that protects cones in rd12 mice. This finding implies that LIF can be used in combination with gene therapy to achieve better therapeutic outcomes for patients with RPE65-associated LCA.

Leukemia inhibitory factor shortens primary cilia by upregulating C-C motif chemokine 2 in human neural stem/progenitor cells.

Primary cilia on neural stem/progenitor cells (NSPCs) play an important role in determining cell fate, although the regulatory mechanisms involved in the ciliogenesis remain largely unknown. In this study, we analyzed the effect of the leukemia inhibitory factor (LIF) for the primary cilia in immortalized human NSPCs. LIF withdrawal elongated the primary cilia length, whereas the addition of LIF shortened it. Microarray gene expression analysis revealed that differentially expressed genes (DEGs) associated with LIF treatment were related with the multiple cytokine signaling pathways. Among the DEGs, C-C motif chemokine 2 (CCL2) had the highest ranking and its increase in the protein concentration in the NSPCs-conditioned medium after the LIF treatment was confirmed by ELISA. Interestingly, we found that CCL2 was a negative regulator of cilium length, and LIF-induced shortening of primary cilia was antagonized by CCL2-specific antibody, suggesting that LIF could influence cilia length via upregulating CCL2. The shortening effect of LIF and CCL2 on primary cilia was also observed in SH-SY5Y cells. The results of the study suggested that the LIF-CCL2 axis may well be a regulator of NSPCs and its primary cilia length, which could affect multiple cellular processes, including NSPC proliferation and differentiation.

Study the effect of recombinant leukemia inhibitory factor on maintenance of pregnancy and frequency of regulatory T cells in abortion-prone mice.

Recurrent spontaneous abortion (RSA) can have a significant impact on a woman's quality of life. Understanding the mechanisms behind abortion is crucial for developing potential treatments. Among various models of abortion, the CBA/J(♀) × DBA/2J(♂) model stands out as the most extensively studied. This model reveals the influence of an altered immune system on resorption during pregnancy. The leukemia inhibitory factor (LIF) holds considerable importance as a secretory glycoprotein essential for successful implantation. Regulatory T cells (Tregs) have been found to produce high levels of LIF in both mice and humans. LIF plays a vital role in the development of Tregs by upregulating the expression of the Foxp3 transcription factor while downregulating the expression of RORγt. To investigate the impact of recombinant LIF (rLIF) on pregnancy maintenance and Treg cell frequency in abortion-prone (AP) mice, a specific recombinant protein was used in this study. The AP group consisted of CBA/J(♀) × DBA/2J(♂) mice, while the control group comprised CBA/J(♀) × BALB/c(♂) mice. Intraperitoneal injections of rLIF were administered to the AP group on the third day of pregnancy, and its effects on Treg cell frequency and pregnancy maintenance were examined during this period. Following rLIF injections on the fourteenth day of pregnancy, the expression of Foxp3 significantly increased in AP mice (p = 0.02,0.008). Additionally, AP mice injected with rLIF demonstrated a significant reduction in resorption rate (p = 0.01) and a notable increase in birth rate (p = 0.01,0.0005). These findings provide new insights into the potential benefits of LIF in treating RSA patients.

An interplay of Progesterone, Leukemia Inhibitor Factor and Interleukin-6 in the window of implantation; Impact on fertility.

BACKGROUND: The process of implantation is crucial for the initiation of conception and hence fertility. In addition to a number of factors, it is regulated by a cross talk of gonadotrophins [Luteinizing Hormone (LH), Follicle Stimulatory Hormone (FSH)], ovarian steroids [Estrogen (Et), Progesterone (Pt)] and cytokines [Leukemia inhibitory factor (LIF) and Interleukin 6 (IL6)]. These biomarkers are chief players of implantation. OBJECTIVE: We aimed to explore the role of gonadotrophins (LH, FSH, LH/FSH ratio), ovarian steroids (Et, Pt) and cytokines (LIF, IL6) in the implantation process. This aim was achieved by comparing these hormones and cytokines in the fertile and infertile groups [Polycystic ovaries (PCOs), endometriosis, unexplained infertility (Uex-IF)] and finding their association in all study groups. METHODS: A case control study conducted from October 2020-March 2023. A total of 135 infertile women (with PCOs, Uex-IF, and endometriosis) and 177 fertile women (matched for age and BMI) were selected. Levels of 'Et', 'Pt', 'LIF' and, 'IL6' were estimated using Enzyme Linked Immunosorbent Assay (ELISA). LH and FSH values were obtained from hospital desk records. The Independent Student'st-test was used to compare fertile and infertile groups. One-way ANOVA test was used to compare more than two groups, and Pearson's chi-square (χ2) test was employed to compare percentages of variables. Pearson correlation analysis was performed to assess the associations and correlations. A p value < 0.05 was considered statistically significant. RESULTS: Significantly higher levels of LIF and IL6 were observed in fertile women compared to infertile women. Pt levels were significantly greater in the fertile group than in the infertile group. The FSH/LH ratio was significantly higher in the fertile group. Among infertile women, PCOs (71%) and Uex-IF (91%) exhibited lower Pt levels than the fertile controls (p < 0.01), but these levels remained within the reference range (RR). Among the fertile group (81%), levels of LIF within the RR were significantly higher compared to those with Uex-IF (49%) and females with endometriosis (37%). Moreover, the highest number of participants (57%) with Uex-IF exhibited IL6 levels significantly below the RR in comparison to the fertile group and infertile groups (PCOS and endometriosis). However, lower levels of IL6 were observed in women with Uex-IF. In the control group, LIF exhibited a significant positive correlation with IL6 (r = 0.370), Pt (r = 0.496), Et (r = 0.403), and LH (r = 0.428). Among women with PCOs, LIF showed a significant positive correlation with IL6 (r = 0.443), Pt (r = 0.607), and LH (r = 0.472). In cases of Uex-IF, LIF demonstrated a significant positive correlation with IL6 (r = 0.727). Females with endometriosis displayed a significant positive correlation between LIF and IL6 (r = 0.535) as well as Pt (r = 0.605). In fertile women, a positive correlation was observed between LH and IL6 (r = 0.197, p = 0.009), LIF (r = 0.428, p = 0.000), Pt (r = 0.238, p = 0.001), and Et (r = 0.356, p = 0.000). Furthermore, a positive correlation was found between LH and LIF (r = 0.472, p = 0.000) in women with PCOs. CONCLUSION: Elevated levels of Pt were found to increase the production of LIF in fertile females. However, infertile females with PCOs and Uex-IF exhibited deficient levels of Pt, supporting its role as a biomarker for successful implantation in infertile women. These females showed decreased levels of gonadotropins as well as reduced LH/FSH ratio and diminished secretion of receptivity marker LIF, in addition to reduced Pt secretion. This suggests that reduced gonadotropin levels contribute to a lower LH/FSH ratio, resulting in decreased Pt secretion and ultimately leading to low levels of LIF, thereby causing impaired implantation in women with PCOs and Uex-IF. The exploration of low levels of LIF in patients with endometriosis requires further investigation. The significantly low levels of IL6 in the Uex-IF group elucidate the role of this cytokine in association with decreased Pt and LIF synthesis within this group.