Optimizing the Expression and Solubilization of an E. coli-Produced Leukemia Inhibitory Factor for Anti-LIF Antibody Production and Use Thereof for Contraception in Mice.

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of the anti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive.

The Poststroke Peripheral Immune Response Is Differentially Regulated by Leukemia Inhibitory Factor in Aged Male and Female Rodents.

BACKGROUND: The goal of this study was to determine whether leukemia inhibitory factor (LIF) promotes anti-inflammatory activity after stroke in a sex-dependent manner. METHODS: Aged (18-month-old) Sprague-Dawley rats of both sexes underwent sham surgery or permanent middle cerebral artery occlusion (MCAO). Animals received three doses of intravenous LIF (125 µg/kg) or PBS at 6, 24, and 48 h before euthanization at 72 h. Spleen weights were measured immediately following euthanization. Western blot was used to measure protein levels of CCL8, CD11b, CXCL9, CXCL10, IL-12 p40, IL-3, and the LIF receptor (LIFR) in spleen tissue. ELISA was used to measure IL-1ß, IL-6, TNFα, and IFNγ in spleen tissue. A Griess Assay was used to indirectly quantify NO levels via measurement of nitrite. Levels of cellular markers and inflammatory mediators were normalized to the baseline (sham) group from each sex. Statistical analysis was performed using two-way ANOVA and followed by Fisher's LSD post hoc test. RESULTS: Aged female rats showed a significantly lower spleen weight after MCAO, but showed a significant increase in spleen size after LIF treatment. This effect was observed in aged male rats, but not to as great of an extent. CD11b levels were significantly higher in the spleens of MCAO+PBS males compared to their female counterparts, but there was no significant difference in CD11b levels between MCAO+LIF males and females. LIF significantly increased CXCL9 after LIF treatment in aged male and female rats. LIFR and IL-3 were upregulated after LIF treatment in aged females. Splenic nitrate increased after MCAO but decreased after LIF treatment in aged females. Splenic nitrate levels did not increase after MCAO but did increase after LIF treatment in aged males. The following cytokines/chemokines were not altered by sex or treatment: TNFα, IL-6, IL-12 p40, CCL8, IFNγ, and CXCL10. CONCLUSIONS: LIF treatment after permanent MCAO induces sex-dependent effects on the poststroke splenic response and the production of proinflammatory cytokines among aged rats.

Contraceptive and molecular function of a novel recombinant vaccine based human leukemia inhibitory factor on Balb/c mice: An experimental in vivo study.

The functional competence of leukemia inhibitory factor (LIF), as immunocontraceptive vaccine in mice, was investigated. Balb/c mice were divided into two groups of vaccinated and controls. The recombinant human LIF (rhLIF) protein and phosphate buffer saline was emulsified with Freund's adjuvant and injected into vaccinated and control groups, respectively. Theinhibition of implantation was evaluated in mice uterine. The concentration of secreted interferon-γ (IFN-γ) and interleukin (IL)-4 were measured in cultured splenocyte of mice stimulated by rhLIF. The expressions of immune responsive gene 1 (IRG-1), cochlin (COCH), amphiregulin(Ar), and heparin-binding EGF-like growth factor (HB-EGF) genes were determined. Mice were assessed for inhibition of fertility after delivery, reversibility of immune response against rhLIF, and survival rate. Active immunization of mice with rhLIF resulted in reduction of the implantation and fertility rate up to 80.49% and 75%, respectively. All mice produced a high titer of anti-rhLIF antibodies in serums and vaginal fluids washes after 16 weeks; however, these antibodies were cleared from vaginal fluid washes after six months. A significant down-regulation in mRNA levels of IRG-1, Ar and HB-EGF was observed in vaccinated group compared to controls; however, no significant change in the expression profile of cochlin gene was detected. The results showed that rhLIF prevented pregnancy in a high percentage of female mice. Although the immunization of female Balb/c mice with rhLIF inhibited fertility and expression of genes associated with this molecule, further studies are needed to support this protein as a suitable candidate for contraceptive vaccine in mammals.

Immunization against leukemia inhibitory factor and its receptor suppresses tumor formation of breast cancer initiating cells in BALB/c mouse.

Immunotherapy is a promising approach for specific targeting of cancer cells. Leukemia inhibitory factor (LIF) regulates several features of cancers and cancer stem cells (CSCs) through binding to LIF receptor (LIFR). In this study, we investigated the consensus of LIF and LIFR immunization on the growth of mouse mammary tumors. For this purpose, mouse LIF and LIFR were designed as truncated proteins, expressed in E. coli and then injected to mice as individual and mixed antigens. The results showed the production of neutralizing antibodies and secretion of interferon-γ and interleukin-2 in response to immunization. In continue, the immunized mice were subjected for tumor formation challenge by inoculation of the breast CSCs derived from MC4-L2 cells. Development of the breast tumors was observed in all the control mice, while the tumors appeared in 75% of animals in the LIF group. LIFR injection, individually or in combination with LIF, strongly inhibited the tumor growth to only 25% of the mice. Moreover, a delay in tumor appearance was observed in the immunized mice compared to the controls. Immunostaining of the tumor sections confirmed the expression of LIF and LIFR. In conclusion, LIF and LIFR might be effective targets for immunotherapy of the tumors that express these proteins.

Production and characterization of recombinant human leukemia inhibitory factor and evaluation of anti-fertility effects of rabbit anti-rhLIF in Balb/c mice.

Human leukemia inhibitory factor (hLIF) is a cytokine of interleukin-6 family. This study aimed to evaluate the recombinant production rate of active hLIF by different vector-host systems under various conditions. Moreover, a rabbit polyclonal antibody (pAb) against recombinant hLIF (rhLIF) was produced and its anti-fertility effects were explored in Balb/c mice. Four different constructs including pET22b/hLIF, pET28b/hLIF, pET32b/hLIF and pColdI/hLIF were designed and transformed into BL21-(DE3), Rosetta-(DE3), Origami-(DE3) and Shuffle T7-(DE3) host cells. The expression level and proliferative effect of rhLIF were measured by SDS-PAGE and MTT assays, respectively. Rabbit pAb to rhLIF was produced and characterized using enzyme-linked immunosorbent assay and western blot techniques. The Balb/c mice were divided into two intervention and control groups. Then, they were intraperitoneally injected by purified rabbit anti-rhLIF and non-immunized rabbit pAb, respectively. After sacrifice on day 7, the number of implantation sites was counted. The rhLIF was successfully expressed by pET32b/hLIF and pColdI/hLIF vectors in all hosts with no significant difference in the rate of their expression. The rhLIF was purified and checked for activity. The results showed that it is functionally active and the produced anti-rhLIF pAb could specifically bind to commercial rhLIF. Passive immunization results showed that anti-rhLIF antibody completely inhibited fertility in all injected Balb/c mice compared to controls. Although previous studies showed expression of rhLIF using various methods, using different vector-host systems ensures us of successful biological active expression of it. The pAb against rhLIF could be a powerful tool for inducing in vivo infertility.

Leukemia inhibitory factor regulates the activation of inflammatory signals in macrophages and trophoblast cells.

The pleiotropic cytokine leukemia inhibitory factor (LIF) is a key gestational factor known to establish dynamic cellular and molecular cross talk at the feto-maternal interface. Previously, we described the regulatory role of the LIF-trophoblast-IL10 axis in the process of macrophage deactivation in response to pro-inflammatory cytokines. However, the direct regulatory effects of LIF in macrophage and trophoblast cell function remains elusive. In this study, we aimed to examine whether and how LIF regulates the behavior of macrophages and trophoblast cells in response to pro-inflammatory stress factors. We found that LIF modulated the activating effects of interferon-gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in macrophages and trophoblast cells by reducing the phosphorylation levels of signal transducer and activator of transcription-1 (Stat1) and -5 (Stat5). Cell activation with IFNγ inhibited cell invasion and migration but this immobilizing effect was abrogated when macrophages and trophoblast cells were deactivated with LIF; macrophage cell motility restitution could in part be explained by the positive effects of LIF in Stat3 activation and matrix metalloproteinase 9 (MMP-9) expression. Pharmacological inhibition of Stat1 and Stat3 indicated that IFNγ-induced Stat1 activation mediated macrophage motility inhibition, and that cell motility in IFNγ-activated macrophages is restored via LIF-induced Stat3 activation and Stat1 inhibition. Moreover, IFNγ-induced TNFα gene expression was also abrogated by LIF through Stat1 inhibition and Stat3 activation. Finally, we have found that cell invasion of trophoblast cells is inhibited when they were cocultured with GM-CSF-differentiated, IFNγ-stimulated macrophages. This effect, however, was inhibited when macrophages were exposed to LIF. Overall, this in vitro study reveals for the first time the anti-inflammatory and pro-gestational activities of LIF by acting directly on macrophages and trophoblast cells.

Leukemia inhibitory factor: Recent advances and implications in biotechnology.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with several functions in health and disease ranging from inflammation to cancer. LIF is also a potential target and/or therapeutic agent for diseases such as multiple sclerosis, stroke and even psychological disorders, where the function of LIF as a neurotrophic factor has only recently been explored. In recent years, a limited number of LIF clinical trials have been completed, which partially explains the shortage of effective applications as a therapeutic agent. With the increasing interest from biotechnology companies producing recombinant LIF, this status quo will certainly change, and the potential impact of LIF in terms of disease diagnosis, treatment and management will be realized.

LIF regulates CXCL9 in tumor-associated macrophages and prevents CD8+ T cell tumor-infiltration impairing anti-PD1 therapy.

Cancer response to immunotherapy depends on the infiltration of CD8+ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8+ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8+ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival.

Leukemia inhibitory factor (LIF) modulates the development of dendritic cells in a dual manner.

Objective: Dendritic cells (DCs) are professional antigen presenting cells majorly modulated by various environmental factors. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine from interleukin-6 family. Previous studies demonstrate that LIF is associated with several tolerogenic events; yet the exact effect of this cytokine on the generation and function of DCs was not explicitly identified. Materials and methods: To clarify the role of LIF in DCs development, immature DCs were differentiated from mouse bone marrow (BM) in a GM-CSF and IL-4 containing medium with or without LIF. Afterwards, in maturation process, the differentiated DCs were exposed to TNF-α in the presence or absence of LIF. Results: Immature DCs differentiated in the presence of LIF, proved a significant enhancement in the expression of MHCII, CD40, or CD86 molecules and in the antigen uptake function. LIF treatment of normal DCs while stimulating for maturation, caused a significant decrement in the expression of phenotypic markers as well as an increment in the antigen uptake function in comparison with TNF-α-only stimulated cells; however, the reduced ability for induction of allogenic T-cell proliferation proved no statistical significance. Conclusions: Our results can reflect a role for LIF in the generation and particularly maturation of DCs. It can be assumed that LIF rather modulates the maturation level, leading to the development of semi-mature and tolerogenic DCs. According to the high levels of LIF in immune-privileged sites like brain and uterine, it seems that the cytokine may account for the formation of local DCs that help the establishment of immunosuppressive environments.

Leukemia Inhibitory Factor-Loaded Nanoparticles with Enhanced Cytokine Metabolic Stability and Anti-Inflammatory Activity.

PURPOSE: To synthesize and assess the in vitro biological activity of nanoparticles containing leukemia inhibitory factor (LIF). These NanoLIF particles are designed to prolong the neuroprotective and anti-inflammatory actions of LIF in future preclinical studies of ischemic stroke. METHODS: LIF was packaged in nanoparticles made of poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) polymer to form LIF-loaded nanoparticles (NanoLIF). The surface of NanoLIF was also modified with the CD11b antibody (CD11b-NanoLIF) targeting activated peripheral macrophages to increase cytokine delivery to inflammatory macrophages. ELISA was used to quantify bioactive cytokine inside and releasing from NanoLIF. NanoLIF biological activity was measured using the M1 murine leukemia cell proliferation assay. RESULTS: NanoLIF and CD11b-NanoLIF had diameters of approximately 30 nm, neutral surface charge, and physicochemical stability retaining biological activity of the cytokine during incubation at 25°C for 12 h. NanoLIF particles released LIF relatively fast from 0 to 6 h after incubation at 37°C followed by slow release from 24 to 72 h according to a two-phase exponential decay model. NanoLIF and CD11b-NanoLIF significantly decreased M1 cell proliferation over 72 h compared to free LIF. CONCLUSIONS: NanoLIF and CD11b-NanoLIF preserved the metabolic stability and biological activity of LIF in vitro. These results are promising to improve the therapeutic potential of LIF in treating neurodegenerative and inflammatory diseases.

TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs.

Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell-intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene Tlr9 (Tlr9ON ) or the control LacZ (LacZON ). When engrafted into mice and treated with tetracycline, Tlr9ON , but not LacZON , tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b+Ly6GHILy6CLO myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9-Tlr9ON tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL-6-type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9-expressing prostate cancer cells and PMN-MDSCs. Antibody-mediated LIF neutralization reduced the percentage of tumor-infiltrating PMN-MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between TLR9 and LIF expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN-MDSCs. Our results suggest that TLR9+ prostate cancers promote immune evasion via LIF-mediated expansion and activation of PMN-MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide-based inhibitors, such as CpG-STAT3dODN, can offer new opportunities for prostate cancer immunotherapy.

Downregulation of genes related to immune and inflammatory response in IVF implantation failure cases under controlled ovarian stimulation.

PROBLEM: Implantation failure (IF) even after the good-quality embryo transfer (ET) is main obstacle in in vitro fertilization (IVF). We aim to study the genomics of endometrial receptivity in IF patients under controlled ovarian stimulation (COS) during which ET is generally practised in IVF. METHOD OF STUDY: Endometrial gene expression profiling in IF patients (n=10) and oocyte donors (n=8) were compared during window of implantation under COS by microarray. Enrichment analysis of microarray data was performed to determine dysregulated pathways. Microarray results were validated by real-time PCR. Localization of genes related to immune response (progestagen-associated endometrial protein (PAEP), leukaemia inhibitory factor (LIF), interleukin-6 signal transducer (IL6ST) was detected by immunohistochemistry. RESULTS: The gene ontology, pathway analysis and enrichment mapping revealed significant downregulation in activation and regulation of immune and inflammation response in IF patients under COS. The lower expression of PAEP, LIF and IL6ST in cases compared to controls by real time and immunohistochemistry suggests the functional importance of these genes. CONCLUSION: Importance of immune and inflammatory response in endometrial receptivity adds on to the current knowledge of gene expression profile in IF under COS. The panel of genes involved in these pathways would be useful in determining further line of treatment for IF during IVF.

Autocrine Loop Involving IL-6 Family Member LIF, LIF Receptor, and STAT4 Drives Sustained Fibroblast Production of Inflammatory Mediators.

Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1ß. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.

miR-17-92 facilitates neuronal differentiation of transplanted neural stem/precursor cells under neuroinflammatory conditions.

BACKGROUND: Neural stem/precursor cells (NSCs) are of particular interest because of their potential application in cell therapy for brain damage. However, most brain injury cases are followed with neuroinflammatory stress, which affects the lineage selection of grafted NSCs by promoting astrocytogenesis, thus hampering the potential for neural replacement. The present study investigated the role of miR-17-92 in protecting against detrimental effects of neuroinflammation on NSC differentiation in cell therapy. METHODS: NSCs were treated with conditioned medium from lesioned astrocytes with/without neutralizing antibodies of leukemia inhibitory factor (LIF) or/and ciliary neurotrophic factor (CNTF), respectively. Afterward, the levels of p-STAT3 and p-JAK2 were determined by western blotting while expression of glial fibrillary acidic protein (GFAP) and ß-tubulin III was assessed by immunostaining. The activation of JAK-STAT pathway and cell differentiation were also evaluated after we overexpressed miR-17-92 in NSCs under different neuroinflammatory conditions. After the transplantation of miR-17-92-overexpressing NSCs into injured mouse cortex, PH3, nestin, GFAP, and NeuN were analyzed by immunostaining. In addition, motor coordination of mice was evaluated by rotarod test. RESULTS: Conditioned medium from lesioned astrocytes activated JAK-STAT pathway and facilitated astrocytic differentiation in NSCs while neutralizing antibodies of LIF and CNTF remarkably attenuated such effects. miR-17-92 cluster repressed the expression of multiple proteins including GP130, CNTFR, JAK2, and STAT3 in JAK-STAT pathway. Overexpression of miR-17-92 in NSCs systematically blocked the activation of JAK-STAT pathway mediated by LIF and CNTF, which facilitated neuronal differentiation in vitro. Furthermore, miR-17-92 increased neuronal generation of grafted NSCs and reduced astrogliosis, which resulted in the improvement of motor coordination of brain-injured mice. CONCLUSIONS: Our results suggest that miR-17-92 promotes neuronal differentiation of grafted NSCs under neuroinflammatory condition via inhibition of multiple proteins in JAK-STAT pathway.

Cytokines of the LIF/CNTF family and metabolism.

The incidence of obesity is increasing worldwide. Obesity is accompanied by a chronic inflammatory state that increases the risk of metabolic diseases such as insulin-resistance and type 2 diabetes. Over the past two decades, interest in immunomodulatory cytokines as potential mediators and/or targets for treatment or prevention of obesity and metabolic syndrome has increased. In this review, we summarize studies that revealed the effects of LIF family cytokines on adipose tissue, energy expenditure and food intake, highlighting the importance of gp130/LIFRß signaling in obesity and obesity-related metabolic diseases.

The Multifaceted Actions of Leukaemia Inhibitory Factor in Mediating Uterine Receptivity and Embryo Implantation.

Embryo implantation is mediated by the combined actions of the ovarian hormones E2 and P4 on the uterus. In addition, the pro-inflammatory cytokine, leukaemia inhibitory factor (LIF), plays a pivotal role in regulating uterine receptivity. LIF is expressed in the endometrial glands and has a robust action on the uterine luminal epithelium (LE). In mice, LIF is induced by nidatory E2 and functions to convert the LE from a non-receptive to an embryo-responsive state. LIF mediates its actions by activating the JAK-STAT pathway specifically in the LE. Activation of JAK-STAT pathway results in the induction of many additional pathways, including some 40 +  transcription factors, many of which initiate a cascade of changes affecting epithelial polarity, epithelial-mesenchymal interactions, angiogenesis, stromal cell decidualization, and inhibiting cell proliferation. This review discusses the role of LIF and the recent analysis of its action on the uterine LE in regulating endometrial receptivity and implantation.

Leukemia inhibitory factor (LIF).

Leukemia inhibitory factor (LIF) is the most pleiotropic member of the interleukin-6 family of cytokines. It utilises a receptor that consists of the LIF receptor ß and gp130 and this receptor complex is also used by ciliary neurotrophic growth factor (CNTF), oncostatin M, cardiotrophin1 (CT1) and cardiotrophin-like cytokine (CLC). Despite common signal transduction mechanisms (JAK/STAT, MAPK and PI3K) LIF can have paradoxically opposite effects in different cell types including stimulating or inhibiting each of cell proliferation, differentiation and survival. While LIF can act on a wide range of cell types, LIF knockout mice have revealed that many of these actions are not apparent during ordinary development and that they may be the result of induced LIF expression during tissue damage or injury. Nevertheless LIF does appear to have non-redundant actions in maternal receptivity to blastocyst implantation, placental formation and in the development of the nervous system. LIF has also found practical use in the maintenance of self-renewal and totipotency of embryonic stem cells and induced pluripotent stem cells.

Analysis of the Effect of Leukemia Inhibitory Factor on Follicular Growth in Cultured Murine Ovarian Tissue.

Leukemia inhibitory factor (LIF) is expressed in the ovary and controls follicular growth. LIF has been reported to accelerate the primordial to primary follicle transition, the growth of cultured preantral follicles, and the maturation of oocytes. Previous reports on factors that regulate follicular growth have largely employed cultured follicles. However, there are several types of follicles and somatic cells in the ovary that are likely to interact with one another to regulate follicular growth. Therefore, a novel approach is essential for understanding the function of factors that regulate follicular growth in the ovary. In this study, we evaluated the function of LIF using cultured ovarian tissue. Ovarian tissue slices were cultured in the presence or absence of recombinant LIF and neutralizing anti-LIF antibody to enable continuous monitoring of follicular growth within the context of the ovary as well as analysis of the process of follicular growth. The results revealed that LIF inhibited the growth of primary, secondary, and antral follicles. Furthermore, we verified the inhibitory function of LIF using the neutralizing antibody, which accelerated follicular growth. These results suggest that LIF is likely to coordinate follicular growth in the ovary. The culture and analysis methods employed in this study are thus effective for clarifying the tissue-level functions of factors that regulate follicular growth within the ovary.

Stimulation of the JAK/STAT pathway by LIF and OSM in the human granulosa cell line COV434.

The development of the follicle and competent oocyte is highly coordinated, requiring interplay among several systems. These implicate endocrine, immune, and metabolic signals, intrafollicular paracrine factors from theca, mural, and cumulus granulosa cells, and the oocyte itself. Granulosa cells play a key role in their interaction. COV434 is one of the few human granulosa cell lines that can be used as an in vitro model for ovarian research. We aimed to evaluate the possible activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway by IL-6-type cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) in COV434 cells. Expression of GP130 (glycoprotein 130), STAT3 (signal transducer and activators of transcription 3), PIAS3 (protein inhibitor of activated STAT 3), and SOCS3 (suppressor of cytokine signaling 3) genes after stimulation with LIF or OSM was assessed using RT-qPCR (real-time PCR). GP130 transcripts were significantly upregulated after incubation with LIF or OSM for 24h. Expression of the STAT3 gene was stimulated only after incubation with LIF, but not OSM. SOCS3 showed significant upregulation for all time periods and the levels of PIAS3 were initially down- and after 24h upregulated. Furthermore, the major signaling components of the JAK/STAT pathway, GP130 and STAT3, and the kinase activation patterns of STAT3, were examined at protein level. We found constitutive protein expression for GP130, STAT3, pSTAT3(ser727) and upregulation of pSTAT3(tyr705) by LIF and OSM. Our results demonstrate the activation of the JAK/STAT pathway by LIF and OSM in human granulosa cells.

Leukemia inhibitory factor tips the immune balance towards regulatory T cells in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), for which current treatments are unable to prevent disease progression. Based on its neuroprotective and neuroregenerating properties, leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is proposed as a novel candidate for MS therapy. However, its effect on the autoimmune response remains unclear. In this study, we determined how LIF modulates T cell responses that play a crucial role in the pathogenesis of MS. We demonstrate that expression of the LIF receptor was strongly increased on immune cells of MS patients. LIF treatment potently boosted the number of regulatory T cells (Tregs) in CD4(+) T cells isolated from healthy controls and MS patients with low serum levels of IL-6. Moreover, IL-6 signaling was reduced in the donors that responded to LIF treatment in vitro. Our data together with previous findings revealing that IL-6 inhibits Treg development, suggest an opposing function of LIF and IL-6. In a preclinical animal model of MS we shifted the LIF/IL-6 balance in favor of LIF by CNS-targeted overexpression. This increased the number of Tregs in the CNS during active autoimmune responses and reduced disease symptoms. In conclusion, our data show that LIF downregulates the autoimmune response by enhancing Treg numbers, providing further impetus for the use of LIF as a novel treatment for MS and other autoimmune diseases.