Cross-reactivity of two human IL-6 family cytokines OSM and LIF explored by protein-protein docking and molecular dynamics simulation.

BACKGROUND: Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are two important pro-inflammatory cytokines of the interleukin-6 (IL-6) family. The two cytokines mediated signaling was recently found to be closely associated with cancer and chronic inflammation, which represent promising therapeutic targets for the treatment of many solid tumors and inflammatory disease. As the most closely related members, cross-reactivity of them may result in undesired activation of off-target cells, leading to toxicity or lack of efficacy of the therapeutic effects. However, the mechanism of the cross-reactivity of OSM and LIF is not well understood. METHODS: In this work, protein-protein docking, molecular dynamics (MD) simulations with explicit solvent and post endpoints binding free energy (BFE) analysis were carried out to further understand the structural and energetic principles of interactions between the two cytokines and the shared receptor LIFR. RESULTS: For the first time, the simulation given a computational model of OSM-LIFR interaction, and provided significant insights into the mechanism of OSM and LIF cross-react with LIFR. The identified common features shared by OSM and LIF bind to LIFR involving 10 "conserved" residues (90% similarity) distributed at the binding site III comprised of AB loop, BC loop and D helix. In addition, 11 shared residues were identified in LIFR contribute 77.85% and 84.63% energies for OSM and LIF binding, which play a critical role in the formation of the two cytokine-receptor complexes. Moreover, the "nonconserved" residues at the same position of cytokines such as Asp41 in OSM and Pro51 in LIF as well as the three residues (Glu338, Asn201 and Glu260) in LIFR were also discovered. CONCLUSIONS: These important information may facilitate the rational design of novel chemical or biological agents with less toxicity and improved efficacy.

Efficient production process of bioactive recombinant human leukemia inhibitory factor in Chinese hamster ovary cells.

The rhLIF is widely used as an essential factor in stem cell cultures for cell therapies. However, all the recombinant LIFs commercially available are expensive, and no commercially available rhLIF meet the standards recommended by USP for use in cell therapies. The current study reports the efficient production of N-glycosylated and bioactive rhLIF in CHO cells. The production rate of established rhLIF-expressing rCHO cells was approximately 0.85 g/l in 12-day fed-batch cultures using a 7.5 l bioreactor. The rhLIF protein was purified via a four-step purification procedure with approximately 57% recovery rate and greater than 99% purity. The purified rhLIF was N-glycosylated and biologically active with an EC50 of 0.167 ng/ml and a specific activity of 0.86 × 103 IU/mg. The purification procedure controlled the levels of process-related impurities below critical levels recommended by USP for cytokines used in cell therapies. The current work is the first production process of N-glycosylated and bioactive rhLIF, which can be applied to large-scale manufacture of GMP-grade rhLIF for use as an ancillary material in cell therapy.

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.

Polymorphism in the 3'-UTR of LIF but Not in the ATF6B Gene Associates with Schizophrenia Susceptibility: a Case-Control Study and In Silico Analyses.

Schizophrenia (SCZ) is a multifactorial disorder caused by environmental and genetic factors. Studies have shown that various single-nucleotide polymorphisms (SNPs) in the binding sites of microRNAs contribute to the risk of developing SCZ. We aimed to investigate whether the variants located in the 3'-UTR region of LIF (rs929271T>G) and ATF6B (rs8283G>A) were associated with increased susceptibility to SCZ in a population from the south-east of Iran. In this case-control study, a total of 396 subjects were recruited. SNPs were genotyped via polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotyping results showed that the G allele of rs929271 significantly increased the risk of SCZ (OR = 1.58 95%CI = 1.19-2.10, p = 0.001). As for rs929271, the GG genotype of co-dominant (OR = 2.54 95%CI = 1.39-4.64, p = 0.002) and recessive (OR = 2.91 95%CI = 1.77-4.80, p < 0.001) models were strongly linked to SCZ. No significant differences were observed between rs8283 polymorphism and predisposition to SCZ. In silico analyses predicted that rs929271 might alter the binding sites of microRNAs, which was believed to have an unclear role in the development of SCZ. Moreover, rs929271 polymorphism changed the LIF-mRNA folding structure. These findings provide fine pieces of evidence regarding the possible effects of LIF polymorphism in the development of SCZ and regulation of the LIF gene targeted by microRNAs.

Optimization of Serum-Free Culture Conditions for Propagation of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) self-renew and produce a large number of differentiated germ cells to maintain normal spermatogenesis. However, the growth factors crucial for SSC self-renewal and the mechanism underlying this process remain unclear. In the present study, a serum-free culture media was used to evaluate the effect of several growth factors on the expression of some SSC markers and self-renewal related genes. The putative SSCs were cultured on buffalo Sertoli cell feeder layer in KO-DMEM +10% KOSR. The colony formation was observed between 7 and 10 days. The putative SSC colonies also expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days, relative mRNA expression study revealed that 20 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of PLZF, TAF4B, and THY1. Furthermore, supplementation of a combination of 20 ng/mL GDNF, 10 ng/mL basic fibroblast growth factor (bFGF), 1000 IU/mL leukemia inhibitory factor (LIF), and 1 ng/mL colony stimulating factor 1 (CSF1) upregulated the expression of PLZF, TAF4B, BCL6B, and ID4 genes. These results demonstrated that our defined culture media in combination with GDNF, bFGF, LIF, and CSF1 well supported SSC self-renewal.

Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour-bearing mice.

BACKGROUND: Cancer cachexia is a metabolic wasting syndrome that is strongly associated with a poor prognosis. The initiating factors causing fat and muscle loss are largely unknown. Previously, we found that leukaemia inhibitory factor (LIF) secreted by C26 colon carcinoma cells was responsible for atrophy in treated myotubes. In the present study, we tested whether C26 tumour-derived LIF is required for cancer cachexia in mice by knockout of Lif in C26 cells. METHODS: A C26 Lif null tumour cell line was made using CRISPR-Cas9. Measurements of cachexia were compared in mice inoculated with C26 vs. C26Lif-/- tumour cells, and atrophy was compared in myotubes treated with medium from C26 vs. C26Lif-/- tumour cells. Levels of 25 cytokines/chemokines were compared in serum of mice bearing C26 vs. C26Lif-/- tumours and in the medium from these tumour cell lines. RESULTS: At study endpoint, C26 mice showed outward signs of sickness while mice with C26Lif-/- tumours appeared healthy. Mice with C26Lif-/- tumours showed a 55-75% amelioration of body weight loss, muscle loss, fat loss, and splenomegaly compared with mice with C26 tumours (P < 0.05). The heart was not affected by LIF levels because the loss of cardiac mass was the same in C26 and C26Lif-/- tumour-bearing mice. LIF levels in mouse serum was entirely dependent on secretion from the tumour cells. Serum levels of interleukin-6 and G-CSF were increased by 79-fold and 68-fold, respectively, in C26 mice but only by five-fold and two-fold, respectively, in C26Lif-/- mice, suggesting that interleukin-6 and G-CSF increases are dependent on tumour-derived LIF. CONCLUSIONS: This study shows the first use of CRISPR-Cas9 knockout of a candidate cachexia factor in tumour cells. The results provide direct evidence for LIF as a major cachexia initiating factor for the C26 tumour in vivo. Tumour-derived LIF was also a regulator of multiple cytokines in C26 tumour cells and in C26 tumour-bearing mice. The identification of tumour-derived factors such as LIF that initiate the cachectic process is immediately applicable to the development of therapeutics to treat cachexia. This is a proof of principle for studies that when carried out in human cells, will make possible an understanding of the factors causing cachexia in a patient-specific manner.

Production of soluble bioactive mouse leukemia inhibitory factor from Escherichia coli using MBP tag.

Embryonic stem cells and induced pluripotent stem cells depend on one of cytokines called leukemia inhibitory factor (LIF) to retain their undifferentiated state and pluripotency. Nevertheless, further progresses of stem cell scientific investigation and its possible application are limited owing to the expense of commercial LIF. Here we introduced a simple, practical and high level expression of MBP-mouse LIF through Escherichia coli system which was bioactive. The mLIF cDNA was inserted into vector of pMAL-C2X in order to generate N-terminal MBP-mLIF recombinant proteins in the cytoplasm of Escherichia coli. MBP-mLIF as a soluble form was expressed. One-step purification through gravitational affinity chromatography was accomplished to acquire high purity (>92%) MBP-mLIF. The MBP-mLIF products specifically suppressed the growth of M1cells in a dose-dependent pattern. MBP-mLIF also was proved the ability to maintain the pluripotency of iPSCs. These outcomes revealed that the N-end MBP tags of the MBP-mLIF did not obstruct mLIF bioactivity. This method to generate recombinant MBP-mLIF is a simple, practical, economical and user-friendly protocol.

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.

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway.

BACKGROUND/AIMS: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. METHODS: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. RESULTS: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. CONCLUSION: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

Phorbol-12-myristate-13-acetate-mediated stabilization of leukemia inhibitory factor (lif) mRNA: involvement of Nucleolin and PCBP1.

Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis-acting elements. A 216 nucleotide containing proximal cis-element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein trans-factors interacting with lif mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent in vivo co-association of both these proteins with lif mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic lif mRNA and gfp reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both lif mRNA and protein levels. Collectively, this report establishes the stabilization of lif mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal cis-element.

Expression, purification and characterization of active untagged recombinant human leukemia inhibitory factor from E.coli.

Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is considered to be a pleiotropic cytokine and functions in both cell proliferation and differentiation. It is widely used in the culture of mouse embryonic stem cells and is implicated in the implantation of mouse model and possibly in humans. Great efforts have been made on the efficient generation of LIF to meet the requirement of this cytokine in biomedical research. However, because of the low expression level in the eukaryotic system and poor purification yields, recombinant human LIF has usually been expressed either as inclusion body or as fusion protein in E. coli (Escherichia coli). Here we introduce a simple method to express hLIF in a soluble form in E. coli and a subsequent purification method. The expression of hLIF was induced at a low temperature (16 °C) and most of the expressed hLIF was observed to be in a soluble form. Then by using three steps of chromatography, which could be easily scaled-up for industrial purposes, active untagged hLIF was purified with similar bioactivity compared to that of the commercial product. The endotoxin level of purified hLIF protein in our method was determined to be lower than 1EU/µg, which was also comparable to the commercial products. Furthermore, as hLIF was expressed in a soluble form, there was no need to develop the denaturation and renaturation methods. The yield of hLIF protein was evaluated to be approximately 0.7 mg hLIF from 1 g wet weight of E.coli in our method.

Acquiring Ground State Pluripotency: Switching Mouse Embryonic Stem Cells from Serum/LIF Medium to 2i/LIF Medium.

Mouse embryonic stem cells (ESCs) derive from the inner cell mass (ICM) of a blastocyst. These cells are pluripotent and thus able to generate both somatic and germinal lineages. It is possible to maintain ESCs in different pluripotent states depending on the in vitro culture conditions. Classically, ESCs are cultured in the presence of serum and LIF, which sustain the naive state of pluripotency but in this metastable state cells exhibit a large degree of heterogeneity. In the last few years, it has been discovered that when ESCs are cultured in a chemically defined medium (without serum), in the presence of LIF and with the addition of two small molecules (in particular the inhibitors of MAPK and Gsk-3 pathways), they reach a ground state of pluripotency where cells are more homogeneous and more "ICM-like." In this protocol, we describe how we culture mouse ESCs and the way we switch them from naive to ground state.

Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (LIF).

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system (CNS). Many nerve axons are insulated by a myelin sheath and their demyelination not only prevents saltatory electrical signal conduction along the axons but also removes their metabolic support leading to irreversible neurodegeneration, which currently is untreatable. There is much interest in potential therapeutics that promote remyelination and here we explore use of leukaemia inhibitory factor (LIF), a cytokine known to play a key regulatory role in self-tolerant immunity and recently identified as a pro-myelination factor. In this study, we tested a nanoparticle-based strategy for targeted delivery of LIF to oligodendrocyte precursor cells (OPC) to promote their differentiation into mature oligodendrocytes able to repair myelin. Poly(lactic-co-glycolic acid)-based nanoparticles of ∼120 nm diameter were constructed with LIF as cargo (LIF-NP) with surface antibodies against NG-2 chondroitin sulfate proteoglycan, expressed on OPC. In vitro, NG2-targeted LIF-NP bound to OPCs, activated pSTAT-3 signalling and induced OPC differentiation into mature oligodendrocytes. In vivo, using a model of focal CNS demyelination, we show that NG2-targeted LIF-NP increased myelin repair, both at the level of increased number of myelinated axons, and increased thickness of myelin per axon. Potency was high: a single NP dose delivering picomolar quantities of LIF is sufficient to increase remyelination. Impact statement Nanotherapy-based delivery of leukaemia inhibitory factor (LIF) directly to OPCs proved to be highly potent in promoting myelin repair in vivo: this delivery strategy introduces a novel approach to delivering drugs or biologics targeted to myelin repair in diseases such as MS.

miRNA-181 regulates embryo implantation in mice through targeting leukemia inhibitory factor.

Embryo implantation is a crucial step in mammalian reproduction. However, little is known regarding the physiological roles of microRNAs in the regulation of embryo implantation. Here we show that a minimum uterine expression of miR-181 is essential for the onset of embryo implantation. Both transient and prolonged transgenic expression of miR-181 led to impaired implantation, which can be rescued by exogenous administration of leukemia inhibitory factor (LIF). Mechanistically, miR-181 is able to directly target LIF and downregulate LIF expression, thereby inhibiting embryo implantation. We also show that miR-181 expression is regulated by the transcriptional factor Emx2, and the Emx2-miR-181 axis plays an important role in regulating embryo implantation. Taken together, these results reveal a novel function of miR-181 in embryo implantation through the regulation of LIF, and also indicate a potential link between miR-181 dysregulation and human embryo implantation defects.

Microfluidic patterning of protein gradients on biomimetic hydrogel substrates.

This protocol describes a versatile microfluidic method to generate tethered protein gradients of virtually any user-defined shape on biomimetic hydrogel substrates. It can be applied to test, in a microenvironment of physiologically relevant stiffness, how cells respond to graded biomolecular signals, for example to elucidate how morphogen proteins affect stem cell fate. The method is based on the use of microfluidic flow focusing to rapidly capture in a step-wise manner tagged biomolecules via affinity binding on the gel surface. The entire patterning process can be performed in <1 h. We illustrate one application of this method, namely, the spatial control of mouse embryonic stem cell self-renewal in response to gradients of the self-renewal-promoting signal leukemia inhibitory factor.

A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.

High-level soluble expression, purification, and functional characterization of the recombinant human leukemia inhibitory factor: a potential general strategy for the recombinant expression of cytokines consisting of four α-helices in a bundle.

The human leukemia inhibitory factor (hLIF) is one of the most important cytokines in the interleukin-6 (IL-6) cytokine family. Numerous studies have demonstrated that hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues. The optimal chemical synthesis of the hLIF gene has been previously reported to increase the expression of the recombinant inclusion body protein in E. coli. However, the required refolding step limits the recovery rate. In this report, a novel strategy was designed to produce a soluble recombinant human LIF (rhLIF) in the prokaryotic system in order to obtain higher yields of the bioactive protein with simpler steps. This optimal hLIF gene was cloned, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. A simple purification procedure is established to purify the recombinant fusion protein from the soluble supernatant of the lysed culture cells. This procedure yields up to 5 mg/L rhLIF with above 95? purity. The strategy allows the protease to release target cytokines without additional N-terminus amino acids, which is an important consideration for maintaining its bioactivity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF. These results present a sound strategy for the soluble production of rhLIF and other homologous tertiary structure cytokines consisting of four α-helices in a bundle for basic research, as well as clinical applications.

The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells.

Human leukemia inhibitory factor (LIF) was immobilized into insect virus-derived microcrystals (polyhedra) to generate LIF polyhedra (LIF-PH) that can slowly release LIF into embryonic stem (ES) cell culture media and thus maintain ES cells in an undifferentiated state. Assays of the biological activities of LIF-PH indicated that a single addition of LIF-PH to the ES cell culture medium can support the proliferation of mouse ES and induced pluripotent stem (iPS) cells continuously for 14 days, and suggest that LIF-PH can be successfully used in the place of a periodic addition of recombinant LIF to the media every 2-3 days. The release of LIF protein from LIF-PH was determined by enzyme-linked immunosorbent assay (ELISA). Maintenance of undifferentiated state of mouse ES and iPS cells cultured with LIF-PH was determined by the detection of pluripotency-related biomarkers Oct3/4 and stage-specific embryonic antigen-1 (SSEA-1) through immunostaining and measurement of alkaline phosphatase activity. In this paper, we propose a closed culture system for mass production of ES and iPS cells that utilize a slow-releasing agent of LIF.

Modulation of CD4+ T lymphocyte lineage outcomes with targeted, nanoparticle-mediated cytokine delivery.

Within the immune system there is an exquisite ability to discriminate between "self" and "non-self" that is orchestrated by antigen-specific T lymphocytes. Genomic plasticity enables differentiation of naive CD4+ T lymphocytes into either regulatory cells (Treg) that express the transcription factor Foxp3 and actively prevent autoimmune self-destruction or effector cells (Teff) that attack and destroy their cognate target. An example of such plasticity is our recent discovery that leukemia inhibitory factor (LIF) supports Treg maturation in contrast to IL-6, which drives development of the pathogenic Th17 effector phenotype. This has revealed a LIF/IL6 axis in T cell development which can be exploited for modulation using targeted cytokine delivery. Here we demonstrate that LIF-loaded nanoparticles (NPs) directed to CD4+ T cells (i) oppose IL6-driven Th17 development; (ii) prolong survival of vascularized heart grafts in mice; and (iii) expand FOXP3+ CD4+ T cell numbers in a non-human primate model in vitro. In contrast, IL-6 loaded nanoparticles directed to CD4+ T cells increase Th17 development. Notably, nanoparticle-mediated delivery was demonstrated to be critical: unloaded nanoparticles and soluble LIF or IL-6 controls failed to recapitulate the efficacy of cytokine-loaded nanoparticles in induction and/or expansion of Foxp3+ cells or Th17 cells. Thus, this targeted nanoparticle approach is able to harness endogenous immune-regulatory pathways, providing a powerful new method to modulating T cell developmental plasticity in immune-mediated disease indications.

[Effect of LIF factor on lipid bilayer].

It has been found that the recombinant protein LIF induces jumps of current in bilayer lipid membranes, which indicates the formation of ionic channels. Some properties of these channels (dependence on voltage, sing of potential, ionic strength of solution, and lipid composition) were studied. A difference between the effects of protein LIF of eukaryotic and prokaryotic origin was shown.