Multiple Sclerosis: LIFNano-CD4 for Trojan Horse Delivery of the Neuro-Protective Biologic "LIF" Into the Brain: Preclinical Proof of Concept.

Multiple sclerosis (MS) is a demyelinating autoimmune disease that attacks the brain, with year-on-year loss of brain volume, starting late teens and becoming manifest late twenties. There is no cure, and current therapies are immunosuppressive only. LIF is a vital stem cell growth factor active throughout life-and essential for health of the central nervous system (CNS), being tolerogenic, myelinogenic, and neuroprotective. Nano-formulation of LIF (LIFNano) using FDA-approved PLGA captures LIF's compound therapeutic properties, increasing potency 1,000-fold when targeted to CD4 (LIFNano-CD4). Moreover, circulating CD4+ lymphocytes are themselves regulated by LIF to express the Treg phenotype, known to release T cell-derived LIF upon engagement with cognate antigen, perpetuating antigen-specific self-tolerance. With the longer-term aim of treating inflammatory lesions of MS, we asked, does LIFNano-CD4 cross the blood-brain barrier (BBB)? We measure pK and pD using novel methodologies, demonstrate crossing of the BBB, show LIF-cargo-specific anti-inflammatory efficacy in the frontal cortex of the brain, and show safety of intravenous delivery of LIFNano-CD4 at doses known to provide efficacious concentrations of LIF cargo behind the BBB.

Mesenchymal stem cells and management of COVID-19 pneumonia.

Human coronavirus, hCoV-19, is highly pathogenic with severe pneumonia associated with rapid virus replication. Arising in Wuhan China December 2019, the current COVID-19 epidemic has rapidly grown with person-to-person infection expanding to become a global health emergency now on pandemic scale. Governments will not be able to minimise both deaths from COVID-19 and the economic impact of viral spread in mitigation of this current COVID-19 pandemic, according to Anderson et al. 2020 [1], Keeping mortality as low as possible will be the highest priority for individuals; hence governments must put in place measures to ameliorate the inevitable economic downturn. The current global picture shows small chains of transmission in many countries and large chains resulting in extensive spread in a few countries, such as Italy, Iran, South Korea, and Japan. Most countries are likely to have spread of COVID-19, at least in the early stages, before any mitigation measures have an impact. The scale of the problem is massive. Here I consider new approaches to improve patient's biological resistance to COVID-19 using stem cells, and how benefit might be scaled and simplified using synthetic stem cells to meet logistical needs within a short time frame.

Neurodegenerative Disease: A Perspective on Cell-Based Therapy in the New Era of Cell-Free Nano-Therapy.

Neurodegenerative diseases (NDD) result in irreversible loss of neurons. Dementia develops when disease-induced neuronal loss becomes sufficient to impair both memory and cognitive functioning and, globally, dementia is increasing to epidemic proportions as populations age. In the current era of regenerative medicine intense activity is asking, can loss of endogenous neurons be compensated by replacement with exogenously derived cells that have either direct, or indirect, neurogenic capacity? But, more recently, excitement is growing around an emerging alternative to the cell-based approach - here nanotechnology for targeted delivery of growth factor aims to support and expand resident central nervous system (CNS) stem cells for endogenous repair. The concept of a high volume, off-the-shelf nano-therapeutic able to rejuvenate the endogenous neuroglia of the CNS is highly attractive, providing a simple solution to the complex challenges posed by cell-based regenerative medicine. The role of inflammation as an underlying driver of NDD is also considered where anti-inflammatory approaches are candidates for therapy. Indeed, cell-based therapy and/or nanotherapy may protect against inflammation to support both immune quiescence and neuronal survival in the CNS - key targets for treating NDD with the potential to reduce or even stop the cascading pathogenesis and disease progression, possibly promoting some repair where disease is treated early. By design, nanoparticles can be formulated to cross the blood brain barrier (BBB) enabling sustained delivery of neuro-protective agents for sufficient duration to reset neuro-immune homeostasis. Proven safe and efficacious, it is now urgent to deliver nano-medicine (NanoMed) as a scalable approach to treat NDD, where key stakeholders are the patients and the global economy.

Multiple Sclerosis and the LIF/IL-6 Axis: Use of Nanotechnology to Harness the Tolerogenic and Reparative Properties of LIF.

Leukaemia inhibitory factor (LIF) plays a critical role in "stemness" versus "differentiation", a property that underpins the core value of LIF as a therapeutic for both the treatment of autoimmune disease and for promoting tissue repair. This value can be realized using nano-engineering technology, where a new generation of tools can, with unprecedented ability, manipulate biological functions. One striking example is the treatment of multiple sclerosis (MS). The underpinning biology is the newly identified LIF/IL-6 axis in T lymphocytes, which can tilt the behaviour between immune tolerance versus immune attack. This LIF/IL-6 axis is ideally suited to nanotherapeutic manipulation, given its inherent mechanistic simplicity of two mutually opposing feed-forward loops that determine either tolerogenic (LIF) or inflammatory (IL-6) immunity. Using LIF that is formulated in biodegradable nanoparticles (LIF-NP) and targeted to CD4+ T cells, the axis is harnessed towards immune tolerance. This has implications for the treatment of autoimmune diseases, where the clinical burden is immense. It encompasses more than 100 diseases and, in the USA alone, costs more than $100 billion in direct health care costs annually. Other properties of LIF include the promotion of healthy neuro-glial interactions within the central nervous system (CNS), where, in addition to MS, LIF-NP therapy is relevant to inflammatory neurodegenerative diseases that represent a large and increasing need within aging populations. Thirdly, LIF is a reparative growth factor that can maintain genomic plasticity. LIF-NP supports the use of stem cell-based therapies in regenerative medicine plus augment therapeutic benefits within the patient. These core properties of LIF are greatly amplified in value by the advantage of being formulated as nanoparticles, namely (i) targeted delivery, (ii) exploitation of endogenous regulatory pathways and (iii) creation of surrogate micro-stromal niches. We discuss LIF-NP as a means to harness endogenous pathways for the treatment of MS, both to reset immune self-tolerance and to promote repair of myelin that is required to support health within the nervous system.

Modelling of a targeted nanotherapeutic 'stroma' to deliver the cytokine LIF, or XAV939, a potent inhibitor of Wnt-ß-catenin signalling, for use in human fetal dopaminergic grafts in Parkinson's disease.

The endogenous reparative capacity of the adult human brain is low, and chronic neurodegenerative disorders of the central nervous system represent one of the greatest areas of unmet clinical need in the developing world. Novel therapeutic strategies to treat them include: (i) growth factor delivery to boost endogenous repair and (ii) replacement cell therapy, including replacing dopaminergic neurons to treat Parkinson's disease (PD). However, these approaches are restricted not only by rapid degradation of growth factors, but also by the limited availability of cells for transplant and the poor survival of implanted cells that lack the necessary stromal support. We therefore hypothesised that provision of a transient artificial stroma for paracrine delivery of pro-survival factors could overcome both of these issues. Using leukaemia inhibitory factor (LIF) - a proneural, reparative cytokine - formulated as target-specific poly(lactic-co-glycolic acid) (PLGA) nano-particles (LIF-nano-stroma), we discovered that attachment of LIF-nano-stroma to freshly isolated fetal dopaminergic cells improved their survival fourfold: furthermore, in vivo, the number of surviving human fetal dopaminergic cells tended to be higher at 3 months after grafting into the striatum of nude rats, compared with controls treated with empty nanoparticles. In addition, we also analysed the effect of a novel nano-stroma incorporating XAV939 (XAV), a potent inhibitor of the developmentally important Wnt-ß-catenin signalling pathway, to investigate whether it could also promote the survival and differentiation of human fetal dopaminergic precursors; we found that the numbers of both tyrosine-hydroxylase-positive neurons (a marker of dopaminergic neurons) and total neurons were increased. This is the first demonstration that LIF-nano-stroma and XAV-nano-stroma each have pro-survival effects on human dopaminergic neurons, with potential value for target-specific modulation of neurogenic fate in cell-based therapies for PD.

Immuno-isolation of pancreatic islet allografts using pegylated nanotherapy leads to long-term normoglycemia in full MHC mismatch recipient mice.

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of "stealth" islets encapsulated within a thin cage of pegylated material of 100-200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice. We first demonstrated that pegylation of the islet surface, plus or minus nanoparticles, improved long-term islet viability in vitro compared to non-pegylated (naked) control islets. Moreover, pegylation of the islets with nanoparticles was compatible with glucose-stimulated insulin secretion and insulin biogenesis. We next looked for functionality of the created "stealth" DBA/2 (H-2(d)) islets in vivo by comparing glycemic profiles across 4 groups of streptozotozin-induced diabetic C57BL/6 (H-2(b)) recipients of (i) naked islets; (ii) pegylated islets; (iii) pegylated islets with nanoparticles (empty); and (iv) pegylated islets with nanoparticles loaded with a cargo of leukemia inhibitory factor (LIF), a factor both promotes adaptive immune tolerance and regulates pancreatic ß cell mass. Without any other treatment, normoglycemia was lost after 17 d (+/-7.5 d) in control group. In striking contrast, recipients in groups (ii), (iii), and (iv) showed long-term (>100 d) normoglycemia involving 30%; 43%, and 57% of the recipients in each respective group. In conclusion, construction of "stealth" islets by pegylation-based nanotherapeutics not only supports islet structure and function, but also effectively isolates the islets from immune-mediated destruction. The added value of nanoparticles to deliver immune modulators plus growth factors such as LIF expands the potential of this novel therapeutic approach to cell therapy for diabetes.

Targeted nanotherapy for induction of therapeutic immune responses.

Nanotechnology permits the design of therapeutic devices with defined structure and molecular composition. Modular designs employing surface-bound ligands provide specific homing devices for loaded cargo, and biocompatible and biodegradable constructs provide surrogate temporary microenvironments. We first present a case for developing 'smart' modular constructs as immunogenic vaccines to prime immune memory against specific pathogens where current vaccines fail. Second, we argue that nanotherapeutic intervention can harness pivotal molecular pathways recently discovered to regulate lineage development between pathogenic TH17 cells associated with autoimmune disease, versus tolerogenic regulatory T cells (Treg). Underpinned by molecular mechanisms that enable exquisitely specific responses in adaptive immunity, targeted nanodevices designed to stimulate either immune aggression or immune tolerance signify the birth of a new era in therapeutics.

A LIF/Nanog axis is revealed in T lymphocytes that lack MARCH-7, a RINGv E3 ligase that regulates the LIF-receptor.

Nanog is a stem cell transcription factor required for self-renewal and for maintaining pluripotency, and Nanog itself is regulated at least in part by leukaemia inhibitory factor (LIF)--a pluripotent cytokine of the IL6 family. MARCH-7 is an E-3 ligase linked to regulation of the LIF-receptor in T lymphocytes and T cells from mice that lack expression of MARCH-7 are hyper-responsive to activation signals and show a five-fold increase in LIF activity. Here we ask, does MARCH-7 influence the expression profile of Nanog during the synchronized entry of T cells into the cell cycle? We discovered that lack of MARCH-7 was permissive for Nanog expression at both transcript and protein levels during G1/S: moreover, addition of exogenous LIF to the MARCH-7 null cells caused a further 13-fold induction of Nanog; other measured transcripts including TGFß, p53 and STAT3 were relatively unchanged. Since lack of MARCH-7 altered responsiveness to activation signals we sought evidence for pre-existing regulatory miR's that might correlate with MARCH-7 gene dose using head-to-head comparisons between MARCH-7 null, heterozygous and wt spleen cells. 34 miRs were found including miR-346 that is known to target LIF transcripts and miR-346 is one of 16 miRs differentially expressed between hESCs and induced hiPSCs. Of the 34 miRs, 12 were known to be temporally regulated in embryonic nerve cells. In summary, in the absence of MARCH-7 a new signaling pathway is unmasked that involves Nanog expression in the T cell lineage. This is the first demonstration that T cells retain responsiveness to a LIF/Nanog axis and that this axis is linked to MARCH-7.

The E3 ligase axotrophin/MARCH-7: protein expression profiling of human tissues reveals links to adult stem cells.

Axotrophin/MARCH-7 was first identified in mouse embryonic stem cells as a neural stem cell gene. Using the axotrophin/MARCH-7 null mouse, we discovered profound effects on T lymphocyte responses, including 8-fold hyperproliferation and 5-fold excess release of the stem cell cytokine leukemia inhibitory factor (LIF). Our further discovery that axotrophin/MARCH-7 is required for targeted degradation of the LIF receptor subunit gp190 implies a direct role in the regulation of LIF signaling. Bioinformatics studies revealed a highly conserved RING-CH domain in common with the MARCH family of E3-ubiquitin ligases, and accordingly, axotrophin was renamed "MARCH-7." To probe protein expression of human axotrophin/MARCH-7, we prepared antibodies against different domains of the protein. Each antibody bound its specific target epitope with high affinity, and immunohistochemistry cross-validated target specificity. Forty-eight human tissue types were screened. Epithelial cells stained strongly, with trophoblasts having the greatest staining. In certain tissues, specific cell types were selectively positive, including neurons and neuronal progenitor cells in the hippocampus and cerebellum, endothelial sinusoids of the spleen, megakaryocytes in the bone marrow, crypt stem cells of the small intestine, and alveolar macrophages in the lung. Approximately 20% of central nervous system neuropils were positive. Notably, axotrophin/MARCH-7 has an expression profile that is distinct from that of other MARCH family members. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Treg versus Th17 lymphocyte lineages are cross-regulated by LIF versus IL-6.

Within the immune system there is an exquisite ability to discriminate between "self" and "non-self" that is orchestrated by T lymphocytes. Discriminatory pathways guide differentiation of these lymphocytes into either regulatory (Treg) or effector (Teff) T cells, influenced by cues from the naïve T cell's immediate micro-environment as it responds to cognate antigen. Reciprocal pathways may lead to commitment of naïve T cells into either the protective tolerance-promoting Treg, or to the pro-inflammatory Th17 effector phenotype. Primary activation of CD4(+) lymphocytes stimulates their release of leukemia inhibitory factor (LIF), and Treg continue to release LIF in response to antigen, implying a role for LIF in tolerance. In contrast, interleukin- 6 (IL-6), although very closely related to LIF, promotes maturation of Th17 cells. Here we show that LIF and IL-6 behave as polar opposites in promoting commitment to the Treg and Th17 lineages. Unlike IL6, LIF supported expression of Foxp3, the Treg lineage transcription factor, and LIF opposed IL6 by suppressing IL-6-induced IL-17A protein release. In striking contrast, we found that IL6 effectively inhibited LIF signalling, repressing transcription of the LIF receptor gp190, and strongly inducing axotrophin/MARCH-7, a novel E3 ubitquitin ligase that we discovered to be active in degradation of gp190 protein. In vivo, anti-LIF treatment reduced donor-specific Treg in recipients of foreign spleen cells. Conversely, a single dose of biodegradable LIF nanoparticles, targeted to CD4, successfully manipulated the LIF/IL6 axis towards development of donor-specific Foxp3(+) Treg. The implications for therapy are profound, harnessing endogenous immune regulation by paracrine delivery of LIF to CD4(+) cells in vivo.

Regulatory transplantation tolerance and "stemness": evidence that Foxp3 may play a regulatory role in SOCS-3 gene transcription.

Immune self-tolerance is controlled by a subset of T lymphocytes that are regulatory (Treg) and epigenetically programmed to suppress autoreactive immune effector cells in vivo. Treg require expression of Foxp3, a transcription factor that not only represses the interleukin-2 gene promoter, but also sequesters key mediators of T-cell signal transduction by complexing with cytoplasmic NFAT and NFkappaB. We have discovered that expression of Foxp3 is linked to two stem cell-related factors, namely leukemia inhibitory factor (LIF) and axotrophin. Because both LIF and axotrophin each influence Foxp3, we now ask if reciprocal cross-talk occurs; for example, does Foxp3 in turn influence LIF and/or axotrophin? We compared the effect of wt-Foxp3 versus mutant DeltaE251-Foxp3, which lacks transcriptional activity, on transcript levels of axotrophin, LIF, and suppressor of cytokine signaling-3 (SOCS-3; a feedback inhibitor of LIF) in the Jurkat human T-cell line. Unexpectedly, a 50-fold increase in SOCS-3 transcripts occurred in the DeltaE251-Foxp3 cells, coincident with a dramatic decrease in LIF transcription. This implies that, either directly or indirectly, transcription of SOCS-3 is negatively regulated by wt-Foxp3. Suppression of SOCS-3 by Foxp3 would support a model wherein Foxp3 promotes LIF signaling in Treg and is further evidence of reciprocity between Foxp3, LIF, and axotrophin.

Evidence for functional inter-relationships between FOXP3, leukaemia inhibitory factor, and axotrophin/MARCH-7 in transplantation tolerance.

In an ex vivo mouse model, regulatory transplantation tolerance is not only linked to Foxp3, but also to release of leukaemia inhibitory factor (LIF) and to expression of axotrophin (also known as MARCH-7), a putative ubiquitin E3 ligase associated with feedback control of T cell activation and of T cell-derived LIF. Given this coordinate correlation with tolerance, we now ask if Foxp3 expression is influenced by LIF or by axotrophin. In spleen cells from allo-rejected mice we found that exogenous LIF reduced interferon gamma release in response to donor antigen by 50%, but LIF had no direct effect on levels of Foxp3 protein in allo-primed cells that were either tolerant, or aggressive, for donor antigen. However, we did find an effect of axotrophin on Foxp3: in the axotrophin null mouse, thymic Foxp3 transcripts were reduced compared to axotrophin wildtype littermates. To test whether these findings in the mouse were of potential significance in man we measured transcript levels of axotrophin and LIF in peripheral blood cell samples collected for a recently published clinical study concerning haematopoietic stem cell recipients. In controls, human peripheral blood CD4+CD25+cells contained significantly more FOXP3 and axotrophin than CD4+CD25-cells. In bone marrow autograft recipients, where peripheral blood cell samples directly represent both the grafted tissue and the immune response, both FOXP3 and axotrophin negatively correlated with graft versus host disease (GVHD). These data suggest that (i) thymic Foxp3+T cell development is influenced by axotrophin; and (ii) clinical auto-GVHD inversely correlates with axotrophin transcript expression as has been previously reported for FOXP3.

Axotrophin and leukaemia inhibitory factor (LIF) in transplantation tolerance.

Immune self-tolerance is controlled by a subset of T lymphocytes that are regulatory (Treg) and epigenetically programmed to suppress auto-reactive immune effector cells in vivo. By extrapolation, donor-specific transplantation tolerance might be controlled by donor-specific Treg that have acquired the appropriate epigenetic program for tolerance. Although such tolerance has yet to be achieved in man, proof of concept comes from mouse models where regulatory transplantation tolerance can be induced within the complex micro-environment of the spleen or draining lymph node. By studying whole spleen cell populations in a murine model of transplantation tolerance we have incorporated a complexity of environmental factors when looking for specific features that characterize tolerance versus aggression. This approach has revealed unexpected patterns of gene activity in tolerance and most notably that a novel stem cell gene, axotrophin, regulates T lymphocyte responsiveness both in terms of proliferation and in release of leukaemia inhibitory factor (LIF). Since LIF is a regulator of stem cells in addition to being a key neuropoietic cytokine, these preliminary results linking both axotrophin and LIF to transplantation tolerance lead us to propose that regulatory pathways encoded during the epigenetic development of Treg cells are related to pathways that regulate fate determination of stem cells.

Leukemia inhibitory factor is linked to regulatory transplantation tolerance.

BACKGROUND: The specific regulation of allo-tolerance in vivo occurs within a complex microenvironment and involves co-operation between a small proportion of different cell types within the spleen or draining lymph node. By analyzing unmanipulated whole spleen cell populations we have aimed to mimic this in vivo situation to identify critical signaling molecules in regulatory allo-tolerance. METHODS: We compared the kinetics of cytokine release and induction of signaling proteins in (BALB/c-tolerant)CBA, versus (BALB/c-rejected)CBA, spleen cells after challenge with BALB/c antigen. RESULTS: The distinguishing features of allo-tolerance were Foxp3 protein expression, LIF release, and increased levels of STAT3. Comparison of isogenic clones of Tr1, Th1, and Th2 cells revealed that only the regulatory Tr1 cells are characterized by both LIF and IL10 release. CONCLUSIONS: Overall, our findings demonstrate that allo-antigen driven signaling events can be detected within a whole spleen cell population and identify a role for LIF in the regulation of transplantation tolerance in vivo.

Leukaemia inhibitory factor (LIF) is functionally linked to axotrophin and both LIF and axotrophin are linked to regulatory immune tolerance.

Axotrophin (axot) is a newly characterised stem cell gene and mice that lack axotrophin are viable and fertile, but show premature neural degeneration and defective development of the corpus callosum. By comparing axot+/+, axot+/- and axot-/- littermates, we now show that axotrophin is also involved in immune regulation. Both T cell proliferation and T cell-derived leukaemia inhibitory factor (LIF) were suppressed by axotrophin in a gene-dose-dependent manner. Moreover, a role for axotrophin in the feedback regulation of LIF is implicated. This is the first evidence that fate determination mediated by LIF maybe qualified by axotrophin.

Transplantation tolerance: gene expression profiles comparing allotolerance vs. allorejection.

An understanding of the molecular basis of immune regulation will allow development of therapies for diseases caused by immune dysregulation and for therapeutic exploitation of the immune response in transplantation of organ grafts or stem cells. To identify critical regulatory factors in immunity, we have used a mouse model wherein infectious regulatory tolerance is inducible by CD4/CD8 blockade in recipients of vascularised heart grafts. Once established, this transplantation tolerance is robust and isolated "tolerant" spleen cells show powerful immune regulatory properties, being able to impose donor-specific allotolerance upon fully immune competent naive recipients. Here, we present a compound comparison of four gene arrays (tolerance vs. rejection, at 48 h, and at 123 h) where a relatively small number of differentially expressed genes occurred. In rejection, there was a strong progressive amplification of IFNgamma and granzyme B mRNAs. In tolerance, both ELKL motif kinase and axotrophin occurred in the group of upregulated genes. Mice lacking ELKL motif kinase develop autoimmune disease, whilst axotrophin is a newly discovered stem cell gene that has only been explored in the context of neural development. This gene expression data is the first to demonstrate a link between axotrophin and regulatory tolerance and, since axotrophin, LIF, STAT3 and c-kit each function in stem cells, we propose that common mechanisms play a central role both in developmental regulation of stem cells, and in immune regulation.