Cardiotrophin-Like Cytokine Factor 1 Exhibits a Myeloid-Biased Hematopoietic-Stimulating Function.

Cardiotrophin-like cytokine factor 1 (CLCF1) is secreted as a complex with the cytokine receptor-like factor 1 (CRLF1). Syndromes caused by mutations in the genes encoding CLCF1 or CRLF1 suggest an important role for CLCF1 in the development and regulation of the immune system. In mice, CLCF1 induces B-cell expansion, enhances humoral responses and triggers autoimmunity. Interestingly, inactivation of CRLF1, which impedes CLCF1 secretion, leads to a marked reduction in the number of bone marrow (BM) progenitor cells, while mice heterozygous for CLCF1 display a significant decrease in their circulating leukocytes. We therefore hypothesized that CLCF1 might be implicated in the regulation of hematopoiesis. To test this hypothesis, murine hematopoietic progenitor cells defined as Lin-Sca1+c-kit+ (LSK) were treated in vitro with ascending doses of CLCF1. The frequency and counts of LSK cells were significantly increased in the presence of CLCF1, which may be mediated by several CLCF1-induced soluble factors including IL-6, G-CSF, IL-1ß, IL-10, and VEGF. CLCF1 administration to non-diseased C57BL/6 mice resulted in a pronounced increase in circulating myeloid cells, which was concomitant with augmented LSK and myeloid cell counts in the BM. Likewise, CLCF1 administration to mice following sub-lethal irradiation or congeneic BM transplantation (BMT) resulted in accelerated LSK recovery along with a sustained increase in BM-derived CD11b+ cells. Altogether, our observations establish an important and unforeseen role for CLCF1 in regulating hematopoiesis with a bias toward myeloid cell differentiation.

Cardiotrophin-like Cytokine Increases Macrophage-Foam Cell Transition.

CLCF1 is a neurotrophic and B cell-stimulating factor belonging to the IL-6 family. Mutations in the gene coding for CLCF1 or its secretion partner CRLF1 lead to the development of severe phenotypes, suggesting important nonredundant roles in development, metabolism, and immunity. Although CLCF1 was shown to promote the proliferation of the myeloid cell line M1, its roles on myeloid activation remain underinvestigated. We characterized the effects of CLCF1 on myeloid cells with a focus on monocyte-macrophage and macrophage-foam cell differentiations. CLCF1 injections in mice resulted in a significant increase in CD11b+ circulating cells, including proinflammatory monocytes. Furthermore, CLCF1 activated STAT3 phosphorylation in bone marrow CD11b+ cells and in bone marrow-derived macrophages (BMDM). BMDM stimulated with CLCF1 produced a large array of proinflammatory factors comprising IL-6, IL-9, G-CSF, GM-CSF, IL-1ß, IL-12, CCL5, and CX3CL1. The pattern of cytokines and chemokines released by CLCF1-treated BMDM led us to investigate the role of CLCF1 in foam cell formation. When pretreated with CLCF1, BMDM presented a marked SR-A1 upregulation, an increase in acetylated-low-density lipoprotein uptake, and an elevated triglyceride accumulation. CLCF1-induced SR-A1 upregulation, triglyceride accumulation, and acetylated-low-density lipoprotein uptake could be prevented using ruxolitinib, a JAK inhibitor, indicating that the effects of the cytokine on myeloid cells result from activation of the canonical JAK/STAT signaling pathway. Our data reveal novel biological roles for CLCF1 in the control of myeloid function and identify this cytokine as a strong inducer of macrophage-foam cell transition, thus bringing forward a new potential therapeutic target for atherosclerosis.

Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity.

The cytokines CLCF1 and CNTF are ligands for the CNTF receptor and the apolipoprotein E (ApoE) receptor sortilin. Both share structural similarities with the N-terminal domain of ApoE, known to bind CNTF. We therefore evaluated whether ApoE or ApoE-containing lipoproteins interact with CLCF1 and regulate its activity. We observed that CLCF1 forms complexes with the three major isoforms of ApoE in co-immunoprecipitation and proximity assays. FPLC analysis of mouse and human sera mixed with CLCF1 revealed that CLCF1 co-purifies with plasma lipoproteins. Studies with sera from ApoE-/- mice indicate that ApoE is not required for CLCF1-lipoprotein interactions. VLDL- and LDL-CLCF1 binding was confirmed using proximity and ligand blots assays. CLCF1-induced STAT3 phosphorylation was significantly reduced when the cytokine was complexed with VLDL. Physiological relevance of our findings was asserted in a mouse model of oxygen-induced retinopathy, where the beneficial anti-angiogenic properties of CLCF1 were abrogated when co-administrated with VLDL, indicating, that CLCF1 binds purified lipoproteins or lipoproteins in physiological fluids such as serum and behave as a "lipocytokine". Albeit it is clear that lipoproteins modulate CLCF1 activity, it remains to be determined whether lipoprotein binding directly contributes to its neurotrophic function and its roles in metabolic regulation.

Interleukin-21 promotes thymopoiesis recovery following hematopoietic stem cell transplantation.

BACKGROUND: Impaired T cell reconstitution remains a major deterrent in the field of bone marrow (BM) transplantation (BMT) due to pre-conditioning-induced damages inflicted to the thymi of recipient hosts. Given the previously reported thymo-stimulatory property of interleukin (IL)-21, we reasoned that its use post-BMT could have a profound effect on de novo T cell development. METHODS: To evaluate the effect of IL-21 on de novo T cell development in vivo, BM derived from RAG2p-GFP mice was transplanted into LP/J mice. Lymphocyte reconstitution was first assessed using a hematological analyzer and a flow cytometer on collected blood samples. Detailed flow cytometry analysis was then performed on the BM, thymus, and spleen of transplanted animals. Finally, the effect of human IL-21 on thymopoiesis was validated in humanized mice. RESULTS: Using a major histocompatibility complex (MHC)-matched allogeneic BMT model, we found that IL-21 administration improves immune reconstitution by triggering the proliferation of BM Lin-Sca1+c-kit+ (LSK) subsets. The pharmacological effect of IL-21 also culminates in the recovery of both hematopoietic (thymocytes) and non-hematopoietic (stromal) cells within the thymi of IL-21-treated recipient animals. Although T cells derived from all transplanted groups proliferate, secrete various cytokines, and express granzyme B similarly in response to T cell receptor (TCR) stimulation, full regeneration of peripheral naïve CD4+ and CD8+ T cells and normal TCRvß distribution could only be detected in IL-21-treated recipient mice. Astonishingly, none of the recipient mice who underwent IL-21 treatment developed graft-versus-host disease (GVHD) in the MHC-matched allogeneic setting while the graft-versus-tumor (GVT) effect was strongly retained. Inhibition of GVHD onset could also be attributed to the enhanced generation of regulatory B cells (B10) observed in the IL-21, but not PBS, recipient mice. We also tested the thymopoiesis-stimulating property of human IL-21 in NSG mice transplanted with cord blood (CB) and found significant improvement in de novo human CD3+ T cell development. CONCLUSIONS: In sum, our study indicates that IL-21 represents a new class of unforeseen thymopoietin capable of restoring thymic function following BMT.

Epstein-Barr virus-induced gene 3 (EBI3) can mediate IL-6 trans-signaling.

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6 trans-signaling, albeit less efficiently than soluble IL-6Rα. In line with this interpretation, co-immunoprecipitation and SPR experiments indicated that EBI3 binds IL-6. An important pro-inflammatory function of IL-6 trans-signaling is to activate blood vessel endothelial cells. We observed that EBI3 in combination with IL-6 could induce the expression of chemokines by human venal endothelial cells. Our results indicate that EBI3 can promote pro-inflammatory IL-6 functions by mediating trans-signaling. These unexpected observations suggest that use of EBI3 as a therapeutic biologic for autoimmune diseases will likely require co-administration of soluble gp130 to prevent the side effects associated with IL-6 trans-signaling. Together with previous studies that demonstrated activation of IL-6R by p28 (IL-30), new findings further suggest a complex interrelation between IL-27 and IL-6.

Renal and Hematological Effects of CLCF-1, a B-Cell-Stimulating Cytokine of the IL-6 Family.

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

Ex vivo generation of murine IL-10-producing B cells by fusokines.

Naïve B cells are crucial components of adaptive immunity. In addition to their capacity to produce immunoglobulins, a minor subset termed regulatory B cells or Bregs has been proven to modulate inflammation through the secretion of soluble mediators. The two main technical difficulties with their clinical use lie in their relatively low abundance in vivo and the scarcity of known methods for their ex vivo expansion. While studying the pharmacological properties of a novel bifunctional granulocyte macrophage-colony-stimulating factor (GM-CSF) and IL-15 fusion transgene (GIFT15) on unfractionated splenocytes in vitro, we observed that the GIFT15 fusokine had a remarkable and unprecedented effect on naïve B cells by converting them into suppressor cells of B-cell ontogeny (hereafter referred to as GIFT15 inducible (i)Bregs). Moreover, GIFT15 promoted iBreg proliferation. We present in this report a detailed protocol using the GIFT15 fusokine as a tool for the ex vivo generation of murine iBregs, which may serve as an immediate remedy to their abundance challenge in the clinic.

The composite cytokine p28/cytokine-like factor 1 sustains B cell proliferation and promotes plasma cell differentiation.

IL-27 is an APC-derived IL-6/IL-12 family composite cytokine with multiple functions such as regulation of Th1, Th17, and regulatory T cell differentiation, B cell proliferation, and Ig class switching. The IL-27 complex is formed by the association of the cytokine p28 with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27 cytokine and soluble receptor subunits p28 and EBI3 can be secreted independently. The p28 subunit has been shown to have IL-27-independent biological activities. We previously demonstrated that p28 can form an alternative composite cytokine with the EBI3 homolog cytokine-like factor 1 (CLF; CRLF1). p28/CLF modulates NK cell activity and CD4 T cell cytokine production in vitro. In this study we used IL-6-dependent plasmacytoma cell line B9 and CD4 T cells from IL-27Rα-deficient mice to demonstrate that p28/CLF activates IL-27-unresponsive cells, indicating that p28/CLF and IL-27 signal through different receptors. The observation that p28/CLF, unlike IL-27, sustains B9 plasmacytoma cell proliferation prompted us to investigate the effects of p28/CLF on mouse B cells. We observed that p28/CLF induces IgM, IgG2c, and IgG1 production and plasma cell differentiation. p28/CLF therefore has the potential to contribute to B and plasma cell function, differentiation, and proliferation in normal and pathological conditions such as Castelman's disease and multiple myeloma.

A polyglutamic acid motif confers IL-27 hydroxyapatite and bone-binding properties.

The p28 subunit of the composite cytokine IL-27 comprises a polyglutamic acid domain, which is unique among type I cytokines. This domain is very similar to the acidic domain known to confer hydroxyapatite (HA)-binding properties and bone tropism to bone sialoprotein. We observed IL-27 binding to HA, in accordance with previous studies reporting successful p28 HA chromatography. The IL-27 polyglutamic acid domain is located in a flexible inter-α helix loop, and HA-bound IL-27 retained biological activity. Using IL-27 alanine mutants, we observed that the p28 polyglutamic acid domain confers HA- and bone-binding properties to IL-27 in vitro and bone tropism in vivo. Because IL-27 is a potent regulator of cells residing in endosteal bone marrow niches such as osteoclasts, T regulatory, memory T, plasma, and stem cells, this specific property could be beneficial for therapeutic applications. IL-27 has potent antitumoral and antiosteoclastogenic activities. It could therefore also be useful for therapies targeting hematologic cancer or solid tumors metastasis with bone tropism. Furthermore, these observations suggest that polyglutamic motifs could be grafted onto other type I cytokine inter-α helix loops to modify their pharmacological properties.

A novel role for STAT5 in DC: Controlling the Th2-response.

Dendritic cells (DC) play a key role in immunity by recognizing and presenting antigens. Cytokines and cytokine-activated transcription factors are fundamental in the regulation of the DC differentiation and their functions. While the role of STAT3 in DC development is well established, the function of STAT5 in DC has yet to be fully elucidated. A recent study published in Nature Immunology by Bell et al., using the DC-specific deletion of Stat5, demonstrated the importance of STAT5 in the induction of a Th2 response in DC. As the activation of this transcription factor is not required for the induction of a Th1 response, the authors further investigated the role STAT5 in the signaling to thymic stromal lymphopoietin (TSLP), a cytokine known to be important for type 2 inflammatory responses. Their results demonstrate the importance of STAT5 activation during TLSP-induced Th2 responses and suggest that DC are a key TLSP target.

The cytokines cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF) and ciliary neurotrophic factor (CNTF) differ in their receptor specificities.

Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor ß (LIFRß) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRß signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRß with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-ß in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.

IL-6 activates STAT5 in T cells.

BACKGROUND: IL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation. METHODS: We analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS). RESULTS: The results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5. CONCLUSIONS: Thus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-ß stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation.

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

The IL-27 p28 subunit binds cytokine-like factor 1 to form a cytokine regulating NK and T cell activities requiring IL-6R for signaling.

IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.