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Interleukin-17 receptor D (IL-17RD), also known as similar expression to Fgf genes (SEF), is proposed to act as a signaling hub that negatively regulates mitogenic signaling pathways, like the ERK1/2 MAP kinase pathway, and innate immune signaling. The expression of IL-17RD is downregulated in certain solid tumors, which has led to the hypothesis that it may exert tumor suppressor functions. However, the role of IL-17RD in tumor biology remains to be studied in vivo. Here, we show that genetic disruption of Il17rd leads to the increased formation of spontaneous tumors in multiple tissues of aging mice. Loss of IL-17RD also promotes tumor development in a model of colitis-associated colorectal cancer, associated with an exacerbated inflammatory response. Colon tumors from IL-17RD-deficient mice are characterized by a strong enrichment in inflammation-related gene signatures, elevated expression of pro-inflammatory tumorigenic cytokines, such as IL-17A and IL-6, and increased STAT3 tyrosine phosphorylation. We further show that RNAi depletion of IL-17RD enhances Toll-like receptor and IL-17A signaling in colon adenocarcinoma cells. No change in the proliferation of normal or tumor intestinal epithelial cells was observed upon genetic inactivation of IL-17RD. Our findings establish IL-17RD as a tumor suppressor in mice and suggest that the protein exerts its function mainly by limiting the extent and duration of inflammation.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10-9, including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into adipocytes, chondrocytes, or osteocytes. MSCs secrete an array of cytokines and express the LIFRβ (leukemia inhibitory factor receptor) chain on their surface. Mutations in the gene coding for LIFRβ lead to a syndrome with altered bone metabolism. LIFRβ is one of the signaling receptor chains for cardiotrophin-like cytokine (CLCF1), a neurotrophic factor known to modulate B and myeloid cell functions. We investigated its effect on MSCs induced to differentiate into osteocytes in vitro. Our results indicate that CLCF1 binds mouse MSCs, triggers STAT1 and -3 phosphorylation, inhibits the up-regulation of master genes involved in the control of osteogenesis, and markedly prevents osteoblast generation and mineralization. This suggests that CLCF1 could be a target for therapeutic intervention with agents such as cytokine traps or blocking mAbs in bone diseases such as osteoporosis.

The Cardiotrophin-like Cytokine Factor 1 (CLCF1) is a cytokine of the IL6 family with important pro-neurotrophic and immuno-modulating functions. However, the mechanisms behind CLCF1 activities and CLCF1 properties in pathological models remain poorly understood. This is partly due to a lack of tools for the study of CLCF1 functions in vivo. Indeed, the complete knock-out of CLCF1 in mice is lethal at P1: underdeveloped motor neurons of the face and jaw prevent the pups from suckling. To overcome this obstacle, we obtained a CLCF1 conditional knock-out mice model generated using the CRISPR-Cas9 technology. We then bred these mice with Vav-Cre mice to induce CLCF1 deletion in hematopoietic cells. We are now conducting immuno-phenotyping experiments of the main immune populations in those animals. Considering the activities of CLCF1 that were previously shown in vitro or in vivo using overexpression models, we hypothesize that the knock-out of CLCF1 in immune cells will lead to decreased numbers of B and myeloid cells. This new CLCF1 conditional knock-out mice model will be a potent tool to confirm and further study the activities, mechanisms, and pathological roles of CLCF1 in vivo. Supported by grants from CIHR (Canadian Institutes of Health Research)

SUMMARYMost loci identified by GWAS have been found in populations of European ancestry (EA). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EA individuals, we identified 5,552 trait-variant associations at P−9, including 71 novel loci not found in EA populations. We also identified novel ancestry-specific variants not found in EA, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional, and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EA-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations, and compared genetic architecture and the impact of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.

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-1β, 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.

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.

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.

Cardiotrophin-like Cytokine Factor 1 (CLCF1) belongs to the IL6 family of cytokines and possesses pro-neurotrophic and immuno-modulating functions. Coding mRNA for CLCF1 has been detected in primary and secondary lymphoid organs (i.e. lymph nodes, spleen and bone marrow), as well as in the lungs and feminine reproductive organs. Modulation of CLCF1’s mRNA levels has been associated with the Th17 polarization in CD4+ T cells. However, little information is available regarding CLCF1 protein levels in these tissues or the nature of the immune cells responsible for its production. This can be explained by a lack of in situ detection options for CLCF1. We have therefore developed a methodology for the detection of human and murine CLCF1 by flow cytometry in permeabilized cells. This technique has been validated using derivatives of the Ba/F3 cell line in which cDNAs coding for human and murine CLCF1 were introduced by transduction with recombinant retroviruses. We are currently using this approach to study CLCF1 production by human and murine immune cells. Preliminary results indicate a production of CLCF1 by Th17 T cells. The development of a method to detect CLCF1 by flow cytometry will be beneficial for the study of CLCF1’s functions in the regulation of the immune response.

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) after renal transplantation is believed to be caused by a circulating factor(s). We detected cardiotrophin-like cytokine factor 1 (CLCF1), a member of the interleukin 6 family, in the plasma from patients with recurrent FSGS. We hypothesized that CLCF1 contributes to the effect of FSGS serum on the glomerular filtration barrier in vitro. Presently, we studied the effect of CLCF1 on isolated rat glomeruli using an in vitro assay of albumin permeability ( P alb ). CLCF1 (0.05–100 ng/mL) increased P alb and caused maximal effect at 5–10 ng/mL ( P P alb was analogous to the effect of FSGS serum. Anti-CLCF1 monoclonal antibody blocked the CLCF1-induced increase in P alb and significantly attenuated the effect of FSGS serum ( P P alb caused by CLCF1 or FSGS serum. Western blot analysis showed that CLCF1 upregulated phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Tyr705) in glomeruli. This effect was diminished by the heterodimer CLCF1-CRLF1. Janus kinase 2 (JAK2) inhibitor BMS-1119543 or STAT3 inhibitor Stattic significantly blocked the effect of CLCF1 or FSGS serum on P alb ( P P alb , the heterodimer CLCF1-CRLF1 may protect the glomerular filtration barrier. We speculate that albuminuria in FSGS is related to qualitative or quantitative changes in the CLCF1-CRLF1 complex, and that JAK2 or STAT3 inhibitors may be novel therapeutic agents to treat FSGS.

Ciliary neurotrophic factor (CNTF) is the most extensively studied member of the cytokine family that signal through intracellular chains of the gp130/LIFRβ receptor. The severe phenotype in patients suffering from mutations inactivating LIFRβ indicates that members of this cytokine family play key, non-redundant roles during development. Accordingly, three decades of research has revealed potent and promising trophic and regulatory activities of CNTF in neurons, oligodendrocytes, muscle cells, bone cells, adipocytes and retinal cells. These findings led to clinical trials to test the therapeutic potential of CNTF and CNTF derivatives for treating neurodegenerative and metabolic diseases. Promising results have encouraged continuation of studies for treating retinal degenerative diseases. Results of some clinical trials showed that side-effects may limit the systemically administrated doses of CNTF. Therefore, therapies being currently tested rely on local delivery of CNTF using encapsulated cytokine-secreting implants. Since the side effects of CNTF might be linked to its ability to activate the alternative IL6Rα-LIFRβ-gp130 receptor, CNTFR-specific mutants of CNTF have been developed that bind to the CNTFRα-LIFRβ-gp130 receptor. These developments may prove to be a breakthrough for therapeutic applications of systemically administered CNTF in pathologies such as multiple sclerosis or Alzheimer's disease. The "designer cytokine approach" offers future opportunities to further enhance specificity by conjugating mutant CNTF with modified soluble CNTFRα to target therapeutically relevant cells that express gp130-LIFRβ and a specific cell surface marker.

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/LIFRβ signaling in obesity and obesity-related metabolic diseases.

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.

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 β (LIFRβ) 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 LIFRβ 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/LIFRβ 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-β 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.

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.

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

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 inin vitroandin vivostudies. 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.

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+CD27− naive, and CD19+CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cε promoter activation in a gene reporter assay, nor germline Cε mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-γ-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-γ production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-γ production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.

Ciliary neurotrophic factor (CNTF) receptor controls a pathway supporting the differentiation and survival of a wide range of neural cell types during development and in adulthood. Cardiotrophin-like cytokine (CLC)–cytokine-like factor 1 (CLF) composite cytokine is a second ligand for the CNTF α-component receptor (CNTFRα). This composite cytokine is built on the structural model of IL-12, with a complex formed by a four-helix bundle type I cytokine, CLC (also referred to asCLCF1), bound to a soluble receptor subunit, CLF (also known asCRLF1). We have reported mutations in the chaperone soluble receptor CLF, causing cold-induced sweating syndrome (CISS). In this study, we studied theCLC-mutated alleles in a patient suffering from a similar disease. This patient was compound heterozygous for two differentCLCmutations. The first allele was inactivated by a stop codon at position 107 (Y107X). In the second allele, a R197L mutation in the CLC-predicted binding site to the CNTFRα was detected. Functional analysis of the mutated protein revealed an incapacity for R197L CLC to bind to CNTFRα and activate the subsequent signaling events. Structural and docking interaction studies showed that the R197L substitution destabilized the contact site between CLC and CNTFRα.

The recently identified IL-6 family member cardiotrophin-like cytokine (also named novel neurotrophin-1 or B cell stimulating factor-3) forms a secreted complex with cytokine-like factor-1 which binds and activates the tripartite ciliary neurotrophic factor receptor. The striking differences between the phenotype of mice in which either the ciliary neurotrophic factor or its receptor are inactivated suggest that the cardiotrophin-like cytokine/cytokine-like factor-1 complex could be the developmentally important ciliary neurotrophic factor receptor ligand. Cardiotrophin-like cytokine is also produced in the immune system and has been reported to activate B cells in vivo and in vitro. B cells do not express the ciliary neurotrophic factor receptor suggesting the existence of an alternative receptor. We produced the cardiotrophin-like cytokine/cytokine-like factor-1 complex tagged with a Bir A biotin ligase AviTag peptide substrate. This cytokine could be efficiently biotinylated in vitro with Bir A. It was subsequently validated as a sensitive tool for ciliary neurotrophic factor receptor detection by flow cytometry and for magnetic-activated cell sorting. It was also shown to allow the detection of a specific receptor by activated B cells. Whereas binding to cells expressing the ciliary neurotrophic factor receptor could be prevented by competition with ciliary neurotrophic factor, binding to B cells was not. The biotinylated cardiotrophin-like cytokine/cytokine-like factor-1 complex therefore represents a new reagent to study ciliary neurotrophic factor and cardiotrophin-like cytokine receptor expression and for the identification of the putative cardiotrophin-like cytokine B cell receptor. It further validates the use of biotin ligase catalysed biotinylation for the detection of cytokine receptors.

MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe191 or Phe121 strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.

The cytokines of the interleukin-6 family are multifunctional proteins that regulate cell growth, differentiation, and other cell functions in a variety of biological systems including the immune, inflammatory, hematopoietic, and nervous systems. One member of this family, ciliary neurotrophic factor (CNTF), displays biological functions more restricted to the neuromuscular axis. We have recently identified two additional ligands for the CNTF receptor complex. Both are composite cytokines formed by cardiotrophin-like cytokine (CLC) associated to either the soluble type I cytokine receptor CLF or the soluble form of CNTF receptor alpha (CNTFRalpha). The present study was aimed at analyzing the interactions between the cytokine CLC and its different receptor chains. For this purpose, we modeled CLC/receptor interactions to define the residues potentially involved in the contact sites. We then performed site-directed mutagenesis on these residues and analyzed the biological interactions between mutants and receptor chains. Importantly, we found that CLC interacts with the soluble forms of CNTFRalpha and CLF via sites 1 and 3, respectively. For site 1, the most crucial residues involved in the interaction are Trp67, Arg170, and Asp174, which interact with CNTFRalpha. Surprisingly, the residues that are important for the interaction of CLC with CLF are part of the conserved FXXK motif of site 3 known to be the interaction site of LIFRbeta. Obtained results show that the Phe151 and Lys154 residues are effectively involved in the interaction of CLC with LIFRbeta. This study establishes the molecular details of the interaction of CLC with CLF, CNTFRalpha, and LIFRbeta and helps to define the precise role of each protein in this functional receptor complex.

IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19+ human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG1 and IgG3 by CD40-activated CD19+CD27− naive human B cells. Although stimulation of CD19+ B cells via CD40 alone induced γ1 and γ3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of Sγ/Sμ switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG1 and IgG3.

Next Section Abstract We describe a novel cytokine receptor named GP130 Like receptor, or GPL, that displays similarities with the interleukin-6 and interleukin-12 family of signaling receptors. Four different isoforms diverging in their carboxyl terminus were isolated, corresponding to proteins encompassing 560, 610, 626, and 745 amino acids. Sequences included a signal peptide of 32 amino acids, followed by a cytokine binding domain containing four conserved cysteines, a WSDWS motif, and a region consisting of three fibronectin type III domain repeats. No immunoglobulin-like module was identified in the GPL sequences. The intracellular part of longer isoforms contained a proline-rich region defining a box1 motif for interaction with the Janus kinases. The Gpl gene is organized in 15 exons and is located on 5q11.2 in tandem with the gp130 gene. Both genes were only separated by 24 kilobases, with opposite transcriptional orientations. The GPL receptor displayed a 28% identity with gp130. Specific GPL transcripts were observed in tissues involved in reproduction. Transcripts were also found in blood cells and in bone marrow, revealing expression of GPL in all of the myelomonocytic lineage, from hematopoietic stem cells to activated dendritic cells. In monocytes and dendritic cells, expression of GPL was strongly up-regulated by interferon-γ, indicating a possible involvement of GPL in Th1-type immune responses. The molecular basis of cell signaling mediated by GPL was studied using chimeric receptors where external portions of α or β interleukin-5 receptor subunits were fused to the internal portion of GPL or of related receptors. Results indicated that association of GPL to the intracellular portions of gp130, or LIF receptor, allowed the signaling cascade.

International audience; Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

Interleukin (IL)-4, which exhibits potent anti-inflammatory activities, is of potential therapeutic value in destructive arthropathies. To further define the response of human joint cells to IL-4, we analyzed the ability of this cytokine to modulate the effects of IL-1beta and growth factors. Freshly isolated chondrocytes, dedifferentiated chondrocytes, and synoviocytes were treated with IL-4 before determination of nitric oxide (NO) and collagenase production in response to IL-1beta, or before proliferation assays in presence of IL-1beta, platelet-derived growth factor (PDGF), or transforming growth factor (TGF)-beta. IL-4 downregulated IL-1beta induced NO production in dedifferentiated chondrocytes and inhibited IL-1beta induced collagenase release, as well as IL-1beta and growth factor induced proliferation in dedifferentiated chondrocytes and synoviocytes. In contrast, IL-4 had no effect in freshly isolated primary chondrocytes and in cartilage explants. The lack of response to IL-4 in primary chondrocytes was associated with impaired signal transduction, as indicated by markedly decreased IL-4 dependent tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-6. It also correlated with differences in the expression pattern of IL-4 receptor (IL-4R) subunits during chondrocyte dedifferentiation. Indeed, whereas the IL-4Ralpha and IL-13Ralpha' subunits were expressed in all cell types, expression of the common receptor gamma chain was restricted to freshly isolated chondrocytes. In conclusion, IL-4 downregulated IL-1beta-induced catabolic events and cell proliferation in dedifferentiated chondrocytes and synoviocytes, but had no effects in freshly isolated chondrocytes. The difference in IL-4 responsiveness between primary and dedifferentiated chondrocytes correlated with changes in proximal signaling events and in the expression pattern of IL-4R subunits during cell dedifferentiation.

The heterodimeric cytokine composed of the soluble ciliary neurotrophic factor receptor (sCNTFR) and the IL-6 family member cardiotrophin-like cytokine (CLC) was recently identified as a new ligand for gp130–leukemia inhibitory factor receptor (LIFR) complex [Plun-Favreau, H., Elson, G., Chabbert, M., Froger, J., deLapeyriere, O., Lelievre, E., Guillet, C., Hermann, J., Gauchat, J. F., Gascan, H. & Chevalier, S. (2001) EMBO J.20, 1692–1703]. This heterodimer shows overlapping biological properties with LIF. Although CLC contains a putative signal peptide and therefore should enter into the classical secretory pathway, the protein has been shown to be retained within transfected mammalian cells, unless coexpressed with either sCNTFR or cytokine like factor (CLF) [Elson, G. C., Lelievre, E., Guillet, C., Chevalier, S., Plun-Favreau, H., Froger, J., Suard, I., de Coignac, A. B., Delneste, Y., Bonnefoy, J. Y., Gauchat, J. F. & Gascan, H. (2000) Nat. Neurosci.3, 867–872]. In the present study, we demonstrate that a fusion protein comprising CLC covalently coupled through a glycine/serine linker to sCNTFR (CC–FP) is efficiently secreted from transfected mammalian cells. CC–FP shows enhanced activities in respect to the CLC/sCNTFR native complex, on a number of cells expressing gp130 and LIFR on their surface. In addition, CC–FP is able to compete with CNTF for cell binding, indicating that both cytokines share binding epitope(s) expressed by their receptor complex. Analysis of the downstream signaling events revealed the recruitment by CC–FP of the signal transducer and activator of transcription (STAT)-3, Akt and mitogen-activated protein (MAP) kinase pathways. The monomeric bioactive CLC/sCNTFR fusion protein is therefore a powerful tool to study the biological role of the recently described cytokine CLC.

1. 6-8 weeks old mouse 2. PBS without Ca 2+ and Mg 2+ (Wisent, catalog number: 311-01-CL) 3. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL) 4. Fetal bovine serum (FBS) (Life Technologies, Gibco ® , catalog number: 12483) 5. Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL) 6. Trypan blue (Life Technologies, Gibco ® , catalog number: 15250-061) 7. Trypsin 0.05%/EDTA 0.53 mM (Wisent, catalog number: 325-042-EL) 8. APC-conjugated anti-CD31 antibody (clone MEC13.3) (BD biosciences, catalog number: 551262) 9. FITC-conjugated anti-CD45 antibody (clone 30-F11) (BD biosciences, catalog number: 553080) 10. APC-conjugated anti-CD44 antibody (clone IM7) (BD biosciences, catalog number: 559250) 11. PE-conjugated anti-CD105 antibody (clone MJ7/18) (BD biosciences, catalog number: 562759) 12. FITC-conjugated anti-CD90 antibody (clone 5E10) (BD biosciences, catalog number: 555595)

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.

CD86 is a costimulatory molecule constitutively expressed by human antigen presenting cells which interacts with CD28 and CTLA-4 expressed by T cells. We have recently reported the identification of an alternatively spliced CD86 mRNA variant (CD86deltaTM) characterized by the deletion of exon 6 which encodes for the transmembrane domain. We report here the identification of an alternatively spliced variant (called CD86deltaEC) expressed by nonstimulated human monocytes and characterized by the deletion of exons 4 and 5 which encode for the extracellular V-like and C-like domains, respectively. The activation of monocytes by IFNgamma (i) induces the preferential expression of the transcript encoding for the membrane form and (ii) increases the expression of CD86 and of the accessory molecules CD40, CD49d and CD54. These results suggest that resting human monocytes may constitutively express different forms of CD86 which can then influence the activation of T cells.

Responsiveness to IL-13 involves at least two chains, IL-4Ralpha and IL-13Ralpha1. Although mouse B cells express IL-4Ralpha, little is known about their expression of IL-13Ralpha chains. To investigate this topic further, we have generated a monoclonal antibody (C41) specific for murine IL-13Ralpha1. Using C41, IL-13Ralpha1 expression was detected on germinal center (GC) B cells by flow cytometry and immunohistochemistry. In addition, IL-13Ralpha1 was observed on follicular dendritic cells, but not interdigitating dendritic cells in the T cell areas. Furthermore, resting B cells also expressed IL-13Ralpha1, and in the presence of IL-13 produced increased amounts of IgM in response to in vitro CD40 stimulation. However, C41 was unable to neutralize this bioactivity. The distribution of IL-13Ralpha1 on murine B cells and during GC reactions suggests a role for IL-13 during B cell differentiation.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of various cell types in the peripheral and central nervous systems. Its receptor complex consists of a non-signaling alpha chain, CNTFR, and two signaling beta chains, gp130 and the leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second, developmentally important ligand. We have identified this factor as a stable secreted complex of cardiotrophin-like cytokine (CLC) and the soluble receptor cytokine-like factor-1 (CLF). CLF expression was required for CLC secretion, and the complex acted only on cells expressing functional CNTF receptors. The CLF/CLC complex activated gp130, LIFR and signal transducer and activator of transcription 3 (STAT3) and supported motor neuron survival. Our results indicate that the CLF/CLC complex is a second ligand for CNTFR with potentially important implications in nervous system development.

CTLA-4, expressed by activated T cells, transduces an inhibitory signal. We show here that PCR amplification of the coding sequence of CTLA-4 in nonstimulated human T lymphocytes results in the amplification of two transcripts of 650 and 550 bp. Sequencing shows that the larger form codes for membrane CTLA-4 and the 550-bp transcript is a spliced variant in which exon 2 coding for the transmembrane region is deleted. This spliced cDNA has been named CTLA-4delTM. The splicing induces a frame shift which results in the addition of 22 extra amino acids before a translational termination. Activation of T cells with phorbol 12-myristate 13-acetate plus ionomycin or anti-CD3 plus anti-CD28 monoclonal antibodies induces a suppression of CTLA-4delTM mRNA expression associated with a preferential expression of the membrane CTLA-4 mRNA, showing that CTLA-4delTM mRNA expression is restricted to nonactivated T cells. A soluble immunoreactive form of CTLA-4 was detected in the serum of 14 / 64 healthy subjects. These results suggest that nonstimulated T cells may constitutively produce a soluble form of CTLA-4 which may have an important role in the regulation of immune homeostasis.

Using the sequence of cosmids derived from chromosome 19p12, we have identified a gene encoding a novel protein, BSMAP (brain-specific membrane-anchored protein) and cloned cDNA encoding the full-length open reading frame. Northern blot analysis revealed that BSMAP mRNA is preferentially expressed at a high level in the brain. BSMAP has a putative transmembrane domain and is predicted to be a type-I membrane glycoprotein. Genomic sequence analysis revealed that the gene encoding BSMAP consists of eight exons spanning approximately 8 kb and lies 6 kb away from the gene encoding CLF-1 in a reverse orientation. Although no candidate genetic disorders were found to map either to this precise region of chromosome 19 or to the syntenic region of the mouse genome, the highly specific expression of BSMAP mRNA suggests a role for the protein in CNS function.

A functional IL-13R involves at least two cell surface proteins, the IL-13Rα1 and IL-4Rα. Using a soluble form of the murine IL-13Rα1 (sIL-13R), we reveal several novel features of this system. The sIL-13R promotes proliferation and augmentation of Ag-specific IgM, IgG2a, and IgG2b production by murine germinal center (GC) B cells in vitro. These effects were enhanced by CD40 signaling and were not inhibited by an anti-IL4Rα mAb, a result suggesting other ligands. In GC cell cultures, sIL-13R also promoted IL-6 production, and interestingly, sIL-13R-induced IgG2a and IgG2b augmentation was absent in GC cells isolated from IL-6-deficient mice. Furthermore, the effects of the sIL-13R molecule were inhibited in the presence of an anti-IL-13 mAb, and preincubation of GC cells with IL-13 enhanced the sIL-13R-mediated effects. When sIL-13R was injected into mice, it served as an adjuvant-promoting production to varying degrees of IgM and IgG isotypes. We thus propose that IL-13Rα1 is a molecule involved in B cell differentiation, using a mechanism that may involve regulation of IL-6-responsive elements. Taken together, our data reveal previously unknown activities as well as suggest that the ligand for the sIL-13R might be a component of the IL-13R complex or a counterstructure yet to be defined.

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R α-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-α, IL-6, and IFN-γ. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

Immunoglobulin (Ig) E production by B cells requires two primary signals provided by T cells, interleukin (IL)-4 or IL-13 and CD40 ligand (CD40L). In addition, costimulatory signals, such as CD23-CD21 interaction, contribute further ensuring a selective control over this production. Recently, CD28, expressed on T cells, has been reported to be involved in this process. The CD28 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on human tonsillar B cells, and their expression is up-regulated by IL-4, IL-13, and/or an anti-CD40 monoclonal antibody (mAb). We have investigated whether signaling via the B7 molecules affects IgE synthesis. Human B cells were stimulated by IL-4 plus anti-CD40 mAb in the presence of different anti-B7 mAbs. Cross-linking of CD86 with IT2.2 potentiated IgE and IgG4 production and e transcripts expression. The production of the other isotypes was not modulated. Conversely, the anti-CD80 and the other anti-CD86 mAbs tested had no effect. The increase of IgE and IgG4 production induced by IT2.2 was accompanied by an increase in proliferation, in cell surface density of CD23, and in CD23 binding to CD21-expressing B cells. In contrast, the expression of other B cell surface molecules such as CD11a, CD30, and CD58 remained unaffected. Since IT2.2 favors CD23-CD21 pairing, we tested whether blocking this interaction affected IT2.2-increased IgE production. The neutralizing anti-CD23 mAb, Mab 25, caused a dose-dependent inhibition of the effect of IT2.2 on IgE synthesis. Finally, IT2.2 potentiation on B cell proliferation and IgE production required the two primary signals, IL-4 and anti-CD40 mAb, since IT2.2 alone or in combination with only one of these stimuli did not show any effect on B cells. This study is the first demonstration of a signaling role for CD86. Together with IL-4 or IL-13 and CD40L, CD86 favors CD23-CD21 pairing and consequently functions as a selective and potent costimulus for human IgE and IgG4 synthesis.

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 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.

Newborns and infants present a higher susceptibility to infection than adults, a vulnerability associated with deficiencies in both the innate and adaptive immune systems. Innate immune receptors are sensors involved in the recognition and elimination of microbes that play a pivotal role at the interface between innate and adaptive immunity. Pentraxin 3 (PTX3), the prototypic long pentraxin, is a soluble pattern recognition receptor involved in the initiation of protective responses against selected pathogens. Because neonates are generally resistant to these pathogens, we suspected that PTX3 may be provided by a maternal source during the early life times. We observed that human colostrum contains high levels of PTX3, and that mammary epithelial cell and CD11b+ milk cells constitutively produce PTX3. Interestingly, PTX3 given orally to neonate mice was rapidly distributed in different organs, and PTX3 ingested during lactation was detected in neonates. Finally, we observed that orally administered PTX3 provided protection against Pseudomonas aeruginosa lung infection in neonate mice. Therefore, breastfeeding constitutes, during the early life times, an important source of PTX3, which actively participates in the protection of neonates against infections. In addition, these results suggest that PTX3 might represent a therapeutic tool for treating neonatal infections and support the view that breastfeeding has beneficial effects on the neonates’ health.