Florence Guilhot

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-γ production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Rα in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Rα-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.

Inhibition of Classic Signaling Is a Novel Function of Soluble Glycoprotein 130 (sgp130), Which Is Controlled by the Ratio of Interleukin 6 and Soluble Interleukin 6 Receptor

Background: IL-6 trans-signaling plays a critical role in chronic inflammation and cancer. Results: The trans-signaling inhibitor sgp130(Fc) also inhibits classic signaling depending on IL-6/sIL-6R ratios. Conclusion: The additional function of sgp130(Fc) suggests that in vivo only low therapeutic concentrations guarantee blockade of trans-signaling but not classic signaling. Significance: The demonstration that the trans-signaling inhibitor can also inhibit classic signaling is central for the field of IL-6 biology. IL-6 trans-signaling via the soluble IL-6 receptor (sIL-6R) plays a critical role in chronic inflammation and cancer. Soluble gp130 (sgp130) specifically inhibits IL-6 trans-signaling but was described to not interfere with classic signaling via the membrane-bound IL-6R. Physiological and most pathophysiological conditions are characterized by a molar excess of serum sIL-6R over IL-6 characterized by free IL-6 and IL-6 found in IL-6·sIL-6R complexes allowing both classic and trans-signaling. Surprisingly, under these conditions, sgp130 was able to trap all free IL-6 molecules in IL-6·sIL-6R·sgp130 complexes, resulting in inhibition of classic signaling. Because a significant fraction of IL-6 molecules did not form complexes with sIL-6R, our results demonstrate that compared with the anti-IL-6R antibody tocilizumab or the anti-trans-signaling monoclonal antibody 25F10, much lower concentrations of the dimeric sgp130Fc were sufficient to block trans-signaling. In vivo, sgp130Fc blocked IL-6 signaling in the colon but not in liver and lung, indicating that the colon is a prominent target of IL-6 trans-signaling. Our results point to a so far unanticipated role of sgp130 in the blockade of classic signaling and indicate that in vivo only low therapeutic concentrations of sgp130Fc guarantee blockade of IL-6 trans-signaling without affecting IL-6 classic signaling.

CC chemokine CCL5 plays a central role impacting infarct size and post-infarction heart failure in mice

AIMS The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event. METHODS AND RESULTS C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function. CONCLUSION Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.

Although IL-6 Trans-Signaling Is Sufficient To Drive Local Immune Responses, Classical IL-6 Signaling Is Obligate for the Induction of T Cell-Mediated Autoimmunity

IL-6–mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6–mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

Background: The IL-6 signaling complex consists of a hexameric structure essential for IL-6 cis- and trans-signaling. Results: mAb 25F10 targets site IIb of IL-6R and disrupts hexamer assembly to selectively block trans-signaling. Conclusion: Cis- and trans-signaling in mice utilize distinct mechanisms to mediate assembly of the IL-6R complex. Significance: Therapeutic targeting of site IIb of IL-6R provides a unique mode of action for IL-6 inhibition. The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.

Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.

Use of a mouse model to identify a blood biomarker for IFNγ activity in pediatric secondary hemophagocytic lymphohistiocytosis.

&NA; Life‐threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon &ggr; (IFN&ggr;) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection‐driven model of sHLH in mice, we observed that the tissue‐derived levels of IFN&ggr; are actually 500‐ to 2000‐fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFN&ggr; gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFN&ggr; activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFN&ggr;‐biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFN&ggr; activity in patients with sHLH.

Interferon-gamma (IFNy) in macrophage activation syndrome (MAS) associated with systemic juvenile idiopathic arthritis (sJIA). High levels in patients and a role in a murine mas model

Interferon-gamma (IFNg) in macrophage activation syndrome (MAS) associated with systemic juvenile idiopathic arthritis (sJIA). High levels in patients and a role in a murine mas model Claudia Bracaglia, Ivan Caiello, Kathy De Graaf, Giovanni D’Ario, Florence Guilhot, Walter Ferlin, Lidia Melli, Giusi Prencipe, Sergio Davi, Grant Schulert, Angelo Ravelli, Alexei Grom, Cristina De Min, Fabrizio De Benedetti

Detection of ε Class Switching and IgE Synthesis in Human B Cells

We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naive or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

NI‐0801, an anti‐chemokine (C‐X‐C motif) ligand 10 antibody, in patients with primary biliary cholangitis and an incomplete response to ursodeoxycholic acid

NI‐0801 is a fully human monoclonal antibody against chemokine (C‐X‐C motif) ligand 10 (CXCL10), which is involved in the recruitment of inflammatory T cells into the liver. The safety and efficacy of NI‐0801 was assessed in patients with primary biliary cholangitis. In this open‐label phase 2a study, patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid received six consecutive intravenous administrations of NI‐0801 (10 mg/kg) every 2 weeks. Patients were followed up for 3 months after the last infusion. Liver function tests, safety assessments, as well as pharmacokinetic and pharmacodynamic parameters were evaluated at different time points throughout the dosing period and the safety follow‐up period. Twenty‐nine patients were enrolled in the study and were treated with NI‐0801. The most frequently reported adverse events included headaches (52%), pruritus (34%), fatigue (24%), and diarrhea (21%). No study drug‐related serious adverse events were reported. NI‐0801 administration did not lead to a significant reduction in any of the liver function tests assessed at the end of the treatment period (i.e., 2 weeks after final NI‐0801 administration) compared to baseline. Conclusion: Despite clear pharmacologic responses in the blood, no therapeutic benefit of multiple administrations of NI‐0801 could be demonstrated. The high production rate of CXCL10 makes it difficult to achieve drug levels that lead to sustained neutralization of the chemokine, thus limiting its targetability. (Hepatology Communications 2018;2:492‐503)