Sébastien Lacroix-Desmazes

T cell-derived IL-22 amplifies IL-1β-driven inflammation in human adipose tissue: relevance to obesity and type 2 diabetes.

Proinflammatory cytokines are critically involved in the alteration of adipose tissue biology leading to deterioration of glucose homeostasis in obesity. Here we show a pronounced proinflammatory signature of adipose tissue macrophages in type 2 diabetic obese patients, mainly driven by increased NLRP3-dependent interleukin (IL)-1β production. IL-1β release increased with glycemic deterioration and decreased after gastric bypass surgery. A specific enrichment of IL-17- and IL-22-producing CD4+ T cells was found in adipose tissue of type 2 diabetic obese patients. Coculture experiments identified the effect of macrophage-derived IL-1β to promote IL-22 and IL-17 production by human adipose tissue CD4+ T cells. Reciprocally, adipose tissue macrophages express IL-17 and IL-22 receptors, making them sensitive to IL-17 and IL-22. IL-22 increased IL-1β release by inducing pro-IL-1β transcription through activation of C-Jun pathways in macrophages. In sum, these human data identified IL-1β and the T-cell cytokine IL-22 as key players of a paracrine inflammatory pathway previously unidentified in adipose tissue, with a pathological relevance to obesity-induced type 2 diabetes. These results provide an additional rationale for targeting IL-1β in obesity-linked type 2 diabetes and may have important implications for the conception of novel combined anti-IL-1β and anti-IL-22 immunotherapy in human obesity.

Molecular basis for bacterial peptidoglycan recognition by LysM domains.

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM–peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

Materno-fetal transfer of preproinsulin through the neonatal Fc receptor prevents autoimmune diabetes

The first signs of autoimmune activation leading to β-cell destruction in type 1 diabetes (T1D) appear during the first months of life. Thus, the perinatal period offers a suitable time window for disease prevention. Moreover, thymic selection of autoreactive T cells is most active during this period, providing a therapeutic opportunity not exploited to date. We therefore devised a strategy by which the T1D-triggering antigen preproinsulin fused with the immunoglobulin (Ig)G Fc fragment (PPI-Fc) is delivered to fetuses through the neonatal Fc receptor (FcRn) pathway, which physiologically transfers maternal IgGs through the placenta. PPI-Fc administered to pregnant PPIB15–23 T-cell receptor–transgenic mice efficiently accumulated in fetuses through the placental FcRn and protected them from subsequent diabetes development. Protection relied on ferrying of PPI-Fc to the thymus by migratory dendritic cells and resulted in a rise in thymic-derived CD4+ regulatory T cells expressing transforming growth factor-β and in increased effector CD8+ T cells displaying impaired cytotoxicity. Moreover, polyclonal splenocytes from nonobese diabetic (NOD) mice transplacentally treated with PPI-Fc were less diabetogenic upon transfer into NOD.scid recipients. Transplacental antigen vaccination provides a novel strategy for early T1D prevention and, further, is applicable to other immune-mediated conditions.

Intravenous immunoglobulin and immune response

Intravenous immunoglobulin (IVIg) is a pooled preparation of polyspecific, polyreactive immunoglobulin (Ig)G molecules from several thousand healthy donors. The immunoglobulin (Ig) molecule has Fab region, which is involved in antigen-binding and the Fc portion, which is involved in effector function. As IVIg is prepared from multiple donors, it contains numerous antibodies directed against a wide range of antigens; consequently, the variable regions on the Ig Fab fragments in IVIg preparations are diverse. The variable region can bind to non-self-antigens (foreign antigens), self-antigens and anti-idiotypic antibodies. IVIg contains a broad spectrum of antibody specificities against bacterial, viral, parasitic and mycoplasma antigens, that are capable of both opsonization and neutralization of microbes and toxins. In addition to its initial use as replacement therapy in primary and secondary immunodeficiencies, IVIg is widely indicated in a large spectrum of autoimmune and inflammatory diseases. One of the first proposed mechanisms of action of IVIg was via Fcγ receptor blockade 1. This study demonstrated that infusion of Fcγ fragments in idiopathic thrombocytopenic purpura/immune thrombocytopenia (ITP) patients increased platelet count, mediated by the blockade of Fcγ receptors 1. It has since been demonstrated in vivo that Fcγ fragments, particularly if sialylated, can exert anti-inflammatory effects 2. This suggests that the clinical benefits of IVIg may be mediated via an Fc pathway; indeed, to date there have been no studies that demonstrate the clinical benefit of Fab fragments alone. However, non-Fc mechanisms have been proposed that provide an insight into the possible molecular mechanisms of action of IVIg, although these do not exclude the potential co-operation of Fab and Fc portion of IgG to elicit the effects of IVIg. One mechanism of action was proposed by Sultan et al., who found that anti-idiotypic antibodies in IVIg were effective in the treatment of autoimmune haemophilia 3. This led to the study and characterization of anti-idiotypic antibodies in IVIg which neutralize pathogenic autoantibodies 4,5. IVIg was found to contain anti-idiotypes against anti-factor VIII, anti-neutrophil cytoplasmic antibody, anti-DNA, anti-thyroglobulin, anti-acetylcholine receptors and anti-neuroblastoma antigens. Furthermore, anti-idiotypic antibodies have been found to play a role in transplantation due to the anti-human leucocyte antigen antibodies. In addition to ITP and haemophilia, IVIg has been found to be effective in several inflammatory and autoimmune diseases. Therefore, anti-inflammatory effects of IVIg were studied and were shown to be mediated in part through anti-complement effects. Dermatomyosis is a condition mediated by C5b/C9 membranolytic attack complexes (MACs) in intramuscular capillaries. The formation of MAC occurs when C3 is hydrolyzed into C3b, which leads to the activation of C5b and the formation of MACs. In a study conducted by Basta et al. 6, IVIg was found to form complexes with C3, preventing MAC formation and deposition in patients with dermatomyosis. This suggests that the clinical benefit of IVIg can be attributed to complement scavenging, demonstrating an additional distinct mechanism of action that may be mediated by F(ab′)2 and whole IVIg, but not Fcγ fragments alone. Anaphylatoxins are complement peptides that are produced when the complement system is activated. A study by Basta et al. implicates F(ab′)2 in the neutralization of anaphylatoxins, such as C3a and C5a 7. IVIg is able to suppress C3a- and C5a-induced release of thromboxane B2 and histamine, which have proinflammatory properties. Moreover, circulatory collapse caused by C5a was prevented in pigs pretreated with F(ab′)2 IVIg. The neutralization of C3a and C5a were observed in cells treated with F(ab′)2 IVIg and whole IVIg and not Fcγ IVIg fragments, suggesting that F(ab′)2 and not Fcγ are implicated in this process. IVIg has also been found to be beneficial in a murine model of brain ischaemia and stroke, via a complement scavenging mechanism. Administration of IVIg, either prior to an ischaemic event or during reperfusion, led to a two- to three-fold improvement in functional outcomes in ischaemia and reperfusion. C3 levels were higher in injured compared to non-injured brain regions. Furthermore, compared with wild-type mice, C5-deficient mice were protected from ischaemia and reperfusion. IVIg decreased C3 and caspase 3 activation, suggesting that IVIg inhibits complement-mediated cell damage via scavenging of complement proteins to elicit beneficial effects 8. In addition to a role in scavenging complement in inflammatory and immune diseases, IVIg has also been shown to alter the cytokine network and mediate the balance between T helper (Th) types. Th cells can be classified into several subsets, such as Th1, Th2, Th17 and regulatory T cells, which produce distinct cytokines. Th1 cells produce cytokines such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, Th2 cells produce IL-4, IL-5, IL-13 and IL-10, Th17 cells produce IL-17, IL-21 and IL-22, and regulatory T cells which are immunosuppressor cells produce TGF-β and IL-10. In a study conducted by Ruiz de Souza et al., peripheral blood monocytes treated with IVIg induced an up-regulation of anti-inflammatory cytokine IL-1 receptor antagonist and down-regulation of several proinflammatory cytokines 9. By the early 2000s there was an increasing focus on the role of dendritic cells and their effect on T cell polarization. Mature dendritic cells can stimulate naive T helper cells (Th0) and polarize them into distinct subsets. Our study demonstrated that both the F(ab′)2 and Fc fragments of IVIg are capable of inhibiting the differentiation and maturation of dendritic cells, suggesting that IVIg is capable of inducing tolerogenic dendritic cell phenotypes 10. As a consequence of IVIg-induced tolerogenic dendritic cells, regulatory T cells are up-regulated. Using a murine model of autoimmune encephalomyelitis (EAE), prophylactic IVIg was found to increase CD4+CD25+forkhead box protein 3 (FoxP3+) regulatory T cells 11. This proliferation of regulatory T cells has also been observed in humans following high-dose IVIg treatment in patients with autoimmune rheumatic disease 12. We recently reported that, in EAE mice, IVIg inhibits the differentiation of CD4+ T cells to Th1 and Th17 cells 13. The down-regulation of Th1 and Th17 cells was observed with a concomitant up-regulation of regulatory T cells, demonstrating the reciprocal regulation mechanism of IVIg. Furthermore, the reciprocal regulation was suggested to be F(ab′)2-dependent due to the comparable inhibition of Th1 and Th17 cells observed in mice treated with F(ab′)2 fragments or IVIg 13. IVIg-induced expansion of regulatory T cells may be due to several mechanisms. Mazer et al. propose that IVIg renders dendritic cells tolerogenic via its interaction with dendritic cell immunoreceptor (DCIR) 14. This leads to increased levels of FoxP3+ regulatory T cells which can attenuate autoimmune disease severity. Another mechanism of action for regulatory T cell expansion is provided recently by our group. Our report suggests that IVIg-induced expansion of regulatory T cells is due to the induction of cyclo-oxygenase 2-dependent prostaglandin E2 production in dendritic cells 15. Inhibition of cyclo-oxygenase 2 enzymatic activity significantly reduced IVIg-mediated regulatory T cell expansion both in vitro and in vivo in EAE mice. This mechanism was dependent on Fab fragments of IVIg but not Fc. Immunomodulatory mechanisms of IVIg in autoimmune conditions are not fully understood, although several mutually non-exclusive effects have been proposed. Individually, each of these mechanisms may participate to a certain extent in the overall effect of IVIg. While some of the effects may rely upon the binding of the Fc moiety of IgG to Fcγ receptors on target cells, others may be primarily dependent on the range of variable regions of IgG.

Impact of Antigen Density on the Binding Mechanism of IgG Antibodies

The density and distribution pattern of epitopes at the surface of pathogens have a profound impact on immune responses. Although multiple lines of evidence highlight the significance of antigen surface density for antibody binding, a quantitative description of its effect on recognition mechanisms is missing. Here, we analyzed binding kinetics and thermodynamics of six HIV-1 neutralizing antibodies as a function of the surface density of envelope glycoprotein gp120. Antibodies that recognize gp120 with low to moderate binding affinity displayed the most pronounced sensitivity to variation in antigen density, with qualitative and substantial quantitative changes in the energetics of the binding process as revealed by non-equilibrium and equilibrium thermodynamic analyses. In contrast, the recognition of gp120 by the antibodies with the highest affinity was considerably less influenced by variations in antigen density. These data suggest that a lower affinity of antibodies permits higher dynamics during the antigen recognition process, which may have considerable functional repercussions. These findings contribute to a better understanding of the mechanisms of antigen recognition by antibodies. They are also of importance for apprehending the impact of antigen topology on immune-defense functions of antibodies.

Methods for Posttranslational Induction of Polyreactivity of Antibodies

An antibody molecule that recognizes multiple unrelated antigens is defined as polyreactive. Polyreactivity is an intrinsic characteristic of immune repertoires. Degenerated antigen binding diversifies the repertoire of specificities, thus contributing to immune defense and immune regulation. Immune repertoire contains also a fraction of immunoglobulins, which acquire polyreactivity only following contact with various protein-destabilizing or pro-oxidative substances. Posttranslational induction of the antibody polyreactivity may have important repercussion for laboratory practice, as well as in cases of pathological conditions accompanied by liberation of large quantities of pro-oxidative substances such as heme, labile iron, or reactive oxygen species. Antibodies with induced polyreactivity have been demonstrated to exert pathogen neutralization and immune regulatory potential in inflammatory conditions, suggesting that this phenomenon may be exploited for design of therapeutic strategies. In this article, we provide description of the basic procedures for uncovering of the cryptic polyreactivity of antibodies by heme, ferrous ions, and acid pH solution.

Inhibitor Formation in Congenital Hemophilia A: an Immunological Perspective

Abstract The immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short‐lived, intravenously administered glycoprotein triggers such a deleterious, long‐lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII‐specific tolerance in preclinical models of hemophilia A.

Relationship between natural and heme-mediated antibody polyreactivity.

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies.