Walter Ferlin

Tolerance to Islet Antigens and Prevention from Diabetes Induced by Limited Apoptosis of Pancreatic β Cells

Crosspresentation of self-antigens by antigen-presenting cells is critical for the induction of peripheral tolerance. As apoptosis facilitates the entry of antigens into the crosspresentation pathway, we sought to prevent the development of autoimmune diabetes by inducing pancreatic beta cell apoptosis before disease onset. Accordingly, young nonobese diabetic (NOD) mice injected with a single low dose of streptozotocin (SZ), a drug cytotoxic for beta cells, exhibited impaired T cell responses to islet antigens and were protected from spontaneous diabetes. Furthermore, beta cell apoptosis was necessary for protection since SZ did not protect RIP-CrmA transgenic NOD mice in which beta cells expressed the caspase inhibitor CrmA. Our results support a model in which apoptosis of pancreatic beta cells induces the development of regulatory cells leading to the tolerization of self-reactive T cells and protection from diabetes.

IL-6 Trans-Signaling Modulates TLR4-Dependent Inflammatory Responses via STAT3

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130F/F knock-in mutant mice displaying hyperactivated IL-6–dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130F/F mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130F/F mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130F/F:Stat3+/− mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130F/F mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.

Neutralization of IFNγ defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice

Hereditary haemophagocytic lymphohistiocytosis (HLH) is a fatal inflammatory disease and treatments currently may lead to serious side effects. There is a pressing need for effective, less toxic treatments for this disease. Previous reports have suggested that interferon γ (IFNγ) has a role in the pathogenesis of HLH. Here, we report that blocking IFNγ had a therapeutic effect in two different murine models of human hereditary HLH (perforin‐deficient and Rab27a‐deficient mice, both infected with lymphocytic choriomeningitis virus). Therapeutic administration of an anti‐IFNγ antibody induced recovery from haemophagocytosis in both genetic models, as evidenced by increased survival in perforin‐deficient mice and correction of blood cytopenia, moderation of body temperature changes, decreased cytokinaemia, restoration of splenic architecture and reduced haemophagocytosis in the liver of both murine models. Involvement of the central nervous system in Rab27a‐deficient mice was prevented by anti‐IFNγ therapy. Hepatic T‐cell infiltrates and virus persisted, with no detectable harm during the time course of these studies. These data strongly suggest that neutralization of IFNγ could be used in humans to safely alleviate the clinical manifestations of haemophagocytosis.

TLR4/MD-2 Monoclonal Antibody Therapy Affords Protection in Experimental Models of Septic Shock

Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 μg) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications.

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.

The induction of a protective response in Leishmania major‐infected BALB/c mice with anti‐CD40 mAb

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4+ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co‐injected with an agonistic anti‐murine CD40 mAb. Anti‐CD40 mAb‐mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/ 6 × 129 mice that were rendered T cell deficient (TCR β–/– × TCR δ–/–) and L. major susceptible. Anti‐CD40 mAb stimulation of L. major‐infected BALB/c mice was accompanied by increased IL‐12 and IFN‐γ production in draining lymphnodes, analyzed either by direct expression, or in an antigen‐specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti‐CD40 mAb‐mediated protection of L. major‐infected BALB/c mice could be reversed by co‐treating the animals with neutralizing anti‐IL‐12 and/or anti‐IFN‐γ mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4+ T cell response to L. major infection when co‐injected with anti‐CD40 mAb. While the CD40‐CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti‐CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. majorinfection and raises the possibility of utilizing this approach for vaccination strategies.

Peptide-MHC Class II Dimers as Therapeutics to Modulate Antigen-Specific T Cell Responses in Autoimmune Diabetes

Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing β cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-Ag7 dimers for use in the nonobese diabetic mouse model of diabetes. I-Ag7 dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4+ T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-Ag7 dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4+ T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4+ T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-γ compared with cells from control I-Ag7 dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-Ag7 dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.

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.