Patrice Decker

Interleukin‐6 Receptor Blockade Enhances CD39+ Regulatory T Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

The rationale for blocking interleukin‐6 (IL‐6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti–IL‐6 receptor (IL‐6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti–IL‐6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA.

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, attacks the neutrophil extracellular traps and is needed for full virulence

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation‐requiring, pH‐stable and heat‐stable nuclease that we named Nuclease A (NucA). Substitution of the histidine148 by alanine reduced nuclease activity of the GBS wild‐type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucAH148A mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence.

The mouse dendritic cell marker CD11c is down-regulated upon cell activation through Toll-like receptor triggering

Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic-polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC.

TLR9 independent interferon α production by neutrophils on NETosis in response to circulating chromatin, a key lupus autoantigen

Objectives Interferon (IFN) α is a key immunoregulatory cytokine secreted by activated plasmacytoid dendritic cells (PDC) that constitute less than 1% of leucocytes. IFNα plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Nevertheless, the natural IFNα inducers in SLE as well as the different IFNα secreting cell types are only partially characterised. Methods Chromatin was purified from calf thymus. Human peripheral blood mononuclear cells (PBMC), neutrophils and mouse bone marrow neutrophils were purified and cultured with different stimuli. IFNα production was estimated by flow cytometry, ELISA and a bioassay, and gene expression by quantitative real time PCR. Neutrophil activation and NETosis were analysed by flow cytometry, ELISA and confocal microscopy. Results Neutrophils produced a bioactive IFNα on stimulation with purified chromatin. IFNα secretion was observed with steady state neutrophils purified from 56 independent healthy individuals and autoimmune patients in response to free chromatin and not chromatin containing immune complexes. Chromatin induced IFNα secretion occurred independently of Toll-like receptor 9 (TLR9). Neutrophil priming by granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor or IFNα was not necessary but PBMC sustained IFNα secretion by neutrophils. PDC were 27 times more efficient than neutrophils but blood neutrophils were 100 times more frequent than PDC. Finally, neutrophil activation by chromatin was associated with NETosis and DNA sensor upregulation. Conclusions Neutrophils have the capability of producing IFNα on selective triggering, and we identified a natural lupus stimulus involved, unveiling a new mechanism involved in SLE. Neutrophils represent another important source of IFNα and important targets for future therapies aimed at influencing IFNα levels.

Nucleosome-induced neutrophil activation occurs independently of TLR9 and endosomal acidification: Implications for systemic lupus erythematosus

The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated whether TLR9 is required for nucleosome‐induced PMN activation. Isolated neutrophils were cultured with nucleosomes, plasma from lupus patients and other stimuli in the presence/absence of various inhibitors. Cells were analyzed by flow cytometry, ELISA and confocal microscopy. We found that nucleosomes circulating in lupus plasma induce the secretion of pro‐inflammatory cytokines by PMN. Nucleosomes activate human PMN independently of unmethylated CpG sequences in nucleosomal DNA, leading to IL‐8/IL‐6/TNF secretion and CD11b up‐regulation. Nucleosomes accumulate in the cytoplasm of PMN upon endocytosis, induce TLR9 up‐regulation and act synergistically with TLR9 ligands. Nucleosome‐induced activation was not inhibited by polymyxin B (PB), chloroquine (CQ), ammonium chloride (AC) or a TLR9 antagonist. Moreover, both PMN isolated from WT and TLR9‐KO mice were activated by nucleosomes, as detected by MIP‐2 secretion and CD11b up‐regulation. Activation occurred therefore independently of endotoxins, endosomal acidification, TLR9 and CpG motifs. TLR9 may thus be differently required in the triggering of nucleosome‐induced innate immunity and anti‐nucleosome B‐cell autoimmunity.

Primary blood neutrophils express a functional cell surface Toll-like receptor 9.

Polymorphonuclear leukocytes (PMNs) represent one of the first lines of defense against pathogens. TLR9 is normally expressed in endosomes/lysosomes where it is activated by pathogen‐derived DNA. Here we show that freshly isolated human and mouse primary PMNs express TLR9 at the cell surface ex vivo. Moreover, surface TLR9 expression is upregulated upon activation of PMNs with different stimuli and not only TLR9 agonists. Importantly, surface TLR9 is processed, active, and functional. TLR9 ligands, oligo‐nucleotides containing unmethylated CpG motifs, indeed bind to surface TLR9 and binding was strongly observed at the cell surface of human cells expressing surface TLR9 and at the surface of WT but not TLR9‐deficient mouse PMNs. Finally, CpG oligonucleotides cross‐linked onto a solid phase and having no access to intracellular TLR9 are able to trigger cell surface TLR9 and induce neutrophil activation, even when endosomal acidification is inhibited. This is the first demonstration of a functional TLR9 expressed at the cell surface of human primary cells. This pathway may be triggered when pathogen‐derived TLR9 ligands cannot reach the endosome, offering a rescue mechanism for neutrophil activation.

Pain and immunity

Chronic neuropathic and inflammatory pain is a major public health problem. Nociceptors undergo sensitization, first in peripheral tissues then in the central nervous sytem, via neuroimmune interactions linking neurons, glial cells (microglia and astrocytes), and immune cells. These interactions may either exacerbate or attenuate the pain and inflammation, which normally reach a state of equilibrium. With more powerful or longer lasting stimuli, specific profiles of microglial and, subsequently, astrocytic activation in the dorsal horn play a key role in neuronal plasticity and transition to chronic pain. Recent insights into the interactions between the nervous system and the immune system suggest a large number of potential therapeutic targets that could be influenced either by targeted inhibition or by directing the neuroimmune response toward the antiinflammatory and analgesic end of its spectrum.

Arthritis models: usefulness and interpretation

Animal models of arthritis are used to better understand pathophysiology of a disease or to seek potential therapeutic targets or strategies. Focusing on models currently used for studying rheumatoid arthritis, we show here in which extent models were invaluable to enlighten different mechanisms such as the role of innate immunity, T and B cells, vessels, or microbiota. Moreover, models were the starting point of in vivo application of cytokine-blocking strategies such as anti-TNF or anti-IL-6 treatments. The most popular models are the different types of collagen-induced arthritis and arthritis in KBN mice. As spontaneous arthritides, human TNF-α transgenic mice are a reliable model. It is mandatory to use animal models in the respect of ethical procedure, particularly regarding the number of animals and the control of pain. Moreover, design of experiments should be of the highest level, animal models of arthritis being dedicated to exploration of well-based novelties, and never used for confirmation or replication of already proven concepts. The best interpretations of data in animal models of arthritis suppose integrated research, including translational studies from animals to humans.

In Vivo Expansion of Activated Foxp3+ Regulatory T Cells and Establishment of a Type 2 Immune Response upon IL-33 Treatment Protect against Experimental Arthritis

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33–treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33–treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39high Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

Developments with investigational Janus kinase inhibitors for rheumatoid arthritis

The evolving therapeutic landscape for rheumatoid arthritis has seen three major breakthroughs over the last decades, each corresponding to a step in the comprehension of the disease [1]. First, understanding of the immunological basis of the disease has led to the systematic and precocious use of immunosuppressants, called conventional disease-modifyingdrugs (csDMARDs), like methotrexate (MTX), that are still the cornerstone of RA treatment. Second, deeper comprehension of disease pathophysiology led to the development of targeted biological treatments (bDMARDs), monoclonal antibodies or soluble receptors, that block pro-inflammatory cytokines like TNF or IL-6, cellular populations, like mature B cells, or cellular interactions that are critical to T-cell activation. The development of orally available small molecules that inhibit intracellular signaling of cytokines and growth factors is the third major advance in the treatment of RA. These compounds are referred to as targeted synthetic DMARDs (tsDMARDs). The jakinibs are a novel family of inhibitors of the Janusassociated kinase (JAK) signaling pathway. The JAK/STAT (signal transducer and activators of transcription) system is a highly evolutionary conserved system that signals downstream type I and type II cytokine receptors (Figure 1) [2]. Tofacitinib, the first developed jakinib, was approved by the FDA in 2012 at the dose of 5 mg twice daily. Originally designed to be a JAK3-selective inhibitor, tofacitinib is considered a pan-JAK inhibitor that inhibits, in descending order of potency, JAK3, 1 and 2. Tofacitinib was developed in a wide phase 3 program, in which it was administered at 5 or 10 mg twice daily. In patients with established RA that were insufficient responders to csDMARDs [3–6] or bDMARds [6,7], tofactinib showed higher rates of clinical response vs. placebo both as monotherapy [6] and as add-on therapy to MTX or other csDMARDs [3–5]. In the latter setting showed similar efficacy to adalimumab as add-on therapy in MTX insufficient responders (to note, the study was not designed to formally test the noninferiority of tofacitinib vs. adalimumab tofacitinib) [3]. Tofacitinib is now approved for clinical use in over 40 countries worldwide, including USA, Japan, Russia, and Switzerland, while it failed to obtain marketing license in the European Union. The reasons for license refusal were unresolved safety concerns, mainly infections, which were dose-dependent, and the fact that the dose of 5 mg did not provide sufficient benefit in terms reduction of structural progression [5]. Nevertheless, a subsequent trial in early csDMARDs naïve RA showed that even at the dose of 5 mg, tofacitinib monotherapy was superior to MTX in preventing radiological progression [8]. In USA, tofacitinib was approved with a warning highlighting the risk of serious infections in particular tuberculosis and malignancy. Pooled analyses of phases 2, 3, and open-label extension studies confirmed the dose-dependency of the infectious risk but suggested that overall risk was comparable to that of RA patients on bDMARDs [9]. Additionally, it was shown that the rates and types of malignancies observed in clinical trials remained stable over time with increasing tofacitinib exposure and were within the expected range of patients with moderate-to-severe RA [10]. There was an increase in the rate of varicella–zoster virus (VZV) infections across the studies, consistent with inhibition of interferon IFN type I and II signaling (Figure 1). Other frequent events like dysplidemia and liver enzyme elevation are reminiscent of those seen on anti-IL-6 treatments [11] and are consistent with JAK1 inhibition. Increased rates of anemia and cytopenia, notably neutropenia, ascribed to inhibition of JAK2 signaling were also observed. To overcome potential limitations of generalized blockade of JAK signaling, other jakinibs with more restricted JAK specificities have been developed. There are currently five jakinibs between phases 2 and 3 of clinical development.