Maxime Touzot

Critical role of IL-21 in modulating TH17 and regulatory T cells in Behçet disease

BACKGROUND Behçet disease (BD) is a chronic systemic inflammatory disorder of unknown etiology. OBJECTIVE To determine the nature of T cells driving inflammatory lesions in BD. METHODS T cell homeostasis and cytokines production were analyzed in peripheral blood and brain inflammatory lesions from 45 adult patients with BD (active and untreated BD [n = 25] and patients in remission [n = 20]) and 20 healthy donors, using Luminex, flow cytometry, immunohistochemistry, and immunofluorescence analysis. RESULTS We found a marked increase in T(H)17 cells and a decrease in the frequency of CD4(+) forkhead box P3(+) regulatory T cells (Tregs) in peripheral blood that were induced by IL-21 production and that correlate with BD activity. The addition of serum from patients with active BD in a sorted CD4(+) T cells culture of healthy donors induced a significant and dose-dependent production of IL-17A and a decrease in forkhead box P3 expression. We demonstrated the presence of IL-21- and IL-17A-producing T cells within the cerebrospinal fluid, brain parenchyma inflammatory infiltrates, and intracerebral blood vessels from patients with active BD and central nervous system involvement. The stimulation of CD4(+) T cells with IL-21 increased T(H)17 and T(H)1 differentiation and decreased the frequency of Treg cells. Conversely, IL-21 blockade with an IL-21R-Fc restored the T(H)17 and Treg homeostasis in patients with BD. CONCLUSION We provided here the first evidence of the critical role of IL-21 in driving inflammatory lesions in BD by promoting T(H)17 effectors and suppressing Treg cells. IL-21 represents a promising target for novel therapy in BD.

Multiparametric analysis of cytokine-driven human Th17 differentiation reveals a differential regulation of IL-17 and IL-22 production

T helper 17 (Th17) cells produce IL-17 but can also make tumor necrosis factor, interleukin (IL)-6, IL-10, IL-21, and IL-22. These cytokines collectively contribute to the functional outcome of the Th response. IL-22 plays a critical role in some Th17-associated diseases, such as psoriasis, but its relationship to IL-17 remains controversial. Here, we used a systematic multiparametric analysis of Th-17-associated cytokines, which revealed the unexpected finding that the regulation pattern of IL-22 was most closely related to interferon-gamma, the prototypical Th1 cytokine, and not to IL-17. To explain this observation, we systematically tested the role of Th1- and Th17-inducing cytokines. We could show that IL-12 and IL-23 induced high levels of IL-22 but no IL-17. Conversely, transforming growth factor-beta inhibited IL-22 production but promoted IL-17. Thus, IL-17 and IL-22 are differentially regulated during cytokine-induced Th cell differentiation. This has important implications for the understanding and pharmacologic manipulation of Th17-associated pathologies.

IFN-α induces IL-10 production and tilt the balance between Th1 and Th17 in Behçet disease.

BACKGROUND AND AIMS Interferon alpha (IFN-α) is an effective treatment for patients with active Behçet disease (BD). Besides its antiviral property, IFN-α is a cytokine with pleiotropic effects that can generate an anti-inflammatory environment or inhibit specific inflammatory T cells such as Th1 and Th17 cells. However, it is not known, in BD patients, whether IFN-α inhibits pro inflammatory T cells, or induces anti-inflammatory properties in T cells. METHODS Total memory CD45RO(+) T cells purified from peripheral blood mononuclear cells (PBMCs) obtained from patients with active BD (N=5), systemic lupus erythematosus (SLE, N=5) or healthy control (HC, N=6) were cultured in vitro with or without IFN-α. Levels of IFN-γ, IL-17, IL-10, IL-6, and TNF-α in the supernatants were analyzed by ELISA, Cytometric Beads Array (CBA) or intracellular cytokine staining. We analyzed the production of IL-10 on the memory subsets Th1 (mTh1), Th2 (mTh2) and Th17 (mTh17). RESULTS IFN-α significantly increased IFN-γ level in memory CD4(+) T cells in BD patients and HC, but not SLE patients. IL17 was not inhibited by IFN-α in BD or in SLE patients. However, IFN-α modulated the pro- and anti-inflammatory cytokines secreted by T cells as it increased the IL-10/IL-6 ratio in BD and HC, but not SLE patients. We further demonstrated that IFN-α increased IL-10 secretion in each memory subset mTh1, mTh2 and mTh17. CONCLUSIONS In BD, IFN-α promotes a regulatory Th1 response through IL-10 secretion. This effect may explain the efficacy of IFN-α in inflammatory disease.

Rituximab in anti-GBM disease: A retrospective study of 8 patients

OBJECTIVES Anti-glomerular basement membrane (GBM) disease is a rare autoantibody-mediated disorder presenting as rapidly progressive glomerulonephritis, and often with pulmonary hemorrhage. Antibody removal with plasmapheresis and immunosuppressive drugs are the cornerstones of the treatment. Data regarding the use of specific B-cell depleting therapy such as rituximab are lacking. METHODS We conducted a retrospective observational study of 8 patients with severe and/or refractory GBM disease that received rituximab therapy. RESULTS Eight patients (2 men, 6 women) with a mean age of 26 ± 13.1 years old were included. Seven had severe renal involvement [median creatinin level was 282 μmol/l, range (65-423)] requiring high immunosuppressive or plasmapheresis dependent, and two had relapse of pulmonary hemorrhage including one with renal failure. Patients received an initial immunosuppressive treatment including steroid and cyclosphosphamide (n = 8) and plasmapheresis (n = 5). Except one late relapse, rituximab therapy was started within two months after diagnosis. All patients except one received 4 weekly dose of rituximab (375 mg(2)). Anti-GBM antibodies were still present in 6/8 patients, at rituximab initiation. Complete remission was observed in 7 out of 8 patients, mostly 3 months after rituximab therapy. After a mean follow-up of 25.6 months (range 4-93), patient and renal survival were 100% and 75% respectively, but rituximab use did not improve GFR. Anti-GBM antibodies remained negative for all patients during follow-up. Only one patient developed a severe bacterial infection but no opportunistic or viral infections were reported. CONCLUSION Rituximab may represent an additional and/or alternative therapy in the induction treatment of anti-GBM disease.

Combinatorial flexibility of cytokine function during human T helper cell differentiation

In an inflammatory microenvironment, multiple cytokines may act on the same target cell, creating the possibility for combinatorial interactions. How these may influence the system-level function of a given cytokine is unknown. Here we show that a single cytokine, interferon (IFN)-alpha, can generate multiple transcriptional signatures, including distinct functional modules of variable flexibility, when acting in four cytokine environments driving distinct T helper cell differentiation programs (Th0, Th1, Th2 and Th17). We provide experimental validation of a chemokine, cytokine and antiviral modules differentially induced by IFN-α in Th1, Th2 and Th17 environments. Functional impact is demonstrated for the antiviral response, with a lesser IFN-α-induced protection to HIV-1 and HIV-2 infection in a Th17 context. Our results reveal that a single cytokine can induce multiple transcriptional and functional programs in different microenvironments. This combinatorial flexibility creates a previously unrecognized diversity of responses, with potential impact on disease physiopathology and cytokine therapy.

Diversification of human plasmacytoid predendritic cells in response to a single stimulus

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1–P3). P1-pDCs (PD-L1+CD80–) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1–CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.Plasmacytoid dendritic cells (pDCs) are known for their copious IFN-I production. Soumelis and colleagues show that functionally and transcriptomically distinct human pDC populations can be generated from a single microbial or cytokine stimulus.

A dive into the complexity of type I interferon antiviral functions

Service d’hepatologie, Hopital Beaujon, Clichy, FranceCOMMENTARY ON:A diverse range of gene products are effectors of the type I inter-feron antiviral response. Schoggins JW, Wilson SJ, Panis M,Murphy MY, Jones CT, Bieniasz P, Rice CM. Nature. 2011 Apr28;472(7344):481–485. Copyright (2011). Abstract reprintedby permission from Macmillan Publishers Ltd.http://www.ncbi.nlm.nih.gov/pubmed/21478870Abstract: The type I interferon response protects cells against invad-ing viral pathogens. The cellular factors that mediate thisdefense arethe products of interferon-stimulated genes (ISGs). Although hun-dreds of ISGs have been identified since their discovery more than25 years ago, only a few have been characterized with respect toantiviral activity. For most ISG products, little is known about theirantiviral potential, their target specificity, and their mechanisms ofaction. Using an overexpression screening approach, here we showthat differentviruses are targeted by unique sets of ISGs. We find thateach viral species is susceptible to multiple antiviral genes, whichtogether encompass a range of inhibitory activities. To conduct thescreen, more than 380 human ISGs were tested for their ability toinhibit the replication of several important human and animalviruses, including hepatitis C virus, yellow fever virus, West Nilevirus, chikungunya virus, Venezuelan equine encephalitis virus, andhuman immunodeficiency virus type-1. Broadly acting effectorsincluded IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (alsoknown as DDX58), MDA5 (also known as IFIH1), and IFITM3,whereas more targeted antiviral specificity was observed withDDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also knownas PBEF1), OASL, RTP4, TREX1, and UNC84B (also known as SUN2).Combined expression of pairs of ISGs showed additive antiviraleffects similar to those of moderate type I interferon doses. Mecha-nistic studies uncovered a common theme of translational inhibitionfor numerous effectors. Several ISGs, including ADAR, FAM46C,LY6E, and MCOLN2, enhanced the replication of certain viruses,highlighting another layer of complexity in the highly pleiotropictype I interferon system. 2011 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.IntroductionType I interferons are a family of major innate immune cytokinesproducedbyhostcellsinresponsetoviralinfection[1].Sincetheirdiscovery 50 years ago, fundamental and biomedical research hasgreatly improved our understanding of their molecular mecha-nisms of action, and led to the development of the first ‘‘cyto-kine-based’’ therapy in the 70s, now licensed worldwide forviral disease, malignant and even immune disorders [1,2].Interferon remains the therapeutic backbone of chronic hepa-titis C. The standard of care, in HCV genotype 1 infected patients,is the addition of direct-acting antivirals (DAAs) with a proteaseinhibitor (telaprevir or boceprevir) to pegylated interferon plusribavirin [3].The type I interferon family is composed of 5 members inhumans: the well described IFN

Using Transcriptional Signatures to Assess Immune Cell Function: From Basic Mechanisms to Immune-Related Disease

Assessing human immune response remains a challenge as it involves multiple cell types in specific tissues. The use of microarray-based expression profiling as a tool for assessing the immune response has grown increasingly over the past decade. Transcriptome analyses provide investigators with a global perspective of the complex molecular and cellular events that unfold during the development of an immune response. In this review, we will detail the broad use of gene expression profiling to decipher the complexity of immune responses from disease biomarkers identification to cell activation, polarisation or functional specialisation. We will also describe how such data-driven strategies revealed the flexibility of immune function with common and specific transcriptional programme under multiple stimuli.

Proliferative lupus nephritis in the absence of overt systemic lupus erythematosus: A historical study of 12 adult patients

Abstract Severe lupus nephritis in the absence of systemic lupus erythematosus (SLE) is a rare condition with an unclear clinical presentation and outcome. We conducted a historical observational study of 12 adult (age >18 years) patients with biopsy-proven severe lupus nephritis or lupus-like nephritis without SLE immunological markers at diagnosis or during follow-up. Excluded were patients with chronic infections with HIV or hepatitis B or C; patients with a bacterial infectious disease; and patients with pure membranous nephropathy. Electron microscopy was retrospectively performed when the material was available. End points were the proportion of patients with a complete response (urine protein to creatinine ratio <0.5 g/day and a normal or near-normal eGFR), partial response (≥50% reduction in proteinuria to subnephrotic levels and a normal or near-normal eGFR), or nonresponse at 12 months or later after the initiation of the treatment. The study included 12 patients (66% female) with a median age of 36.5 years. At diagnosis, median creatinine and proteinuria levels were 1.21 mg/dL (range 0.5–11.6) and 7.5 g/day (1.4–26.7), respectively. Six patients had nephrotic syndrome and acute kidney injury. Renal biopsy examinations revealed class III or class IV A/C lupus nephritis in all cases. Electron microscopy was performed on samples from 5 patients. The results showed mesangial and subendothelial dense deposits consistent with LN in 4 cases, and a retrospective diagnosis of pseudo-amyloid fibrillary glomerulonephritis was made in 1 patient. Patients received immunosuppressive therapy consisting of induction therapy followed by maintenance therapy, similar to treatment for severe lupus nephritis. Remission was recorded in 10 patients at 12 months after the initiation of treatment. One patient reached end-stage renal disease. After a median follow-up of 24 months, 2 patients relapsed. Lupus nephritis in the absence of overt SLE is a nosological entity requiring careful etiological investigation, including systematic electron microscopy examination of renal biopsies to rule out fibrillary glomerulonephritis. In this series, most patients presented with severe glomerulonephritis, which was highly similar to lupus nephritis at presentation and in terms of response to immunosuppressive therapy.