Jean-Philippe Girard

The IL-1-Like Cytokine IL-33 Is Constitutively Expressed in the Nucleus of Endothelial Cells and Epithelial Cells In Vivo: A Novel ‘Alarmin’?

Background Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1α and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo. Methodology/Principal Findings Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands. Conclusions/Significance Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype ‘alarmin’ HMGB1, as an endogenous ‘danger’ signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection.

The pro-Th2 cytokine IL-33 directly interacts with invariant NKT and NK cells to induce IFN-gamma production.

IL‐33 has recently been identified as a cytokine endowed with pro‐Th2 functions, raising the question of its effect on invariant natural killer T cell (iNKT), which are potent IL‐4 producers. Here, we report a two‐fold increase of iNKT‐cell counts in spleen and liver after a 7‐day treatment of mice with IL‐33, which results from a direct effect, given that purified iNKT cells express the T1/ST2 receptor constitutively and respond to IL‐33 by in vitro expansion and functional activation. Conversely to the expected pro‐Th2 effect, IL‐33 induced a preferential increase in IFN‐γ rather than IL‐4 production upon TCR engagement that depended on endogenous IL‐12. Moreover, in combination with the pro‐inflammatory cytokine IL‐12, IL‐33 enhanced IFN‐γ production by both iNKT and NK cells. Taken together these data support the conclusion that IL‐33 can contribute as a co‐stimulatory factor to innate cellular immune responses.

Molecular mimicry between IL‐33 and KSHV for attachment to chromatin through the H2A–H2B acidic pocket

Interleukin‐33 (IL‐33) is an IL‐1‐like ligand for the ST2 receptor that stimulates the production of Th2‐associated cytokines. Recently, we showed that IL‐33 is a chromatin‐associated factor in the nucleus of endothelial cells in vivo. Here, we report the identification of a short IL‐33 chromatin‐binding peptide that shares striking similarities with a motif found in Kaposi sarcoma herpesvirus LANA (latency‐associated nuclear antigen), which is responsible for the attachment of viral genomes to mitotic chromosomes. Similar to LANA, the IL‐33 peptide docks into the acidic pocket formed by the H2A–H2B dimer at the nucleosomal surface and regulates chromatin compaction by promoting nucleosome–nucleosome interactions. Taken together, our data provide important new insights into the nuclear roles of IL‐33, and show a unique example of molecular mimicry of a chromatin‐associated cytokine by a DNA tumour virus. In addition, the data provide, to the best of our knowledge, the first demonstration of the existence of non‐histone cellular factors that bind to the acidic pocket of the nucleosome.

Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules

While patrolling the body in search of foreign antigens, naive lymphocytes continuously circulate from the blood, through the lymph nodes, into the lymphatic vessels and back to the blood. This process, called lymphocyte recirculation, provides the body with effective immune surveillance for foreign invaders and for alterations to the body’s own cells. However, the mechanisms that regulate lymphocyte recirculation during homeostasis remain incompletely characterized. Here we show that dendritic cells (DCs), which are well known for their role in antigen presentation to T lymphocytes, control the entry of naive lymphocytes to lymph nodes by modulating the phenotype of high endothelial venules (HEVs), which are blood vessels specialized in lymphocyte recruitment. We found that in vivo depletion of CD11c+ DCs in adult mice over a 1-week period induces a reduction in the size and cellularity of the peripheral and mucosal lymph nodes. In the absence of DCs, the mature adult HEV phenotype reverts to an immature neonatal phenotype, and HEV-mediated lymphocyte recruitment to lymph nodes is inhibited. Co-culture experiments showed that the effect of DCs on HEV endothelial cells is direct and requires lymphotoxin-β-receptor-dependent signalling. DCs express lymphotoxin, and DC-derived lymphotoxin is important for lymphocyte homing to lymph nodes in vivo. Together, our results reveal a previously unsuspected role for DCs in the regulation of lymphocyte recirculation during immune surveillance.

Neutralisation of the interleukin-33/ST2 pathway ameliorates experimental colitis through enhancement of mucosal healing in mice

Objective Inflammatory bowel diseases (IBD) have been intrinsically linked to a deregulated cytokine network, but novel therapeutic principles are urgently needed. Here we identify the interleukin (IL)-33 and its receptor ST2 as key negative regulators of wound healing and permeability in the colon of mice. Design Expression of IL-33 and ST2 was determined by qRT-PCR, ELISA, immunohistochemistry and western-blot analysis. Wild-type and St2−/− mice were used in wound healing experiments and in two experimental models of IBD triggered by 2,4,6-trinitrobenzene sulphonic acid or dextran sodium sulphate (DSS). Neutralisation of ST2 was performed by using a specific blocking antibody. Results Nuclear localisation and enhanced expression of IL-33 in myofibroblasts and enterocytes was linked to disease involvement independently of inflammation, while the expression of ST2 was primarily restricted to the colonic epithelia. In two experimental models of IBD, genetic ablation of ST2 significantly improved signs of colitis, while a sustained epithelial expression of the cyto-protective factor connexin-43 was observed in DSS-treated St2-deficient mice. Unexpectedly, absence of ST2 in non-hematopoietic cells was sufficient to protect against colitis. Consistently, specific inhibition of endogenous ST2-mediated signalling by treatment with neutralising antibody improved DSS-induced colitis. In addition, IL-33 treatment impaired epithelial barrier permeability in vitro and in vivo, whereas absence of ST2 enhanced wound healing response upon acute mechanical injury in the colon. Conclusions Our study unveiled a novel non-hematopoietic function of IL-33 in epithelial barrier function and wound healing. Therefore, blocking the IL-33/ST2 axis may represent an efficient therapy in IBD.

Direct interaction between causative genes of DYT1 and DYT6 primary dystonia

Primary dystonia is a movement disorder characterized by sustained muscle contractions and in which dystonia is the only or predominant clinical feature. TOR1A(DYT1) and the transcription factor THAP1(DYT6) are the only genes identified thus far for primary dystonia. Using electromobility shift assays and chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR), we demonstrate a physical interaction between THAP1 and the TOR1A promoter that is abolished by pathophysiologic mutations. Our findings provide the first evidence that causative genes for primary dystonia intersect in a common pathway and raise the possibility of developing novel therapies targeting this pathway. Ann Neurol 2010

High endothelial venules (HEVs) in human melanoma lesions: Major gateways for tumor-infiltrating lymphocytes.

The presence of tumor-infiltrating lymphocytes (TILs) is a strong prognostic parameter for local dissemination and overall survival in melanoma. Lymphocyte migration from blood into peripheral tissues is mainly regulated by vascular endothelium. However, the blood vessels and mechanisms governing the recruitment of TILs in melanoma tumors remain poorly understood. Here, we show that high endothelial venules (HEVs), specialized blood vessels for lymphocyte extravasation into lymphoid tissues, are frequently found in melanoma tumors and are associated with high levels of lymphocyte infiltration. The analysis of 225 primary melanomas revealed that lymphocytes specifically infiltrated HEV-rich areas of melanoma tumors and that the density of MECA-79+ HEVs was variable among patients and strongly correlated with CD3+, CD8+ and CD20+ TIL densities. Inflammatory (CCL5, CXCL9, CXCL10 and CXCL11) and lymphoid (CCL21, CCL19 and CXCL13) chemokines as well as TH1 and naïve T-cell genes were overexpressed in melanoma samples with high densities of tumor HEVs. Mature dendritic cells (mDCs) were frequently found around tumor HEVs and densities of HEVs and DC-LAMP+ mDCs within tumor stroma were strongly correlated. DCs which maintain HEVs in lymph nodes, may thus also contribute to the regulation of HEVs in melanomas. Finally, we found significantly higher densities of tumor HEVs in melanomas with tumor regression, low Clark level of invasion and thin Breslow thickness (all p < 0.001). The strong association between tumor HEVs, TILs, mDCs and clinical parameters of melanoma, supports a critical role for HEVs in limiting malignant melanoma development through both naïve and effector T-lymphocyte recruitment and activation.

Desirable cell death during anticancer chemotherapy

The concept of immunogenic chemotherapy that has recently emerged relies upon the capacity of a cytotoxic compound to trigger a cell‐death modality. This modality elicits cross‐priming by dendritic cells of tumor antigen‐specific T cells that will contribute to the tumoricidal activity of the compound and protect the host against relapse. In contrast, most anticancer drugs elicit nonimmunogenic apoptosis that is not accompanied with an immunizing property. This review will discuss some molecular and metabolic changes required at the level of the tumor that must engage key pathways at the level of the host for the induction of Tc1 polarized–protective T cell responses during chemotherapy. We will summarize the immune adjuvants that can boost the immunogenicity of cell death to augment the efficacy of chemotherapy.

Androgen signaling negatively controls group 2 innate lymphoid cells

Prevalence of asthma is higher in women than in men, but the mechanisms underlying this sex bias are unknown. Group 2 innate lymphoid cells (ILC2s) are key regulators of type 2 inflammatory responses. Here, we show that ILC2 development is greatly influenced by male sex hormones. Male mice have reduced numbers of ILC2 progenitors (ILC2Ps) and mature ILC2s in peripheral tissues compared with females. In consequence, males exhibit reduced susceptibility to allergic airway inflammation in response to environmental allergens and less severe IL-33–driven lung inflammation, correlating with an impaired expansion of lung ILC2s. Importantly, orchiectomy, but not ovariectomy, abolishes the sex differences in ILC2 development and restores IL-33–mediated lung inflammation. ILC2Ps express the androgen receptor (AR), and AR signaling inhibits their differentiation into mature ILC2s. Finally, we show that hematopoietic AR expression limits IL-33–driven lung inflammation through a cell-intrinsic inhibition of ILC2 expansion. Thus, androgens play a crucial protective role in type 2 airway inflammation by negatively regulating ILC2 homeostasis, thereby limiting their capacity to expand locally in response to IL-33.

The Alarmin Concept Applied to Human Renal Transplantation: Evidence for a Differential Implication of HMGB1 and IL-33

The endogenous molecules high mobility group box 1 (HMGB1) and interleukin-33 (IL-33) have been identified as alarmins, capable of mediating danger signals during tissue damage. Here, we address their possible role as innate-immune mediators in ischemia-reperfusion injury (IRI) following human kidney transplantation. We analysed serum and urinary HMGB1 and IL-33 levels, all determined by enzyme-linked immunosorbent assay, in a cohort of 26 deceased renal transplant recipients. Urinary HMGB1 and IL-33 levels were significantly increased as soon as 30 min after reperfusion, as compared to those before treatment. Moreover, both serum and urinary IL-33 (but not HMGB1) increase was positively correlated with cold ischemia time, from 30 min to 3 days post-transplantation. In vitro, human umbilical vein endothelial cells subjected to hypoxia conditions released both HMGB-1 and IL-33, while only the latter was further increased upon subsequent re-oxygenation. Finally, we postulate that leukocytes from renal recipient patients are targeted by both HMGB1 and IL-33, as suggested by increased transcription of their respective receptors (TLR2/4 and ST2L) shortly after transplantation. Consistent with this view, we found that iNKT cells, an innate-like T cell subset involved in IRI and targeted by IL-33 but not by HMGB1 was activated 1 hour post-transplantation. Altogether, these results are in keeping with a potential role of IL-33 as an innate-immune mediator during kidney IRI in humans.