Claudine Blin-Wakkach

Identification of adipose tissue dendritic cells correlated with obesity-associated insulin-resistance and inducing Th17 responses in mice and patients.

T-cell regulation in adipose tissue provides a link between inflammation and insulin resistance. Because of alterations in adipose tissue T-cell composition in obesity, we aimed to identify the antigen-presenting cells in adipose tissue of obese mice and patients with insulin resistance. Dendritic cells (DCs) and T cells were studied in mice and in two cohorts of obese patients. In lean mice, only CD11c+ DCs were detected in adipose tissue. Adoptive transfer of naive CD4+ T cells in Rag1−/− mice led to a predominant Th1 response in adipose tissue. In contrast, during obesity DCs (human CD11c+CD1c+ and mouse CD11chighF4/80low) accumulated in adipose tissue. CD11chighF4/80low DCs from obese mice induced Th17 differentiation. In patients, the presence of CD11c+CD1c+ DCs correlated with the BMI and with an elevation in Th17 cells. In addition, these DCs led to ex vivo Th17 differentiation. CD1c gene expression further correlated with homeostatic model assessment-insulin resistance in the subcutaneous adipose tissue of obese patients. We show for the first time the presence and accumulation of specific DCs in adipose tissue in mouse and human obesity. These DCs were functional and could be important regulators of adipose tissue inflammation by regulating the switch toward Th17 cell responses in obesity-associated insulin resistance.

Bone marrow microenvironment controls the in vivo differentiation of murine dendritic cells into osteoclasts

Finding that activated T cells control osteoclast (OCL) differentiation has revealed the importance of the interactions between immune and bone cells. Dendritic cells (DCs) are responsible for T-cell activation and share common precursors with OCLs. Here we show that DCs participate in bone resorption more directly than simply through T-cell activation. We show that, among the splenic DC subsets, the conventional DCs have the higher osteoclastogenic potential in vitro. We demonstrate that conventional DCs differentiate into functional OCLs in vivo when injected into osteopetrotic oc/oc mice defective in OCL resorptive function. Moreover, this differentiation involves the presence of activated CD4(+) T cells controlling a high RANK-L expression by bone marrow stromal cells. Our results open new insights in the differentiation of OCLs and DCs and offer new basis for analyzing the relations between bone and immune systems.

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.

Characterization of a Novel Bipotent Hematopoietic Progenitor Population in Normal and Osteopetrotic Mice

Several reports indicate that osteoclasts and B‐lymphocytes share a common progenitor. This study focuses on the characterization of this bipotent progenitor from the bone marrow of the osteopetrotic oc/oc mouse, where the bipotent progenitor population is amplified, and of normal mice.

Bone marrow Th17 TNFα cells induce osteoclast differentiation, and link bone destruction to IBD

Objective Under both physiological and pathological conditions, bone volume is determined by the rate of bone formation by osteoblasts and bone resorption by osteoclasts. Excessive bone loss is a common complication of human IBD whose mechanisms are not yet completely understood. Despite the role of activated CD4+ T cells in inflammatory bone loss, the nature of the T cell subsets involved in this process in vivo remains unknown. The aim of the present study was to identify the CD4+ T cell subsets involved in the process of osteoclastogenesis in vivo, as well as their mechanism of action. Design CD4+ T cells were studied in IL10−/− mice and Rag1−/− mice adoptively transferred with naive CD4+CD45RBhigh T cells, representing two well-characterised animal models of IBD and in patients with Crohns disease. They were phenotypically and functionally characterised by flow cytometric and gene expression analysis, as well as in in vitro cocultures with osteoclast precursors. Results In mice, we identified bone marrow (BM) CD4+ T cells producing interleukin (IL)-17 and tumour necrosis factor (TNF)-α as an osteoclastogenic T cell subset referred to as Th17 TNF-α+ cells. During chronic inflammation, these cells migrate to the BM where they survive in an IL-7-dependent manner and where they promote the recruitment of inflammatory monocytes, the main osteoclast progenitors. A population equivalent to the Th17 TNF-α+ cells was also detected in patients with Crohns disease. Conclusions Our results highlight the osteoclastogenic function of the Th17 TNF-α+ cells that contribute to bone loss in vivo in IBD.

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFκB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

Inflammatory Blood Monocytes Contribute to Tumor Development and Represent a Privileged Target To Improve Host Immunosurveillance

Progressing tumors in humans and mice are frequently infiltrated by a highly heterogeneous population of inflammatory myeloid cells that contribute to tumor growth. Among these cells, inflammatory Gr-1+ monocytes display a high developmental plasticity in response to specific microenvironmental signals, leading to diverse immune functions. These observations raise the question of the immune mechanisms by which inflammatory monocytes may contribute to tumor development. In this study, we found that adoptive transfer of normal inflammatory Gr-1+ monocytes in tumor-bearing mice promotes tumor growth. In this tumoral environment, these monocytes can differentiate into tolerogenic dendritic cells (DCs) that produce IL-10 and potently induce regulatory T cell responses in vivo. Moreover, diverting the differentiation of Gr-1+ monocytes into tolerogenic DCs by forced expression of IL-10 soluble receptor and IL-3 in tumor cells improves host immunosurveillance by reducing the regulatory T cell frequency and by inducing immunogenic DCs in the tumor. As a consequence, tumor growth is strongly reduced. Our findings indicate that Gr-1+ monocytes represent a valuable target for innovative immunotherapeutic strategies against cancer.

Characterization of IL-10-Secreting T Cells Derived from Regulatory CD4+CD25+ Cells by the TIRC7 Surface Marker

Natural CD25+CD4+ regulatory T cells (Treg) are essential for self-tolerance and for the control of T cell-mediated immune pathologies. However, the identification of Tregs in an ongoing immune response or in inflamed tissues remains elusive. Our experiments indicate that TIRC7, T cell immune response cDNA 7, a novel membrane molecule involved in the regulation of T lymphocyte activation, identifies two Treg subsets (CD25lowTIRC7+ and CD25highTIRC7−) that are characterized by the expression of Foxp3 and a suppressive activity in vitro and in vivo. We also showed that the CD25lowTIRC7+ subset represents IL-10-secreting Tregs in steady state, which is accumulated intratumorally in a tumor-bearing mice model. Blockade of the effect of IL-10 reversed the suppression imposed by the CD25lowTIRC7+ subset. Interestingly, these IL-10-secreting cells derived from the CD25highTIRC7− subset, both in vitro and in vivo, in response to tumoral Ags. Our present results strongly support the notion that, in the pool of natural Tregs, some cells can recognize foreign Ags and that this recognition is an essential step in their expansion and suppressive activity in vivo.

Role of osteoclasts in the hematopoietic stem cell niche formation

In mammals, the bone marrow (BM) is the major site for hematopoiesis, and its colonization by hematopoietic stem cells (HSCs) is preceded by and requires endochondral ossification and invasion of blood vessels. There, HSCs are found in niches located in the trabecular regions of the bone in association with bone-lining osteoblasts (OBLs) (endosteal niche) or with vascular area (perivascular niche). Transplantation and homing assays in genetic mutant mice and in vitro assays have identified OBLs as the main components of the endosteal niche and mesenchymal progenitors as components of the perivascular niche (reviewed in ref. 1). The exact function of these niches is still a matter of debate, but it has been suggested that the endosteal niche could be involved in HSC maintenance by providing a quiescent environment, whereas the perivascular niche could harbor mobilized HSCs and could allow proliferation and differentiation. Osteoblasts are tightly coupled with the bone-resorbing osteoclasts (OCLs) in terms of differentiation and activity. Given the role of OBLs in the establishment and the regulation of the HSC niches, the question arose of a possible role of OCLs in these processes. Modulation of OCL activity in adult mice has been shown to favor HSC mobilization in response to mobilizing signals, indicating that OCLs are important regulators of the HSC niche. Moreover, extramedullary hematopoiesis is a hallmark of the loss of OCL activity in murine osteopetrotic models. However, until very recently, no data were available about the precise role of OCLs in the initial establishment of the HSC niche. Using newborn osteopetrotic oc/ oc mice in which OCLs are present but inactive, we reported very recently that Role of osteoclasts in the hematopoietic stem cell niche formation

Inflammatory osteoclasts prime TNFα‐producing CD4+ T cells and express CX3CR1

Bone destruction is a hallmark of chronic rheumatic diseases. Although the role of osteoclasts in bone loss is clearly established, their implication in the inflammatory response has not been investigated despite their monocytic origin. Moreover, specific markers are lacking to characterize osteoclasts generated in inflammatory conditions. Here, we have explored the phenotype of inflammatory osteoclasts and their effect on CD4+ T cell responses in the context of bone destruction associated with inflammatory bowel disease. We used the well‐characterized model of colitis induced by transfer of naive CD4+ T cells into Rag1–/– mice, which is associated with severe bone destruction. We set up a novel procedure to sort pure osteoclasts generated in vitro to analyze their phenotype and specific immune responses by FACS and qPCR. We demonstrated that osteoclasts generated from colitic mice induced the emergence of TNFα‐producing CD4+ T cells, whereas those generated from healthy mice induced CD4+FoxP3+ regulatory T cells, in an antigen‐dependent manner. This difference is related to the osteoclast origin from monocytes or dendritic cells, to their cytokine expression pattern, and their environment. We identified CX3CR1 as a marker of inflammatory osteoclasts and we demonstrated that the differentiation of CX3CR1+ osteoclasts is controlled by IL‐17 in vitro. This work is the first demonstration that, in addition to participating to bone destruction, osteoclasts also induce immunogenic CD4+ T cell responses upon inflammation. They highlight CX3CR1 as a novel dual target for antiresorptive and anti‐inflammatory treatment in inflammatory chronic diseases.