Abstract
Th17 cells are critical effectors in inflammation and tissue damage such as bone erosion, but the mechanisms regulating their activation in this process are not fully understood. In this study, we considered the cooperation between cytokine receptors and integrin pathways in Th17-osteoclast function. We found that human Th17 cells coexpress IL-7R and the collagen-binding integrin α2β1 (CD49b), and IL-7 increases their adhesion to collagen via α2β1 integrin. In addition, coengagement of the two receptors in human Th17 cells cooperatively enhanced their IL-17 production and their osteoclastogenic function. The functional cooperation between IL-7R and α2β1 integrin involves activation of the JAK/PI3K/AKT (protein kinase B) and MAPK/ERK pathways. We also showed that IL-7–induced bone loss in vivo is associated with Th17 cell expansion. Moreover, blockade of α2β1 integrin with a neutralizing mAb inhibited IL-7–induced bone loss and osteoclast numbers by reducing Th17 cell numbers in the bone marrow and reducing the production of IL-17 and the receptor activator of NF-κB ligand. Thus, the cooperation between IL-7R and α2β1 integrin can represent an important pathogenic pathway in Th17-osteoclast function associated with inflammatory diseases.
Introduction
T helper 17 cells are a subset of Th cells that are broadly implicated in the development of inflammatory diseases (1). After their differentiation, Th17 cells migrate to target tissues where their reactivation leads to the production of IL-17, which orchestrates the inflammatory response leading to tissue damage. In this regard, Th17 cells are critical effectors in bone erosion associated with inflammatory diseases like rheumatoid arthritis (RA), colitis, and periodontitis (2, 3). They promote, from myeloid precursors, the development of osteoclasts (osteoclastogenesis), the cells responsible for bone erosion. IL-17 stimulates fibroblasts and osteoblasts to produce receptor activator of NF-κB ligand (RANKL), which is a crucial cytokine for the development of osteoclasts (4, 5). Th17 cells are also capable of producing high levels of RANKL (6), and IL-17 can directly act on myeloid precursors and osteoclasts to promote osteoclastogenesis (7, 8). Currently, the molecular pathways that promote the production of IL-17 by Th17 cells at inflammatory sites are not fully understood.
In addition to the TCR, effector/memory T cells also respond to cytokines, which are critical to sustain the chronic inflammatory response. IL-7 is an important cytokine involved in T cell survival and development, and as such has been shown to promote autoimmune diseases (9). In addition, in RA, IL-7 stimulates contact-dependent activation of arthritic T cells and macrophages leading to TNF and metalloproteinase production (10, 11). IL-7–aggravated joint inflammation in collagen-induced arthritis is also associated with expansion of Th1 and Th17 cells, although the mechanism by which it affects effector T cells is unclear (12). IL-7 is also an important osteoclastogenic cytokine. It upregulates RANKL production in human nonpolarized effector/memory T cells (13, 14) and in vivo it leads to T cell–mediated bone loss (15–17). Furthermore, IL-7/IL-7R signaling axis enhances macrophage differentiation toward multinucleated osteoclasts (18, 19). Despite these findings, the role and the mechanisms by which IL-7 regulates Th17 cell activation and osteoclast function are still poorly understood.
Besides cytokines, inflammatory tissues are rich in extracellular matrix (ECM) components, and T cells express several ECM receptors, among which are the very late Ags 1–6 that constitute the β1 subfamily of integrins (20, 21). In addition to their roles in cell adhesion and migration, β1 integrins and especially collagen-binding integrins also provide costimulatory signals for TCR activation of effector T cells (21, 22). We have recently shown that α2β1 integrin is the major collagen-binding integrin expressed by human Th17 cells and that collagen enhanced TCR-mediated IL-17 production (23).
Whether integrins and cytokine receptors interact to modulate Th17 cell responses and to induce tissue injury remains unclear. In this study, we have considered the cooperation between IL-7R and α2β1 integrin in Th17-mediated bone loss. Our results showed that the two receptors are coexpressed on human Th17 cells. IL-7 increased Th17 cell adhesion to collagen, and coengagement of the two receptors led to additive/synergistic production of IL-17. This occurred via activation of the JAK/PI3K/AKT (protein kinase B) and MAPK/ERK pathways. We further found that blockade of α2β1 integrin inhibited IL-7–induced bone loss in mice by reducing Th17 cell numbers and production of IL-17 and RANKL. Thus, the cooperation between IL-7R and α2β1 integrin can represent an important pathogenic pathway in Th17-mediated bone erosion associated with inflammatory diseases.
Materials and Methods
Reagents and Abs
αMEM, FBS, glutamine, and antibiotics were from Wisent (St. Bruno, QC, Canada); X Vivo-15 medium was from Lonza Technologies (Basel, Switzerland). BSA, PMA, ionomycin, collagen type I (Col I), and the tartrate-resistant acid phosphatase (TRAP) detection kit were from Sigma-Aldrich (St. Louis, MO). The MEK/ERK inhibitor PD98059, the PI3K/AKT inhibitor wortmannin, and the JAK inhibitor (Pan+ T cells and CD16−
Human Th17 differentiation, stimulation, and analysis by flow cytometry
Human polarized Th17 cells were generated from peripheral blood naive CD4+ T cells cultured for 6 d in the presence of CD3/CD28 beads and cytokines (TGF-β, IL-1β, IL-6, and IL-23) as we previously described (23) except that IL-6 was used at 40 ng/ml. Th17 cells were then activated for 12 h with coated Col I (20 μg/ml) and soluble human IL-7 (10 ng/ml) in the presence of 5 μg/ml GolgiPlug containing brefeldin A to block cytokine secretion. Subsequently, the cells were washed and first stained with anti–α2 integrin-PE and anti–IL-7Rα–Alexa 488. The cells were washed, fixed/permeabilized with a CytoFix/CytoPerm kit, stained for intracellular IL-17 using anti–IL-17–Alexa 647, and analyzed by flow cytometry (BD FACSCalibur). Cells stained with isotypic Abs were used as controls.
Cell adhesion assays
Cell adhesion assays were performed in 96-well plates as described previously (23). A 96-well microtiter plate (TC plate, flat bottom; Falcon) was coated with 20 μg/ml collagen in PBS overnight at 4°C. The wells were then washed with PBS and blocked for 1 h at 37°C with 1% BSA. Triplicates of 2.5 × 105 cells in 0.1 ml X-vivo medium containing or not IL-7 (10 ng/ml) were added to the wells and were incubated for 6 h at 37°C. In some cases, 10 μg/ml blocking anti-α2β1 integrin mAb or control IgG was added to the cells 1 h before cellular activation. After incubation, the cells were washed with PBS and the adherent cells were recovered and counted microscopically by two readers in blinded manner.
Cytokine ELISA assays
Human polarized Th17 cells were also activated with IL-7 and collagen in the absence of GolgiPlug, and after 24 h, IL-17 and RANKL productions were measured in cell-free supernatants by ELISA assays according to the manufacturer’s instructions.
In vitro osteoclast differentiation and resorption activity
The ability of collagen- and IL-7–treated Th17 cells to induce the development of osteoclasts in vitro was tested in a coculture assay with human monocytes used as osteoclast precursors. Human CD16− monocytes, which have the ability to differentiate into osteoclasts, were isolated from the peripheral blood of healthy volunteers by immunomagnetic negative selection. Monocytes (1 × 105 cells) were seeded in 48-well plates and were activated with M-CSF (100 ng/ml) in αMEM supplemented with FBS glutamine and antibiotics. Two hours later, polarized human Th17 cells (105 cells) that had been activated or not for 12 h with 10 ng/ml IL-7, 10 μg/ml coated Col I, or IL-7+Col I were washed twice and added to the monocytic cultures. Culture media and activated Th17 cells were replaced every 2 d with fresh media and freshly activated Th17 cells. At the end of the experiment (day 15), osteoclast development was visualized by TRAP staining kit according to the manufacturer’s instructions. After staining, multinucleated (≥4 nuclei) TRAP+ cells were considered as osteoclasts. To evaluate osteoclast resorption activity, we first seeded monocytes in Osteo Assay 96-well plates coated with a mineralized matrix and then activated with M-CSF and cocultured with activated Th17 cells as described earlier. Resorption lacunae resulting from osteoclast activity on the mineralized matrix were evaluated after osteoclast lysis with a bleach solution followed by three washes using distilled water as described previously (24). The number of osteoclasts and resorption surface area per well were evaluated by an Eclipse TE300 microscope (Nikon) connected to a digital camera (DS-Fi1; Nikon) using the 100× and 20× objectives, respectively. Total resorption areas were quantified using the ImageJ analysis software (National Institutes of Health, Bethesda, MD).
Measurement of STAT-5, AKT, and ERK phosphorylation by immunoblot
Human Th17 cells were activated with immobilized Col I and soluble IL-7. The cells were lysed, and phosphorylation levels of STAT5, AKT, and ERK were determined by immunoblot analysis using specific Abs as we previously described (25).
Mice and IL-7–induced bone loss
Six- to 8-wk-old female C57BL/6 mice (Charles River Laboratories, Senneville, QC, Canada) were used. Water and food were provided ad libitum. All procedures involving animals were conducted according to the requirements of and with the approval of the Laval University Animal Protection Committee. Groups of 10 mice were daily injected i.p. for 30 d with vehicle (PBS) or 10 μg/kg body weight recombinant human IL-7 to induce bone loss as previously described (15–17). The anti-α2β1 integrin blocking mAb (Ha1/29) and isotypic control mAb (Ha4/8) (50 μg/mouse) were injected i.p. every 2 d starting at day 2 after the first IL-7 injection. At day 30, mice were sacrificed; bones and bone marrow cells were recovered for analysis. Blood was also collected and serum fractions were prepared for cytokine measurements using ELISA kits.
Bone analyses by microcomputerized tomography and TRAP assay
Trabecular bone loss was examined by microcomputerized tomography (micro-CT) analysis and by determination of osteoclasts using the TRAP assay. Femurs were fixed in 4% paraformaldehyde and scanned with high-resolution micro-CT scanner (SkyScan 1072; Antwerp, Belgium). In brief, image acquisition was performed at 45 kV/222 μA at resolution of 5.63 μm per pixel with step rotation of 0.9° for 180° and step exposure of 2.87 s. Three-dimensional reconstructions were enabled by the three-dimensional creator software (NRecon [v1.6.1.3] and CT-Analyzer [v1.8.1.2]) supplied with the instrument. Trabecular bone indices such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular pattern factor (Tb.pf) were measured in the distal femur, 2.25 mm starting from growth plate.
Femurs were then decalcified in 10% EDTA and were embedded in paraffin and sliced at equivalent sections through the center of the bone. Five-micrometer sections were prepared and stained for TRAP activity using the Sigma-Aldrich kit. The sections were counterstained with methyl green and examined histologically. Ten fields from each distal femur area were randomly chosen and observed microscopically at 400× magnification, and the related pictures were analyzed by two observers in a blinded manner. Osteoclasts were identified by the red staining corresponding to TRAP activity. The number of TRAP+ cells per section was calculated by counting the osteoclasts present in the 10 fields analyzed for each femur. The mean values of TRAP+ cells per section were then calculated for the different treated groups. These experiments (micro-CT and TRAP) were carried out in the McGill Bone Center platform (McGill University, Montréal, QC).
Analysis of bone inflammatory Th17 cells
Bone marrow was flushed from femurs and tibias and passed through 40-μm cell strainers. The resulting cell suspensions were activated with PMA (50 ng/ml) and ionomycin (1 μM) for 6 h in the presence of 5 μg/ml GolgiPlug containing brefeldin A to capture IL-17–producing cells. The cells were first stained with anti-α2 integrin-PE and anti-CD4-FITC Abs and then fixed/permeabilized with a CytoFix/CytoPerm kit and stained for intracellular IL-17 using anti–IL-17-Alexa 647 Ab. The cells were then analyzed by flow cytometry (BD FACSCalibur). Cells stained with isotypic Abs were used as controls.
Statistical analysis
Statistical analysis was performed by the Student t test (two-tailed, two-sample equal variance). Results with p values <0.05 were considered significant.
Results
IL-7R and α2β1 integrin are coexpressed on human Th17 cells
We examined the expression of IL-7R (CD127; the IL-7Rα-chain) and α2 integrin chain (CD49b) on human polarized Th17 cells. The flow cytometry results indicate that a high proportion of cells express both IL-7R and α2 integrin (Fig. 1A). We then assessed whether the two receptors can be coexpressed on IL-17–producing cells. The cells were restimulated with PMA and ionomycin to capture IL-17, and the expression of IL-7R and α2 integrin was determined. The vast majority of IL-17–producing cells (Th17) express both IL-7R and α2 integrin (Fig. 1B, top panel). Expression analysis on human Th17 cells polarized from five different blood donors indicated that between 69 and 95% of the cells are positive for both receptors (Fig. 1B, lower panel). As expected, virtually all the cells express the β1 integrin chain (CD29), which partners with α2 integrin (CD49b) in forming the α2β1 integrin (data not shown). These results indicate that human Th17 cells coexpress IL-7R and α2β1 integrin.
IL-7R and α2β1 integrin are coexpressed on human Th17 cells. (A) Polarized human Th17 cells were stained with anti–α2 integrin-PE and anti–IL-7Rα–Alexa 488 and analyzed by flow cytometry. (B) Polarized human Th17 cells were activated with PMA+ionomycin for 4 h in the presence of brefeldin A. The cells were washed and first stained with anti–α2 integrin-PE and anti–IL-7R–Alexa 488 Abs. The cells were then washed, fixed/permeabilized, and stained for intracellular IL-17 using an anti–IL-17–Alexa 647 and analyzed by flow cytometry. α2β1/IL-7R double-positive cells were gated on the IL-17+ population. The flow cytometry profiles are representative of five different samples. The histogram represents percentages of human Th17 cells coexpressing α2β1 integrin and IL-7R from five different blood donors.
IL-7 increases adhesion of human polarized Th17 cells to collagen
Retention of inflammatory T cells is regulated by integrin-mediated adhesion to ECM, which is important for T cell persistence in inflammatory tissues. Therefore, we tested whether IL-7 regulates adhesion of polarized human Th17 cells to collagen. Polarized human Th17 cells attach to collagen and their stimulation with IL-7 increased their attachment by 2- to 3-fold (Fig. 2A). Adhesion of the cells was significantly reduced by a blocking anti-α2 integrin mAb (P1E6), but not with control IgG, which is in agreement with α2β1 integrin being the major collagen-binding integrin expressed by human Th17 cells (23).
IL-7R/α2β1 signaling enhances adhesion of human Th17 cells and IL-17 production. (A) IL-7 enhances human polarized Th17 cell adhesion to collagen (Col) via α2β1 integrin. The cells were activated or not with IL-7 (10 ng/ml) and seeded in wells that had been coated with Col. They were allowed to attach for 6 h at 37°C and, in some cases, 10 μg/ml inhibitory anti-α2 integrin mAb or control IgG was added to the wells as indicated. After PBS washing, adherent cells were recovered and counted microscopically. Results are mean values ± SEM (n = 5). *p < 0.05 between IL-7– and IL-7+IgG–treated compared with nontreated (NT, -) or IL-7+anti–α2 mAb–treated samples. (B) Col cooperatively with IL-7 enhances IL-17 production. Human polarized Th17 cells were left NT or activated in wells coated with Col in the presence or absence of soluble IL-7 for 12 h in the presence of brefeldin A. The cells were then washed, fixed/permeabilized, stained with an anti–IL-17–Alexa 647 or isotypic control Abs, and analyzed by flow cytometry. Upper panel shows a representative flow cytometry profile and lower panel represents mean values of IL-17+ cells from five independent experiments performed with Th17 cells derived from five different blood donors (n = 5). (C) IL-17 levels were monitored by ELISA in cell-free supernatants (n = 5). *p < 0.05 compared with NT samples, **p < 0.05 compared with Col- or IL-7–treated samples.
Ligation of IL-7R and α2β1 integrin activates human Th17 cells
We then investigated the functional interactions between IL-7R and α2β1 in human Th17 cell activation. The cells were cultured on immobilized collagen with or without IL-7, and the number of IL-17–producing cells was detected by flow cytometry. IL-7 and collagen are both capable of stimulating the production of IL-17, as 3–4% of IL-17+ cells were detected in response to each stimulus (Fig. 2B, upper panel). However, the combination of IL-7+collagen led to the detection of 13.4% of IL-17–producing cells. Analysis of Th17 cells derived from five different blood donors indicated that IL-7 and collagen are equal in inducing IL-17 production, and the combination of both led to a 2- to 3-fold increase than with each stimulus alone (Fig. 2B, lower panel). We obtained similar results when we assessed IL-17 production in cell culture supernatants by ELISA (Fig. 2C). The stimulatory effects of IL-7 and collagen were not due to changes in cell proliferation or apoptosis because comparable cell numbers with similar viability were recovered after 24 h of stimulation under all experimental conditions (data not shown). In addition, the neutralizing anti-α2 and anti-β1 integrin Abs blocked the effect of collagen (data not shown). Together, these results show that IL-7R and α2β1 integrin cooperate additively/synergistically to induce the production of IL-17 from human Th17 cells.
IL-7R and α2β1 integrin cooperatively induce osteoclastogenesis in vitro
Th17 are crucial cells in inducing the development of osteoclasts, the cells responsible for bone erosion associated with inflammatory diseases (2, 3). In addition, activated human Th17 cells can induce the development of osteoclasts from monocytes in vitro (7, 8). Accordingly, we examined whether activation of human Th17 cells via IL-7R and α2β1 integrin can trigger their osteoclastogenic function. Human polarized Th17 cells were activated with IL-7 and collagen and then tested for their ability to induce the development of osteoclasts (TRAP+ and multinucleated cells) from human monocytes in a coculture system. We found that IL-7 and collagen-activated Th17 cells are able to induce the formation of osteoclasts (Fig. 3A, 3B). Importantly, IL-7+collagen–activated Th17 cells were the most potent in this process. There is a 2-fold increase in the number of osteoclasts induced by IL-7+collagen–activated Th17 cells than with IL-7– or collagen-activated Th17 cells (Fig. 3B). Monocyte-derived osteoclasts obtained after coculture with IL-7–, collagen-, or IL-7+collagen–activated Th17 cells are functionally active because they were able to resorb a mineralized matrix mimicking the bone matrix (Fig. 3C, 3D). Th17 cells activated with IL-7 or collagen are equal in inducing osteoclast activity. However, Th17 cells activated with IL-7+collagen are more potent in increasing the activity of osteoclasts. In addition to the production of IL-17 (Fig. 2), we also found that IL-7+collagen cooperatively enhanced the production of RANKL from human polarized Th17 cells (Supplemental Fig. 1). Together these results indicate that the cooperation between IL-7R and α2β1 integrin in Th17 cells is functionally relevant.
Collagen (Col) and IL-7 cooperatively stimulate Th17-mediated osteoclastogenesis. Human Th17 cells were stimulated with 10 ng/ml IL-7, 10 μg/ml coated Col, or IL-7+Col before being cocultured with monocytes in the presence of M-CSF (100 ng/ml). Th17 cells and medium were replaced every 2 d. (A) After 15 d, the cultures were washed and the adherent cells stained for TRAP activity as described in Materials and Methods. Representative images (original magnification ×1000) showing the formation of TRAP+ multinucleated cells representing osteoclasts, which are indicated by arrows. (B) Quantification of osteoclast formation induced by activated Th17 cells. After TRAP staining, the total numbers of TRAP+ cells with at least four nuclei were counted as osteoclasts. Results are mean values ± SEM (n = 5). *p < 0.05 compared with monocytes cultured with nonactivated Th17 cells, **p < 0.05 compared with monocytes cultured with IL-7– or Col-treated Th17 cells. (C) Resorption activity of osteoclasts induced by activated Th17 cells. Monocytes were plated in Osteo Assay 96 wells coated with mineralized matrix and cocultured with IL-7–, Col-, or IL-7+Col–stimulated Th17 cells for 15 d as indicated earlier. Differentiated osteoclasts were then lysed and the matrix surface was observed by microscopy. Representative images (original magnification ×200) showing the formation of resorption lacunae. (D) Quantification of total resorption area per well was carried out using the ImageJ analysis software, and the results are expressed in square millimeter (mm2) of resorption. Results represent mean values ± SEM (n = 3). *p < 0.05 compared with monocytes cultured with nonactivated Th17 cells, **p < 0.05 compared with monocytes cultured with IL-7– or Col-treated Th17 cells.
Involvement of JAK/PI3K/AKT and MAPK/ERK pathways in IL-7R/α2β1 integrin functional cooperation
To gain more insights into how IL-7R and α2β1 cooperate to activate Th17 cells, we examined the signaling pathways involved downstream of these two receptors. IL-7 signaling is associated with activation of JAK/STAT and PI3K/AKT pathways (26), and STAT-5 is the major isoform activated by IL-7 in human T cells (25, 26). In addition, integrins are known to activate MAPK/ERK and PI3K/AKT pathways. Therefore, using specific inhibitors, we examined the implication of these pathways. Basal adhesion of human polarized Th17 cells to collagen was reduced by their treatment with the PI3K/AKT and MAPK/ERK inhibitors (wortmannin and PD98059, respectively), but not the JAK inhibitor (Pan-JAK) (Fig. 4A). In addition to the PI3K/AKT and MAPK/ERK pathways, the effect of IL-7 was also associated with JAK signaling because the Pan-JAK inhibitor significantly reduced IL-7–induced Th17 cell adhesion (Fig. 4A).
Role of JAK/STAT, PI3K/AKT, and MAPK/ERK signaling pathways in α2β1/IL-7R cross talk. (A) The JAK, PI3K/AKT, and MAPK/ERK inhibitors reduce adhesion of human polarized Th17 cells to collagen. The cells were preincubated for 1 h with 20 μM Pan-JAK (JAK inhibitor), 100 nM wortmannin (PI3K/AKT inhibitor), or 10 μM PD98059 (MAPK/ERK inhibitor) and then seeded on collagen in the presence or absence of IL-7. Results represent mean values ± SEM from five independent experiments (n = 5). *p < 0.05 compared with nontreated (NT) or Pan-JAK–treated samples, **p < 0.05 compared with NT samples stimulated with IL-7. (B) The pan-JAK, PI3K/AKT, and MAPK/ERK inhibitors abolish IL-7– and collagen (Col)-induced IL-17 production. Human polarized Th17 cells were preincubated or not for 1 h with pan-JAK, wortmannin, or PD98059. The cells were then left NT or stimulated with coated Col, soluble IL-7, or IL-7+Col in the presence of brefeldin A for 12 h. IL-17+ cells were determined by intracellular staining and flow cytometry. Mean values ± SEM of IL-17+ cells from five independent experiments (n = 5). *p < 0.05 compared with the corresponding values in control cells that were not treated with inhibitors. (C) IL-7/collagen–induced activation of STAT-5 (p-STAT-5), AKT (p-AKT), and ERK (p-ERK). Th17 cells were stimulated as indicated for 60 min. The cells were then washed and lysed using RIPA buffer, and activation of STAT-5 (left panel), AKT (middle panel), and ERK (right panel) was determined by immunoblot analysis using specific Abs recognizing the phosphorylated forms. The blots were stripped and reprobed with anti–β-actin, anti-AKT, and anti-ERK2 Abs as indicated to ensure equal loading. The numbers indicated on the top of the bands represent densitometry analysis and are expressed as fold increase of the ratio between total p-STAT-5 and β-actin, p-AKT and total AKT, and p-ERK and ERK2. The presented blots are representative of three independent experiments.
Next, we assessed the effects of these inhibitors on IL-17 production. Treatment of human polarized Th17 cells with the JAK and PI3K/AKT inhibitors reduced the numbers of IL-17+ cells in IL-7– and in IL-7+collagen–activated cells, but not in cells activated only with collagen (Fig. 4B, upper panels). However, the MAPK/ERK inhibitor (PD98059) inhibited the numbers of IL-17+ cells in collagen-activated cells and in collagen+IL-7–activated cells, but not in cells activated with IL-7 alone (Fig. 4B, lower panel). We then examined by immunoblot the activation of STAT-5 and PI3K/AKT (activated downstream of IL-7/IL-7R) and MAPK/ERK pathways. IL-7–induced STAT-5 phosphorylation is reduced by collagen, which by itself had only a small effect (Fig. 4C, left panel). IL-7, either alone or in combination with collagen, increased AKT phosphorylation, whereas collagen alone had no effect (Fig. 4C, middle panel). However, only collagen strongly induced ERK phosphorylation (Fig. 4C, right panel). Together these results indicate that the functional cooperation between α2β1 integrin and IL-7R involves interactions of multiple signaling pathways.
IL-7–induced bone loss in vivo is associated with α2β1 integrin–positive Th17 cells
We then sought to determine whether the functional cooperation between IL-7R and α2β1 integrin occurs in vivo. To this end, we used the model of IL-7–induced bone loss in mice. This model has been shown to be associated with T cells and RANKL (16, 17), thus mimicking inflammatory bone loss occurring in RA, which is also associated with increased IL-7 levels (11, 27, 28). However, the role of Th17 cells in this model is unclear. In this study, we confirmed that injection of IL-7 induces bone loss in mice after 30 d, as measured by micro-CT analysis. Three-dimensional reconstructions of trabecular BV in the distal femurs indicated that IL-7 treatment induces trabecular bone damage (Supplemental Fig. 2A, top panel). This concurred with a decrease in BV/TV ratio and in Tb.N, and with an increase in Tb.pf (Supplemental Fig. 2A, lower panel). Furthermore, IL-7–treated mice also showed high numbers of osteoclasts compared with mice injected only with PBS (Supplemental Fig. 2B).
In addition, we found that treatment of mice with IL-7 led to an increase of CD4+ T cell numbers in the bone marrow (5.27% in PBS-treated mice versus 7.66% in IL-7–treated mice; Fig. 5A, 5B), and a 1.5-fold increase in Th17 (CD4+/IL-17+) cell numbers was observed upon IL-7 treatment (Fig. 5A, 5B). Interestingly, the majority of Th17 cells in both PBS- and IL-7–treated mice express α2 integrin, and the number of α2 integrin–positive Th17 cells also increased with IL-7 treatment (Fig. 5A, 5B). The increased Th17 cell numbers in IL-7–treated mice correlated with increased serum levels of both IL-17 and RANKL (Fig. 5C).
IL-7–induced bone loss in mice is associated with Th17 cell expansion. Bone marrow cells were recovered from PBS- or IL-7–treated mice and stimulated with PMA+ionomycin in the presence of brefeldin A. The cells were then stained with anti–CD4-FITC and anti–α2 integrin-PE Abs. The cells were washed, fixed/permeabilized, stained with anti–IL-17–Alexa 647 Ab, and analyzed by flow cytometry. Cells stained with isotypic Abs were used as control (Iso). (A) Representative flow cytometry profiles showing the expression of α2 integrin in Th17 cells from PBS- (top panels) and IL-7–treated mice (lower panels). α2 integrin/IL-17+ cells shown in right panels were gated on the CD4+ cells shown in left panels. (B) Quantification of CD4+ cells, Th17 cells, and Th17/α2 integrin double-positive population numbers in the bone marrow of PBS- and IL-7–treated mice. (C) IL-7 enhances IL-17 and RANKL production in vivo. ELISA measurements of IL-17 (left panel) and RANKL (right panel) in serum from PBS- and IL-7–treated mice. Results in (B) and (C) are the mean values ± SEM (n = 10). *p < 0.05 compared with the corresponding values in PBS-treated mice.
Blockade of α2β1 integrin reduces IL-7–induced bone loss by affecting Th17 cells
The earlier results suggested that α2β1 integrin could regulate IL-7–induced bone loss. Thus, we tested the effect of Ha1/29 mAb directed against α2 integrin chain on IL-7–induced bone loss. The Ha1/29 mAb has shown potent blocking activity in vivo (29, 30). Three-dimensional reconstructions of trabecular BV indicated that the anti-α2β1 mAb reduced trabecular bone damage associated with IL-7 treatment compared with mice treated with a control IgG mAb (Fig. 6A). This concurred with increased BV/TV ratio, Tb.N, and with a decrease in Tb.pf (Fig. 6B). Furthermore, the anti-α2β1 mAb also decreased osteoclast numbers in IL-7–treated mice compared with the control IgG (Fig. 6C). We then examined whether the effect of the anti-α2β1 mAb occurred at the level of Th17 cells. To this end, we measured Th17 cell numbers in the bone marrow of IL-7–treated mice and found that the treatment with anti-α2β1 mAb reduced total CD4+ T cell numbers as well as Th17 (CD4+/IL-17+) numbers by ∼40% (Fig. 7A, 7B). This correlated with a 55% reduction of IL-17 levels in the serum of IL-7–injected mice treated with anti-α2β1 mAb (Fig. 7C). Similarly, RANKL levels were reduced by ∼40% by the anti-α2β1 mAb in IL-7–treated mice (Fig. 7D). Together, these results show that inhibition of IL-7–induced bone loss by the blocking anti-α2β1 mAb is associated with reduced Th17 activity.
Blockade of α2β1 integrin inhibits IL-7–induced bone loss in vivo. Femurs were recovered and prepared for micro-CT and histology analyses as described in Materials and Methods. (A) Representative three-dimensional reconstruction of micro-CT images of trabecular bone from IL-7–injected mice treated with control IgG or anti-α2β1 mAbs. (B) Histomorphometric analysis of trabecular bone from IL-7–injected mice treated with control IgG or anti-α2β1 mAbs. (C) Representative TRAP staining (red staining, corresponding to osteoclasts) of distal femur sections from IL-7–injected mice treated with control IgG or anti-α2β1 mAbs at original magnification ×400 (left panels). Quantification of osteoclast numbers in IL-7–injected mice treated with control IgG or anti-α2β1 mAbs (right panel). (B and C) Data are the mean values ± SEM (n = 10). *p < 0.05 compared with the corresponding values in control IgG mAb-treated mice.
Blockade of α2β1 integrin decreases Th17 cell numbers and IL-17 and RANKL secretion in vivo in IL-7–treated mice. Total bone marrow cell suspensions from IL-7–injected mice treated with control IgG or anti-α2β1 mAbs were prepared, stimulated with PMA+ionomycin in the presence of brefeldin A, and stained with conjugated isotypic control mAbs (Iso) or with anti–CD4-FITC and anti–IL-17–Alexa 647 Abs. After washing with PBS, the cells were analyzed by flow cytometry. (A) Representative flow cytometry profiles from each experimental group are shown. (B) Mean percentage of CD4+ T cells and CD4/IL-17+ cells (Th17 cells) in control IgG- and anti–α2β1 mAb–treated mice. ELISA measurements of IL-17 (C) and RANKL (D) levels in sera from IL-7–injected mice treated with control IgG or anti-α2β1 mAbs. (B–D) Results represent mean values ± SEM (n = 10). *p < 0.05 compared with the corresponding values in control IgG-treated mice.
Discussion
The mechanisms by which Th17 cells induce tissue injury are not well understood. In this study, we identified a cooperative effect between cytokine receptors and integrin pathways in the capacity of Th17 cells to induce bone erosion. We showed that IL-7R and α2β1 integrin were coexpressed on human Th17 cells and that IL-7 increases their adhesion to collagen. Ligation of IL-7R and α2β1 integrin on human Th17 cells led to additive production of IL-17 and RANKL, and to the development of functionally active osteoclasts. Furthermore, IL-7–induced bone loss in mice is associated with increased Th17 cells in the bone marrow and IL-17 and RANKL serum levels, and blockade of α2β1 integrin with a neutralizing mAb reduced IL-7–induced bone loss by affecting Th17 cells. Together, these findings suggest that at sites of inflammation (bone tissue), effector Th17 cells can be reactivated independently from the TCR and that cooperation between IL-7R and α2β1 integrin can be sufficient to drive Th17-osteoclast function.
The human Th17 cells used in this study have a memory phenotype (CD45RA− CD45RO+) and as such express high levels of IL-7R. In addition, these cells also express the collagen-binding integrin α2β1, which is associated with effector/memory T cells, but not with naive T cells (21, 22). The coexpression of the two receptors on human Th17 cells is not due to activation and polarizing conditions in vitro. Indeed, we also found that in RA, which is associated with bone erosion, a significant portion of ex vivo human RA synovial Th17 cells (26–50%) coexpress IL-7R and α2β1 integrin (Supplemental Fig. 3).
Our results showed that activation of IL-7R led to increased adhesion of polarized human Th17 cells, suggesting that IL-7 could be an important signal contributing to the retention of Th17 cells in the inflammatory sites. Although we have not tested whether IL-17+ cells adhered to collagen, this is likely to be the case because IL-7R and α2β1 integrin are coexpressed on the majority of the cells and collagen alone or in combination with IL-7 led to increased IL-17 production. The majority of studies addressing T cell adhesion focused on the TCR as an activator. However, a previous study reported that IL-7 can induce adhesion of resting T cells to ECM (31) and IL-7 also enhanced T cell adhesion to endothelium through LFA-1 during sepsis (32). In this study, we further showed that the basal and IL-7–stimulated adhesion of human Th17 cells to collagen was regulated by the MAPK/ERK and PI3K/AKT pathways. The IL-7 effect also involved activation of the JAK pathway. The MAPK/ERK and PI3K/AKT pathways were shown to regulate cell adhesion to ECM (33). The implication of JAK downstream of IL-7 is likely to be important for the increased activation of the PI3K/AKT pathway, which lies downstream of JAK (26). However, it is not excluded that additional signaling pathways can be involved because IL-7 has been shown to activate Pyk2, a member of the focal adhesion kinase family (34).
In addition to cell adhesion, the two receptors cooperatively enhanced the production of osteoclastogenic cytokines (IL-17 and RANKL), thus promoting Th17-osteoclast function. The cell signaling experiments indicated that the functional cooperation between IL-7R and α2β1 integrin involves interactions of multiple signaling pathways, which then converge to additively enhance IL-17 production. IL-7–induced IL-17 production is dependent on JAK and downstream activation of PI3K/AKT. The fact that PI3K/AKT is necessary for IL-7–induced IL-17 is consistent with the ability of IL-7 to increase AKT phosphorylation (26) (Fig. 4C). α2β1 integrin stimulated IL-17 production via MAPK/ERK, but not via PI3K/AKT, and contributed to the effect of IL-7 likely by reducing STAT-5 phosphorylation, which can be a negative regulator of IL-17 production by Th17 cells (35).
The implication of ERK downstream of collagen in IL-17 production is supported by studies showing that MAPK/ERK inhibitors inhibited Th17 development and attenuated colitis (36) and experimental autoimmune encephalomyelitis (37). The PI3K/AKT pathway has been associated with Th17 cell development (38) and with IL-17 expression by CCR6+ human memory T cells (39). Thus, targeting MAPK/ERK along with the JAK/PI3K/AKT pathways may be beneficial for inhibiting Th17-mediated bone loss.
Our study also provided in vivo evidence for the role of IL-7R/α2β1 integrin cooperation in Th17-mediated osteoclastogenesis. We showed that the two receptors interact in inducing bone loss in vivo, via Th17 cells. IL-7 treatment led to expansion of Th17 cells in mice, which is consistent with the proliferative/survival function of IL-7 on T cells (9) and with the role of Th17 cells in bone loss. Moreover, we found that blockade of α2β1 integrin reduced IL-7–induced bone loss, and this was associated with reduction of Th17 cell numbers in the bone marrow and reduced levels of IL-17 and RANKL in IL-7–treated mice. This could be explained by three nonexclusive mechanisms. First, α2β1 integrin can be important for Th17 cell retention in the bone marrow, which is rich in collagen. In support, it was reported that α2β1 is required for the migration and retention of effector/memory CD4+ T cells to the bone marrow (40). In fact, these cells were shown to reside near bone marrow stromal cells, which produce both collagen and IL-7 (40, 41). In addition, IL-7 enhanced α2β1-mediated adhesion of human Th17 cells to collagen (Fig. 2A). Second, IL-7 is a critical cytokine for effector/memory T cell survival, IL-7 levels produced by bone marrow stromal cells increase in bone loss–associated diseases like RA (11, 27, 28), and IL-7 has been involved with survival of memory CD4+ T cells in the bone marrow niche in colitis (42). Thus, α2β1 can provide an additional help for maintaining the survival of Th17 cells in the bone marrow of IL-7–treated mice. In this regard, we have previously shown that engagement of α2β1 integrin with collagen inhibited Fas-mediated apoptosis of human effector T cells (43). Finally, α2β1 integrin can also enhance IL-17 production in vivo as supported by our in vitro studies with human Th17 cells and by the fact that anti-α2β1 mAb reduced IL-17 levels in IL-7–treated mice. Thus, it is plausible that α2β1 integrin enhances the attachment and retention of Th17 cells to collagen-rich areas, and its ligation together with the engagement of IL-7R would lead to enhanced cell survival and IL-17 production and subsequently to bone loss. The reduced levels of RANKL and osteoclast numbers by α2β1 integrin mAb in IL-7–treated mice can be attributed at least to the reduction of IL-17 levels because IL-17 is a strong inducer of RANKL in stromal cells and osteoblasts (4, 5, 44).
Although IL-7/IL-7R signaling has recently been involved in myeloid cells of RA (18), it is unlikely that the blockade of IL-7–induced bone loss by anti-α2β1 mAb occurs at the level of myeloid cells because α2β1 integrin expression is not associated with neutrophils or monocytic/macrophage lineage (45–47), which constitutes the main source of osteoclast precursors. In addition, previous studies and recent evidence from the α2β1 integrin-deficient mice indicated that although osteoclasts may express α2β1 integrin, it seems that their function depends more on αvβ3 integrin rather than on α2β1 and other integrins (48, 49).
Th1 cells via IFN-γ have been reported to inhibit osteoclastogenesis (3, 50), but their role in bone loss is still controversial. They are protective in collagen-induced arthritis (3, 50) but seem to favor bone loss in other models of inflammation (51). We found that IL-7 induced expansion of Th1/α2β1+ cells (50% of Th1 express α2β1 integrin versus 80% of Th17 that express α2β1) in the bone marrow, and the blocking anti-α2β1 integrin mAb reduced their numbers (26 versus 40% reduction of Th17 cells; data not shown). These results are in agreement with previous studies showing that IL-7 can also regulate Th1 cells (12, 52), which also express α2β1 integrin, and collagen costimulates the production of IFN-γ (53). In contrast, we have not detected Th1/Th17 cells in the bone marrow and these cells were not induced upon treatment with IL-7 (data not shown). The role of Th1 cells in IL-7–induced bone loss is unclear and deserves further investigation. However, our study indicates that the net effect of α2β1 integrin is the enhancement of bone loss in IL-7–treated mice, which occurs at least through Th17 cells known to be the T cell subset involved in inflammatory bone erosion.
Cytokines are important in sustaining chronic inflammation. Our results showed that the functional role of IL-7 on Th17 cells and bone loss is regulated by α2β1 integrin. We have recently shown that blockade of α2β1 integrin reduced the development of collagen-induced arthritis by inhibiting Th17 cells (54). This indicates that α2β1 integrin can be important for both Ag- and cytokine receptor-dependent responses, thus emphasizing the critical role of α2β1 integrin in Th17 cells and in the effector phase of inflammatory diseases.
It is likely that the IL-7R/α2β1 integrin double-positive T cell population is the most pathogenic subpopulation driving tissue damage. In support, the vast majority of human Th17 cells (Fig. 1) and a significant portion of human RA synovial Th17 cells (26–50%) coexpress IL-7R and α2β1 integrin (Supplemental Fig. 3). Moreover, the IL-7R/α2β1 integrin CD4+ T cell population expanded in IL-7–treated mice and the numbers of CD4+/IL-7R+ T cells were reduced in the bone marrow upon anti-α2β1 mAb treatment (Supplemental Fig. 4).
In summary, our study provides evidence of coordination between integrin and cytokine receptors pathways in Th17-mediated bone erosion. Further understanding of the mechanisms by which IL-7R and α2β1 integrin regulate Th17 functions is likely to bring new insights into the pathogenic mechanisms of inflammatory diseases and may lead to the development of new therapeutic avenues.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank the Bio-Imaging platform of the Centre Hospitalier Universitaire de Québec Research Center.
Footnotes
This work was supported by Arthritis Society of Canada Grant SOG-12-01 (to F.A.). The funding sources play no role in the conception of the study or the manuscript content.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- AKT
- protein kinase B
- Alexa
- Alexa Fluor
- BV
- bone volume
- Col I
- collagen type I
- ECM
- extracellular matrix
- micro-CT
- microcomputerized tomography
- RA
- rheumatoid arthritis
- RANKL
- receptor activator of NF-κB ligand
- Tb.N
- trabecular number
- Tb.pf
- trabecular pattern factor
- TRAP
- tartrate-resistant acid phosphatase
- TV
- tissue volume.
- Received February 25, 2015.
- Accepted August 20, 2015.
- Copyright © 2015 by The American Association of Immunologists, Inc.
References
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