The novel role of IL-7 ligation to IL-7 receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis

Abstract

Although the role of IL-7 and IL-7R has been implicated in the pathogenesis of rheumatoid arthritis (RA), the majority of the studies have focused on the effect of IL-7/IL-7R in T cell development and function. Our novel data, however, document that patients with RA and greater disease activity have higher levels of IL-7, IL-7R, and TNF-α in RA monocytes, suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells that is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant that attracts IL-7R(+) monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen-induced arthritis (CIA) was therapeutically treated with anti-IL-7 Ab or IgG control. Anti-IL-7 Ab treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers, suggesting that both direct and indirect pathways might contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in mice treated with anti-IL-7 Ab compared with the control group. To our knowledge, we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts, and vascularization in the CIA effector phase.

Figure 1. IL-7 and IL-7R expression correlates with DAS28 and TNF-α levels in RA monocytes and ligation of RA synovial fluid IL-7 to myeloid IL-7R promotes chemotaxis

Linear regression analysis was used to compare expression of TNF-α with IL-7 (n=76) (A), IL-7R (n=76) (B) mRNA levels and DAS28 (n=76) (C) in RA monocytes. Correlation was also calculated for DAS28 score (n=76) and expression levels of IL-7 (D) and/or IL-7R (E) in RA monocytes. The mRNA expression levels in RA monocytes are shown as fold increase above NL PB monocytes and are normalized to GAPDH. F. Concentration of IL-7 was quantified by ELISA in RA, OA and NL STs (n=10) as well as in G. SFs from RA and OA patients (n=18). H. 12 RA SFs were pre-incubated with anti-IL-7 antibody (10 μg/ml) or control IgG for 1h prior to performing monocyte chemotaxis in response to the RA SFs. I. Monocytes were incubated with antibody to IL-7R (10 μg/ml) as well as IgG control for 1h prior to performing monocyte chemotaxis in response to 6 RA SFs. Values demonstrate mean ± SE.* represents p <0.05.

Figure 2. IL-7 induces monocyte migration through ligation to IL-7R and activation of AKT1/PI3K and ERK pathways

A. IL-7 monocyte chemotaxis was performed in a Boyden chemotaxis chamber with varying concentration of IL-7, n=3. B. Monocytes were incubated with anti-IL-7R antibody (10 μg/ml) or control IgG for 1h thereafter chemotaxis was performed in response to IL-7 (10 and 50 ng/ml), n=3. C. Chemotaxis was performed using monocytes negatively selected for CD14+CD16+/− and CD14+CD16− in response to IL-7 (1 and 50 ng/ml), n=3. D. To determine the mechanism of IL-7 in monocytes, cells were stimulated with IL-7 (10 ng/ml) for 0–65 minutes, and the cell lysates were probed for p-ERK, pAKT1 and pSTAT1, pSTAT3 and STAT5, n=3. E. To examine signaling pathways associated with IL-7 monocyte migration, cells were preincubated with 1 and 5 μM of the identified chemical inhibitors for ERK (U0126), PI3K (LY294002; LY), STAT3 (WP1066; WP) or 10 and 50 μM of STAT5 inhibitor (573108 STAT5 inhibitor; S5i) prior to performing chemotaxis in the Boyden chamber (2h), n=3. F. Monocytes were treated with DMSO or inhibitors for PI3K/AKT (LY294002; 5μM) and p38 (SB203580; 5μM) in order to examine the impact of these pathways on FMLP (1μM) as well as of CCL2 (0.9 nM), CCL5 (1.01 nM), IL-17 (0.667 nM) or IL-7 (0.58 nM) induced monocyte migration, n=3–5. Values demonstrate mean ± SE.* represents p <0.05.

Figure 3. Inhibition of AKT and ERK cascades suppresses IL-7 mediated monocyte homing, and like in RA IL-7R is expressed in CIA ST lining and sublining macrophages and sublining endothelial cells

A. NL monocytes were transfected with Ctl or DN-AKT plasmid at 2.5 μg for 48h. Cells were either untreated or stimulated with 100 ng/ml of IL-7 for 30 and 60 min and probed for pAKT, AKT and actin, n=3. B. Migration of Ctl or DN-AKT transfected NL monocytes was examined in response to 10 ng/ml IL-7 or PBS and response to FMLP was only tested in Ctl transfected monocytes, n=3. C. THP-1 cells were transfected with 100 nM scrambled (ctl si) or ERK siRNA (ERK si) for 48h and subsequently transfected cells were probed for ERK and actin, n=3. D. Migration of THP-1 control or ERK knockdown cells were examined in response to 10 ng/ml IL-7 or PBS and the ability of FMLP to attract cells was only examined in control knockdown cells, n=3. E. Polycarbonate membranes harvested from NL monocytes that migrated in response to PBS, FMLP (1 μM) or IL-7 (10 ng/ml) were fixed, stained with anti-CD68 antibody (1:100 dilution) and visualized with a secondary antibody labeled with Alexa 594 (1:500 dilution). F. The total number of migrated cells was quantified in 15 HPFs based on double positive DAPI (blue) and CD68 (red) staining. G. Ankles harvested from PBS control or CIA induced mice were stained with anti-IL-7R antibody. H. IL-7R positive immunostaining was scored on a 0–5 scale on synovial tissue lining and sublining macrophages (mac) as well as sublining endothelial cells (endo), n=5–7 (original magnification x200). I. IL-7 levels were determined in ankles harvested from PBS injected control or CIA mice by ELISA, n=5. Values demonstrate mean ± SE.* represents p <0.05.

Figure 4. Therapeutic treatment of anti-IL-7 antibody ameliorates CIA pathology and bone erosion

A. Changes in joint circumference was recorded in CIA mice that were treated with IgG or anti-IL-7 antibody (100 μg/injection) i.p. on days 26, 29, 33, 36, 40 and 42, n=10–11 mice. B. Effect of anti-IL-7 antibody treatment on inflammation, lining thickness, and bone erosion was scored on a 0–5 scale, n=8 and C. demonstrates representative ankle H&E staining (original magnification x 200). D. TRAP staining of CIA mice treated with IgG or anti-IL-7 antibody was scored on a 0–5 scale, n=8 and E. shows representative ankle TRAP staining (original magnification x 200) and arrows demonstrate TRAP+ cells. F. mRNA concentration of RANKL and Cathepsin K was determined in CIA ankles treated with IgG or anti-IL-7 antibody employing real-time RT-PCR, n=5. The data are shown as fold increase above IgG treated CIA ankles and are normalized to GAPDH. Values are mean ± SE. * indicates p<0.05.

Figure 5. Neutralization of IL-7 reduces potent monocyte chemoattractants and CIA monocyte homing

Changes in TNF-α (A), CCL2 (B) and CCL5 (C) expression levels in ankle homogenates (n=7) and/or sera (n=10) from CIA mice treated with IgG control or anti-IL-7 antibody were determined by ELISA. D. STs from CIA mice treated with IgG or anti-IL-7 antibody were harvested on day 43 and immunostained with anti-F480 antibody (original magnification x 200) and arrows demonstrate F480+ cells. E. Macrophage staining was quantified on a 0–5 scale, n=5–7. Values are mean ± SE. * indicates p<0.05.

Figure 6. MIP-2 and joint vascularization is reduced in CIA mice treated with anti-IL-7 antibody compared to the control group

Changes in MIP-2 (A), Ang-1 (B), CXCL1 (C), bFGF (D) and VEGF (E) expression levels in ankle homogenates (n=7) from CIA mice treated with IgG control or anti-IL-7 antibody were determined by ELISA. F. Hemoglobin levels are quantified in CIA ankles harvested from control and anti-IL-7 antibody treatment groups on day 43 and results are demonstrated as hemoglobin (g/dl)/ joint weight (mg/ml). G. STs from CIA mice treated with IgG or anti-IL-7 antibody were harvested and immunostained with anti-VWF antibody (original magnification x 200) and cells positive for VWF staining are shown by arrows. (H) Endothelial immunostaining was quantified on a 0–5 scale, n=5–7. Values are mean ± SE. * indicates p<0.05.

Figure 7. Spleen TH-1 cells, TH-17 cells and TH-17 promoting cytokines as well as joint CD3 immunostaining were unaffected by anti-IL-7 antibody treatment and schematic representation of the mechanism that contributes to IL-7 mediated pathogenesis in RA and CIA

A. Percent spleen CD3+, CD4+, TH-1 and TH-17 positive cells were determined by Flow cytometry analysis in control IgG and anti-IL-7 antibody treatment in CIA. Changes in IL-6 (B), IL-1β (C), IL-17 (D) expression levels in ankle homogenates (n=7) from CIA mice treated with IgG control or anti-IL-7 antibody were quantified by ELISA. E. STs from CIA mice treated with IgG or anti-IL-7 antibody were immunostained with anti-CD3 antibody (original magnification x 200) and cells positive for CD3 staining are demonstrated by arrows. (F) T cell immunostaining was quantified on a 0–5 scale, n=5–7. G. Schematic representation demonstrates that IL-7 is capable of modulating monocyte homing in RA joint as well as contributing to CIA myeloid cell trafficking and function, bone erosion and vascularization.