Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo

Abstract

There have been many attempts to visualize the inflamed joints using multiphoton microscopy. However, due to the hypervascular and multilayered structure of the inflamed synovium, intravital imaging of the deep synovial tissue has been difficult. Here, we established original intravital imaging systems to visualize synovial tissue and pathological osteoclasts at the pannus–bone interface using multiphoton microscopy. Combined with fluorescence-labeling of CTLA-4 Ig, a biological agent used for the treatment of rheumatoid arthritis, we identified that CTLA-4 Ig was distributed predominantly within the inflamed synovium and bound to CX 3 CR1 + macrophages and CD140a + fibroblasts 6 h after injection, but not to mature osteoclasts. Intravital imaging of blood and lymphatic vessels in the inflamed synovium further showed that extravasated CTLA-4 Ig was immediately drained through lymphatic vessels under acute arthritic conditions, but the drainage activity was retarded under chronic conditions. These results indicate that this intravital synovial imaging system can serve as a platform for exploring the dynamics of immune cells, osteoclasts, and biological agents within the synovial microenvironment in vivo.

Updating the pathophysiology of arthritic bone destruction: identifying and visualizing pathological osteoclasts in pannus

“…This probe is composed of a bisphosphonate group and boron-dipyrromethene (BODIPY) dye, which has large molar absorption coefficients, high environmental stability, and environment-independent fluorescence quantum yields. After subcutaneous injection of this probe, intravital imaging of the pannus showed that pathological osteoclasts are actively resorbing the bone matrix in the tiny resorption pits without migrating on the bone surface [15]. This contrasts with the physiological osteoclasts in the BM, which are in close contact with osteoblasts and slowly migrate on the bone surface [18].…”

“…A wide field of view can be achieved by tile scan imaging, and living immune cells, blood vessels, and collagen fibers were directly visualized in the synovial microenvironment of the CIA model in vivo (Figure 2) [15]. Combined with fluorescence labeling of CTLA-4 Ig, a biological agent used to treat RA, we tracked the intravital distribution of CTLA-4 Ig under arthritic conditions and revealed that CTLA-4 Ig predominantly distributes within the inflamed synovium, binding to CX 3 CR1 þ macrophages and CD140a þ fibroblasts immediately after intravenous injection (Figure 2).…”

“…Mature osteoclasts resorbing bone at the pannus-bone interface can be observed with multi-photon microscopy at the third metacarpophalangeal joint of the front paw [15]. We used mice expressing tdTomato in the cytosol of osteoclasts (TRAP-tdTomato mice) [16] and visualized bone tissue by SHG.…”

“…We used mice expressing tdTomato in the cytosol of osteoclasts (TRAP-tdTomato mice) [16] and visualized bone tissue by SHG. Pathological osteoclasts were observed making small 50-lm-diameter resorption pits [15], which was unique to arthritic joints (Figure 3). Intravital images of the pannus À bone interface of CX 3 CR1-EGFP/TRAP-tdTomato double transgenic mice showed that some of AtoMs labeled with CX 3 CR1-EGFP merge with TRAP-tdTomato, indicating that these cells are in transition from OPs to mature osteoclasts (Figure 3).…”

Arthritis-associated osteoclastogenic macrophages (AtoMs) participate in pathological bone erosion in rheumatoid arthritis

“…In this review, we discuss the origin and characteristics of newly identified arthritis-associated osteoclastogenic macrophages (AtoMs) [10] in synovial tissues. We also introduce the multiphoton intravital imaging systems established in our laboratory to visualize synovial tissues and pathological osteoclasts at the pannus-bone interface [11].…”