Cetuximab-Toxin Conjugate and NPe6 with Light Enhanced Cytotoxic Effects in Head and Neck Squamous Cell Carcinoma In Vitro.

BACKGROUND: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and irradiation of a specific wavelength to exert cytotoxic effects. To enhance the antitumor effect against head and neck squamous cell carcinoma (HNSCC), we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin (IT) and a PS for PDT and light irradiation. In our prior study, we demonstrated that an immunotoxin (IT) consisting of an anti-ROBO1 antibody conjugated to saporin, when used in combination with the photosensitizer (PS) disulfonated aluminum phthalocyanine (AlPcS2a) and irradiated with light at the appropriate wavelength, resulted in increased cytotoxicity against head and neck squamous cell carcinoma (HNSCC) cells. ROBO1 is a receptor known to be involved in the progression of cancer. In this study, we newly investigate the iTAP targeting epidermal growth factor receptor (EGFR) which is widely used as a therapeutic target for HNSCC. METHODS: We checked the expression of EGFR in HNSCC cell lines, SAS, HO-1-u-1, Sa3, and HSQ-89. We analyzed the cytotoxicity of saporin-conjugated anti-EGFR antibody (cetuximab) (IT-Cmab), mono-L-aspartyl chlorin e6 (NPe6, talaporfin sodium), and light (664 nm) irradiation (i.e., iTAP) in SAS, HO-1-u-1, Sa3, and HSQ-89 cells. RESULTS: EGFR was expressed highly in Sa3, moderately in HO-1-u-1, SAS, and nearly not in HSQ-89. Cmab alone or IT-Cmab alone did not show cytotoxic effects in Sa3, HO-1-u-1, and HSQ-89 cells, which have moderate or low expression levels of EGFR protein. However, the iTAP method enhanced the cytotoxicity of IT-Cmab by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression. CONCLUSION: Our study is the first to report on the iTAP method using IT-Cmab and NPe6 for HNSCC. The cytotoxic effects are enhanced in cell lines with moderate levels of EGFR protein expression, but not in nonexpressing cell lines, which is expected to expand the range of therapeutic windows and potentially reduce complications.

The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts.

Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.

Plasma cells promote osteoclastogenesis and periarticular bone loss in autoimmune arthritis.

In rheumatoid arthritis (RA), osteoclastic bone resorption causes structural joint damage as well as periarticular and systemic bone loss. Periarticular bone loss is one of the earliest indices of RA, often preceding the onset of clinical symptoms via largely unknown mechanisms. Excessive osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL) expressed by synovial fibroblasts causes joint erosion, whereas the role of RANKL expressed by lymphocytes in various types of bone damage has yet to be elucidated. In the bone marrow of arthritic mice, we found an increase in the number of RANKL-expressing plasma cells, which displayed an ability to induce osteoclastogenesis in vitro. Genetic ablation of RANKL in B-lineage cells resulted in amelioration of periarticular bone loss, but not of articular erosion or systemic bone loss, in autoimmune arthritis. We also show conclusive evidence for the critical contribution of synovial fibroblast RANKL to joint erosion in collagen-induced arthritis on the arthritogenic DBA/1J background. This study highlights the importance of plasma-cell RANKL in periarticular bone loss in arthritis and provides mechanistic insight into the early manifestation of bone lesion induced by autoimmunity.

Stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution.

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions1,2. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes1-3. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.

OPG Production Matters Where It Happened.

Osteoprotegerin (OPG) is a circulating decoy receptor for RANKL, a multifunctional cytokine essential for the differentiation of tissue-specific cells in bone and immune systems such as osteoclasts, medullary thymic epithelial cells (mTECs), and intestinal microfold cells (M cells). However, it is unknown whether OPG functions only at the production site or circulates to other tissues acting in an endocrine fashion. Here we explore the cellular source of OPG by generating OPG-floxed mice and show that locally produced OPG, rather than circulating OPG, is crucial for bone and immune homeostasis. Deletion of OPG in osteoblastic cells leads to severe osteopenia without affecting serum OPG. Deletion of locally produced OPG increases mTEC and M cell numbers while retaining the normal serum OPG level. This study shows that OPG limits its functions within the tissue where it was produced, illuminating the importance of local regulation of the RANKL system.

Photosensitizer With Illumination Enhances In Vivo Antitumor Effect of Anti-ROBO1 Immunotoxin on Maxillary Sinus Squamous Cell Carcinoma.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of cancer worldwide. Our study focused on the axon guidance receptor roundabout guidance receptor 1 (ROBO1) as a target for monoclonal antibody therapy of HNSCC. We previously showed that saporin-conjugated anti-ROBO1 (B5209B) immunotoxin (IT-ROBO1) enhanced cytotoxic effects on HNSCC cells in combination with the photosensitizer aluminum phthalocyanine disulphonate (AlPcS2a) and illumination. We examined the effects of this combination therapy in a mouse xenograft model. MATERIALS AND METHODS: IT-ROBO1 was intraperitoneally administered to HSQ-89 (derived from Japanese maxillary sinus squamous carcinoma, RCB0789; RIKEN, Tsukuba, Japan) xenografted mice. After 3 days, AlPcS2a was injected subcutaneously around the tumor and the area was illuminated at 650 nm for 30 min. The growth of the tumor was evaluated and the effects on the tumor were examined. RESULTS: Pronounced anti-tumor effects were elicited by the administration of IT-ROBO1 and AlPcS2a with light illumination on tumor size and pathological characteristics. CONCLUSION: The results showed that photosensitizer treatment with illumination robustly enhanced the antitumor effect of the IT-ROBO1 immunotoxin.

Saponin Facilitates Anti-Robo1 Immunotoxin Cytotoxic Effects on Maxillary Sinus Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide. The standard treatment of surgery, chemotherapy, and radiotherapy can result in long-term complications which lower the patient's quality of life, such as eating disorders, speech problems, and disfiguring or otherwise untoward cosmetic issues. Antibody therapy against cancer-specific antigens is advantageous in terms of its lesser side effects achieved by its greater specificity, though the antitumor activity is still usually not enough to obtain a complete cure. Robo1, an axon guidance receptor, has received considerable attention as a possible drug target in various cancers. We have shown previously the enhanced cytotoxic effects of saporin-conjugated anti-Robo1 immunotoxin (IT-Robo1) on the HNSCC cell line HSQ-89 in combination with a photochemical internalization technique. Considering the light source, which has only limited tissue penetrance, we examined the drug internalization effect of saponin. Treatment with saponin facilitated significant cytotoxic effects of IT-Robo1 on HSQ-89 cells. Saponin exerts its own nonspecific cytotoxicity, which may cover the actual extent of the internalization effect. We thus examined whether a flashed treatment with saponin exerted a significant specific cytotoxic effect on cancer cells. The combination of an immunotoxin with saponin also exhibited a significant tumor-suppressive effect on mice HSQ-19 xenografts. These results suggest the utility of saponin treatment as an enhancer of immunotoxin treatment in cancer.

Host defense against oral microbiota by bone-damaging T cells.

The immune system evolved to efficiently eradicate invading bacteria and terminate inflammation through balancing inflammatory and regulatory T-cell responses. In autoimmune arthritis, pathogenic TH17 cells induce bone destruction and autoimmune inflammation. However, whether a beneficial function of T-cell-induced bone damage exists is unclear. Here, we show that bone-damaging T cells have a critical function in the eradication of bacteria in a mouse model of periodontitis, which is the most common infectious disease. Bacterial invasion leads to the generation of specialized TH17 cells that protect against bacteria by evoking mucosal immune responses as well as inducing bone damage, the latter of which also inhibits infection by removing the tooth. Thus, bone-damaging T cells, which may have developed to stop local infection by inducing tooth loss, function as a double-edged sword by protecting against pathogens while also inducing skeletal tissue degradation.

Osteoimmunology: The Conceptual Framework Unifying the Immune and Skeletal Systems.

The immune and skeletal systems share a variety of molecules, including cytokines, chemokines, hormones, receptors, and transcription factors. Bone cells interact with immune cells under physiological and pathological conditions. Osteoimmunology was created as a new interdisciplinary field in large part to highlight the shared molecules and reciprocal interactions between the two systems in both heath and disease. Receptor activator of NF-κB ligand (RANKL) plays an essential role not only in the development of immune organs and bones, but also in autoimmune diseases affecting bone, thus effectively comprising the molecule that links the two systems. Here we review the function, gene regulation, and signal transduction of osteoimmune molecules, including RANKL, in the context of osteoclastogenesis as well as multiple other regulatory functions. Osteoimmunology has become indispensable for understanding the pathogenesis of a number of diseases such as rheumatoid arthritis (RA). We review the various osteoimmune pathologies, including the bone destruction in RA, in which pathogenic helper T cell subsets [such as IL-17-expressing helper T (Th17) cells] induce bone erosion through aberrant RANKL expression. We also focus on cellular interactions and the identification of the communication factors in the bone marrow, discussing the contribution of bone cells to the maintenance and regulation of hematopoietic stem and progenitors cells. Thus the time has come for a basic reappraisal of the framework for understanding both the immune and bone systems. The concept of a unified osteoimmune system will be absolutely indispensable for basic and translational approaches to diseases related to bone and/or the immune system.

LOX Fails to Substitute for RANKL in Osteoclastogenesis.

Osteoclasts are the exclusive bone-resorbing cells that have a central role in bone homeostasis as well as bone destruction in cancer and autoimmune disease. Both mouse and human genetic studies have clearly proven that receptor activator of NF-κB ligand (RANKL; encoded by the Tnfsf11 gene) and its receptor RANK are essential for osteoclastogenesis. Although there have been several reports on RANKL-independent osteoclastogenesis, previous studies have never provided in vivo evidence showing RANKL can be substituted by other molecules using RANKL- or RANK-deficient genetic backgrounds. Thus, to date, there is no clear evidence of RANKL-independent osteoclastogenesis and no molecule has ever been proven capable of inducing osteoclast differentiation more efficiently than RANKL. Recently, lysyl oxidase (LOX), the enzyme that mediates collagen cross-linking, has been shown to induce human osteoclasts in the absence of RANKL and has a stronger osteoclastogenic activity than RANKL. Here, we investigated the effect of LOX on osteoclast differentiation using RANKL- and RANK-deficient cells to strictly explore RANKL-independent osteoclastogenesis. CD14+ human peripheral blood cells as well as osteoclast precursor cells derived from wild-type, RANKL- and RANK-deficient mice were treated with RANKL and/or LOX in short-term (3 days) or long-term (3 weeks) experimental settings. LOX treatment alone did not result in the formation of tartrate-resistant acid phosphatase (TRAP)+ cells or resorption pits in either short-term or long-term culture. In combination with RANKL, long-term treatment with LOX synergistically promoted osteoclastogenesis in cells derived from wild-type mice; however, this was abrogated in RANKL-deficient cells. Long-term treatment with LOX stimulated RANKL expression in mouse bone marrow stromal cells via the production of reactive oxygen species (ROS). Furthermore, LOX injection failed to rescue the phenotype of RANKL-deficient mice. These results suggest that LOX has the ability to induce RANKL expression on stromal cells; however, it fails to substitute for RANKL in osteoclastogenesis. © 2016 American Society for Bone and Mineral Research.

[Mechanism of bone destruction and the contribution of T lymphocytes].

Rheumatoid arthritis (RA), one of the most common autoimmune diseases, is characterized by inflammation and bone destruction in the joints. Abnormal activation of the immune system leads to RANKL-dependent osteoclast differentiation, which ultimately results in bone destruction in RA. A newly identified Th17 subset induces osteoclastogenesis potently by upregulating RANKL on synovial fibroblasts, indicating a synergy between T-synovial fibroblast plays a primary role in the bone destruction. Immune-regulating factors, such as CTLA-4 highly expressed on regulatory T cells, are identified as new bone-regulating factors and can be attractive therapeutic targets for bone destruction in RA. The mechanism by which T cells contribute to the RA pathogenesis will help understand the etiology of RA and develop therapeutic approach against it.

RANKL expressed on synovial fibroblasts is primarily responsible for bone erosions during joint inflammation.

OBJECTIVE: RANKL is mainly expressed by synovial fibroblasts and T cells within the joints of rheumatoid arthritis patients. The relative importance of RANKL expression by these cell types for the formation of bone erosions is unclear. We therefore aimed to quantify the contribution of RANKL by each cell type to osteoclast differentiation and bone destruction during inflammatory arthritis. METHODS: RANKL was specifically deleted in T cells (Tnfsf11(flox/Δ) Lck-Cre), in collagen VI expressing cells including synovial fibroblasts (Tnfsf11(flox/Δ) Col6a1-Cre) and in collagen II expressing cells including articular chondrocytes (Tnfsf11(flox/Δ) Col2a1-Cre). Erosive disease was induced using the collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA) models. Osteoclasts and cartilage degradation were assessed by histology and bone erosions were assessed by micro-CT. RESULTS: The inflammatory joint score during CAIA was equivalent in all mice regardless of cell-targeted deletion of RANKL. Significant increases in osteoclast numbers and bone erosions were observed in both the Tnfsf11(flox/Δ) and the Tnfsf11(flox/Δ) Lck-Cre groups during CAIA; however, the Tnfsf11(flox/Δ) Col6a1-Cre mice showed significant protection against osteoclast formation and bone erosions. Similar results on osteoclast formation and bone erosions were obtained in CIA mice. The deletion of RANKL on any cell type did not prevent articular cartilage loss in either model of arthritis used. CONCLUSIONS: The expression of RANKL on synovial fibroblasts rather than T cells is predominantly responsible for the formation of osteoclasts and erosions during inflammatory arthritis. Synovial fibroblasts would be the best direct target in RANKL inhibition therapies.

Inhibition of the TNF Family Cytokine RANKL Prevents Autoimmune Inflammation in the Central Nervous System.

The central nervous system (CNS) is an immunologically privileged site protected from uncontrolled access of T cells by the blood-brain barrier (BBB), which is breached upon autoimmune inflammation. Here we have shown that receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) on T cells regulates C-C type chemokine ligand 20 (CCL20) production by astrocytes and T cell localization in the CNS. Importantly, mice specifically lacking RANKL in T cells were resistant to experimental autoimmune encephalomyelitis (EAE) due to altered T cell trafficking. Pharmacological inhibition of RANKL prevented the development of EAE without affecting the peripheral immune response, indicating that RANKL is a potential therapeutic target for treating autoimmune diseases in the CNS.

[Mechanism of bone and cartilage damage in rheumatoid arthritis].

Rheumatoid arthritis(RA)is an autoimmune diseases characterized by inflammation and destruction of bone and cartilage. Bone destruction in RA is triggered by abnormal activation of immune system and osteoclasts induced by RANKL. Advances in osteoimmunology clarified that immune-factors such as inflammatory cytokines and antibodies promote not only inflammation but also bone destruction in RA. Importantly, a newly identified Th17 subset induces osteoclastogenesis potently by upregulating RANKL on synovial fibroblasts, indicating a synergy between T-synovial fibroblast plays a primary role in the inflammatory bone destruction. Recently, novel bone-regulating factors are identified and can be attractive therapeutic targets for destruct ion of bone and cartilage in RA.

HIV-associated Burkitt lymphoma in a Japanese patient with early submandibular swelling.

BACKGROUND: Patients infected with the human immunodeficiency virus (HIV) are at risk of developing malignancies and have an increased susceptibility to infection. HIV-associated Burkitt lymphoma (BL) is relatively rare in developed countries, but remains prevalent in developing counties and is sometimes compounded by the fact that patients may be unaware that they are HIV-positive. CASE PRESENTATION: A 37-year-old Japanese man was referred to our department for diagnosis and management of submandibular swelling. He was unaware that he was HIV-positive at the initial visit. Here, we describe our diagnostic approach, in which we used hematological and immunological investigations, biopsy, fluorescence-activated cell sorting and fluorescence in situ hybridization to confirm the diagnosis of HIV-associated BL. The patient has no risk factors for HIV infection, and the source of infection remains unclear. CONCLUSIONS: In this case, submandibular swelling was the first clinical sign of pathology and the patient's HIV-positive status only became evident later. It is highly likely that BL was triggered by HIV infection.

[Bone and cartilage destruction in rheumatoid arthritis].

Rheumatoid arthritis is an inflammation-mediated bone disease characterized by local joint inflammation which results from systemic immune responses. It is essential to clarify the mechanisms by which inflammation elicits bone destruction for the establishment of novel therapeutic strategies. Advances in osteoimmunology, in addition to the development of a various kind of genetically-modified mice and animal models of RA, have greatly contributed to our understanding of these mechanisms. Recently, Th17 cells have been shown to contribute not only to the initiation and amplification of inflammation in RA, but also to bone destruction by enhancing osteoclast differentiation through the interaction with synovial fibroblasts. Thus, Th17-synovial fibroblasts interaction is considered to be a promising therapeutic target for RA.

[Animal models for bone and joint disease. Osteoimmunology and animal models for rheumatoid arthritis].

Bone homeostasis is maintained by not only bone cells but also by various types of cells. Particularly, the skeletal system has an apparent relationship with the immune system, in that immune cells are generated in the bone marrow and that osteoclasts which play a crucial role in bone destruction differentiate from monocyte/macrophage lineage cells. In addition, investigation into rheumatoid arthritis (RA) has highlighted the relevance of the interplay between the bone and immune systems and promoted a new research field of 'osteoimmunology'. Here, we summarize how various animal models for RA contributed, and can contribute to the progress in osteoimmunology and increasing understanding of RA development and treatment.

Preferential generation of follicular B helper T cells from Foxp3+ T cells in gut Peyer's patches.

Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers of Peyer's patches through a process that requires the presence of CD4+ follicular B helper T(TFH) cells. The nature of these T(FH) cells in Peyer's patches has been elusive. Here, we demonstrate that suppressive Foxp3+CD4+ T cells can differentiate into TFH cells in mouse Peyer's patches. The conversion of Foxp3+ T cells into TFH cells requires the loss of Foxp3 expression and subsequent interaction with B cells. Thus, environmental cues present in gut Peyer's patches promote the selective differentiation of distinct helper T cell subsets, such as TFH cells.

Heparanase expression in B16 melanoma cells and peripheral blood neutrophils before and after extravasation detected by novel anti-mouse heparanase monoclonal antibodies.

Degradation of extracellular matrix is associated with extravasation of metastatic tumor cells and inflammatory cells. Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, is a key enzyme for the matrix degradation, yet its involvement in extravasation and invasion during pathological processes was not fully clarified in vivo. In the present study, we examined heparanase expression in mouse experimental models, lung metastasis of melanoma and skin infiltration of neutrophils. Sixteen novel monoclonal antibodies specific for mouse heparanase were established by enzyme-linked immunosorbent assay with a recombinant mouse proheparanase, immunocytochemical staining of B16F10 melanoma cells cultured in vitro, and immunoprecipitation of the lysate of heparanase transfectant cells. Heparanase expression in metastatic nodules of B16F10 melanoma cells and in neutrophils localized in the inflamed skin was immunohistochemically detected using a monoclonal antibody RIO-1 that recognized the C-terminus of mouse heparanase. Homogeneous and strong heparanase staining was observed in 46% of the lung micrometastases of B16F10 melanoma cells. The staining was intensely positive on the invasive front of larger established metastasis nodules, but it was weak or heterogeneous inside the nodules. Heparanase expression in skin-infiltrating neutrophils was examined after inducing local inflammation with croton oil. The monoclonal antibody stained a significant portion of neutrophils inside and along the blood vessels, whereas it did not stain dermal neutrophils located distant from the vasculatures. The present study strongly suggests that both melanoma cells and neutrophils transiently express heparanase before and during the invasive process in vivo.