Yusuke Murakami

Toll-like receptor 7 (TLR7) is an endosomal sensor that responds to both pathogen-derived and self-derived single-stranded RNA (ssRNA). Responses of TLR7 to self-derived ssRNA have been implicated in the development of autoimmune diseases, such as systemic lupus erythematosus (SLE). TLR7 antagonists and inhibitory anti-TLR7 monoclonal antibodies (mAbs) can protect lupus-prone NZBWF1 mice from lethal nephritis. However, less is known about TLR7 dependence and activation in human SLE, as both TLR7 and TLR8 respond to ssRNA in humans. Here, we analyzed public databases and found that TLR7 gene signature scores consistently elevated across datasets, races, and SLEDAI scores compared to TLR8, suggesting a deeper involvement of TLR7 in SLE pathogenesis. To specifically inhibit human TLR7 responses, we developed inhibitory mAbs against human TLR7. Utilizing an inhibitory clone, we generated the humanized mAb, DS-7011a. DS-7011a effectively inhibited TLR7-mediated responses in plasmacytoid dendritic cells (pDCs) and B cells. Furthermore, DS-7011a was internalized in a TLR7-dependent manner and accumulated in B cells, pDCs, conventional dendritic cells (cDCs), and monocytes/macrophages. In this study, we describe the generation and preclinical development of DS-7011a, which has the potential to be a therapeutic option for the treatment of SLE.

Although the cause of multiple sclerosis (MS) is not fully known, environmental and lifestyle factors are considered significant risk factors for its development and progression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we found that dietary high salt (HS) intake significantly exacerbated the clinical scores of myelin oligodendrocyte glycoprotein-induced EAE mice in the acute phase, but not in the chronic phase. During the acute phase of EAE, HS diet intake selectively promoted neutrophil infiltration into the spinal cord without affecting T cell, B cell, and dendritic cell infiltration. The HS diet-induced exacerbation of clinical scores and microglial activation were improved by the pharmacological inhibition of neutrophil chemotaxis with SB225002, a selective CXC chemokine receptor 2 inhibitor. In addition, the pharmacological inhibition of microglial activation with minocycline markedly ameliorated clinical scores of HS diet-fed EAE mice. Compared with normal diet-fed mice, the levels of thrombin (a serine protease involved in microglial activation) and protease activated receptor 1 (PAR-1), a thrombin receptor, were increased in the spinal cords of the HS diet-fed group. Blockade of thrombin signaling with vorapaxar, a selective blocker of PAR-1, significantly improved EAE symptoms in the HS diet group. Collectively, our findings suggest that excessive salt intake promotes EAE induction via the activation of neutrophils and microglia in the spinal cord. Dietary salt restriction might be a promising strategy to prevent developing or relapsing MS.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies and damage to multiple organs. Glomerulonephritis, a manifestation involving glomerular deposition of immune complexes and complement components, significantly contributes to disease morbidity. Although the endosomal single-stranded RNA sensor TLR7 is known to drive glomerulonephritis by promoting autoantibody production in B cells, the contribution of macrophage TLR7 responses to glomerulonephritis remains poorly understood. Here, we have examined Tlr7‒/‒ NZBWF1 mice and found that TLR7-deficiency ameliorates lupus nephritis by abolishing autoantibody production against RNA-associated antigens, C3 deposition, and macrophage accumulation in glomeruli. Furthermore, TLR7 signaling increased CD31 expression on glomerular endothelial cells and Ly6Clow macrophages but not on T and B cells, suggesting that CD31 mediates TLR7-dependent migration of monocyte into glomeruli. Compared to their splenic counterparts, glomerular macrophages produced IL-1β in a TLR7-dependent manner. In addition, single cell RNA sequencing (scRNA-seq) of glomerular macrophages revealed that TLR7 signaling induced expression of lupus associated genes including those encoding Chitinase 3 like 1, ferritin heavy chain 1, IKKε, and complement factor B (CfB). Although serum CfB did not increase in NZBWF1 mice, TLR7-dependent CfB protein expression was detected in glomerular macrophages. In addition, TLR7 signaling promoted C3 cleavage and deposition predominantly on mesangial cells. These findings suggest that TLR7 responses in glomerular macrophages accelerates the progression of glomerulonephritis in NZBWF1 mice.

BACKGROUND: Oxaliplatin, in combination with 5-fluorouracil and leucovorin, is a standard treatment for colorectal cancer and shows high efficacy. However, oxaliplatin induces side effects, such as chemotherapy-induced peripheral neuropathy and myelosuppression, which may lead to dose reduction, temporary drug withdrawal, or discontinuation. Lecithinized superoxide dismutase (PC-SOD) is a drug delivery system formulation with improved blood stability and tissue affinity for SOD. A phase II clinical trial of PC-SOD for chemotherapy-induced peripheral neuropathy has been conducted, and its efficacy has been confirmed for certain parameters. METHODS: In this study, we focused on myelosuppression, a major side effect of oxaliplatin, and aimed to elucidate the preventive effect of PC-SOD in a murine model of myelosuppression. RESULTS: Oxaliplatin administration decreased the white blood cell, platelet, and red blood cell counts and hemoglobin levels in the whole blood of mice. PC-SOD treatment significantly restored the oxaliplatin-dependent reduction in white blood cell count (day 10). The gene expression of cytokines involved in hematopoietic progenitor cell differentiation and proliferation, including colony-stimulating factor (CSF)2, CSF3, interleukin (IL)-3, IL-4, IL-5, IL-6, IL-9, and stem cell factor, was also decreased by oxaliplatin administration. In contrast, PC-SOD treatment markedly restored the gene expression of these cytokines. In vivo imaging analysis showed that oxaliplatin treatment enhanced reactive oxygen species (ROS) production in the femur and tibia, whereas PC-SOD significantly suppressed this production. Furthermore, analysis of mouse-derived bone marrow cells revealed that PC-SOD suppressed oxaliplatin-induced cytotoxicity and ROS production in vitro. CONCLUSION: These results suggest that PC-SOD exerts an antioxidant effect and prevents oxaliplatin-induced myelosuppression, particularly in a murine model of leukopenia.

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) reside in the endosomal compartment of innate immune cells, such as macrophages and dendritic cells. NAs transported to the endosomal compartment are degraded by DNases and RNases. Degradation products, including single-stranded DNA, oligoRNA, and nucleosides, are recognized by TLR7, TLR8, and TLR9 to drive the defense responses against pathogens. NA degradation influences endosomal TLR responses by generating and degrading TLR ligands. TLR ligand accumulation because of impaired NA degradation causes constitutive TLR activation, leading to autoinflammatory and autoimmune diseases. Furthermore, some genes associated with these diseases promote endosomal TLR responses. Therefore, endosomal TLRs are promising therapeutic targets for TLR-mediated inflammatory diseases, and novel drugs targeting TLRs are being developed.