Hiroki Mitoma

Transmembrane TNF-α: structure, function and interaction with anti-TNF agents

Transmembrane TNF-α, a precursor of the soluble form of TNF-α, is expressed on activated macrophages and lymphocytes as well as other cell types. After processing by TNF-α-converting enzyme (TACE), the soluble form of TNF-α is cleaved from transmembrane TNF-α and mediates its biological activities through binding to Types 1 and 2 TNF receptors (TNF-R1 and -R2) of remote tissues. Accumulating evidence suggests that not only soluble TNF-α, but also transmembrane TNF-α is involved in the inflammatory response. Transmembrane TNF-α acts as a bipolar molecule that transmits signals both as a ligand and as a receptor in a cell-to-cell contact fashion. Transmembrane TNF-α on TNF-α-producing cells binds to TNF-R1 and -R2, and transmits signals to the target cells as a ligand, whereas transmembrane TNF-α also acts as a receptor that transmits outside-to-inside (reverse) signals back to the cells after binding to its native receptors. Anti-TNF agents infliximab, adalimumab and etanercept bind to and neutralize soluble TNF-α, but exert different effects on transmembrane TNF-α-expressing cells (TNF-α-producing cells). In the clinical settings, these three anti-TNF agents are equally effective for RA, but etanercept is not effective for granulomatous diseases. Moreover, infliximab induces granulomatous infections more frequently than etanercept. Considering the important role of transmembrane TNF-α in granulomatous inflammation, reviewing the biology of transmembrane TNF-α and its interaction with anti-TNF agents will contribute to understanding the bases of differential clinical efficacy of these promising treatment modalities.

Differential response of macrophage subpopulations to myelin degradation in the injured rat sciatic nerve.

Molecular mechanisms of myelin removal by macrophages were explored by examining the immunophenotypes of macrophages following injury of rat sciatic nerve, using a combined method of immunohistochemistry and confocal laser microscopy. In the crush injury model, the involvement in myelin clearance of a cytoplasmic antigen specific for monocytes/macrophages, ED1, was evident. The obvious recruitment of ED1-immunoreactive (-ir) cells was detected first at the crush injury site and then in the distal stump within which Wallerian degeneration had occurred. Double labelling revealed that the ED1-ir cells, except for monocyte-like round cells, always phagocytosed myelin basic protein-ir myelin debris. On the other hand, the expression of ED2, a surface antigen specific for resident macrophages, was significantly different; ED2-ir cells also increased while myelin removal was progressing from day 3 to day 7, but only some of the cells were engaged in myelin phagocytosis. The poor capacity of myelin phagocytosis by ED2-ir cells was supported by the transection model, in which the proximal stump was ligated to suppress regeneration. ED2 may be involved in events other than myelin removal, providing a local environment conducive to axonal regeneration. Our findings thus seem to suggest that ED1 is one of the most reliable markers for cells carrying out myelin phagocytosis, whereas ED2 may participate in entirely different functions. The expression of complement receptor type 3, OX42, was similar to that of ED1 in terms of the swift recruitment of immunopositive cells, their distribution with close association to myelin debris and their high phagocytotic capacity. This supports previously reported in vitro evidence that myelin phagocytosis by macrophages may be complement-mediated.

Association of MBL gene polymorphisms with major bacterial infection in patients treated with high-dose chemotherapy and autologous PBSCT

A growing body of evidence indicates that genetic factors are involved in an increased risk of infection. We investigated whether mannose-binding lectin (MBL) gene polymorphisms that cause low levels of MBL are associated with the occurrence of major infections in patients, mainly bearing hematological malignancies, after high-dose chemotherapy (HDT) rescued by autologous peripheral blood stem cell transplantation (auto-PBSCT). A retrospective evaluation of 113 patients treated with HDT and auto-PBSCT revealed that the low-producing genotypes, B/B and B/LXA, were associated with major bacterial infection (P=0.0016, OR 7.9). We next performed a nation-wide large-scale study to assess the allele frequency of the MBL coding mutation in a total of 2623 healthy individuals in Japan. The frequency of allele B was estimated to be ∼0.2, almost the same in seven different areas of Japan. This common occurrence suggests that MBL deficiency may play an important role in the clinical settings of immunosuppression.

A functional M196R polymorphism of tumour necrosis factor receptor type 2 is associated with systemic lupus erythematosus: a case–control study and a meta-analysis

Objectives: To perform a case–control study of a functional M196R polymorphism of tumour necrosis factor receptor type 2 (TNF-RII) in a Japanese population and a meta-analysis of all published reports on the polymorphism to investigate the association of the M196R polymorphism of TNF-RII with systemic lupus erythematosus (SLE). Methods: The functional M196R polymorphism of TNF-RII was genotyped by using polymerase chain reaction combined with the subsequent single-strand conformation polymorphism (PCR—SSCP) analysis for screening, followed by nucleotide sequencing for confirmation. A total of 331 patients and 359 controls were subjected to a case–control study. A meta-analysis of the available case–control studies including all published data as well as our own data was performed to investigate the association of the functional M196R polymorphism of TNF-RII with SLE. Results: Our case–control study did not show any significant association of a functional M196R polymorphism of TNF-RII with SLE, although there was a trend towards association. A meta-analysis of seven case–control studies in eight different ethnic populations including our own showed that 196M/R and 196R/R genotypes combined was significantly associated with an increased risk of SLE (odds ratio (OR) 1.29, 95% confidence interval (CI) 1.04 to 1.60; p = 0.02). Stratification by ethnicity showed a more significant association in Asians, including Japanese, Korean and Vietnamese (OR 1.40, 95% CI 1.10 to 1.78; p = 0.006). The effect of the 196R allele on SLE was not clear in Caucasians. Conclusions: The 196R allele of the functional M196R polymorphism of TNF-RII is a risk factor for SLE, especially in the Asian population.

Human NLRP3 inflammasome senses multiple types of bacterial RNAs

Significance The innate immune system has evolved to protect the host from potential pathogens. The nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome is one of the platforms that can sense pathogenic bacteria, which can cause fatal bacterial infections. Here we show that all three types of bacteria-derived RNA—mRNA, tRNA, and rRNAs—as well as synthetic 20-guanosine ssRNA, are capable of activating the NLRP3 inflammasome and inducing human macrophages to secrete inflammatory cytokines. Interestingly, only bacterial mRNA is able to activate the murine Nlrp3 inflammasome. Therefore human macrophages may have evolved in a unique fashion, adapting to the bacterial environment. This study could provide important clues for developing efficient medicines for bacterial infections and various immunotherapies such as anticancer vaccines. Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA’s 5′-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.

The Cytotoxic Effects of Certolizumab Pegol and Golimumab Mediated by Transmembrane Tumor Necrosis Factor α

Background:Anti–tumor necrosis factor &agr; (anti-TNF-&agr;) agents have been successfully applied for the treatment of rheumatoid arthritis, Crohns disease, and other chronic inflammatory diseases. Not only the neutralization of soluble TNF-&agr; but also the effect on transmembrane TNF-&agr; is important mechanisms of action of anti-TNF-&agr; agents. This study investigated the cytotoxic effects of new anti-TNF-&agr; agents, certolizumab pegol and golimumab, which are mediated by transmembrane TNF-&agr;. Methods:Transmembrane TNF-&agr;–expressing Jurkat T cells that did not express TNF receptors were used. The binding ability of each anti-TNF-&agr; agent to transmembrane TNF-&agr;, antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and the apoptotic effect were examined. Results:Certolizumab pegol and golimumab bound to transmembrane TNF-&agr;. Golimumab induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, which was comparable to infliximab and adalimumab. However, certolizumab pegol did not induce antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. Certolizumab pegol directly induced nonapoptotic cell death in transmembrane TNF-&agr;–expressing cells. Golimumab induced a weaker apoptotic effect than infliximab and adalimumab. Conclusions:The cytotoxic effects of anti-TNF-&agr; agents on TNF-&agr;–expressing cells are considered to be associated with the clinical effect of these agents on granulomatous diseases. The direct cytotoxic effect of certolizumab pegol on TNF-&agr;–producing cells may contribute to its clinical efficacy in Crohns disease. Golimumab may be less effective for granulomatous diseases.

Analysis of immune reconstitution after autologous CD34+ stem/progenitor cell transplantation for systemic sclerosis: predominant reconstitution of Th1 CD4+ T cells

OBJECTIVE The aim of this study is to evaluate the mechanism of long-term effect of autologous haematopoietic stem cell transplantation (ASCT) in treatment of SSc. METHODS Eleven patients (three males and eight females) with SSc were enrolled. Blood mononuclear cells were harvested after mobilization treatment with CYC and G-CSF. CD34+ haematopoietic stem/progenitor cell fractions were purified and cryopreserved. Patients were transplanted with > 2 × 10(6)/kg autologous CD34+ cells after high-dose CYC (50 mg/kg for 4 days) conditioning. Immune reconstitution was evaluated serially by analysing lymphocyte subpopulations for 36 months. RESULTS Progressive improvement of skin sclerosis has been observed for 3 years in most of the patients. The serum level of anti-Scl-70, an auto-antibody specific to SSc, was progressively decreased after ASCT. Improvement of skin sclerosis was significantly associated with the change in the serum anti-Scl-70 level after ASCT at 36 months. Serum levels of KL-6 and surfactant protein D, indicators for interstitial pneumonia activity, were also significantly decreased. The number of CD8+ T cells immediately recovered within a month after ASCT, while the number of CD4+ T cells remained low for >36 months post-transplant. The majority of CD4+ cells were memory but not naïve T cells, and regulatory CD4+ T cells were not recovered. Th1/Th2 ratio was significantly increased after ASCT. CONCLUSIONS ASCT with purified CD34+ cells was effective in controlling the disease activity of SSc. Th1/Th2 ratio was significantly increased for at least 3 years after ASCT.

The E3 Ubiquitin Ligase Tripartite Motif 33 Is Essential for Cytosolic RNA–Induced NLRP3 Inflammasome Activation

NLRP3 is a key component of caspase-activating macromolecular protein complexes called inflammasomes. It has been found that DHX33 is a cytosolic dsRNA sensor for the NLRP3 inflammasome, which induces caspase-1–dependent production of IL-1β and IL-18 upon activation. However, how the cytosolic dsRNAs induce the interaction between DHX33 and the NLRP3 inflammasome remains unknown. In this study, we report that TRIM33, a member of the tripartite motif (TRIM) family, can bind DHX33 directly and induce DHX33 ubiquitination via the lysine 218 upon dsRNA stimulation. Knocking down of TRIM33 abolished the dsRNA-induced NLRP3 inflammasome activation in both THP-1–derived macrophages and human monocyte-derived macrophages. The ubiquitination of DHX33 by TRIM33 is lysine 63 specific and is required for the formation of the DHX33–NLRP3 inflammasome complex.

Molecular mechanisms of action of anti-TNF-α agents – Comparison among therapeutic TNF-α antagonists

ABSTRACT Tumor necrosis factor (TNF)‐&agr; is a potent pro‐inflammatory and pathological cytokines in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases. Anti‐TNF‐&agr; therapy has been established as an efficacious therapeutic strategy in these diseases. In clinical settings, three monoclonal anti‐TNF‐&agr; full IgG1 antibodies infliximab, adalimumab, and golimumab, PEGylated Fab′ fragment of anti‐TNF‐&agr; antibody certolizumab pegol, extracellular domain of TNF receptor 2/IgG1‐Fc fusion protein etanercept, are almost equally effective for rheumatoid arthritis. Although monoclonal full IgG1 antibodies are able to induce clinical and endoscopic remission in inflammatory bowel diseases, certolizumab pegol without Fc portion has been shown to be less effective for inflammatory bowel diseases compared to full IgG1 antibodies. In addition, there are no evidences that etanercept leads clinical remission in inflammatory bowel diseases. Besides the common effect of anti‐TNF‐&agr; agents on neutralization of soluble TNF‐&agr;, each anti‐TNF‐&agr; agent has its own distinctive pharmacological properties which cause the difference in clinical efficacies. Here we focus on the distinctions of action of anti‐TNF‐&agr; agents especially in following points; (1) blocking ability against ligands, transmembrane TNF‐&agr; and lymphotoxin, (2) effects toward transmembrane TNF‐&agr;‐expressing cells, (3) effects toward Fc&ggr; receptor‐expressing cells, (4) degradation and distribution in inflamed tissue. Accumulating evidence will give us the idea how to modify anti‐TNF‐&agr; agents to enhance the clinical efficacy in inflammatory diseases.

Clinical significance of SNORA42 as an oncogene and a prognostic biomarker in colorectal cancer

Purpose Despite recent advances in colorectal cancer (CRC) treatment, the prognosis of patients suffering from this malignancy still remains substandard, and metastatic recurrence following curative surgery is the leading cause of mortality. Therefore, it is imperative to identify prognostic markers to predict the clinical outcome of CRC patients. Recent evidence revealed the new role of small nucleolar RNAs (snoRNAs) in oncogenesis. Herein, we systematically evaluated dysregulation of snoRNAs in CRC and clarified their biomarker potential and biological significance in CRC. Experimental design We analysed expression levels of 4 snoRNAs in 274 colorectal tissues from 3 independent cohorts and 6 colon cancer cell lines. The functional characterisation for the role of SNORA42 in CRC was investigated through a series of in vitro and in vivo experiments. Results In the screening phase, expression levels of all four snoRNAs were significantly elevated in CRC tissues than in corresponding normal mucosa. In the clinical validation cohort, increased SNORA42 expression was an independent prognostic factor for overall survival and disease-free survival, and was a risk factor for distant metastasis. SNORA42 expression negatively correlated with overall survival in an additional independent cohort and identified the patients with high risk for recurrence and poor prognosis in stage II CRC. Furthermore, in vitro and in vivo analyses showed that SNORA42 overexpression resulted in enhanced cell proliferation, migration, invasion, anoikis resistance and tumorigenicity. Conclusions SNORA42 appears to be a novel oncogene and could serve as a promising predictive biomarker for recurrence and prognosis in patients with CRC.