Norimoto Kobayashi

Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes

UNLABELLED : Familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome are dominantly inherited autoinflammatory diseases associated to gain-of-function NLRP3 mutations and included in the cryopyrin-associated periodic syndromes (CAPS). A variable degree of somatic NLRP3 mosaicism has been detected in ≈35% of patients with CINCA. However, no data are currently available regarding the relevance of this mechanism in other CAPS phenotypes. OBJECTIVE To evaluate somatic NLRP3 mosaicism as the disease-causing mechanism in patients with clinical CAPS phenotypes other than CINCA and NLRP3 mutation-negative. METHODS NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. Apoptosis-associated Speck-like protein containing a CARD (ASC)-dependent nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. RESULTS A variable degree (5.5-34.9%) of somatic NLRP3 mosaicism was detected in 12.5% of enrolled patients, all of them with a MWS phenotype. Six different missense variants, three novel (p.D303A, p.K355T and p.L411F), were identified. Bioinformatics and functional analyses confirmed that they were disease-causing, gain-of-function NLRP3 mutations. All patients treated with anti-interleukin1 drugs showed long-lasting positive responses. CONCLUSIONS We herein show somatic NLRP3 mosaicism underlying MWS, probably representing a shared genetic mechanism in CAPS not restricted to CINCA syndrome. The data here described allowed definitive diagnoses of these patients, which had serious implications for gaining access to anti-interleukin 1 treatments under legal indication and for genetic counselling. The detection of somatic mosaicism is difficult when using conventional methods. Potential candidates should benefit from the use of modern genetic tools.

TIM-4, a Receptor for Phosphatidylserine, Controls Adaptive Immunity by Regulating the Removal of Antigen-Specific T Cells

Adaptive immunity is characterized by the expansion of an Ag-specific T cell population following Ag exposure. The precise mechanisms, however, that control the expansion and subsequent contraction in the number of Ag-specific T cells are not fully understood. We show that T cell/transmembrane, Ig, and mucin (TIM)-4, a receptor for phosphatidylserine, a marker of apoptotic cells, regulates adaptive immunity in part by mediating the removal of Ag-specific T cells during the contraction phase of the response. During Ag immunization or during infection with influenza A virus, blockade of TIM-4 on APCs increased the expansion of Ag-specific T cells, resulting in an increase in secondary immune responses. Conversely, overexpression of TIM-4 on APCs in transgenic mice reduced the number of Ag-specific T cells that remained after immunization, resulting in reduced secondary T cell responses. There was no change in the total number of cell divisions that T cells completed, no change in the per cell proliferative capacity of the remaining Ag-specific T cells, and no increase in the development of Ag-specific regulatory T cells in TIM-4 transgenic mice. Thus, TIM-4–expressing cells regulate adaptive immunity by mediating the removal of phosphatidylserine-expressing apoptotic, Ag-specific T cells, thereby controlling the number of Ag-specific T cells that remain after the clearance of Ag or infection.

IL-10 enhances B-cell IgE synthesis by promoting differentiation into plasma cells, a process that is inhibited by CD27/CD70 interaction

Interleukin‐10 (IL‐10) is a major regulatory cytokine of inflammatory responses that is considered to play an important role in specific immunotherapy. However, whether IL‐10 enhances or inhibits B‐cell IgE production has remained a matter of contention. To clarify the effect of IL‐10 on IgE synthesis in the presence of IL‐4 and CD40 signalling, we examined B‐cell proliferation, germline ɛ transcripts and plasma cell differentiation. In addition, the effect of CD27 signalling on IgE synthesis in the presence of IL‐10, IL‐4 and CD40 signalling was investigated. IL‐10 facilitated the production of IgE in mononuclear cells and highly purified B‐cells, enhanced B‐cell proliferation and, most importantly, promoted the generation of plasma cells. However, IL‐10 did not enhance expression of germline ɛ transcripts. The addition of CD27 signalling through the use of CD32–CD27 ligand (CD70) double transfectants significantly diminished the B‐cell proliferation, IgE synthesis and plasma cell differentiation enhanced by IL‐10. IL‐10 enhances B‐cell IgE production by promoting differentiation into plasma cells. CD27/CD70 interactions under IL‐10 and sufficient CD40 cosignalling exert the opposite effect on IgE synthesis. The results of this study indicate that precautions are critical when planning immunotherapy using IL‐10 in IgE‐related allergic diseases.

Thalidomide as an Immunotherapeutic Agent: The Effects on Neutrophil- Mediated Inflammation

Thalidomide was developed in the 1950s as a sedative drug and withdrawn in 1961 because of its teratogenic effects, but has been rediscovered as an immuno-modifying drug. It has been administered successfully for the treatment of erythema nodosum leprosum, aphthous ulceration in HIV disease, inflammatory bowel diseases, and multiple myeloma. So far, investigations into the mode of action of thalidomide have focused on lymphocytes and vascular endothelial cells and have shown that this agent inhibits the production of tumor necrosis factor (TNF)-alpha and is an inhibitor of tumor angiogenesis. Recently, other immunological effects of this drug have been gaining attention, including attenuation of neutrophil activation and inhibition of myelo-proliferative responses. In autoimmune diseases, inflammation is characterized by an influx of granulocytes, and the association of granulocytes with gastrointestinal ulcer formation or rheumatic arthritis has been well documented. The suppressive effect of thalidomide on the activation of the nuclear transcription factor NF-(kappa)B may explain these effects of thalidomide. NF-(kappa)B is retained in the cytoplasm with I(kappa)B(alpha), and is activated by a wide variety of inflammatory stimuli including TNF, IL-1 and endotoxin followed by its translocation to the nucleus. Constitutive activation of NF-(kappa)B has been detected in various inflammatory diseases, while angiogenesis and organogenesis also require NF-(kappa)B activation. Thalidomide, on the other hand, has been shown to selectively suppress NF-(kappa)B activation induced by inflammatory mediators. NF-(kappa)B is known to be located downstream of proliferative and/or survival signaling induced by growth factors, which regulate anti-apoptotic genes. Myeloid cells in vitro, however, have been found to proceed to apoptosis as the result of the treatment with thalidomide and subsequent inactivation of NF-(kappa)B. These findings are consistent with clinical symptoms that showed the recovery from leukocytosis and/or neutrophilia after the administration of thalidomide. These findings shed new light on the anti-inflammatory properties of thalidomide and suggested that they may inhibit granulocyte-mediated tissue injury.

Clinical and laboratory features of fatal rapidly progressive interstitial lung disease associated with juvenile dermatomyositis

OBJECTIVE Rapidly progressive interstitial lung disease (RP-ILD) is a rare but potentially fatal complication of JDM. The aim of this study was to establish markers for the prediction and early diagnosis of RP-ILD associated with JDM. METHODS The clinical records of 54 patients with JDM were retrospectively reviewed: 10 had RP-ILD (7 died, 3 survived), 19 had chronic ILD and 24 were without ILD. Routine tests included a high-resolution CT (HRCT) scan of the chest and measurement of serum levels of creatine phosphokinase, ferritin and Krebs von den Lungen-6 (KL-6). Anti-melanoma differentiation-associated gene 5 (MDA5) antibodies and IL-18 levels were measured by ELISA. RESULTS No differences were found in the ratio of juvenile clinically amyopathic DM between the three groups. Initial chest HRCT scan findings were variable and could not distinguish between RP-ILD and chronic ILD. Anti-MDA5 antibodies were positive in all 8 patients with RP-ILD and 10 of 14 with chronic ILD, but none of the patients without ILD. Serum levels of anti-MDA5 antibody, ferritin, KL-6 and IL-18 were significantly higher in the RP-ILD group than in the chronic ILD and non-ILD groups. Serum levels of IL-18 positively correlated with serum KL-6 (R = 0.66, P < 0.001). CONCLUSION High serum levels of IL-18, KL-6, ferritin and anti-MDA5 antibodies (e.g. >200 units by ELISA) are associated with RP-ILD. These can be used as an indication for early intensive treatment. Both alveolar macrophages and autoimmunity to MDA5 are possibly involved in the development of RP-ILD associated with JDM.

Superoxide dismutase (SOD) as a potential inhibitory mediator of inflammation via neutrophil apoptosis.

Superoxide dismutase (SOD) is supposed to be an effective agent for neutrophil-mediated inflammation in the area of critical medicine. We investigated the involvement of SOD in the regulation of neutrophil apoptosis. Exogenously added SOD effectively induced neutrophil apoptosis, and the fluorescence patterns determined using annexin-V and the 7-AAD were similar to those seen in Fas-mediated neutrophil apoptosis. Neutrophils are short-lived leukocytes that need to be removed safely by apoptosis. The clearance of apoptotic neutrophils from sites of inflammation is a crucial determinant of the resolution of inflammation. Catalase inhibited the neutrophil apoptosis and caspase-3 activation. Spontaneous apoptosis, hydrogen peroxide and anti-Fas antibody-induced apoptosis of neutrophils were accelerated in Downs syndrome patients, in whom the SOD gene is overexpressed. Hydrogen peroxide was thought to be a possible major mediator of ROS-induced neutrophil apoptosis in caspase-dependent manner. Neutrophil apoptosis represents a crucial step in the mechanism governing the resolution of inflammation and has been suggested as a possible target for the control of neutrophil-mediated tissue injury. SOD may be a potential inhibitory mediator of neutrophil-mediated inflammation.

Neutrophil-mediated inflammation in respiratory syncytial viral bronchiolitis

Abstract  Background : The involvement of neutrophil‐mediated inflammation may play an important role in the pathogenesis of acute respitory syncytial virus bronchiolitis. However, no measurable marker is sensitive enough to assess neutrophil‐mediated inflammation in the airways. Released neutrophil elastase (NE) in intraluminal airways has been reported to induce pulmonary inflammation. The aim of this study was to determine whether the amount of urinary trypsin inhibitor (UTI) in serum, a degenerate induced by NE, reflects the degree of airway inflammation in children with respiratory syncytial viral (RSV) bronchiolitis and whether the severity of inflammation is evaluated. The pre‐α‐/inter‐α‐trypsin inhibitor is assumed to be precursors of the UTI. When NE degrades these inhibitors, UTI is liberated.

Novel heterozygous C243Y A20/TNFAIP3 gene mutation is responsible for chronic inflammation in autosomal-dominant Behçet's disease

Objective Although Behçets disease (BD) is a chronic inflammatory disorder of uncertain aetiology, the existence of familial BD with autosomal-dominant traits suggests that a responsibility gene (or genes) exists. We investigated a Japanese family with a history of BD to search for pathogenic mutations underlying the biological mechanisms of BD. Methods 6 patients over 4 generations who had suffered from frequent oral ulcers, genital ulcers and erythaema nodosum-like lesions in the skin were assessed. Whole-exome sequencing was performed on genomic DNA, and cytokine production was determined from stimulated mononuclear cells. Inflammatory cytokine secretion and Nod2-mediated NF-κB activation were analysed using the transfected cells. Results By whole-exome sequencing, we identified a common heterozygous missense mutation in A20/TNFAIP3, a gene known to regulate NF-κB signalling, for which all affected family members carried a heterozygous C243Y mutation in the ovarian tumour domain. Mononuclear cells obtained from the proband and his mother produced large amounts of interleukin 1β, IL-6 and tumour necrosis factor α (TNF-a) on stimulation as compared with those from normal controls. Although inflammatory cytokine secretion was suppressed by wild-type transfected cells, it was suppressed to a much lesser extent by mutated C243Y A20/TNFAIP3-transfected cells. In addition, impaired suppression of Nod2-mediated NF-κB activation by C243Y A20/TNFAIP3 was observed. Conclusions A C243Y mutation in A20/TNFAIP3 was likely responsible for increased production of human inflammatory cytokines by reduced suppression of NF-κB activation, and may have accounted for the autosomal-dominant Mendelian mode of BD transmission in this family.

Differential Effects of Short-Acting β2-Agonists on Human Granulocyte Functions

Background: β₂-Adrenergic agonists play a pivotal role in the management of bronchial asthma. Although the major effect of short-acting β₂-agonists on the airway is relaxation of smooth muscles, they may also have several effects on surrounding immunomodulatory cells. Methods: We examined whether widely used short-acting β₂-agonists differ in their ability to modulate granulocyte functions, such as superoxide anion (O₂^–) production and degranulation. Results: Procaterol (PC), a full agonist, significantly inhibited both O₂^– production by granulocytes (neutrophils and eosinophils) and their degranulation at the clinically relevant concentrations, whereas salbutamol and tulobuterol (partial agonists) showed smaller effects. PC inhibited N-formyl methionyl-leucyl-phenylalanine-induced O₂^– production and peroxidase release, but failed to inhibit responses induced by phorbol 12-myristate 13-acetate and/or opsonized zymosan. Exposure to 5 × 10^–8M PC for 120 min resulted in approximately 50% inhibition of O₂^– production and degranulation of neutrophils. The effects of β₂-agonists were more obvious in neutrophils than in eosinophils. A selective β₂-receptor antagonist, ICI-118551, reversed the inhibitory effect of β₂-agonists (PC, salbutamol, tulobuterol B) on N-formyl methionyl-leucyl-phenylalanine-induced O₂^– production. Conclusions: These results suggest that β₂-agonists had an inhibitory effect on granulocyte functions, mainly mediated viareceptors and their efficacy. Our observations support that β₂-agonists with a rapid onset of action and high intrinsic efficacy (short-acting and full agonists) may be optimal for the rescue therapy against acute asthma attack and sedation of its airway inflammation in an early phase.

Polarization of Th1/Th2 in human CD4^+ T cells separated by CD62L : analysis by transcription factors

Background:  T‐cell surface antigens that differentiate clearly between Th1 and Th2 have not been identified. Discrimination of Th1/Th2 subpopulations by CD62L expression has been reported. We investigated the expression of transcription factors that regulate Th1/Th2 cytokine synthesis in human CD4+ T‐cell subpopulations separated by CD45RO and CD62L, and compared the ratio of CD62L+ to CD62L− cells between healthy individuals and patients with allergic diseases.