Th17/IL-17A axis is critical for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc): SSc patients with high levels of serum IL-17A exhibit reduced lung functions and increased prevalence of PAH.

BACKGROUND: It is thought that systemic sclerosis (SSc) might be a T helper 17 (Th17) cell-driven autoimmune disease. Noticeably, pulmonary arterial hypertension (PAH) is a leading cause of death in patients with SSc. Here, we investigated the association between serum Th17-related cytokines and prevalence of PAH in SSc patients. METHODS: This study included 72 SSc patients and 51 healthy controls (HC). We determined clinical manifestations, immunophenotypes including Th subsets in peripheral blood lymphocytes, and the serum levels of interleukin (IL)-17A, IL-17A/F, IL-17B. IL-17C, IL-17D. IL-1ß, IL-6, IL-21, IL-22, and IL-23. RESULTS: The frequency of Th17 cells was significantly increased in SSc patients compared to HC and was positively correlated with the modified Rodnan skin scores. Furthermore, the serum levels of IL-17A, IL-17D, IL-1ß, and IL-6 were significantly increased in SSc patients compared to HC. SSc patients with detected IL-17A showed high levels of IL-17A/F, IL-1ß, IL-6, and IL-22, and high frequency of Th17 cells. Interestingly, these patients exhibited the reduced lung functions and increased prevalence of PAH significantly compared to patients with undetected IL-17A. Similarly, SSc patients with detected IL-17A and high IL-6 (≥1.2 pg/mL) exhibited the decreased lung functions and increased prevalence of PAH compared to patients with undetected IL-17A and low IL-6. CONCLUSION: We found that SSc patients with high levels of serum IL-17A or both IL-17A and IL-6 show reduced lung functions and high prevalence of PAH. Consequently, it is highly probable that Th17/IL-17A axis is critical for the prevalence of PAH in SSc patients.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

T follicular helper (Tfh) cells and T peripheral helper (Tph) cells produce interleukin (IL)-21 and are thought to contribute to follicular and extra-follicular B-cell activation, respectively, in autoimmune diseases. It is known that programmed cell death-1 (PD-1)-positive CXCR5+ Tfh-like cells are differentiated from human naive CD4+ T cells by IL-12 plus transforming growth factor (TGF)-ß. However, it remains unclear what cytokines are required for Tph differentiation. In this study, we found that interferon (IFN)-α and IFN-ß reduce the frequency of Tfh-like cells under the IL-12 plus TGF-ß condition, whereas they promote generation of PD-1+CXCR5-CD4+ T cells and secretion of IL-21, IFN-γ and CXCL13. Intracellular cytokine staining and T-cell-B-cell co-culture studies indicated that IFN-α promotes generation of IL-21+IFN-γ +CXCR5-CD4+ T cells thereby enhancing B-cell helper function. By IFN-α treatment, the mRNA levels of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB and PRDM1 were significantly up-regulated but BCL6 mRNA expression was down-regulated, suggesting a Tph-related gene expression pattern. On the other hand, IL-2-neutralization increased mRNA levels of IL21, CXCL13 and CXCR5, retained BCL6, but showed no clear effect on IFNG or PRDM1. RNA sequencing analyses revealed that PD-1hiCXCR5-CD4+ T cells prepared from in vitro culture show a Tph-related gene expression pattern similar with that of PD-1hiCXCR5- Tph cells obtained from the blood of patients with systemic lupus erythematosus. From our findings, it is highly probable that type I IFNs play a key role in differentiation of Tph cells and trigger Tph cell expansion in autoimmune diseases.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 -CD4+ T cells and enhance the secretion of IL-21, IFN-γ and CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5- Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

Distinct impact of malignancy and allergy on the clinical and immunological features of IgG4-related disease.

OBJECTIVES: To clarify the clinical and immunological characteristics of IgG4-RD based on the underlying diseases. METHODS: Consecutive patients with IgG4-RD treated at Keio University Hospital between 2010 and 2021 were divided according to the presence of malignancy or allergy into three groups. The clinical characteristics and 56 immune cell subsets in the peripheral blood were compared among the groups. RESULTS: Among 123 patients, 18 (14.6%) had malignancy including 4 with allergy (malignancy group), 57 (46.3%) had allergy alone (allergy group), and 48 (39.0%) had neither (idiopathic group). In the malignancy group, the patients were older (70.1 vs. 54.4 vs. 64.9 years, p<0.001), male-dominant (83.3 vs. 42.1 vs. 54.2%, p=0.008), and had smoking habits (77.8 vs. 42.1 vs. 43.8%, p=0.02). They also had significant involvement of the aorta/large vessels (33.3 vs. 7.0 vs. 20.8%, p=0.02), while the patients in the allergy group tended to have orbital/lacrimal gland involvement. Remission and relapse rates were not different between the groups; however, overall survival was significantly poorer in the malignancy group (p=0.02). Comprehensive immunophenotyping of the peripheral blood revealed that the increase in CXCR5+CD2-double negative T cells and the decrease in naive CD8 T cells were characteristic of the malignancy group. CONCLUSIONS: The clinical and immunological phenotypes of IgG4-RD differ among those with underlying diseases.

Lymphadenopathy in IgG4-related disease: a phenotype of severe activity and poor prognosis, with eotaxin-3 as a new biomarker.

OBJECTIVE: To clarify relevant proteins and clinical characteristics of a phenotype of IgG4-related disease (IgG4-RD) with lymphadenopathy. METHODS: We enrolled patients newly diagnosed with IgG4-RD in our department between January 2000 and June 2018 and performed proteomic analysis to measure serum concentrations of 1305 proteins. We extracted proteins overexpressed in patients with IgG4-RD with lymphadenopathy by comparing between those with lymphadenopathy, those without lymphadenopathy and healthy controls. We further reviewed all the patients with IgG4-RD in our institution and investigated the characteristics and prognosis of the patients with IgG4-RD with lymphadenopathy. RESULTS: Eighty-five patients with IgG4-RD were enrolled, of which, 55% had lymphadenopathy. Proteomic analysis in 31 patients with IgG4-RD and 6 healthy controls revealed that eotaxin-3 was a potential serum biomarker in the patients with lymphadenopathy versus those without lymphadenopathy and healthy controls. A cohort of 85 patients with IgG4-RD demonstrated that patients with lymphadenopathy showed a significantly higher serum IgG4, IgG4:IgG ratio, IgG4-RD responder index and eosinophilia (P < 0.001 for all), irrelevant of the extent to which organ involvement developed. Patients with lymphadenopathy treated with glucocorticoid alone relapsed with significantly higher rates than those without lymphadenopathy (P = 0.03). CONCLUSION: Lymphadenopathy in IgG4-RD represents a phenotype associated with high disease activities, eosinophilia and relapsing disease. Eotaxin-3 is a novel biomarker related to IgG4-RD with lymphadenopathy.

Significant association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis.

OBJECTIVES: To elucidate the association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis (AAV). METHODS: Peripheral blood from 36 patients with active AAV and 18 healthy controls was examined for numbers of circulating T cells, B cells, NK cells, dendritic cells, monocytes and granulocytes using flow cytometry. These immuno-phenotyping data were subjected to cluster analysis and principal components analysis to divide AAV patients into subgroups. Associated organ involvement or therapeutic prognosis were assessed for each subgroup. RESULTS: AAV patients had higher proportions of plasma cells, plasmablasts, activated T cells, CD14++ CD16+ monocytes, eosinophils and neutrophils than healthy controls. Immuno-phenotyping findings were similar between patients with microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis. Cluster analysis indicated that AAV patients could be divided into three subgroups according to peripheral immune cell numbers: antibody production-related (n = 9), cytotoxic activity-related (n = 4) and neutrocytosis/lymphocytopenia-related (n = 23). The antibody production-related or cytotoxic activity-related group was associated with CNS involvement, and the neutrocytosis/lymphocytopenia-related group was associated with high incidence of kidney involvement. Incidence of severe infection was markedly higher in the neutrocytosis/lymphocytopenia-related group than the other two groups. Incidence of disease relapse was comparable among the three groups. CONCLUSION: Patients with active AAV can be divided into three subgroups based on immuno-phenotyping. These results may provide a hint to understanding disease pathophysiology and prognosis, and determining appropriate treatment.

IL-17-Producing Vγ4+ γδ T Cells Require Sphingosine 1-Phosphate Receptor 1 for Their Egress from the Lymph Nodes under Homeostatic and Inflammatory Conditions.

Conventional αß T cells require sphingosine 1-phosphate (S1P) receptor 1 (S1P1) for circulation through the lymph nodes (LN); however, it is unclear whether γδ T cells use similar mechanisms. In this study, we found that treatment with fingolimod (FTY720, 1 mg/kg, orally) markedly reduced not only conventional CD4 T cells but also circulating γδ T cells (Vγ4(+) and Vγ4(-) subsets) in the blood of mice. In contrast, IL-17(+)Vγ4(+), IL-17(+)Vγ4(-), and IL-17(-)Vγ4(-) subsets were significantly accumulated in the LN after 6 h of FTY720 treatment. By skin application of a synthetic TLR7/8 agonist, Vγ4(+) γδ T cells (IL-17(+) and IL-17(-) subsets) were accumulated and expanded in the draining LN (DLN), whereas the IL-17(+) subset predominantly migrated to the inflamed skin. FTY720 induced a marked sequestration of IL-17-producing Vγ4(+) γδ T cells in the DLN and inhibited their infiltration into the inflamed skin. Similarly, FTY720 inhibited infiltration of Vγ4(+) γδ T cells into the CNS by their sequestration into the DLN in experimental autoimmune encephalomyelitis. Vγ4(+) γδ T cells expressed a significant level of S1P1 and showed a migratory response toward S1P. FTY720 treatment induced almost complete downregulation of S1P1 expression and S1P responsiveness in Vγ4(+) γδ T cells. Our findings strongly suggest that IL-17-producing Vγ4(+) γδ T cells require S1P1 for their egress from the LN under homeostatic and inflammatory conditions. Consequently, inhibition of S1P1-dependent egress of pathogenic IL-17-producing Vγ4(+) γδ T cells from the DLN may partly contribute the clinical therapeutic effects of FTY720 in relapsing multiple sclerosis.

Involvement of Neuroinflammation during Brain Development in Social Cognitive Deficits in Autism Spectrum Disorder and Schizophrenia.

Development of social cognition, a unique and high-order function, depends on brain maturation from childhood to adulthood in humans. Autism spectrum disorder (ASD) and schizophrenia have similar social cognitive deficits, although age of onset in each disorder is different. Pathogenesis of these disorders is complex and contains several features, including genetic risk factors, environmental risk factors, and sites of abnormalities in the brain. Although several hypotheses have been postulated, they seem to be insufficient to explain how brain alterations associated with symptoms in these disorders develop at distinct developmental stages. Development of ASD appears to be related to cerebellar dysfunction and subsequent thalamic hyperactivation in early childhood. By contrast, schizophrenia seems to be triggered by thalamic hyperactivation in late adolescence, whereas hippocampal aberration has been possibly initiated in childhood. One of the possible culprits is metal homeostasis disturbances that can induce dysfunction of blood-cerebrospinal fluid barrier. Thalamic hyperactivation is thought to be induced by microglia-mediated neuroinflammation and abnormalities of intracerebral environment. Consequently, it is likely that the thalamic hyperactivation triggers dysregulation of the dorsolateral prefrontal cortex for lower brain regions related to social cognition. In this review, we summarize the brain aberration in ASD and schizophrenia and provide a possible mechanism underlying social cognitive deficits in these disorders based on their distinct ages of onset.

A phenotypic drug discovery study on thienodiazepine derivatives as inhibitors of T cell proliferation induced by CD28 co-stimulation leads to the discovery of a first bromodomain inhibitor.

A phenotypic screening of thienodiazepines derived from a hit compound found through a binding assay targeting co-stimulatory molecules on T cells and antigen presenting cells successfully led to the discovery of a thienotriazolodiazepine compound (7f) possessing potent immunosuppressive activity. A chemical biology approach has succeeded in revealing that 7f is a first inhibitor of epigenetic bromodomain-containing proteins. 7f is expected to become an anti-cancer agent as well as an immunosuppressive agent.

FTY720 stimulated ROS generation and the Sty1/Atf1 signaling pathway in the fission yeast Schizosaccharomyces pombe.

Fingolimod hydrochloride (FTY720) is the first-in-class immune modulator known as sphingosine 1-phosphate (S1P) receptor agonists. FTY720 has also been reported to exert a variety of physiological functions such as antitumor effect, angiogenesis inhibition, and Ca2+ mobilization. Here, we show that FTY720 treatment induced reactive oxygen species (ROS) accumulation, and investigated the effect of FTY720 on the stress-activated MAP kinase Spc1/Sty1, a functional homologue of p38 MAPK, using a Renilla luciferase reporter construct fused to the CRE, which gives an accurate measure of the transcriptional activity of Atf1 and thus serves as a faithful readout of the Spc1/Sty1 MAPK signaling in response to oxidative stresses. FTY720 stimulated the CRE responses in a concentration-dependent manner, which was markedly reduced by deletion of the components of the Spc1/Sty1 MAPK pathway. The blockade of ROS production by NAC (N-acetyl-L-cysteine) significantly reversed the FTY720-induced ROS accumulation, subsequent activation of the Spc1/Sty1 MAPK pathway, and inhibition of cell proliferation. Cells lacking the components of the Spc1/Sty1 MAPK exhibited higher sensitivity to FTY720 and higher ROS levels upon FTY720 treatment than in wild-type cells. Thus, our results demonstrate the usefulness of fission yeast for elucidating the FTY720-mediated signaling pathways involving ROS.

S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via up-regulation of S1P receptor 1.

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Longitudinal analysis at pre- and post-flare of T peripheral helper and T follicular helper subsets in patients with systemic lupus erythematosus.

OBJECTIVE: We focused to analyze the time-course changes at pre- and post-flare of T peripheral helper (Tph) cells and circulating T follicular helper (Tfh) cells in the blood of patients with systemic lupus erythematosus (SLE) with lupus low disease activity state (LLDAS) before flare. METHODS: This study included inactive (n=29) and active (n=55) patients with SLE. Tph subsets, Tfh subsets, CD11chi B cells, and plasma cells in the blood were determined by flow cytometry. The blood levels of cytokines including interferons (IFNs) were measured by electrochemiluminescence assay or cytokine beads array. RESULTS: Active SLE patients exhibited the increased frequency of Tph1, Tph2, Tfh1, and Tfh2 subsets when compared to inactive patients, but no clear changes in the other subsets. During the treatment with medications, Tph1, Tph2, and Tfh2 subsets were significantly reduced along with disease activity and Tph1 and Tph2 subsets were positively correlated with SLE disease activity index (SLEDAI). The time course analysis of patients at pre- and post-flare revealed that in the patients at LLDAS before flare, Tph subsets and Tfh subsets were relatively low levels. At the flare, Tph cells, particularly Tph1 and Tph2 subsets, were increased and correlated with SLEDAI. Furthermore, the blood levels of IFN-α2a, IFN-γ, and IFN-λ1 were low in the patients with LLDAS before flare but these IFNs, particularly IFN-λ1, were increased along with flare. CONCLUSION: Increased frequency of Tph1 and Tph2 subsets and elevated levels of serum IFN-λ1 are presumably critical for triggering of flare in SLE.

Cytotoxic Tph subset with low B-cell helper functions and its involvement in systemic lupus erythematosus.

T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. However, the role of Tph subsets is not fully elucidated. Here, we investigate the immunological functions of Tph subsets and their involvement in systemic lupus erythematosus (SLE). We have defined four Tph subsets (Tph1: CXCR3+CCR6-, Tph2: CXCR3-CCR6-, Tph17: CXCR3-CCR6+, and Tph1-17: CXCR3+CCR6+) and performed RNA sequencing after cell sorting. Tph1 and Tph17 subsets express substantial levels of IL21, indicating B cell helper functions. However, Tph2 and Tph1-17 subsets express low IL21. Interestingly, we have found Tph2 subset express high levels of CX3CR1, GZMB, PRF1, GLNY, S1PR5, TBX21, EOMES, ZNF863, and RUNX3, indicating a feature of CD4+ cytotoxic T lymphocytes. In SLE patients, the frequency of Tph1 and Tph2 subsets are significantly increased and positively correlated with SLE disease activity indexes. Tph1 cells expansion has been observed in patients with cutaneous and musculoskeletal manifestations. On the other hand, Tph2 cell expansion has been found in patients with lupus nephritis in addition to the above manifestations. Our findings imply that Tph1 and Tph2 subsets exert distinct immunological functions and are contributed to the complexity of clinical manifestations in SLE.

Human mesenchymal stem cells inhibit osteoclastogenesis through osteoprotegerin production.

OBJECTIVE: Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy. METHODS: Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers. RESULTS: The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium. CONCLUSION: The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA.

Sphingosine 1-phosphate receptor type 1 regulates egress of mature T cells from mouse bone marrow.

Sphingosine 1-phosphate (S1P) and its receptor, S1P receptor type 1 (S1P(1)), are essential for lymphocyte egress from secondary lymphoid organs (SLO). Fingolimod (FTY720), the S1P receptor modulator, inhibits lymphocyte egress from SLO and decreases circulating lymphocytes; however, it also induces a significant decrease in the number of peripheral blood lymphocytes in alymphoplasia (aly/aly) mice lacking SLO. In this study, we demonstrated that the administration of FTY720 induced sequestration of mature lymphocytes, particularly T cells, into the bone marrow (BM) in aly/aly mice, implying that the reduction of circulating lymphocytes in these mice by FTY720 was due to inhibition of lymphocyte egress from the BM. Since sequestration of mature T cells into the BM was also induced in normal mice by selective S1P(1) agonist or S1P lyase inhibitor, it is suggested that S1P(1) expression and the S1P gradient play an important role in egress of mature T cells from the BM. Prophylactic administration of FTY720 to ovalbumin (OVA)-immunized mice significantly inhibited footpad swelling induced by OVA challenging with a marked reduction of OVA-specific T(h) cells in the BM, indicating that immunomodulation by FTY720 is likely due to reduced circulation of antigen-specific T(h) cells. On the other hand, OVA-specific T(h) cells, like naive T cells, were also sequestered into the BM and SLO of OVA-immunized mice by a short exposure of FTY720 after OVA challenging. These results suggest that the S1P-S1P(1) axis plays a regulatory role in egress of mature T cells including antigen-specific T(h) cells from the BM.

Proton Pump Inhibitor and Tacrolimus Uses are Associated With Hypomagnesemia in Connective Tissue Disease: a Potential Link With Renal Dysfunction and Recurrent Infection.

Background: Low levels of serum magnesium perturb renal tubular cell function and lymphocytes, resulting in renal deterioration and an imbalance in mononuclear cells. This study investigated the mechanism and influence of hypomagnesemia in patients with connective tissue disease. Methods: We retrospectively evaluated patients with connective tissue disease and available serum magnesium data who visited Keio University Hospital in 2019. Patients were divided into two groups: those with (serum magnesium < 1.8 mg/dl) and those without hypomagnesemia; their rates of hospitalization for severe infection and cumulative renal deterioration were compared. Patients' fractions of lymphocytes and natural killer and dendritic cell subsets, as measured by fluorescence-activated cell sorting (FACS) analysis, were also compared. Results: Among 284 patients, hypomagnesemia was detected in 63 (22.2%). Multivariate analysis revealed that the use of proton pump inhibitors [odds ratio (OR), 1.48; p = 0.01] and tacrolimus (OR, 6.14; p < 0.01) was independently associated with hypomagnesemia. In addition, the renal deterioration rate was significantly higher in tacrolimus and/or proton pump inhibitor users with hypomagnesemia (p = 0.01). The hospitalization rate for severe infection was also higher in patients with hypomagnesemia (p = 0.04). FACS analysis showed lower CD8+ T cell, CD19+ B cell, natural killer cell, and dendritic cell counts in patients with hypomagnesemia (p = 0.03, p = 0.02, p = 0.02, and p = 0.03, respectively). Conclusion: The use of tacrolimus and proton pump inhibitors may be associated with hypomagnesemia and lead to poor renal outcomes and severe infection in patients with connective tissue disease.

C5a promotes migration, proliferation, and vessel formation in endothelial cells.

OBJECTIVES: The goal of this paper is to investigate the effects of activated complement C5a on vascular endothelium during vessel formation. METHODS: A human microvascular endothelial cell line (HMEC-1) derived from post-capillary venules in skin was used to measure DNA synthesis, proliferation and cell-cycle progression. In vitro ring-shaped formation by the cells was assessed by using type I collagen gel matrix and a cell-migration assay using the Chemotaxicell chamber. A Matrigel plug assay was performed to confirm the effect of C5a in vivo. RESULTS: C5a progressed the cell cycle of HMEC-1 into G2/M phases, and induced DNA synthesis and proliferation in a dose-dependent manner. C5a efficiently induced migration and ring-shaped structure formation both in vitro and in vivo. Furthermore, a C5a receptor antagonist (W-54011) suppressed all HMEC-1 activities including proliferation and migration. CONCLUSIONS: Proliferation, migration, and ring-shaped formation by HMEC-1 cells was induced by C5a. The actions were efficiently inhibited by a specific antagonist against C5a. Our results implicated C5a in vessel formation and as a potent target for management of inflammatory diseases.

Discovery of fingolimod based on the chemical modification of a natural product from the fungus, Isaria sinclairii.

Fingolimod is a first-in-class of sphingosine-1-phosphate (S1P) receptor modulator and is widely used a therapeutic drug for multiple sclerosis (MS), autoimmune disease in the central nervous system. About 25 year ago, a natural product, myriocin was isolated from culture broths of the fungus Isaria sinclairii. Myriocin, a rather complex amino acid having three successive asymmetric centers, was found to show a potent immunosuppressive activity in vitro; however, it induced a strong toxicity in vivo. To find out a less toxic immunosuppressive candidate, the chemical structure of myriocin was simplified to a nonchiral symmetric 2-substituted-2-aminoproane-1,3-diol framework. Finally, a highly potent immunosuppressant, fingolimod was found by the extensive chemical modification and pharmacological evaluation using skin allograft model in vivo. Throughout the analyses of the mechanism action of fingolimod, it is revealed that S1P receptor 1 (S1P1) plays an essential role in lymphocyte circulation and that the molecular target of fingolimod is S1P1. Phosphorylated fingolimod acts as a "functional" antagonist at S1P1, modulates lymphocyte circulation, and shows a potent immunosuppressive activity. Fingolimod significantly reduced the relapse rate of MS in the clinical studies and has been approved as a new therapeutic drug for MS in more than 80 countries.

Longitudinal immune cell monitoring identified CD14++ CD16+ intermediate monocyte as a marker of relapse in patients with ANCA-associated vasculitis.

BACKGROUND: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease that affects small- to medium-sized blood vessels. Despite treatments having been improved, patients often experience disease relapses. It remains unclear how the immune cells involve in the development of vasculitis and how they fluctuate over the course of treatment. In this study, we aimed to identify the immune subsets and serum cytokines associated with disease relapse by comprehensive immuno-phenotyping in AAV patients. METHODS: We reviewed consecutive patients (n = 29) from Keio University Hospital who had been newly diagnosed with AAV from January 2015 to February 2019 and chronologically followed until 52 weeks. Numbers of circulating T cells, B cells, monocytes, and granulocytes were analyzed by flow cytometry (FACS). Serum levels of cytokines were measured by electrochemiluminescence enzyme immunoassay. Clinical information was obtained from patients' records and association with time-course changes in immuno-phenotypes and serum levels of cytokines were assessed. RESULTS: Comprehensive immuno-phenotyping data from 161 samples from 29 AAV patients at diagnosis; at weeks 4, 12, 24, and 52 of treatment; and at time of major relapse were examined. FACS analysis from patients with relapse revealed that CD14++ CD16+ intermediate monocytes and plasma cells concomitantly changed associated with disease relapse, which were independent from treatment regimen, ANCA status, or disease phenotype. In particular, the number of CD14++ CD16+ intermediate monocytes at relapse was significantly higher than that in remission or in healthy controls. Serum cytokine measurement revealed that changes of monocyte-derived proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α were associated with disease status. CONCLUSIONS: Chronological changes in CD14++ CD16+ intermediate monocyte counts can be a marker of disease relapse in AAV patients.

[New therapeutic approach for autoimmune diseases by the sphingosine 1-phosphate receptor modulator, fingolimod (FTY720)].

Fingolimod (FTY720) is the first substance in the new immunomodulator class called sphingosine 1-phosphate (S1P) receptor modulators. We isolated an immunosuppressive natural product, myrocin, from the culture broth of Isaria sinclairii, a kind of vegetative wasp. The chemical modification of myriocin yielded a new compound, FTY720, which has more potent immunosuppressive activity and less toxicity than myriocin. FTY720 has been shown to be highly effective in experimental allograft models and autoimmune disease models such as autoimmune encephalomyelitis, collagen-induced arthritis, and lupus nephritis. The most striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunosuppressive activity in these models. FTY720 is rapidly converted to FTY720-phosphate (FTY720-P) by sphingosine kinases. FTY720-P acts as a potent agonist at S1P receptor type 1 (S1P(1)), internalizes S1P(1) on lymphocytes, and inhibits the migration of lymphocytes toward S1P. It is highly likely that the reduction of peripheral blood lymphocytes by FTY720 is due to the inhibition of S1P(1)-dependent lymphocyte egress from secondary lymphoid organs and thymus. Recently, it has been reported that FTY720 exerted considerable therapeutic effects in a placebo-controlled clinical trail involving patients with relapsing multiple sclerosis. Patients who received FTY720 orally had a significant reduction in the clinical disease activity, the number of lesions in the central nervous system, and the relapse rates. Since FTY720 possesses a new mechanism of action that has not been observed with other immunosuppressive agents, it is believed that FTY720 provides a new therapeutic approach for autoimmune diseases including multiple sclerosis.