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.

Distinct features between HLA-DR+ and HLA-DR- PD-1hi CXCR5- T peripheral helper cells in seropositive rheumatoid arthritis.

OBJECTIVES: PD-1hi CXCR5- T peripheral helper (Tph) cells are newly identified pathogenic CD4 helper T cells in RA. We evaluated the usefulness of Tph cell subsets as biomarkers of RA. METHODS: RA patients who visited our rheumatology department between May 2015 and September 2017 and met the 2010 ACR/EULAR classification criteria were included. We compared the correlation of DAS28-ESR between Tph cell subsets and 40 immune cell subsets. We also explored which subsets reflected the chronological changes in the disease activity after treatment. RESULTS: Thirty-four seropositive RA patients, 11 seronegative RA patients and 34 healthy controls were included. Tph cell subsets that correlated with the DAS28-ESR were HLA-DR+ Tph cells (rs = 0.50, P = 0.002), HLA-DR- Tph cells (rs = 0.39, P = 0.03) and Tph1 cells (rs = 0.41, P = 0.02). Among the other 40 immune cell subsets, HLA-DR+ Th1-17 cells (rs = 0.38, P = 0.03), activated B cells (rs = -0.35, P = 0.04), plasma cells (rs = 0.43, P = 0.01) and CD14++ CD16+ monocytes (rs = 0.36, P = 0.04) correlated, but not strongly as HLA-DR+ Tph cells. However, MTX treatment reduced the proportion of HLA-DR+ Tph cells independently of the disease activity. In contrast, HLA-DR- Tph cells accurately reflected the change in the DAS28-ESR during MTX treatment. CONCLUSION: HLA-DR+ Tph cells were decreased with MTX treatment, independent of the disease activity, while HLA-DR- Tph cells reflected the disease activity accurately during the treatment.

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.

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.

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.

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.

Significant association between clinical characteristics and changes in peripheral immuno-phenotype in large vessel vasculitis.

BACKGROUND: Large vessel vasculitis (LVV) is a type of vasculitis characterized by granulomatous inflammation of medium- and large-sized arteries. Clinical assessment of acute phase reactants has been conventionally used to diagnose and monitor diseases; however, accurate assessment of vascular disease activity status can be difficult. In this study, we investigated comprehensive immuno-phenotyping to explore useful biomarkers associated with clinical characteristics. METHODS: Consecutive patients with newly diagnosed LVV who visited our institution between May 2016 and May 2019 were enrolled. The number of circulating T cells, B cells, natural killer cells, dendritic cells, monocytes, and granulocytes was examined and chronologically followed. Baseline and time-course changes in immuno-phenotyping associated with disease activity were assessed. RESULTS: Comprehensive immuno-phenotyping data from 90 samples from each of 20 patients with LVV were compared with those from healthy controls (HCs). The number of helper T (Th), follicular helper T (Tfh), CD8+ T, CD14++ CD16+ monocytes, and neutrophils were higher in patients with giant cell arteritis (GCA) and/or Takayasu arteritis (TAK) than in HCs. Among them, the number of CD8+ T and CD8+ Tem were higher in patients with TAK than in GCA. Notably, memory CD4+ and CD8+ T cells in patients with TAK remained high even in the remission phase. Further analysis revealed that the number of Th1, Th17, and Tfh cells was associated with disease relapse in GCA and TAK and that the number of CD8+ T cells was associated with relapse in TAK. Th1, Th17, and Tfh cells decreased after treatment with biologic agents, while CD8+ T cells did not. CONCLUSIONS: Our results from peripheral immuno-phenotyping analysis indicate that the numbers of Th and Tfh cells changed along with the disease condition in both GCA and TAK, while that of CD8+ T cells did not, especially in TAK. Treatment with biologic agents decreased the proportion of Th and Tfh cells, but not CD8+ T cells, in the patients. Chronological immuno-phenotyping data explained the difference in therapeutic response, such as reactivities against biologics, between GCA and TAK.

Diffusion of macrolide antibiotics through the outer membrane of Moraxella catarrhalis.

We reported previously that the high susceptibility of Moraxella catarrhalis to macrolide antibiotics and other hydrophobic antimicrobial agents was related to the hydrophobicity of the cell surface. Electrophoretic analysis of lipopolysaccharide (LPS) extracted from M. catarrhalis revealed a deep rough-type profile similar to that of an LPS Re type mutant of Salmonella typhimurium, which also exhibits high susceptibility to macrolides. Moreover, treatment of 32P-labeled cells of M. catarrhalis by phospholipase C induced the release of radioactive materials. These results suggested that hydrophobic agents such as macrolides readily access the cell surface exposed by the deep rough-type LPS and phospholipids, and permeate into the cell interior through the lipid bilayer. In fact, M. catarrhalis cells rapidly accumulated large amounts of the macrolide antibiotics, erythromycin and rokitamycin, whereas no accumulation of the macrolides was observed in cells having smooth-type or Rc type LPS under the same conditions.