Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization.

Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements, with mechanistic target of rapamycin (mTOR) kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here we show that an mTOR-Semaphorin 6D (Sema6D)-Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1hi macrophages and prevents development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity, and metabolism via PPARγ.

DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome.

Mitochondrial DNA (mtDNA) leakage into the cytoplasm can occur when cells are exposed to noxious stimuli. Specific sensors recognize cytoplasmic mtDNA to promote cytokine production. Cytoplasmic mtDNA can also be secreted extracellularly, leading to sterile inflammation. However, the mode of secretion of mtDNA out of cells upon noxious stimuli and its relevance to human disease remain unclear. Here, we show that pyroptotic cells secrete mtDNA encapsulated within exosomes. Activation of caspase-1 leads to mtDNA leakage from the mitochondria into the cytoplasm via gasdermin-D. Caspase-1 also induces intraluminal membrane vesicle formation, allowing for cellular mtDNA to be taken up and secreted as exosomes. Encapsulation of mtDNA within exosomes promotes a strong inflammatory response that is ameliorated upon exosome biosynthesis inhibition in vivo. We further show that monocytes derived from patients with Behçet's syndrome (BS), a chronic systemic inflammatory disorder, show enhanced caspase-1 activation, leading to exosome-mediated mtDNA secretion and similar inflammation pathology as seen in BS patients. Collectively, our findings support that mtDNA-containing exosomes promote inflammation, providing new insights into the propagation and exacerbation of inflammation in human inflammatory diseases.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

The proliferative activity levels of each immune cell population evaluated by mass cytometry are linked to the clinical phenotypes of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, and many peripheral immune cell populations (ICPs) are thought to be altered according to the course of the disease. However, it is unclear which ICPs are associated with the clinical phenotypes of SLE. We analyzed peripheral blood mononuclear cells (PBMCs) of 28 SLE patients using mass cytometry and identified 30 ICPs. We determined the proliferative activity of ICPs by measuring the proportion of cells expressing specific markers and Ki-67 among CD45+ cells (Ki-67+ proportion). We observed an increased Ki-67+ proportion for many ICPs of SLE patients and examined the association between their Ki-67+ proportions and clinical findings. The Ki-67+ proportions of five ICPs [classical monocyte (cMo), effector memory CD8+ T cell (CD8Tem), CXCR5- naive B cell (CXCR5- nB), and CXCR5- IgD-CD27- B cell (CXCR5- DNB)] were identified as clinically important factors. The SLE Disease Activity Index (SLEDAI) was positively correlated with cMo and plasma cells (PC). The titer of anti-DNA antibodies was positively correlated with cMo, CXCR5- nB, and CXCR5- DNB. The C4 level was negatively correlated with CXCR5- DNB. The bioactivity of type I interferon was also positively correlated with these ICPs. Fever and renal involvement were associated with cMo. Rash was associated with CD8Tem and CXCR5- DNB. On the basis of the proliferative activity among five ICPs, SLE patients can be classified into five clusters showing different SLE phenotypes. Evaluation of the proliferative activity in each ICP can be linked to the clinical phenotypes of individual SLE patients and help in the treatment strategy.

SLE stratification based on BAFF and IFN-I bioactivity for biologics and implications of BAFF produced by glomeruli in lupus nephritis.

OBJECTIVE: B-cell activating factor (BAFF) is implicated in SLE pathogenesis. Blocking BAFF signalling has contributed to reducing glucocorticoid dosage and preventing organ damage. However, clinical characteristics of patients who may benefit from this therapy are not yet fully elucidated. Therefore, we identified patients with high BAFF-bioactivity to investigate their clinical characteristics and BAFF-producing cells. METHODS: We established the reporter cell for BAFF and investigated the clinical characteristics of SLE patients with high BAFF-bioactivity. We identified BAFF-expressing kidney cells using publicly available scRNA-seq data and immunohistological analysis. SLE patients were stratified based on the bioactivity of BAFF and type-I IFN (IFN-I) to identify associated characteristic clinical manifestations. RESULTS: SLE patients, especially patients with LN, had significantly higher serum BAFF-bioactivity than healthy controls (HC) and non-LN patients. Additionally, single-cell-RNA-seq data and immunohistological analysis of kidney samples from LN patients revealed that BAFF is expressed in glomerular macrophages and mesangial cells. Notably, BAFF bioactivity was elevated in the urine of LN patients compared with that of non-LN patients, while no IFN-I bioactivity was detected in the urine. Furthermore, SLE stratification based on bioactivities of serum BAFF and IFN-I revealed the clinical characteristics of patients: high BAFF represented patients with LN and high IFN-I represented patients with blood and skin manifestations. CONCLUSIONS: Monitoring urinary BAFF-bioactivity may be valuable in diagnosing LN. Furthermore, stratification based on serum BAFF and IFN-I bioactivities may allow the identification of appropriate patients for biologics targeting BAFF and IFN-I.

First-line osimertinib in EGFR mutation-positive non-small cell lung cancer patients with poor performance status.

Background: The efficacy of osimertinib as a first-line treatment for patients with poor performance status (PS) remains unclear. Patients & methods: This multicenter retrospective study evaluated patients treated with osimertinib between 2018 and 2020, with PS 2-4. Results: Among 36 patients with PS 2, the median progression-free survival (PFS), 1-year PFS, median overall survival (OS) and 1-year OS were 14.5 months, 65.4%, 18.1 months and 72.7%, respectively. Among 20 patients with PS 3-4, the median PFS, 1-year PFS, median OS and 1-year OS were 3.0 months, 27.1%, 5.0 months and 46.1%, respectively. Conclusion: Osimertinib was not as efficacious as other EGFR-tyrosine kinase inhibitors.

Lay abstract Osimertinib is a first-line pharmacotherapy for patients with EGFR mutation-positive non-small cell lung cancer. However, the efficacy of osimertinib as a first-line treatment for patients with poor performance status (PS) remains unclear. This study evaluated 56 patients classified as PS 2, 3 and 4 (36, 14 and 6 patients, respectively) treated with osimertinib between 2018 and 2020. Osimertinib efficacy was lower than that of other EGFR­tyrosine kinase inhibitors. This study is the first to report using osimertinib as a first-line treatment in patients with poor PS.

Long-term efficacy of immune checkpoint inhibitors in non-small cell lung cancer patients harboring MET exon 14 skipping mutations.

INTRODUCTION: MET exon 14 skipping mutation, observed in 3-4% of non-small cell lung cancer (NSCLC), is emerging as a targetable alteration. In recent years, immune checkpoint inhibitors (ICI) have been effective in treating several NSCLCs. Our research aimed to investigate the characteristics of patients with NSCLCs harboring MET exon 14 mutations and their response to ICI in Japan. METHODS: Among the 1954 consecutive NSCLCs diagnosed at Saitama Cancer Center between 2010 and 2019, MET exon 14 skipping mutations were detected in 68 (3.5%) NSCLCs. We evaluated their characteristics such as programmed cell death ligand 1 (PD-L1) expression. RESULTS: Median age of patients with NSCLCs harboring MET exon 14 skipping mutations was 73 years. PD-L1 was highly expressed in 17 (70.8%) of the 24 patients examined. Seven patients received ICI monotherapy, and three out of seven had a remarkable treatment response, resulted in objective response rate (ORR) of 42.9% and progression-free survival of 24.7 months. Three patients with donor splice-site mutations showed a long-term treatment response, despite the fact that two with acceptor splice-site mutations demonstrated no response and experienced early disease progression with ICI monotherapy. CONCLUSION: Our results indicated that patients with NSCLCs harboring MET exon 14 mutations presented with a high rate of positive PD-L1 expression. ICI treatment showed a high ORR and long-term efficacy for NSCLCs harboring MET exon 14 mutations. Variants of MET exon 14 splice-site mutations may be associated with ICI response.

Pathological and therapeutic implications of eosinophil-derived semaphorin 4D in eosinophilic chronic rhinosinusitis.

BACKGROUND: Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis. Clinical markers for ECRS disease activity and treatment strategies have not been sufficiently established. Although semaphorins are originally identified as neuronal guidance factors, it is becoming clear that they play key roles in immune regulation and inflammatory diseases. OBJECTIVE: We sought to investigate the pathological functions and therapeutic potential of semaphorin 4D (SEMA4D) in ECRS. METHODS: Serum soluble SEMA4D levels in patients with paranasal sinus diseases were measured by ELISA. The expression of SEMA4D in blood cells and nasal polyp tissues was assessed by flow cytometry and immunohistochemistry, respectively. Generation of soluble SEMA4D was evaluated in matrix metalloproteinase-treated eosinophils. Endothelial cells were stimulated with recombinant SEMA4D, followed by eosinophil transendothelial migration assays. Allergic chronic rhinosinusitis was induced in mice using Aspergillus protease with ovalbumin. The efficacy of treatment with anti-SEMA4D antibody was evaluated histologically and by nasal lavage fluid analysis. RESULTS: Serum soluble SEMA4D levels were elevated in patients with ECRS and positively correlated with disease severity. Tissue-infiltrated eosinophils in nasal polyps from patients with ECRS stained strongly with anti-SEMA4D antibody. Cell surface expression of SEMA4D on eosinophils from patients with ECRS was reduced, which was due to matrix metalloproteinase-9-mediated cleavage of membrane SEMA4D. Soluble SEMA4D induced eosinophil transendothelial migration. Treatment with anti-SEMA4D antibody ameliorated eosinophilic infiltration in sinus tissues and nasal lavage fluid in the ECRS animal model. CONCLUSIONS: Eosinophil-derived SEMA4D aggravates ECRS. Levels of serum SEMA4D reflect disease severity, and anti-SEMA4D antibody has therapeutic potential as a treatment for ECRS.

Eosinophil-derived neurotoxin enhances airway remodeling in eosinophilic chronic rhinosinusitis and correlates with disease severity.

Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) that is characterized by intractable nasal polyp formation. Eosinophil-derived neurotoxin (EDN) is an eosinophil granule protein that is closely related to allergic inflammation, but the pathological implications of EDN in ECRS remain unknown. In this study, we evaluated the function of EDN in ECRS pathogenesis and assessed its potential as a disease activity marker. Serum EDN levels were significantly higher in patients with ECRS than in those with other nasal and paranasal diseases, and were positively correlated with clinical disease activity. Production of EDN from isolated human eosinophils was induced by stimulation with IL-5 in vitro. Human nasal epithelial cells were stimulated with EDN, and the resultant changes in gene expression were detected by RNA sequencing. Pathway analysis revealed that the major canonical pathway affected by EDN stimulation was 'regulation of the epithelial-mesenchymal transition pathway'; the only gene in this pathway to be up-regulated was matrix metalloproteinase 9 (MMP-9). Consistent with this, immunostaining analysis revealed intense staining of both EDN and MMP-9 in nasal polyps from patients with ECRS. In conclusion, our data demonstrate that serum EDN level is a useful marker for the evaluation of ECRS severity. Furthermore, EDN induces production of MMP-9 from the nasal epithelium, which may be involved in the pathogenesis of ECRS.

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.

Lamtor1 Is Critically Required for CD4+ T Cell Proliferation and Regulatory T Cell Suppressive Function.

Mechanistic target of rapamycin complex (mTORC)1 integrates intracellular sufficiency of nutrients and regulates various cellular functions. Previous studies using mice with conditional knockout of mTORC1 component proteins (i.e., mTOR, Raptor, and Rheb) gave conflicting results on the roles of mTORC1 in CD4+ T cells. Lamtor1 is the protein that is required for amino acid sensing and activation of mTORC1; however, the roles of Lamtor1 in T cells have not been investigated. In this article, we show that Lamtor1-deficient CD4+ T cells exhibited marked reductions in proliferation, IL-2 production, mTORC1 activity, and expression of purine- and lipid-synthesis genes. Polarization of Th17 cells, but not Th1 and Th2 cells, diminished following the loss of Lamtor1. Accordingly, CD4-Cre-driven Lamtor1-knockout mice exhibited reduced numbers of CD4+ and CD8+ T cells at rest, and they were completely resistant to experimental autoimmune encephalomyelitis. In contrast, genetic ablation of Lamtor1 in Foxp3+ T cells resulted in severe autoimmunity and premature death. Lamtor1-deficient regulatory T cells survived ex vivo as long as wild-type regulatory T cells; however, they exhibited a marked loss of suppressive function and expression of signature molecules, such as CTLA-4. These results indicate that Lamtor1 plays essential roles in CD4+ T cells. Our data suggest that Lamtor1 should be considered a novel therapeutic target in immune systems.

Pretreatment of ceramic membrane microfiltration in wastewater reuse: A comparison between ozonation and coagulation.

To determine the most efficient pretreatment for ceramic membrane filtration (CMF) of primary clarifier effluent (PE), the effectiveness of ozonation and coagulation was investigated from the viewpoint of both virus removal and mitigation of membrane fouling. Our results showed virus removal by coagulation to be more efficient as a CMF pretreatment, whereas ozonation showed better efficiency when used as a CMF posttreatment. The effect of ozonation and coagulation on ceramic membrane fouling was investigated during short-term operation. With the use of coagulation before CMF (PACl + CMF), irreversible fouling resistance was 0.5 × 1011 m-1 at a dosage of 150 mg/L of polyaluminum chloride (PACl), which was 10 times lower than when ozonation was used as a pretreatment to CMF (O3+CMF) (0.7 × 1012 m-1 at 50 mg-O3/L). This result indicates coagulation to be more efficient than ozonation for mitigating ceramic membrane fouling. Based on these results, the process sustainability of PACl + CMF was then investigated during longer-term operation. At a dosage of 150 mg/L of PACl, the PACl + CMF process could be sustainably operated for 120 h without any need for chemically enhanced backwashing, which was twice as long as for PACl dosages of 50 and 100 mg/L. Coagulation is thus a more efficient pretreatment for CMF of PE from the viewpoint of both virus removal and mitigation of ceramic membrane fouling. The hygienic safety of reclaimed water can be further improved if ozonation is used as a CMF posttreatment.

IL-6 inhibitor for the treatment of rheumatoid arthritis: A comprehensive review.

Tocilizumab (TCZ) is an interleukin-6 (IL-6) inhibitor used for the treatment of rheumatoid arthritis (RA). It was developed in 2008, and its effectiveness is supported by evidence from all over the world based on its first decade of use. Although the overall efficacy and safety profiles of TCZ are similar to those of tumor necrosis factor (TNF) inhibitors, TCZ displays certain differences. The most notable advantage of TCZ is its usefulness as a monotherapy. Additionally, TCZ is favorable in the improvement of systemic inflammatory symptoms such as anemia and fatigue. The low immunogenicity of TCZ contributes favorably to long-term drug retention. Due to frequent relapse after TCZ cessation, TCZ use should be tapered beyond remission. During TCZ therapy, C-reactive protein (CRP) is unable to recognize disease activity and the severity of infection. The most common adverse events (AEs) are infection and abnormalities in laboratory findings including dyslipidemia, neutropenia, thrombocytopenia, and abnormality of liver enzymes. TCZ obscures the symptoms of infection. Therefore, stealth infections without obvious CRP elevation can sometimes cause severe damage to patients. Lower intestinal perforation is an uncommon but serious AE in TCZ therapy. Further clinical investigations will continue to refine the IL-6 inhibitory strategy.

Apoptosis-derived membrane vesicles drive the cGAS-STING pathway and enhance type I IFN production in systemic lupus erythematosus.

OBJECTIVE: Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS-STING pathway in the IFN-I-producing cascade driven by SLE serum. METHODS: We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction. RESULTS: IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells. CONCLUSIONS: AdMVs in SLE serum induce IFN-I production through activation of the cGAS-STING pathway. Thus, blockade of the cGAS-STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis.

OBJECTIVES: Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis. METHODS: Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D-/- mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays. RESULTS: Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D's intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation. CONCLUSIONS: Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

Expression and localization of aquaporin-4 in sensory ganglia.

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4(-/-) mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4(-/-) mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.