C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.

Tfh cells with NLRP3 inflammasome activation are essential for high-affinity antibody generation, germinal centre formation and autoimmunity.

OBJECTIVE: NLRP3 inflammasome regulates T cell responses. This study examined the roles of NLRP3 inflammasome activation in the regulation of T follicular helper (Tfh) cells during humoral response to T dependent antigens and in systemic lupus erythematosus (SLE). METHODS: NLRP3 inflammasome activation of Tfh cells was studied in B6, MRL/lpr and NZM2328 mice and in SLE patients and healthy controls using a fluorescence-labelled caspase-1 inhibitor probe. MCC950, a selective inhibitor of NLRP3, was used to investigate the relation between NLRP3 inflammasome activation and germinal centre (GC) reaction, Ab responses to immunisation, and autoantibody production. RESULTS: NLRP3 inflammasome activation in Tfh cells after immunisation was identified in B6 mice. MCC950 inhibited humoral responses to sheep red blood cell and NP-CGG with reduction of the GC reaction. B6 mice with lymphoid cell-specific deletion of NLRP3 or Casp1 mounted suboptimal humoral responses with impaired GC formation and defective affinity maturation. In MRL/lpr and NZM2328 mice, inhibition of NLRP3 activation suppressed NLRP3 activated Tfh cell expansion as well as attenuated lupus-like phenotypes. Tfh cells with activated NLRP3 inflammasome exhibited increased expression of molecules for Tfh cell function and differentiation, and had greater ability to activate B cells. In SLE patients, disease activity was positively correlated with an increase in the activated NLRP3+ Tfh population and this population was markedly reduced in response to therapy. CONCLUSIONS: The activation of NLRP3 inflammasome in Tfh cells is an integral part of responses to immunisation. The activated NLRP3+ Tfh population is essential for optimal humoral responses, GC formation and autoimmunity.

Clinical treatment outcomes and their changes in extremely preterm twins: a multicenter retrospective study in Guangdong Province, China. / è¶ æœªæˆç†ŸåŒèƒŽå„¿ä¸´åºŠæ•‘æ²»ç»“å±€åŠå˜åŒ–åˆ†æžï¼šä¸€é¡¹å¹¿ä¸œçœå¤šä¸­å¿ƒå›žé¡¾æ€§ç ”ç©¶.

OBJECTIVES: To investigate the clinical treatment outcomes and the changes of the outcomes over time in extremely preterm twins in Guangdong Province, China. METHODS: A retrospective analysis was performed for 269 pairs of extremely preterm twins with a gestational age of <28 weeks who were admitted to the department of neonatology in 26 grade A tertiary hospitals in Guangdong Province from January 2008 to December 2017. According to the admission time, they were divided into two groups: 2008-2012 and 2013-2017. Besides, each pair of twins was divided into the heavier infant and the lighter infant subgroups according to birth weight. The perinatal data of mothers and hospitalization data of neonates were collected. The survival rate of twins and the incidence rate of complications were compared between the 2008-2012 and 2013-2017 groups. RESULTS: Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of severe asphyxia and smaller head circumference at birth (P<0.05). The mortality rates of both of the twins, the heavier infant of the twins, and the lighter infant of the twins were lower in the 2013-2017 group compared with the 2008-2012 group (P<0.05). Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of pulmonary hemorrhage, patent ductus arteriosus (PDA), periventricular-intraventricular hemorrhage (P-IVH), and neonatal respiratory distress syndrome (NRDS) and a higher incidence rate of bronchopulmonary dysplasia (P<0.05). CONCLUSIONS: There is a significant increase in the survival rate over time in extremely preterm twins with a gestational age of <28 weeks in the 26 grade A tertiary hospitals in Guangdong Province. The incidences of severe asphyxia, pulmonary hemorrhage, PDA, P-IVH, and NRDS decrease in both the heavier and lighter infants of the twins, but the incidence of bronchopulmonary dysplasia increases. With the improvement of diagnosis and treatment, the multidisciplinary collaboration between different fields of fetal medicine including prenatal diagnosis, obstetrics, and neonatology is needed in the future to jointly develop management strategies for twin pregnancy.

JAK/STAT signaling controls the fate of CD8+CD103+ tissue-resident memory T cell in lupus nephritis.

Autoimmune mediated inflammation and renal damage in lupus nephritis (LN) depends partly on the infiltration of lymphocytes in glomeruli and renal interstitium. Here we identified a population of CD8+ T cells with a CD103+-phenotype in the healthy kidneys of human and mouse. These cells were typically CD69+CD103+ tissue-resident memory T cells (TRM) in the kidney. CD8+ TRM cells were expanded in the kidneys of patients with LN or MRL/lpr mice. The expansion of renal CD8+ TRM cells correlated significantly with kidney disease activity. These cells were active in producing cytokines, perforin and granzyme B in the kidney of MRL/lpr mice. Importantly, renal CD8+ TRM cells underwent proliferation and self-renewal to maintain a stable TRM pool in the kidney of MRL/lpr mice, contributing to renal inflammation and damage. JAK/STAT signaling in the MRL/lpr mice was required for renal TRM self-renewal as well as maintenance of effector functions. Targeting JAK/STAT signaling by tofacitinib effectively suppressed effector functions and impaired the survival of renal TRM cells in the kidney, contributing to improved kidney function in MRL/lpr mice. These results provided evidences that renal CD8+ TRM cells play a role in the pathogenesis of LN. They could serve as a therapeutic target for LN.

Acute progressive stroke with middle cerebral artery occlusion caused by idiopathic hypereosinophilic syndrome: a case report.

BACKGROUND: Idiopathic hypereosinophilic syndrome (IHES) is associated with various organ system dysfunctions. Neurologic abnormalities have been previously noted in this syndrome. Cerebral infarction secondary to occlusion of large cerebral artery is rarely reported. Here we described a patient with IHES presented progressive multiple cerebral infarctions caused by bilateral middle cerebral artery occlusion. CASE PRESENTATION: A 55-year-old Chinese woman presented to our hospital with acute onset of right limbs weakness and slurred speech. Laboratory tests showed a significant eosinophilia of 5.29 × 109/L (normal, < 0.5), 49.9% of leukocytes. Brain magnetic resonance imaging (MRI) revealed multiple acute cerebral ischemic lesions. Magnetic resonance angiography (MRA) demonstrated stenosis in horizontal segment of right middle cerebral artery. A pretibial skin biopsy revealed eosinophilic infiltration around the capillaries in deep dermis and adipose tissue. The patient was given oral dual anti platelet agents and intravenous methylprednisolone. However, one week later, the patient presented significant neurological deterioration with right-sided hemiparesis and totally motor aphasia. Brain MRI and computed tomography perfusion (CTP) demonstrated new acute cerebral ischemia in left hemisphere. Digital subtraction angiography (DSA) revealed left middle cerebral artery completely occluded. The patient received a high-dose of intravenous methylprednisolone 500 mg per day and the eosinophil count quickly fell to normal within 2 days. She was transferred to a rehabilitation center and her neurological symptoms improved with modified Ranking Scale from 4 to 2. CONCLUSIONS: IHES is one of the rare causes of acute ischemic stroke with large cerebral artery occlusion. An early high-dose of corticosteroids therapy should be considered in cases of IHES patients. Our case study is benefit to clinical diagnosis and treatment of cerebral infarction with IHES.

Innate lymphoid cell disturbance with increase in ILC1 in systemic lupus erythematosus.

The innate lymphoid cell (ILC) is a group of effector cells with diverse important cellular functions in both health and disease states. In comparison with healthy controls, there were increases in circulating ILC in SLE patients. The proportion of ILC1 significantly increased with significant decreases of ILC2 in SLE patients and ILC3 in SLE patients with moderate to severe activity. IL-12, IL-18, IL-25, IL-33, IL-23, IL-1ß and IFN-γ were significantly increased in SLE patients. Moreover, IL-12, IL-18 and IL-1ß but not IFN-γ correlated significantly with SLEDAI. Successful treatments rapidly reduced them and with certain normalization of the ILC subsets. In addition to increases in ILC1 numbers, ~ 80% of the ILC1 in SLE patients were positive for synthesis of IFN-γ. Plasma from SLE patients were shown to be potent in inducing ILC1. Thus, increased circulating ILC1 might contribute to the pathogenesis of SLE through mounting type 1 immune response.

Pathogenesis of lupus nephritis: RIP3 dependent necroptosis and NLRP3 inflammasome activation.

RIP3 activation leads to activation of necroptosis and the NLRP3 inflammasome pathways. The activation of RIP3 in lupus nephritis (LN) has not been investigated. In this study, RIP3 and necroptosis pathway activations were demonstrated in podocytes in renal biopsies from patients with class IV LN and in the diseased kidneys from lupus-prone NZM2328 and MRL/lpr mice. RIP3 activation was accompanied with the activation of MLKL, the effector molecule of the necroptosis pathway, and activation of caspase-1, the effector of the NLRP3 inflammasome pathway. Podocyte activation of RIP3 was detected readily with the development of LN in NZM2328 mice, suggesting this activation may play a significant role in the pathogenesis of LN. GSK872, a RIP3 specific inhibitor, inhibited the development of LN in MRL/lpr mice with down-regulation of RIP3 activation in podocytes, decreased the splenic sizes and weights and anti-dsDNA antibody titers. IgG from pooled sera of diseased NZM2328 mice succumbing to LN induced both the necroptosis pathway and NLRP3 inflammasome activation in a podocyte cell line and this activation was specifically blocked by GSK872. These results indicate that the necroptosis pathway and the RIP3 dependent NLRP3 inflammasome pathway are activated in podocytes during LN. Inhibition of RIP3 kinase may be a novel therapeutic approach to treat LN and systemic lupus erythematosus (SLE).

Short-term outcomes of extremely preterm infants at discharge: a multicenter study from Guangdong province during 2008-2017.

BACKGROUND: An increasing number of extremely preterm (EP) infants have survived worldwide. However, few data have been reported from China. This study was designed to investigate the short-term outcomes of EP infants at discharge in Guangdong province. METHODS: A total of 2051 EP infants discharged from 26 neonatal intensive care units during 2008-2017 were enrolled. The data from 2008 to 2012 were collected retrospectively, and from 2013 to 2017 were collected prospectively. Their hospitalization records were reviewed. RESULTS: During 2008-2017, the mean gestational age (GA) was 26.68 ± 1.00 weeks and the mean birth weight (BW) was 935 ± 179 g. The overall survival rate at discharge was 52.5%. There were 321 infants (15.7%) died despite active treatment, and 654 infants (31.9%) died after medical care withdrawal. The survival rates increased with advancing GA and BW (p < 0.001). The annual survival rate improved from 36.2% in 2008 to 59.3% in 2017 (p < 0.001). EP infants discharged from hospitals in Guangzhou and Shenzhen cities had a higher survival rate than in others (p < 0.001). The survival rate of EP infants discharged from general hospitals was lower than in specialist hospitals (p < 0.001). The major complications were neonatal respiratory distress syndrome, 88.0% (1804 of 2051), bronchopulmonary dysplasia, 32.3% (374 of 1158), retinopathy of prematurity (any grade), 45.1% (504 of 1117), necrotizing enterocolitis (any stage), 10.1% (160 of 1588), intraventricular hemorrhages (any grade), 37.4% (535 of 1431), and blood culture-positive nosocomial sepsis, 15.7% (250 of 1588). The multivariate logistic regression analysis indicated that improved survival of EP infants was associated with discharged from specialist hospitals, hospitals located in high-level economic development region, increasing gestational age, increasing birth weight, antenatal steroids use and a history of premature rupture of membranes. However, twins or multiple births, Apgar ≤7 at 5 min, cervical incompetence, and decision to withdraw care were associated with decreased survival. CONCLUSIONS: Our study revealed the short-term outcomes of EP infants at discharge in China. The overall survival rate was lower than the developed countries, and medical care withdrawal was a serious problem. Nonetheless, improvements in care and outcomes have been made annually.

RIP3 dependent NLRP3 inflammasome activation is implicated in acute lung injury in mice.

BACKGROUND: NLRP3 inflammasome is involved in the inflammatory responses during acute lung injury (ALI). RIP3 triggered NLRP3 inflammasome activation independent of necroptosis induction has recently been documented. In this study, the role of RIP3 in the activation of NLRP3 inflammasome in the development of ALI was investigated. METHODS: A selective RIP3 inhibitor GSK872 was used to investigate the roles of RIP3 in NLRP3 inflammasome activation in the lipopolysaccharide (LPS) induced ALI mouse model. The mechanism of NLRP3 inflammasome activation was investigated in the human monocytic cell line THP-1. NLRP3 inflammasome and necroptosis were measured by flow cytometry or western blot. RIP3-NLRP3 interaction was interrogated using immunoprecipitation and the Duolink® In situ detection. RESULTS: Significant upregulation of both necroptosis and NLRP3 inflammasome pathways were observed in the lungs of mice with LPS induced ALI. GSK872 significantly suppressed the activation of necroptosis and NLRP3 activation with reduction of IL-1ß and IL-18 production and inflammatory cells infiltration, resulting in a significant amelioration of lung injury. These two processes were shown to be active in interstitial macrophages and CD11b+ monocyte-macrophages/dendritic cells. In THP-1 cells, RIP3 and NLRP3 interaction was enhanced by LPS/ATP stimulation resulting in IL-1ß and IL-18 production. This RIP3-NLRP3 interaction was significantly inhibited by GSK872. CONCLUSION: Taking together, these results show that RIP3 participates in the NLRP3 inflammasome activation in infiltrating macrophages in ALI induced by LPS. This process plays a significant pathogenic role in LPS-induced lung injury.

The ratio of circulating follicular T helper cell to follicular T regulatory cell is correlated with disease activity in systemic lupus erythematosus.

Follicular T regulatory (Tfr) cells inhibit follicular T helper (Tfh) cells mediated B cell responses. Tfh cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). However, the role of Tfr cells in SLE remains unclear. The frequency of circulating Tfr and Tfh cells were examined in SLE patients and healthy controls. The frequency of circulating Tfr cell decreased and Tfh/Tfr ratio increased in SLE patients. Serum anti-dsDNA antibody level positively correlated with frequency of Tfh cells and Tfh/Tfr ratios but negatively correlated with the frequency of Tfr cells. Moreover, the frequency of Tfr and Tfh/Tfr ratio but not that of Tfh was correlated with diseases activity. In addition, increase in Tfr cell numbers and decrease in the Tfh/Tfr ratios were observed with successful treatments. Thus, Tfr cells should be considered as a biomarker for SLE and their role in the pathogenesis of SLE warrants further investigation.

Anti-dsDNA antibodies bind to TLR4 and activate NLRP3 inflammasome in lupus monocytes/macrophages.

BACKGROUND: NLRP3 inflammasome has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The activation of NLRP3 inflammasome results in the production of IL-1ß and the subsequent inflammation. Anti-dsDNA antibodies (anti-dsDNA Abs) play critical roles in the development and progression of SLE. However, the mechanism of NLRP3 inflammasome activation in SLE is still not known. This study investigated the activation of NLRP3 inflammasome stimulated by anti-dsDNA Abs in monocytes/macrophages from SLE patients. METHODS: Monocytes/macrophages from SLE patients or healthy controls were stimulated with anti-dsDNA Ab-positive serum or purified anti-dsDNA Abs. Activation of inflammasome was measured by flow cytometry or Western blot. Anti-dsDNA Abs isolated from active SLE patients were injected into female (NZB × NZW) F1 mice and the activation of NLRP3 inflammasome and the frequencies of Th17 and Treg were examined. RESULTS: The activity of caspase-1 was significantly increased in active SLE patients and was correlated with serum levels of anti-dsDNA Abs and disease activities. The concentrations of IL-1ß and IL-17A were also significantly higher in SLE patients compared to healthy controls. Anti-dsDNA Ab-positive serum rather than healthy serum or RF (rheumatoid factor)-positive serum stimulated the activation of caspase-1 in monocytes. Anti-dsDNA Abs bound to TLR4 on macrophages and induced the production of ROS. Mitochondria-targeting antioxidant Mito-TEMPO, IκB kinase inhibitor peptide or TLR4 siRNA inhibited the activation of NLRP3 inflammasome and the secretion of IL-1ß induced by anti-dsDNA Abs. Injection of anti-dsDNA Abs into (NZB × NZW) F1 mice resulted in increased caspase-1 activation and production of IL-1ß and IL-17A. The Th17/Treg cell ratio also significantly increased following anti-dsDNA Ab injection. CONCLUSIONS: Anti-dsDNA Abs activated NLRP3 inflammasome in monocytes/macrophages from SLE patients by binding to TLR4 and inducing the production of mitochondrial ROS.

Myeloid-derived suppressor cells are proinflammatory and regulate collagen-induced arthritis through manipulating Th17 cell differentiation.

Myeloid-derived suppressor cells (MDSC) and Th17 cells were found to expand in collagen-induced arthritis (CIA) significantly. Two subsets of MDSC, polymorphonuclear (PMN) and mononuclear (MO), were detected and their ratios varied during the development of CIA. The depletion of MDSC in vivo resulted in suppression of T-cell proliferation and decreased IL-17A and IL-1ß production. The adoptive transfer of MDSC restored the severity of arthritis and Th17 cell differentiation. The depletion of MDSCs on day 35 resulted in arthritis amelioration without reaching a significant difference. Furthermore, MDSCs from CIA mice had higher production of IL-1ß and promoted Th17 cell differentiation. The expansion of MDSCs in the peripheral blood of rheumatoid arthritis (RA) patients was in correlation with increased Th17 cells and disease activity DAS28. These results support the hypothesis that MDSC may play a significant proinflammatory role in the pathogenesis of CIA and RA by inducing Th17 development in an IL-1ß-dependent manner.

Anti-dsDNA antibodies induce inflammation via endoplasmic reticulum stress in human mesangial cells.

BACKGROUND: Anti-dsDNA antibodies play an important role in the pathogenesis of lupus nephritis (LN). Endoplasmic reticulum (ER) stress is a physical reaction under stressful condition and can cause inflammation when stimulation is sustained. This study investigated the roles of ER stress in anti-dsDNA antibody-induced inflammation response in human mesangial cells (HMCs). METHOD: Anti-dsDNA antibodies isolated from LN patients were used to stimulate HMCs. The expression of GRP78, PERK, p-PERK, p-eIF2α, ATF4, p-IRE1α, ATF6 and CHOP in HMCs was measured by western blot. NF-κB activation was detected by examining nuclear translocation of NF-κB p65. The expression and production of IL-1ß, TNF-α and MCP-1 were examined by qPCR and ELISA. RESULTS: Flow cytometry and cellular ELISA showed that anti-dsDNA antibodies can bind to HMCs. The binding was not inhibited by blockage of Fc receptor. Anti-dsDNA antibody stimulation significantly enhanced the expression of GRP78, p-PERK, p-eIF2α and ATF4 in HMCs. However, no significant increase in the expression of p-IRE1α and ATF6 was found. In addition, anti-dsDNA antibodies also significantly increased the activation of NF-κB and upregulated the expression of IL-1ß, TNF-α and MCP-1, which were suppressed by pretreatment of HMCs with chemical ER stress inhibitor 4-PBA. Transfection of specific ATF4 siRNA also significantly reduced the activation of NF-κB and expression of proinflammatory cytokines. CONCLUSION: Anti-dsDNA antibodies induce NF-κB activation and inflammation in HMCs via PERK-eIF2α-ATF4 ER stress pathway.

Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts.

Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP(+) osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11-7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.

Low serum adiponectin levels are associated with reduced insulin sensitivity and lipid disturbances in short children born small for gestational age.

BACKGROUND: Being born as small for gestational age (SGA) has an increased risk of developing metabolic/cardiovascular disturbances in later life. The role of adiponectin in the metabolic disturbance in SGA children remained undefined. OBJECTIVE: The aim of this study was to investigate the association between serum levels of adiponectin and insulin sensitivity as well as lipid profile in short children born SGA at prepubertal ages. PATIENTS AND METHODS: Serum levels of adiponectin, fasting glucose, insulin, IGF-I, IGFBP-1, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), apolipoprotein A-I (ApoA-I) and Apo B were measured in 30 prepubertal short children born SGA. Insulin resistance (IR) and ß-cell function were assessed using the method of homeostatic model (HOMA). Data were compared to those of 30 short appropriate for gestational age (AGA) children matched for age, gender, height and body mass index, and correlation analysis was performed. RESULTS: Short SGA children had significantly higher levels of fasting insulin, HOMA-IR and HOMA-ß but lower levels of adiponectin than short AGA controls. No significant differences in the level of IGFBP-1 and IGF-I were found between the two groups. Serum levels of TC, TG, Apo B and Apo B/ApoA-I ratio were significantly higher in SGA, with 33% of hypercholesteraemia and 23% of hyperglyceridaemia. Stepwise multiple regression analysis revealed that serum adiponectin level was negatively correlated with HOMA-IR and TG and was positively correlated with birthweight SDS in SGA children. CONCLUSIONS: These findings suggest that low serum adiponectin levels are associated with reduced insulin sensitivity and unfavourable lipid profiles in short children born SGA at prepubertal ages.

P2X7 blockade attenuates murine lupus nephritis by inhibiting activation of the NLRP3/ASC/caspase 1 pathway.

OBJECTIVE: The NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and purinergic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7 /NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN). METHODS: MRL/lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ASC/caspase 1 inflammasome pathway, and ratio of Th17 cells to Treg cells were evaluated. P2X7 -targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα). RESULTS: Significant up-regulation of P2X7 /NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1ß (IL-1ß), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1ß and IL-17 and the Th17:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease. CONCLUSION: Activation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1ß production and enhanced Th17 cell polarization. Thus, targeting of the P2X7 /NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus.

Serum procalcitonin and C-reactive protein for differentiating bacterial infection from disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: To study the clinical value of procalcitonin (PCT) and C-reactive protein (CRP) in differentiating bacterial infection from disease activity in systemic lupus erythematosus (SLE) patients. METHOD: PCT and CRP in active SLE patients complicated with and without bacterial infection were retrospectively studied. Bacterial infection was diagnosed by positive culture results or typical symptoms and signs combined with positive response to antibiotics. Disease activity of SLE was assessed by systemic lupus erythematosus disease activity index (SLEDAI). RESULT: One hundred and fourteen active SLE patients were recruited, 47 of which were with bacterial infection and 67 were non-infected. PCT and CRP levels were significantly elevated in patients with bacterial infection (P < 0.05). The ideal cutoff value for PCT was 0.38 ng/ml, at which the sensitivity (74.5%) and specificity (95.5%) combined the best. The negative predictive value and positive predictive value to detect bacterial infection were 84.2% and 92.1%, respectively. PCT but not the CRP level in the septic patients was significantly higher than that of non-septic ones. Meanwhile, in patients with SLEDAI score of > 10, both PCT and CRP levels were higher in patients with bacterial infection, but the difference was only statistically significant for PCT (P < 0.05). PCT was significantly reduced after anti-bacterial treatment. CONCLUSION: PCT test is superior to CRP test in detecting superimposed bacterial infection in active SLE patients. The levels of PCT are correlated with the severity of bacterial infection and can be used to monitor the response to antibiotic treatment.

VISTA Deficiency Exacerbates the Development of Pulmonary Fibrosis by Promoting Th17 Differentiation.

Background: Interstitial lung disease (ILD), characterized by pulmonary fibrosis (PF), represents the end-stage of various ILDs. The immune system plays an important role in the pathogenesis of PF. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an immune checkpoint with immune suppressive functions. However, its specific role in the development of PF and the underlying mechanisms remain to be elucidated. Methods: We assessed the expression of VISTA in CD4 T cells from patients with connective tissue disease-related interstitial lung disease (CTD-ILD). Spleen cells from wild-type (WT) or Vsir -/- mice were isolated and induced for cell differentiation in vitro. Additionally, primary lung fibroblasts were isolated and treated with interleukin-17A (IL-17A). Mice were challenged with bleomycin (BLM) following VISTA blockade or Vsir knockout. Moreover, WT or Vsir -/- CD4 T cells were transferred into Rag1 -/- mice, which were then challenged with BLM. Results: VISTA expression was decreased in CD4 T cells from patients with CTD-ILD. Vsir deficiency augmented T-helper 17 (Th17) cell differentiation in vitro. Furthermore, IL-17A enhanced the production of inflammatory cytokines, as well as the differentiation and migration of lung fibroblasts. Both VISTA blockade and knockout of Vsir increased the percentage of IL-17A-producing Th17 cells and promoted BLM-induced PF. In addition, mice receiving Vsir -/- CD4 T cells exhibited a higher percentage of Th17 cells and more severe PF compared to those receiving WT CD4 T cells. Conclusion: These findings demonstrate the significant role of VISTA in modulating the development of PF by controlling Th17 cell differentiation. These insights suggest that targeting VISTA could be a promising therapeutic strategy for PF.

TIGIT regulates CD4+ T cell immunity against polymicrobial sepsis.

Background: Sepsis is one of the major causes of death and increased health care burden in modern intensive care units. Immune checkpoints have been prompted to be key modulators of T cell activation, T cell tolerance and T cell exhaustion. This study was designed to investigate the role of the negative immune checkpoint, T cell immunoglobulin and ITIM domain (TIGIT), in the early stage of sepsis. Method: An experimental murine model of sepsis was developed by cecal ligation and puncture (CLP). TIGIT and CD155 expression in splenocytes at different time points were assessed using flow cytometry. And the phenotypes of TIGIT-deficient (TIGIT-/-) and wild-type (WT) mice were evaluated to explore the engagement of TIGIT in the acute phase of sepsis. In addition, the characteristics were also evaluated in the WT septic mice pretreated with anti-TIGIT antibody. TIGIT and CD155 expression in tissues was measured using real-time quantitative PCR and immunofluorescence staining. Proliferation and effector function of splenic immune cells were evaluated by flow cytometry. Clinical severity and tissue injury were scored to evaluate the function of TIGIT on sepsis. Additionally, tissue injury biomarkers in peripheral blood, as well as bacterial load in peritoneal lavage fluid and liver were also measured. Results: The expression of TIGIT in splenic T cells and NK cells was significantly elevated at 24 hours post CLP.TIGIT and CD155 mRNA levels were upregulated in sepsis-involved organs when mice were challenged with CLP. In CLP-induced sepsis, CD4+ T cells from TIGIT-/- mice shown increased proliferation potency and cytokine production when compared with that from WT mice. Meanwhile, innate immune system was mobilized in TIGIT-/- mice as indicated by increased proportion of neutrophils and macrophages with potent effector function. In addition, tissue injury and bacteria burden in the peritoneal cavity and liver was reduced in TIGIT-/- mice with CLP induced sepsis. Similar results were observed in mice treated with anti-TIGIT antibody. Conclusion: TIGIT modulates CD4+ T cell response against polymicrobial sepsis, suggesting that TIGIT could serve as a potential therapeutic target for sepsis.

Fetal and neonatal outcome in severe alloimmunization managed with intrauterine transfusion: 18-year experience in a tertiary referral hospital in China.

Background: Hemolytic disease of the fetus and newborn (HDFN) due to red cell alloimmunization, is an important cause of fetal and neonatal morbidity and mortality. However, fetal and neonatal outcome of HDFN managed with intrauterine transfusion (IUT) in China are unknown. In addition, fetal and neonatal outcomes according to the type of maternal red cell alloantibodies involved and outcomes of hydrops fetalis are also unclear. Objectives: The objective of this study was to evaluate fetal and neonatal outcomes of severe red-cell alloimmunization treated by IUT, to compare the outcomes according to the type of antibody, and to investigate the perinatal and postnatal outcomes of hydrops fetalis due to red cell alloimmunization. Methods: A retrospective study of pregnancies affected by HDFN and managed with IUT at a tertiary care university hospital in China between January 2001 and December 2018 was performed. Fetal and neonatal outcomes were investigated, and comparison of outcomes depending on the type of antibody and comparison of outcome between hydrops fetalis and fetuses without hydrops were also conducted. Results: 244 IUTs were performed in 81 fetuses from 80 pregnancies. Anti-RhD was the major etiology of HDFN requiring IUT (71.6%). The fetal survival rate was 90.1%. The survival rate of the hydropic fetuses was significantly lower than those of the non hydropic fetuses (61.2% vs. 95.6%) (P = 0.002**). Compared with non hydropic fetuses, hydropic fetuses had significantly lower gestational age and lower hemoglobin level at first IUT. The neonatal survival rate was 98.6%. Exchange transfusions were required in 26% of the neonates. 30.1% of neonates had late anemia and required top-up transfusions, and hydropic fetuses required more late top-up transfusions than fetuses without hydrops. No significant difference in fetal and neonatal outcomes was found among the four subgroups stratified by the antibody involved. Conclusion: Our study demonstrates that IUT is an effective and safe therapy for severe HDFN at our institution. Early detection and treatment of hydrops is critical for perinatal outcomes. Particular attention should be paid to late postnatal anemia in affected neonates and top-up transfusion is still commonly needed.