Proinflammatory Cytokines Monocyte Research Articles

Safe and potent anti-CD19 CAR T-cells with shRNA-IL-6 gene silencing element in patients with refractory or relapsed B-cell acute lymphoblastic leukemia.

Severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) have limited the widespread use of chimeric antigen receptor T (CAR T)-cell therapy. We designed a novel anti-CD19 CAR (ssCART-19) with a small hairpin RNA (shRNA) element to silence the interleukin-6 (IL-6) gene, hypothesizing it could reduce sCRS and ICANS by alleviating monocyte activation and proinflammatory cytokine release. In a post hoc analysis of two clinical trials, we compared ssCART-19 with common CAR T-cells (cCART-19) in relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). Among 87 patients, 47 received ssCART-19 and 40 received cCART-19. Grade ≥3 CRS occurred in 14.89% (7/47) of the ssCART-19 group versus 37.5% (15/40) in the cCART-19 group (p = 0.036). ICANS occurred in 4.26% (2/47) of the ssCART-19 group (all grade 1) compared to 15% (2/40) of the cCART-19 group. Patients in the ssCART-19 group showed comparable rates of treatment response (calculated with rates of complete remission and incomplete hematological recovery) were 91.49% (43/47) for ssCART-19 and 85% (34/40) for cCART-19 (p = 0.999). With a median follow-up of 21.9 months, cumulative nonrelapse mortality was 10.4% for ssCART-19 and 13.6% for cCART-19 (p = 0.33). Median overall survival was 37.17 months for ssCART-19 and 32.93 months for cCART-19 (p = 0.40). Median progression-free survival was 24.17 months for ssCART-19 and 9.33 months for cCART-19 (p = 0.23). These data support the safety and efficacy of ssCART-19 for r/r B-ALL, suggesting its potential as a promising therapy.

Discovery of a 6-Aminobenzo[b]thiophene 1,1-Dioxide Derivative (K2071) with a Signal Transducer and Activator of Transcription 3 Inhibitory, Antimitotic, and Senotherapeutic Activities.

6-Nitrobenzo[b]thiophene 1,1-dioxide (Stattic) is a potent signal transducer and activator of the transcription 3 (STAT3) inhibitor developed originally for anticancer therapy. However, Stattic harbors several STAT3 inhibition-independent biological effects. To improve the properties of Stattic, we prepared a series of analogues derived from 6-aminobenzo[b]thiophene 1,1-dioxide, a compound directly obtained from the reduction of Stattic, that includes a methoxybenzylamino derivative (K2071) with optimized physicochemical characteristics, including the ability to cross the blood-brain barrier. Besides inhibiting the interleukin-6-stimulated activity of STAT3 mediated by tyrosine 705 phosphorylation, K2071 also showed cytotoxicity against a set of human glioblastoma-derived cell lines. In contrast to the core compound, a part of K2071 cytotoxicity reflected a STAT3 inhibition-independent block of mitotic progression in the prophase, affecting mitotic spindle formation, indicating that K2071 also acts as a mitotic poison. Compared to Stattic, K2071 was significantly less thiol-reactive. In addition, K2071 affected cell migration, suppressed cell proliferation in tumor spheroids, exerted cytotoxicity for glioblastoma temozolomide-induced senescent cells, and inhibited the secretion of the proinflammatory cytokine monocyte chemoattractant protein 1 (MCP-1) in senescent cells. Importantly, K2071 was well tolerated in mice, lacking manifestations of acute toxicity. The structure-activity relationship analysis of the K2071 molecule revealed the necessity of the para-substituted methoxyphenyl motif for antimitotic but not overall cytotoxic activity of its derivatives. Altogether, these results indicate that compound K2071 is a novel Stattic-derived STAT3 inhibitor and a mitotic poison with anticancer and senotherapeutic properties that is effective on glioblastoma cells and may be further developed as an agent for glioblastoma therapy.

Plac8-ERK pathway modulation of monocyte function in sepsis

Sepsis, a life-threatening condition caused by infection, is characterized by the dysregulation of immune responses and activation of monocytes. Plac8, a protein, has been implicated in various inflammatory conditions. This study aimed to investigate the effect of Plac8 upregulation on monocyte proliferation and activation in sepsis patients. Peripheral blood samples were collected from healthy individuals and sepsis patients. Monocytes were stimulated with lipopolysaccharide (LPS) to create an in vitro sepsis model, while a murine sepsis model was established using cecal ligation and puncture (CLP). The levels of monocyte markers, proliferation index (PI), and pro-inflammatory cytokines were assessed using flow cytometry and qPCR, respectively. Plac8 and phosphorylated ERK protein levels were determined by western blot, and TNF-α, IL-6, and IL-10 levels were quantified using ELISA. The CCK-8 assay was used to evaluate PBMC proliferation and activation. The results showed that Plac8 was highly expressed in sepsis models, promoting the survival, proliferation, and activation of monocytes. Plac8 upregulation activated the ERK pathway, leading to increased phosphorylation of ERK protein and elevated levels of CD14, CD16, TNF-α, IL-6, Plac8, and IL-10. In sepsis mice, Plac8 overexpression similarly activated the ERK pathway and promoted the survival, proliferation, and activation of monocytes. In conclusion, the upregulation of Plac8 enhances the activation of the ERK pathway and promotes monocyte proliferation and activation in sepsis patients.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75mg/h) monotherapy or co-solvent only (50% hydroxypropyl-β-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

The Inflammation Spectrum of Monocytes in Relation to Obesity and Severity of Type 2 Diabetes (T2D), A Case-Control Study

Introduction. Increased monocyte and macrophage inflammatory state and pro-inflammatory cytokine production are linked to type 2 diabetes (T2D). Research design and Methods. This is a case-control study aimed to examine the expression of 23 monocyte genes related to inflammation, adhesion, and repair in individuals with mild (mean HbA1c 7.3%, illness duration 5.6 years) and severe type 2 diabetes (mean HbA1c 8.4%, disease duration 14.2 years) compared also with lean and obese controls. In addition, we determined a set of serum inflammatory cytokines and growth factors. Results. The monocytes of mild T2D patients (who were in general overweight/obese) showed overexpression of a subset of genes related to adhesion (CD9), vascular repair and growth (HGF). The monocytes of the severe T2D patients showed in contrast an upregulation of many of the pro-inflammatory genes, without a significantly increased expression of the repair gene HGF and the adhesion gene CD9. Serum cytokine expression in the severe T2D patients supported the increased inflammatory state of the patients showing high levels of IL-6, IL-1β, and TNF-α. Conclusions. This study, therefore, shows a pro-inflammatory gene expression profile of monocytes of severe T2D patients, while patients with mild T2D did not show such monocyte profile.

Inflammatory markers for improved recurrent UTI diagnosis in postmenopausal women.

Recurrent urinary tract infection (rUTI) severely impacts postmenopausal women. The lack of rapid and accurate diagnostic tools is a major obstacle in rUTI management as current gold standard methods have >24-h diagnostic windows. Work in animal models and limited human cohorts have identified robust inflammatory responses activated during UTI. Consequently, urinary inflammatory cytokines secreted during UTI may function as diagnostic biomarkers. This study aimed to identify urinary cytokines that could accurately diagnose UTI in a controlled cohort of postmenopausal women. Women passing study exclusion criteria were classified into no UTI and active rUTI groups, and urinary cytokine levels were measured by immunoassay. Pro-inflammatory cytokines IL-8, IL-18, IL-1β, and monocyte chemoattractant protein-1 were significantly elevated in the active rUTI group, and anti-inflammatory cytokines IL-13 and IL-4 were elevated in women without UTI. We evaluated cytokine diagnostic performance and found that an IL-8, prostaglandin E2, and IL-13 multivariable model had the lowest misclassification rate and highest sensitivity. Our data identify urinary IL-8, prostaglandin E2, and IL-13 as candidate biomarkers that may be useful in the development of immunoassay-based UTI diagnostics.

Efficacy and Safety of Interleukin-6-Knockdown CD19-Targeted CAR T Cells(ssCART-19) for Relapsed/Refractory B-ALL

Background： Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are significant challenges in the use of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for patients with relapsed or refractory B-type acute lymphoblastic leukemia (r/r B-ALL). These complications limit the widespread adoption of CAR-T therapy. To address this issue, we developed a novel approach using anti-CD19 CAR T-cells incorporating a small hairpin RNA (shRNA) element to silence the interleukin-6 (IL-6) gene (ssCART-19). By reducing IL-6 expression levels in ssCART-19 cells, we aimed to decrease monocyte activation and pro-inflammatory cytokine release, thereby mitigating the incidence of severe CRS and neurotoxicity. In this phase 1/2 clinical trial conducted at two centers, we compared the efficacy and safety of ssCART-19 to classical CART-19 cells (cCART-19) in patients with r/r B-ALL (NCT03919240). Methods： Peripheral blood mononuclear cells were collected from patients through leukapheresis. ssCART-19 cells were generated by transducing anti-CD19 CAR with a shRNA-IL-6 gene silencing element into bulk peripheral T lymphocytes using a lentivirus vector. In contrast, cCART-19 cells were only transduced with an anti-CD19 CAR. Both cell types contained 4-1BB and CD3ζ co-stimulatory domains. Lymphodepletion (fludarabine at 30 mg/m 2/day and cyclophosphamide at 300 mg/m 2/day) was conducted on days -5, -4, and -3 before infusion. Each patient received a dose of 5×10 6CAR +/kg. The primary endpoint was safety, with efficacy as the secondary endpoint for both ssCART-19 and cCART-19. Results: A total of 121 patients who had received at least two prior lines of treatment were screened for this study. A total of 47 patients received ssCART-19 and 40 patients received cCART-19 were included in the analysis. The median patient age of group ssCART-19 and group cCART-19 was 33(9-64) years and 24(2-68) years. At enrollment, ssCART-19 patients and cCART-19 patients had a median percentage of BM blasts of 4.0(0.01-86.0) % and 4.0(0.02-98.0) % by flow cytometry. ssCART-19 patients and cCART-19 patients had a median of 3 (2-8) and 4（2-9）prior lines of therapy(table 1). The transfection efficiency was 44.66% (range: 13.70-84.66%) for ssCART-19 and 39.98% (range: 14.0-65.0%) for cCART-19. Adverse events within 28 days included grade 3-4 neutropenia in 14 patients (29.79%), thrombocytopenia in 17 patients (36.17%), grade 1-2 CRS in 25 patients (53.20%), grade 3-4 CRS in 7 patients (14.89%), and grade 1 ICANS in 2 patients (4.26%) in the ssCART-19 group. In the cCART-19 group, adverse events within 28 days included grade 3-4 neutropenia in 20 patients (50%), thrombocytopenia in 18 patients (45%), grade 1-2 CRS in 19 patients (47.5%), grade 3-4 CRS in 15 patients (37.5%), grade 1-2 ICANS in 4 patients (10%), and grade 3 ICANS in 2 patients (5%). The incidence of neutropenia, thrombocytopenia, grade 3-4 CRS, and ICANS in the ssCART-19 group was significantly lower than that in the cCART-19 group (p < 0.05)(Table 2). Furthermore, the peak levels of IL-6, IL-2 and TNF-α were significantly lower in the ssCART-19 group compared to the cCART-19 group (p < 0.05)(Fig.1). No significant differences were observed between the two groups in terms of the start time, peak value, duration and killing efficacy of CART cells (p > 0.05)(Fig.2). As of the data cutoff on December 25, 2020, the median follow-up time was 12 months (range: 0.5-41.33). On day 28, with 91.49% of patients achieving complete remission (CR) or CR with incomplete hematological recovery (CRi) in ssCART-19, compared to 85% in the cCART-19 group. Median overall survival (OS) was not available for ssCART-19, while it was 32.93 months for cCART-19 (p=0.575). The median progression-free survival (PFS) was 14.17 months for ssCART-19 and 15.33 months for cCART-19 (p=0.339). Although no significant differences were observed in terms of 3-year OS and PFS, the 3-month PFS was higher in the ssCART-19 group compared to the cCART-19 group (82.3% vs 66.9%, p=0.045)(Fig.3). Conclusion： Our clinical studies demonstrated the safety and high efficacy of ssCART-19 with IL-6 gene silencing in patients with relapsed/refractory B-ALL. These findings support the potential of ssCART-19 as a promising therapeutic approach for this challenging patient population.

Identification and elucidation of cross talk between SLAM Family Member 7 (SLAMF7) and Toll-like receptor (TLR) pathways in monocytes and macrophages

To further elucidate the expression, regulation and function of Signaling Lymphocytic Activation Molecule Family (SLAMF) protein members in human monocytes and macrophages. Un-differentiated monocytic THP-1 cell (u-THP-1) and differentiated THP-1 macrophage (d-THP-1) were used as culture models in the study. Responses of cells to the differentiation agents phorbol ester (25 ng/ml) and TLR (Toll-like receptor) ligands were assessed. RT-PCR and Western blot analysis were used to determine mRNA and protein level. Pro-inflammatory cytokine mRNA expression levels and phagocytosis were used as functional markers. Data analyzed using t-test, one-way or two-way ANOVA followed by post hoc test. SLAMFs were differentially expressed in THP-1 cells. Differentiation of u-THP-1 to d-THP-1 led to significantly higher SLAMF7 mRNA and protein levels than other SLAMF. In addition, TLR stimuli increased SLAMF7 mRNA expression but not protein expression. Importantly, SLAMF7 agonist antibody and TLR ligands synergistically increased the mRNA expression levels of IL-1β, IL-6 and TNF-α, but had no effect on phagocytosis. SLAMF7 knocked-down in d-THP-1 significantly lowered TLR-induced mRNA expressions of pro-inflammatory markers. SLAM family proteins are differentially regulated by differentiation and TLRs. SLAMF7 enhanced TLR-mediated induction of pro-inflammatory cytokines in monocytes and macrophages but not phagocytosis.

Microfluidic-to-macrofluidic: A simple in vitro model of atherosclerosis induced by fluidic stimulation.

Atherosclerosis is the narrowing of the arteries due to the formation of fatty plaques, which is the main cause of myocardial infarction and stroke. It is important to develop an in vitro model that can combine multiple-type cell co-culture, vessel wall-like structure, and fluid condition to simulate the processes of atherosclerosis. Herein, we used a simple microfluidic chip made of three polydimethylsiloxane layers to co-culture endothelial and smooth muscle cells in a flat rectangular microchannel. After being connected with a circulating culture medium driven by a peristaltic pump, the flat microchannel was deformed to a tunnel-like macrochannel. The fluid pressure and shear stress applied on the cells in the deformed macrochannel can be varied by adjusting the circulating flow rate and the thickness of the middle layer. Under three levels of the pressure (65, 131, and 196 mm Hg) or shear stress (0.99, 4.78, and 24 dyne/cm2) conditions, a series of atherosclerosis-related events, including endothelial cell junction, pro-inflammatory cytokine secretion, monocyte adhesion, and lipid accumulation, were investigated. The atherosclerosis-related results showed that the medium pressure or shear stress exhibited a relatively weak pro-atherosclerotic effect in a V-shaped trend. To demonstrate the potential in drug screen, the effects of three well-known anti-atherosclerotic drugs (atorvastatin, tetramethylpyrazine, and high-density lipoprotein) on the lipid accumulation and pro-inflammatory cytokine secretion were evaluated under a strong pro-atherosclerotic fluid condition (65 mm Hg, 0.99 dyne/cm2). This in vitro model of atherosclerosis has shown great potential in drug screen application.

Ceramide present in cholangiocarcinoma-derived extracellular vesicle induces a pro-inflammatory state in monocytes

Cholangiocarcinoma (CCA) is a rare cancer characterized by a global increasing incidence. Extracellular vesicles (EV) contribute to many of the hallmarks of cancer through transfer of their cargo molecules. The sphingolipid (SPL) profile of intrahepatic CCA (iCCA)-derived EVs was characterized by liquid chromatography-tandem mass spectrometry analysis. The effect of iCCA-derived EVs as mediators of inflammation was assessed on monocytes by flow cytometry. iCCA-derived EVs showed downregulation of all SPL species. Of note, poorly-differentiated iCCA-derived EVs showed a higher ceramide and dihydroceramide content compared with moderately-differentiated iCCA-derived EVs. Of note, higher dihydroceramide content was associated with vascular invasion. Cancer-derived EVs induced the release of pro-inflammatory cytokines in monocytes. Inhibition of synthesis of ceramide with Myriocin, a specific inhibitor of the serine palmitoyl transferase, reduced the pro-inflammatory activity of iCCA-derived EVs, demonstrating a role for ceramide as mediator of inflammation in iCCA. In conclusion, iCCA-derived EVs may promote iCCA progression by exporting the excess of pro-apoptotic and pro-inflammatory ceramides.

PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMCPCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMCPCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMCPCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMCPCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.

P2X7 is expressed on human innate-like T lymphocytes and mediates susceptibility to ATP-induced cell death.

Extracellular ATP activates the P2X7 receptor, leading to inflammasome activation and release of pro-inflammatory cytokines in monocytes. However, a detailed analysis of P2X7 receptor expression and function in the human T cell compartment has not been reported. Here, we used a P2X7-specific nanobody to assess cell membrane expression and function of P2X7 on peripheral T lymphocyte subsets. The results show that innate-like T cells, which effectively react to innate stimuli by secreting high amounts of pro-inflammatory cytokines, have the highest expression of P2X7 in the human T cell compartment. Using Tγδ cells as example for an innate-like lymphocyte population, we demonstrate that these cells are more sensitive to P2X7 receptor activation than conventional T cells, affecting fundamental cellular mechanisms like calcium signaling and ATP-induced cell death. The increased susceptibility of innate-like T cells to P2X7-mediated cell death provides a mechanism to control their homeostasis under inflammatory conditions. Understanding the expression and function of P2X7 on human immune cells is essential to assume the benefits and consequences of newly developed P2X7-based therapeutic approaches.

DISSEMINATED HERPES ZOSTER VIRUS INFECTION FOLLOWING BARICITINIB IN COVID-19

DISSEMINATED HERPES ZOSTER VIRUS INFECTION FOLLOWING BARICITINIB IN COVID-19

HbS promotes TLR4-mediated monocyte activation and proinflammatory cytokine production in sickle cell disease

AbstractMonocytes are considered crucial actors of inflammation in sickle cell disease (SCD), being responsible for an increased production of proinflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6. Although a role of free heme released by intravascular hemolysis has been suspected, the mechanisms underlying monocyte activation in patients with SCD remain unknown. Using purified human hemoglobin (Hb), we demonstrate herein, that cell-free HbS, unlike HbA or heme, is responsible for a major enhancement in the expression of proinflammatory cytokines by human monocytes. This effect was found mediated by direct interaction with the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex, resulting in the activation of both the nuclear factor-κB (NF-κB) and type I interferon pathways. In Townes SCD mice, injection of HbS, unlike HbA, was responsible for an increased production of proinflammatory cytokines, which was prevented by the TLR4 inhibitor, TAK-242. Our results reveal a novel mechanism of monocyte activation and systemic inflammation in SCD, which opens new promising therapeutic perspectives targeting the HbS-TLR4 interaction.

Effect of aging on monocyte phagocytic and inflammatory functions, and on the ex vivo inflammatory responses to lipopolysaccharide, in horses

Studies investigating age-related changes in the function of monocytes are currently limited for horses. Thus, the main goal of this study was to determine the effect of aging on monocyte phagocytic capacity and pro-inflammatory cytokine responses to bacterial lipopolysaccharide (LPS). A second goal of this work was to examine the effect of aging on the inflammatory cytokine responses to LPS in a whole blood ex vivo model. Seven healthy young adult (4–6 years of age) and seven healthy senior horses (>20 years of age) were enrolled. Phagocytosis of E. coli, and pro-inflammatory cytokine (TNFα) responses to LPS, were measured in monocytes by flow cytometry. Gene expressions of pro-inflammatory cytokines (TNFα, IL-1β, IL-6, IL-8, IL-18, CCL-5, CCL-2) were measured in peripheral blood mononuclear cells (PBMCs) and whole blood by RT-qPCR post incubation for 2 h or 6 h with a low (0.01 µg/mL) or a high (1 µg/mL) dose of LPS. Two sets of statistical models were applied to compare the age groups, one adjusted, and one unadjusted for the horses’ body condition scores (BCS). The percentage of monocytes that phagocytosed E. coli after 2 h of incubation was significantly lower in senior compared to young adult horses in the BCS-adjusted model. In the senior group, the expression of IL-1β in 2 h-0.01 µg/mL LPS-stimulated PBMCs was significantly higher than in the young adult group (BCS-adjusted and unadjusted models). In senior horses, expressions of IL-8 and IL-6 in whole blood samples stimulated for 6 h with 0.01 µg/mL LPS and for 2 h with 1 µg/mL LPS, respectively, were significantly lower than in young adult horses (BCS-adjusted models). The results of this study suggest that the phagocytic function of monocytes, as well as their IL-1β response to LPS may be altered in senior horses. In addition, the whole blood IL-8 and IL-6 gene expression responses to LPS may be insufficient in senior horses. While investigation of the effect of BCS on monocyte functions and whole blood pro-inflammatory LPS-responses was not a major goal of this work, it appears that adiposity may play a role in innate immune cell function, as significant differences between the age groups were often not apparent until the models were adjusted for BCS.

Bone bruising severity after anterior cruciate ligament rupture predicts elevation of chemokine MCP-1 associated with osteoarthritis

PurposeAnterior cruciate ligament rupture is associated with characteristic bone contusions in approximately 80% of patients, and these have been correlated with higher pain scores. Bone bruising may indicate joint damage that increases inflammation and the likelihood of posttraumatic osteoarthritis. We sought to characterize the severity of bone bruising following acute anterior cruciate ligament injury and determine if it correlates with synovial fluid and serum levels of the proinflammatory chemokine monocyte chemoattractant protein-1 associated with posttraumatic osteoarthritis.MethodsThis was a retrospective analysis of data collected prospectively from January 2014 through December 2016. All patients who sustained an acute ligament rupture were evaluated within 15 days of injury, obtained a magnetic resonance imaging study, and underwent bone-patellar-tendon-bone autograft reconstruction were offered enrollment. The overall severity of bone bruising on magnetic resonance imaging was graded (sum of 0–3 grades in 13 sectors of the articular surfaces). Serum and synovial fluid levels of monocyte chemoattractant protein-1 were measured within 14 days of injury, and serum levels were again measured 6 and 12 months following surgery. Separate univariate linear regression models were constructed to determine the association between monocyte chemoattractant protein-1 and bone bruising severity at each time point.ResultsForty-eight subjects were included in this study. They had a mean age of 21.4 years and were 48% female. Median overall bone bruising severity was 5 (range 0–14). Severity of bone bruising correlated with higher synovial fluid concentrations of monocyte chemoattractant protein-1 preoperatively (R2 = 0.18, p = 0.009) and with serum concentrations at 12 months post-reconstruction (R2 = 0.12, p = 0.04).ConclusionsThe severity of bone bruising following anterior cruciate ligament rupture is associated with higher levels of the proinflammatory cytokine monocyte chemoattractant protein-1 in synovial fluid acutely post-injury and in serum 12-months following anterior cruciate ligament reconstruction. This suggests that severe bone bruising on magnetic resonance imaging after ligament rupture may indicate increased risk for persistent joint inflammation and posttraumatic osteoarthritis.Level of evidenceIII ― retrospective cohort study.

Concomitant pyroptotic and apoptotic cell death triggered in macrophages infected by Zika virus.

Zika virus (ZIKV) is a positive-sense RNA flavivirus and can cause serious neurological disorders including microcephaly in infected fetuses. As a mosquito-borne arbovirus, it enters the bloodstream and replicates in various organs. During pregnancy, it can be transmitted from the blood of the viremic mother to the fetus by crossing the placental barrier. Monocytes and macrophages are considered the earliest blood cell types to be infected by ZIKV. As a first line defense, these cells are crucial components in innate immunity and host responses and may impact viral pathogenesis in humans. Previous studies have shown that ZIKV infection can activate inflammasomes and induce proinflammatory cytokines in monocytes. In this report, we showed that ZIKV could infect and induce cell death in human and murine macrophages. In addition to the presence of cleaved caspase-3, indicating that apoptosis was involved, we identified the cleaved caspase-1 and gasdermin D (GSDMD) as well as increased secretion of IL-1β and IL-18. This suggests that the inflammasome was activated and that may lead to pyroptosis in infected macrophages. The pyroptosis was NLRP3-dependent and could be suppressed in the macrophages treated with shRNA to target and knockdown caspase-1. It was also be inhibited by an inhibitor for caspase-1, indicating that the pyroptosis was triggered via a canonical approach. Our findings in this study demonstrate a concomitant occurrence of apoptosis and pyroptosis in ZIKV-infected macrophages, with two mechanisms involved in the cell death, which may have potentially significant impacts on viral pathogenesis in humans.

ROCK inhibitor fasudil reduces the expression of inflammatory factors in LPS-induced rat pulmonary microvascular endothelial cells via ROS/NF-\u03baB pathway

BackgroundInflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated.MethodsLPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer’s instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65.ResultsFasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB.ConclusionsThe present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway.

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes.

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of β2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1β cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.

Prunella vulgaris L. Attenuates Experimental Autoimmune Thyroiditis by Inhibiting HMGB1/TLR9 Signaling.

BackgroundPrunella vulgaris L. (PV) has been used to treat autoimmune thyroiditis (AIT), but the underlying mechanism remains unknown. The present study was designed to evaluate the effect of PV on AIT and explore the role of high-mobility group box-1 (HMGB1) signaling in PV-mediated effects in vivo and in vitro.MethodsIn the present study, bioactive components of PV were identified using UPLC-ESI-MS. The protective effects and potential mechanisms critical for the anti-inflammatory and immunomodulatory effects of PV in AIT were investigated in a rat model of thyroglobulin-induced experimental autoimmune thyroiditis (EAT) and in lipopolysaccharide (LPS)-induced thyroid follicular cells (TFCs).ResultsThe main bioactive compound identified in PV was rosmarinic acid. The thyroid volume, thyroiditis inflammation score and serum thyroglobulin antibody levels of EAT rats were attenuated by PV treatment (P<0.01). In addition, PV significantly reduced the elevated levels of the proinflammatory cytokines TNF-α, IL-6, IL-1β and monocyte chemoattractant protein-1 (MCP-1) both in vivo (P<0.01) and in vitro (P<0.05). PV downregulated HMGB1 mRNA and protein expression, reduced HMGB1 secretion, and inhibited TLR9 signaling pathways (TLR9 and MyD88) in PV-treated EAT rats and TFCs. Moreover, PV reversed the increases in the numbers of splenic Th1, Th2, and Th17 cells. Finally, our results acquired following administration of ethyl pyruvate, an HMGB1 inhibitor, to splenocytes cultured in vitro supported the hypothesis that the HMGB1/TLR9 pathway is involved in the PV-mediated reductions in Th1, Th2 and Th17 cells.ConclusionPV decreased the activity of the TLR9/MyD88 pathway and proinflammatory cytokines through HMGB1. In addition, we are the first to show that PV attenuated the HMGB1-induced increases in Th1, Th2 and Th17 cells in AIT models. These findings provide new evidence for the potential therapeutic value of PV as a treatment for AIT and other autoimmune diseases.