Monocytic Cell Line Research Articles

Luteolin Mitigates Acute Lung Injury Through Immune Modulation and Antinecroptosis Effects by Targeting the BTK and FLT3 Signaling Pathways.

Overactive immune responses and lung cell damage exacerbate acute lung injury (ALI). Luteolin, a flavonoid commonly found in traditional herbs, shows potential as an anti-ALI agent in pharmacological and clinical research, although its biological mechanism is not fully understood. This study aims to investigate whether luteolin can ameliorate ALI through its immune-modulatory and antinecroptosis mechanisms. We found that luteolin significantly inhibits the cellular activity of the FLT3-dependent monocyte cell line MOLM-13 and BTK-dependent B-cell line TMD-8. Through molecular docking and HTRF detection, it was confirmed that luteolin inhibits BTK and FLT3 enzyme activity by binding to their kinase domains, with IC50 values of 0.78 and 0.35 μM, respectively. In a TNF-α-induced lung epithelial cell injury model, luteolin reduced the increased expression of IL1B, IL6, and CXCL8 mRNAs by blocking the necroptosis signal TNF-α/BTK/MLKL. Furthermore, using a Balb/c mouse ALI model with intratracheal LPS infusion (5 mg/kg), it was observed that luteolin improved lung function and pathology, regulated immune cell infiltration, and reduced cell death in pulmonary tissues by inhibiting BTK and FLT3 protein phosphorylation. In conclusion, luteolin acts as a natural BTK and FLT3 inhibitor, effectively preventing ALI both in vivo and in vitro through its immune-modulating and antinecroptosis properties.

P1302 OCTN1 variants shape innate immunity and predict individual response to vedolizumab in ulcerative colitis patients: from preclinical models to AI

Abstract Background Limited evidence is available about predictors of response to specific medications for ulcerative colitis. Variants of OCTN1, an organic cation transporter, could modulate the inflammatory response and potentially predict individual response to immune-modulatory therapy. Methods We first assessed the ability of OCTN1 to differentially modulate IL1β secretion under different stimuli in a human leukemia monocytic cell line (THP-1) and in human peripheral blood mononuclear cells. We therefore prospectively enrolled a cohort of UC patients starting either anti-TNFα or vedolizumab, determined their OCTN1 genotype and evaluated their response to therapy after six months, as well as steroid free therapy persistence after 2 years. Finally, we performed an integrated machine learning based analysis including serum cytokines and gut microbiota analysis. Results OCTN1-deficient THP1 cells showed reduced IL-1β secretion in response to peptidoglycan (PGN) and Fusobacterium nucleatum (Fn), whilst primary monocytes bearing OCTN1 503F variant displayed an increased production of IL-1β in response to PGN and live bacteria. We therefore enrolled 90 patients, of which 50 starting anti-TNFα and 40 vedolizumab. At the multivariate analysis, a significant reduction of clinical response after six months was observed in patients with 503F variant treated with vedolizumab. The same trend was observed for steroid free therapy persistence after two years. The machine learning analysis revealed that changes in bacterial composition, notably the relative abundance of Gemellaceae, followed by Lachnospiracea, were significant predictors of lack of two-year steroid-free therapy persistence. Conclusion Mutated OCNT1 genotype (503F) could favor an increased IL1β secretion from human leukocytes, thus suggesting a worse disease course and lower response to vedolizumab as compared to 503L. ovel machine learning data analysis provides further insights into such topic.

C-type natriuretic peptide suppresses VEGFa gene expression by attenuating IL6-STAT3 signal pathway in primary synovial fibroblasts from rat knee.

C-type natriuretic peptide (CNP) can be a new disease-modifying anti-osteoarthritis drug (DMOAD) candidate because intraarticular injection of CNP attenuates both articular cartilage degradation and persistent pain in a rat knee arthritis model. This study aimed to elucidate the underlying molecular mechanisms by which CNP protects the knee joint from osteoarthritic changes. Gene expression analyses indicated that CNP did not interfere with the expression of IL1β -responsive genes in rat primary synovial fibroblasts or the monocytic cell line, RAW264.7cells. In contrast, total RNA sequence analyses indicated that CNP negatively regulated the IL6-STAT3 signaling pathway and VEGFa gene expression in rat synovial fibroblasts. As previously indicated, IL6 induced phosphorylation of 705Tyr residue of STAT3 and its nuclear translocation to activate VEGFa gene expression; however, in this study, we showed that CNP induced phosphorylation of 727Ser residue and inhibited IL6-induced nuclear translocation of STAT3. Since the IL6 pathway has been shown to accelerate articular cartilage degradation and induce knee pain, our data suggest that CNP can act as a DMOAD by negatively regulating IL6-mediated proinflammatory signals in the knee joint.

Functional Characteristics of the Crosstalk Between Vocal Fold Fibroblasts and Macrophages-The Role of Vibration in Vocal Fold Inflammation.

This in vitro study investigated the interaction between human vocal fold fibroblasts (hVFF) and macrophages under the influence of cigarette smoke extract (CSE) and vibration as potential regulators of vocal fold (VF) inflammation. Experimental in vitro pilot study. Immortalized hVFF were cultured in flexible-bottomed cell culture plates, treated with CSE, and subjected to static or dynamic conditions in a phonomimetic bioreactor. For coculture, unstimulated orlipopolysaccharide/IFNγ-stimulated THP-1 (human leukemia monocytic cell line) macrophages were added in inserts for a final 24hours of vibration period. We measured messenger ribonucleic acid (mRNA) (quantitative polymerase chain reaction [qPCR]) and protein levels (Western Blot, ELISA, and LUMINEX®) of hVFF and analyzed the results using two- and three-way ANOVA with post hoc tests. Under inflammatory stimulation, we observed a reduction of collagen (COL) type 1A1, 1A2, and 3A1, and increased gene expression of COL4A1, matrix metallopeptidase 2, and vascular endothelial growth factor A in hVFF. Additionally, the pro-inflammatory markers cyclooxygenase (COX) 1 and 2, interleukin (IL) 1β, IL-6, and IL-8 were upregulated. CSE increased COX1 and COX2 levels, whereas vibration reduced CSE-induced increases of COL4A1 and COX2 in pro-inflammatory stimulated hVFF. This study indicates that vibration may mitigate CSE-induced inflammatory damage in the hVFF, thereby offering new insights into the cellular crosstalk that underlies the pathophysiology of VF inflammation in smoking-related voice disorders.

15-Deoxy-Δ-12,14-Prostaglandin J2 Represses Immune Escape of Lung Adenocarcinoma by Polarizing Macrophages Through Epidermal Growth Factor Receptor/Ras/Raf Pathway.

Lung adenocarcinoma (LUAD) is a widespread and deadly form of cancer. Prostaglandin 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) possesses antioxidant, anti-inflammatory, and anticancer properties. However, it is unclear whether this effect on LUAD progression stems from its ability to influence macrophage polarization. By utilizing 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, colony formation, transwell assays, and enzyme linked immunosorbent assay (ELISA), we investigated how 15d-PGJ2 affects A549 cell viability, proliferation, apoptosis, and invasion, as well as levels of interleukin (IL)-4, IL-13, and IL-17. Human monocytic cell line THP-1 was induced into M2 macrophages using phorbol 12-myristate 13-acetate and IL-4/IL-13, followed by treatment with 15d-PGJ2. The study employed flow cytometry to observe the polarization of macrophages, quantitative reverse transcription polymerase chain reaction (qRT-PCR) to identify epidermal growth factor receptor (EGFR) expression, western blot for identifying expression of macrophage marker proteins, and examining EGFR/rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf) activation. In a coculture setup, CD8+ T cells were shown to have a proliferation capacity by carboxifluorescein diacetate succinimidyl ester (CFSE), a killing ability by lactate dehydrogenase, and an analysis of their interferon gamma and tumor necrosis factor alpha levels by ELISA. 15d-PGJ2 reduced invasion capacity and expression of inflammatory cytokines, lowered A549 cell viability in a dose-dependent way, and promoted apoptosis. 15d-PGJ2 facilitated the transition of M2 macrophages to the M1 type, inhibited Ras/Raf pathway activation, reduced EGFR expression in macrophages, and stimulated CD8+ T cells to enhance anti-tumor immunity. 15d-PGJ2 repressed M2 macrophage polarization and LUAD immune evasion by targeting the EGFR/Ras/Raf pathway in macrophages.

Role of TLR4 activation and signaling in bone remodeling, and afferent sprouting in serum transfer arthritis

BackgroundIn the murine K/BxN serum transfer rheumatoid arthritis (RA) model, tactile allodynia persists after resolution of inflammation in male and partially in female wild type (WT) mice, which is absent in Toll-like receptor (TLR)4 deficient animals. We assessed the role of TLR4 on allodynia, bone remodeling and afferent sprouting in this model of arthritis.MethodsK/BxN sera were injected into male and female mice with conditional or stable TLR4 deletion and controls. Paw swelling was scored and allodynia assessed by von Frey filaments. At day 28, synovial neural fibers were visualized with confocal microscopy and bone density assayed with microCT. Microglial activity and TLR4 dimerization in spinal cords were examined by immunofluorescence and flow cytometry.ResultsIn the synovium, K/BxN injected WT male and female mice showed robust increases in calcitonin gene related-peptide (CGRP+), tyrosine hydroxylase (TH)+ and GAP43+ nerve fibers. Trabecular bone density by microCT was significantly decreased in K/BxN WT female but not in WT male mice. The number of osteoclasts increased in both sexes of WT mice, but not in Tlr4-/- K/BxN mice. We used conditional strains with Cre drivers for monocytes/osteoclasts (lysozyme M), microglia (Tmem119 and Cx3CR1), astrocytes (GFAP) and sensory neurons (advillin) for Tlr4f/f disruption. All strains developed similar arthritis scores after K/BxN serum injection with the exception being the Tlr4Tmem119 mice which showed a reduction. Both sexes of Tlr4Lyz2, Tlr4Tmem119 and Tlr4Cx3cr1 mice displayed a partial reversal of the chronic pain phenotype but not in Tlr4Avil, and Tlr4Gfap mice. WT K/BxN male mice showed increases in spinal Iba1, but not GFAP, compared to Tlr4-/- male mice. To determine whether spinal TLR4 was indeed activated in the K/BxN mice, flow cytometry of lumbar spinal cords of WT K/BxN male mice was performed and revealed that TLR4 in microglia cells (CD11b+ /TMEM119+) demonstrated dimerization (e.g. activation) and a characteristic increase in lipid rafts.ConclusionThese results demonstrated a complex chronic allodynia phenotype associated with TLR4 in microglia and monocytic cell lineages, and a parallel spinal TLR4 activation. However, TLR4 is dispensable for the development of peripheral nerve sprouting in this model.

The Anticancer Activity of Cannabinol (CBN) and Cannabigerol (CBG) on Acute Myeloid Leukemia Cells.

Several cannabis plant-derived compounds, especially cannabinoids, exhibit therapeutic potential in numerous diseases and conditions. In particular, THC and CBD impart palliative, antiemetic, as well as anticancer effects. The antitumor effects include inhibition of cancerous cell growth and metastasis and induction of cell death, all mediated by cannabinoid interaction with the endocannabinoid system (ECS). However, the exact molecular mechanisms are still poorly understood. In addition, their effects on leukemia have scarcely been investigated. The current work aimed to assess the antileukemic effects of CBN and CBG on an acute monocytic leukemia cell line, the THP-1. THP-1 cell viability, morphology and cell cycle analyses were performed to determine potential cytotoxic, antiproliferative, and apoptotic effects of CBN and CBG. Western blotting was carried out to measure the expression of the proapoptotic p53. Both CBN and CBG inhibited cell growth and induced THP-1 cell apoptosis and cell cycle arrest in a dose- and time-dependent manner. CBN and CBG illustrated different dosage effects on THP-1 cells in the MTT assay (CBN > 40 μΜ, CBG > 1 μM) and flow cytometry (CBN > 5 μM, CBG > 40 μM), highlighting the cannabinoids' antileukemic activity. Our study hints at a direct correlation between p53 expression and CBG or CBN doses exceeding 50 μM, suggesting potential activation of p53-associated signaling pathways underlying these effects. Taken together, CBG and CBN exhibited suppressive, cell death-inducing effects on leukemia cells. However, further in-depth research will be needed to explore the molecular mechanisms driving the anticancer effects of CBN and CBG in the leukemia setting.

Triboelectric multilayered electrospun co-polyamide-based wound dressings with inherent antibacterial properties

Abstract Wound healing is a challenging problem to healthcare and society, requiring the development of advanced materials that can enhance tissue rejuvenation and prevent infection. This article presents the design and synthesis of a multilayered copolyamide based electrospun membrane for wound healing applications. The dressing is engineered using a combination of copolymerisation and electrospinning techniques. The unique porous architecture, hydrophilicity, and adequate mechanical and thermal stability make the developed materials ideal candidates for skin regeneration. The inherent antibacterial effectiveness of the membrane was investigated on a range of pathogens that cause wound infections. The cytocompatibility of the membrane was studied on human monocyte cell lines, which play a vital role in immune response and skin regeneration. This multilayered membrane embodies a promising strategy for promoting wound care consequences through its combination of biocompatibility, structural integrity, and inherent antibacterial properties. The potential impact of this research on the field of wound healing is significant and inspiring. Graphical abstract

Humanized anti-CD11d monoclonal antibodies suitable for basic research and therapeutic applications.

Immunomodulatory agents targeting the CD11d/CD18 integrin are in development for the treatment of several pathophysiologies including neurotrauma, sepsis, and atherosclerosis. Murine anti-human CD11d therapeutic antibodies have successfully improved neurological and behavioral recovery in rodent neurotrauma models. Here, we present the progression of CD11d-targeted agents with the development of humanized anti-CD11d monoclonal antibodies. Primary human leukocytes and the THP-1 monocytic cell line were used to determine the binding of the CD11d antibodies, determine binding affinities, and assess outside-in signaling induced by CD11d antibody binding. In addition, a rat model of spinal cord injury was employed to demonstrate that the humanized monoclonal antibodies retained their therapeutic function in vivo. These determinations were made using a combination of flow cytometry, western blotting, immunohistochemistry, biochemical assays, and a locomotor behavioral assessment. Flow cytometric analysis demonstrated that the humanized anti-CD11d clones bind both human monocytes and neutrophils. Using a THP-1 model, the humanized anti-CD11d-2 clone was then determined to bind both the active and inactive CD11d/CD18 conformations without inducing inflammatory cell signaling. Finally, an investigation using anti-CD11d-2 as a detection tool uncovered a mismatch between total and surface-level CD11d and CD18 expression that was not altered by CK2 inhibition. By developing humanized anti-CD11d monoclonal antibodies, new tools are now available to study CD11d biology and potentially treat inflammation arising from acute neurotrauma via CD11d targeting.

Inflammatory Response of THP1 and U937 Cells: The RNAseq Approach.

THP1 and U937 are monocytic cell lines that are common bioassays to reflect monocyte and macrophage activities in inflammation research. However, THP-1 is a human monocytic leukemia cell line, and U937 originates from pleural effusion of histiocytic lymphoma; thus, even though they serve as bioassay in inflammation research, their response to agonists is not identical. Consequently, there has yet to be a consensus about the panel of strongly regulated genes in THP1 and U937 cells representing the inflammatory response to LPS and IFNG. Therefore, we have performed an RNAseq of THP1 and U937 exposed to LPS and IFNG to identify the most sensitive genes and the unique properties of each individual cell line. When applying a highly stringent threshold, we could identify 43, 8 up and 94, 103 down-regulated genes in THP1 and U937 cells, respectively. In THP1 cells, among the most strongly up-regulated genes are CCL1, CXCL2, CXCL3, IL1A, IL1B, IL6, and PTGES. In U937 cells, the strongest up-regulated genes include CSF2, CSF3, CXCL2, CXCL5, CXCL6, IL1A, IL19, IL36G, IL6, ITGA1, ITGA2, and PTGS2. Even though THP1 is considerably less responsive than U937, there are genes commonly upregulated by LPS and IFNG, including the CCL1, CCL3, CCL20, CXCL2, CXCL3, CXCL8, as well as IL1A, IL1B, IL23A, IL6, and genes of prostaglandin synthesis PTGES and PTGS2. Downregulated genes are limited to NRGN and CD36. This head-to-head comparison revealed that THP1 is less responsive than U937 cells to LPS and IFNG and identified a panel of highly regulated genes that can be applied in bioassays in inflammation research. Our data further propose bulk RNAseq as a standard method in bioassay research.

MiR-130a-3p enhances autophagy through the YY1/PI3K/AKT/mTOR signaling pathway to regulate macrophage polarization and alleviate diabetic retinopathy.

Diabetic retinopathy (DR) is a common complication of diabetes that can lead to poor vision and blindness. This study aimed to explore the mechanism of action of miR-130a-3p in DR progression. In this study, we administered a single intraperitoneal injection of 100 mg/kg streptozotocin (STZ) to construct a DR mouse model, and induced a human monocyte cell line (THP-1) to differentiate into M0 macrophages, after which the M0 macrophages were cultured with 30 mM high glucose (HG) as a model of inflammation. The relative gene and protein levels were validated by RT-qPCR and western blotting. Macrophage polarization and retinal damage in the mice were tested using ELISA, MDC staining, immunofluorescence staining, and HE staining. The results revealed that the expression of miR-130a-3p was low in M1 macrophages, whereas the expression of miR-130a-3p was high in M2 macrophages, and the level of miR-130a-3p was reduced after HG treatment of macrophages. The overexpression of miR-130a-3p attenuated HG- or STZ-induced inflammation, promoted macrophage autophagy, inhibited M1 polarization of macrophages, and attenuated the progression of DR. In addition, YY1 was the downstream target gene of miR-130a-3p, and overexpression of miR-130a-3p inhibited YY1 expression. However, overexpression of YY1 weakened the effect of miR-130a-3p mimic. After further treatment with the PI3K/Akt/mTOR pathway activator 740 Y-P, the effect of YY1 knockdown was weakened, macrophage autophagy was inhibited, and M1 polarization and inflammation were promoted. miR-130a-3p inhibited the activation of the PI3K/Akt/mTOR pathway by downregulating YY1 expression, thus facilitating macrophage autophagy, inhibiting M1 polarization and the inflammatory response of macrophages, and finally attenuating the progression of DR. The results of this study provide theoretical support for the use of miR-130a-3p as a new target for the treatment of DR.

Distinct immune microenvironment of venous tumor thrombus in hepatocellular carcinoma at single-cell resolution.

Portal vein tumor thrombus (PVTT) worsens the prognosis of hepatocellular carcinoma by increasing intrahepatic dissemination and inducing portal vein hypertension. However, the immune characteristics of PVTT remain unclear. Therefore, this study aims to explore the immune microenvironment in PVTT. Time-of-flight mass cytometry (CyTOF) revealed that macrophages and monocytes were the dominant immune cell type in PVTT, with a higher proportion than in primary tumor (PT) and blood (54.1% vs. 26.3% and 9.1%, p<0.05). The differentially enriched clustering of inhibitory and regulatory immune cells in PVTT indicated an immune-suppressive environment. According to the single-cell RNA sequencing (scRNA-seq), TAM-C5AR1 was characterized by leukocyte chemotaxis and was the most common subpopulation in PVTT (36.7%). Multiple immunofluorescence staining showed that the C5aR+ TAM/Mφ were enriched in PVTT compared to both PT and liver, and positively correlated with C5a (r=0.559, p<0.001). Notably, THP-1 (monocyte cell line) was recruited by CSQT2 (PVTT cell line) and exhibited up-regulation of CD163, CD206, and PD-L1 upon stimulation. C5aR antagonist could reverse this. C5aR+ TAMs could also inhibit Granzyme B in CD8+ T cells. High infiltration of C5aR+ TAMs in PVTT correlated with poor differentiation (p<0.009), and was a risk factor for overall survival (p=0.003) and for re-formation of PVTT after resection (p=0.007). TAMs, especially C5aR+ TAMs, were enriched in PVTT. C5aR+ TAMs contribute to the development of PVTT and poor prognosis by reshaping the immunosuppressive environment.

LINC01270 Regulates the NF-κB-Mediated Pro-Inflammatory Response via the miR-326/LDOC1 Axis in THP-1 Cells.

Long intergenic noncoding (LINC)01270 is a 2278 bp transcript belonging to the intergenic subset of long noncoding (lnc)RNAs. Despite increased reports of LINC01270's involvement in different diseases, evident research on its effects on inflammation is yet to be achieved. In the present study, we investigated the potential role of LINC01270 in modulating the inflammatory response in the human monocytic leukemia cell line THP-1. Lipopolysaccharide treatment upregulated LINC01270 expression, and siRNA-mediated suppression of LINC01270 enhanced NF-κB activity and the subsequent production of cytokines IL-6, IL-8, and MCP-1. Interestingly, the knockdown of LINC01270 downregulated expression of leucine zipper downregulated in cancer 1 (LDOC1), a novel NF-κB suppressor. An analysis of the LINC01270/micro-RNA (miRNA)/protein interactome profile identified miR-326 as a possible mediator. Synthetic RNA agents that perturb the interaction among LINC01270, miR-326, and LDOC1 mRNA mitigated the changes caused by LINC01270 knockdown in THP-1 cells. Additionally, a luciferase reporter assay in HEK293 cells further confirmed that LINC01270 knockdown enhances NF-κB activation, while its overexpression has the opposite effect. This study provides insight into LINC01270's role in modulating inflammatory responses to lipopolysaccharide stimulation in THP-1 cells via the miR-326/LDOC1 axis, which negatively regulates NF-κB activation.

Chimeric antigen receptor macrophages targeting c-MET(CAR-M-c-MET) inhibit pancreatic cancer progression and improve cytotoxic chemotherapeutic efficacy

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors. Macrophages are abundant in the tumor microenvironment, making them an attractive target for therapeutic intervention. While current immunotherapies, including immune checkpoint inhibition (ICI) and chimeric antigen receptor T (CAR-T) cells, have shown limited efficacy in pancreatic cancer, a novel approach involving chimeric antigen receptor macrophages (CAR-M) has, although promising, not been explored in pancreatic cancer. In this study, we first investigated the role of CAR-M cells targeting c-MET in pancreatic cancer.MethodsThe effectiveness and rationality of c-MET as a target for CAR-M in pancreatic cancer were validated through bioinformatic analyses and immunohistochemical staining of samples from pancreatic cancer patients. We utilized flow cytometry and bioluminescence detection methods to demonstrate the specific binding and phagocytic killing effect of CAR-M on pancreatic cancer cells. Additionally, we observed the process of CAR-M engulfing pancreatic cancer cells using confocal microscopy and a long-term fluorescence live cell imaging system. In an in situ tumor model transplanted into NOD/SCID mice, we administered intraperitoneal injections of CAR-M to confirm its inhibitory function on pancreatic cancer. Furthermore, we validated these findings in human monocyte-derived macrophages (hMDM).ResultsBioinformatics and tumor tissue microarray analyses revealed significantly higher expression levels of c-MET in tumor tissues, compared to the paired peritumoral tissues, and higher c-MET expression correlated with worse patient survival. CAR-M cells were engineered using human monocytic THP-1 cell line and hMDM targeting c-MET (CAR-M-c-MET). The CAR-M-c-MET cells demonstrated highly specific binding to pancreatic cancer cells and exhibited more phagocytosis and killing abilities than the pro-inflammatory polarized control macrophages. In addition, CAR-M-c-MET cells synergized with various cytotoxic chemotherapeutic drugs. In a NOD/SCID murine model, intraperitoneally injected CAR-M-c-MET cells rapidly migrated to tumor tissue and substantially inhibited tumor growth, which did not lead to obvious side effects. Cytokine arrays and mRNA sequencing showed that CAR-M-c-MET produced higher levels of immune activators than control macrophages.ConclusionsThis study provides compelling evidence for the safety and efficacy of CAR-M therapy in treating pancreatic cancer. The results demonstrate that CAR-M-c-MET significantly suppresses pancreatic cancer progression and enhances the effectiveness of cytotoxic chemotherapy. Remarkably, no discernible side effects occur. Further clinical trials are warranted in human pancreatic cancer patients.

P53 Guselkumab binding to CD64+ IL-23–producing myeloid cells enhances potency for neutralizing IL-23 signalling

Abstract IL-23 is implicated in the pathogenesis of psoriasis (PsO), and myeloid cells expressing FcγRI (CD64) are the primary source of IL-23 in lesional PsO skin tissue. The incidence and prevalence of psoriatic arthritis increases with PsO severity, and joint disease activity is positively correlated with frequency of peripheral CD64+ monocytes. We evaluated: CD64 and IL-23 expression in PsO patient skin biopsies; binding of anti-IL-23p19 subunit IgG1 monoclonal antibodies (mAbs) guselkumab [(GUS) fully human, native Fc-region] and risankizumab [(RZB) humanized, mutated Fc-region] to CD64; and functional consequences of CD64 binding by anti-IL-23p19 subunit mAbs in in vitro assays. CD64, IL-23p19 and IL-23p40 subunits mRNA transcripts were analysed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes and IL-23–secreting inflammatory monocytes, and capture of endogenously secreted IL-23, were evaluated by flow cytometry. Internalization of IL-23 and GUS/RZB within CD64+ macrophages was assessed with live-cell confocal imaging. GUS and RZB potency for inhibiting IL-23 was determined in a co-culture of THP-1 cells (CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). IL-23p19 mRNA transcript expression in the co-culture was measured by qPCR. Expression of CD64, IL-23p19, and IL-23p40 mRNA transcripts was increased in lesional vs. non-lesional PsO skin, as were the abundance of myeloid cell types co-expressing CD64/IL-23p19 mRNA transcripts. In in vitro assays, GUS (not RZB) demonstrated Fc-mediated binding to CD64 on IFNγ-primed monocytes. Moreover, CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS (not RZB) bound to the CD64+ macrophages and mediated IL-23 internalization to low pH intracellular compartments. GUS and RZB equipotently inhibited signalling by IL-23 present in THP-1-conditioned medium. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency vs. RZB for inhibiting IL-23 signalling. GUS did not alter IL-23p19 mRNA transcript expression in the co-culture. These findings were consistent with previous observations of CD64+ myeloid cells as a key source of IL-23 in lesional PsO skin tissue. GUS binding to CD64 on IL-23-producing cells likely contributed to the enhanced potency of GUS vs. RZB in inhibiting IL-23 signalling in the co-culture assay. These in-vitro data support a hypothesis for optimal localization of GUS in inflamed tissues, where CD64+ IL-23-producing myeloid cells are increased and in proximity to IL-23-responsive lymphoid cells, enhancing GUS neutralization of IL-23 at its source of production.

A Graphene-Based Bioactive Product with a Non-Immunological Impact on Mononuclear Cell Populations from Healthy Volunteers.

We previously described GMC, a graphene-based nanomaterial obtained from carbon nanofibers (CNFs), to be biologically compatible and functional for therapeutic purposes. GMC can reduce triglycerides' content in vitro and in vivo and has other potential bio-functional effects on systemic cells and the potential utility to be used in living systems. Here, immunoreactivity was evaluated by adding GMC in suspension at the biologically functional concentrations, ranging from 10 to 60 µg/mL, for one or several days, to cultured lymphocytes (T, B, NK), either in basal or under stimulating conditions, and monocytes that were derived under culture conditions to pro-inflammatory (GM-MØ) or anti-inflammatory (M-MØ) macrophages. All stirpes were obtained from human peripheral mononuclear cells (PBMCs) from anonymized healthy donors. The viability (necrosis, apoptosis) and immunological activity of each progeny was analyzed using either flow cytometry and/or other analytical determinations. A concentration of 10 to 60 µg/mL GMC did not affect lymphocytes' viability, either in basal or active conditions, during one or more days of treatment. The viability and expression of the inflammatory interleukin IL-1β in the monocyte cell line THP-1 were not affected. Treatments with 10 or 20 µg/mL GMC on GM-MØ or M-MØ during or after their differentiation process promoted phagocytosis, but their viability and the release of the inflammatory marker activin A by GM-MØ were not affected. A concentration of 60 µg/mL GMC slightly increased macrophages' death and activity in some culture conditions. The present work demonstrates that GMC is safe or has minimal immunological activity when used in suspension at low concentrations for pre-clinical or clinical settings. Its biocompatibility will depend on the dose, formulation or way of administration and opens up the possibility to consider GMC or other CNF-based biomaterials for innovative therapeutic strategies.

Incorporating immune cell surrogates into a full-thickness tissue equivalent of human skin to characterize dendritic cell activation

In the past decades studies investigating the dendritic cell (DC) activation have been conducted almost exclusively in animal models. However, due to species-specific differences in the DC subsets, there is an urgent need for alternative in vitro models allowing the investigation of Langerhans cell (LC) and dermal dendritic cell (DDC) activation in human tissue. We have engineered a full-thickness (FT) human skin tissue equivalent with incorporated LC surrogates derived from the human myeloid leukemia-derived cell line Mutz-3, and DDC surrogates generated from the human leukemia monocytic cell line THP-1. Topical treatment of the skin models encompassing Mutz-LCs only with nickel sulfate (NiSO4) or 1-chloro-2,4-dinitrobenzene (DNCB) for 24 h resulted in significant higher numbers of CD1a positive cells in the dermal compartment, suggesting a sensitizer-induced migration of LCs. Remarkably, exposure of the skin models encompassing both, LC and DDC surrogates, revealed an early sensitizer-induced response reflected by increased numbers of CD1a positive cells in the epidermis and dermis after 8 h of treatment. Our human skin tissue equivalent encompassing incorporated LC and DDC surrogates allows the investigation of DC activation, subsequent sensitizer identification and drug discovery according to the principles of 3R.

Uncovering the Significance of JNK/AKT Axis in the Autophagic Regulation of Leishmania major Infection.

The role of autophagy in host induced by infection of parasites of the Leishmania genus remains inadequately understood. Leishmania parasites modulate host macrophages to promote its survival by inducing autophagy response in the host cell. In this study, we conducted an investigation of L. major infection, focusing on host autophagy processes where we reconstructed two mathematical models elucidating autophagy induction and inhibition processes and its impact on parasite survival. Our models presented systems modulatory dynamics of the parasite-mediated host autophagy. Our work highlighted the pivotal role of signaling molecules associated with the immune response which included signaling induced by Toll-like receptor (TLR), specifically through regulation of JNK and AKT. Both molecules emerged as key regulators of host autophagy process, highlighting that JNK/AKT signaling axis may be a potential avenue for innovative therapeutic approaches in targeting leishmaniasis. Also, ATG16L complex was identified as a critical determinant in shaping the course of leishmanial infection through formation of autophagosomes. Through invitro analyses in differentiated human monocyte cell line, we observed an increase in nitric oxide synthase (iNOS) concentration upon autophagy inhibition, while autophagy induction resulted in decreased iNOS concentration. This suggested that autophagy induction favors parasite survival in the host, potentially by providing a nutrient source that may be advantageous for the parasite. Inhibition of host autophagy promoted parasite elimination. Hence, our work proposed an avenue for strategically blocking host autophagy which enumerates a targeted approach for combating leishmaniasis.

Involvement of Mediterranean fever gene mutations in colchicine-responsive enterocolitis: a retrospective cohort study

The involvement of Mediterranean fever (MEFV) gene mutations in patients with inflammatory bowel disease unclassified (IBDU) remains unclear. This study aimed to determine the clinical characteristics and responsiveness to colchicine in Japanese patients with IBDU carrying MEFV mutations. In this retrospective cohort study, we examined MEFV mutations using gene analysis, clinical information, and colchicine responsiveness. Furthermore, we examined cytokine production in exon 2-mutated THP-1cells (a monocytic cell line) and microbiome analysis. Of the 396 patients diagnosed with IBDU, 60.1% had MEFV mutations. Exon 2 mutations were the most common (83.7%). Among patients with available clinical information, 43.3% of patients with IBDU had typical Familial Mediterranean fever (FMF). The efficacy of colchicine in patients with IBDU carrying MEFV mutations was 84.6%. Significant differences were noted in the production of inflammatory; cytokines between THP-1cells with and without MEFV mutations. Microbial compositions differed between patients with IBDU carrying MEFV mutations and patients with IBD and healthy controls. Patients with IBDU carrying MEFV mutations responded well to colchicine treatment. A notable subset of patients met the criteria for typical FMF. Alterations in intestinal microbiota may contribute to disease pathogenesis. This work was supported by the Japan Agency for Medical Research and Development (21ek0410057h0003), a grant from the Uehara Memorial Foundation, and the Health and Labour Sciences Research Grants for research on intractable diseases from the Ministry of Health, Labour and Welfare (MHLW) of Japan (Investigation and Research for Intractable Inflammatory Bowel Disease; Grant Number 20316729).

Enhanced capture of preeclampsia-derived extracellular vesicles from maternal plasma by monocytes and T lymphocytes.

Released from trophoblast and other fetal cells, placental extracellular vesicles (EVs) reach the maternal peripheral blood and modulate immune responses. Increased EVs in plasma of preeclampsia (PE) patients indicate their involvement in the etiology of this condition. This study addresses the uptake of plasma EVs by peripheral blood mononuclear cells (PBMCs) and explores the underlying internalization mechanisms. Plasma EVs were isolated from women with normotensive pregnancy (EVNP) and those with PE (EVPE), and characterized by cryo-transmission electron microscopy, nanoparticle tracking analysis, Western blotting, flow cytometry, and micro bicinchoninic acid assay (micro-BCA). To investigate whether the origin of PBMCs affects uptake, samples from males, pregnant women, and non-pregnant women were included. Primary PBMCs and macrophages derived from the human leukemia monocytic cell line THP-1 were incubated with PKH-stained EVs, and uptake was assessed by flow cytometry and confocal microscopy. Key molecules involved in monocyte differentiation and macrophage function were evaluated in EV-treated cells using LEGENDplex™ assay and real-time polymerase chain reaction (RT-PCR). Independent of the PBMC source, EVs were mostly captured by monocytes and in a lower proportion by T lymphocytes. Capture of EVPE was higher than of EVNP in primary T lymphocytes, monocytes, and THP-1-derived macrophages. After inhibition by Wortmannin and Cytochalasin D, EV internalization by THP-1-derived macrophages was significantly inhibited but not completely abolished. No defined polarization profile of treated THP-1-derived macrophages could be identified. These findings provide evidence of EV modifications in PE, which enhance their uptake by monocytes and other immune cells, mainly through phagocytosis and endocytosis.