Induction Of Interleukin Research Articles

Rab7l1 plays a role in regulating surface expression of toll like receptors and downstream signaling in activated macrophages

Rab GTPases are known for controlling intracellular membrane traffic in a GTP-dependent manner. Rab7l1, belonging to family of Rab GTPases, is important for both endosomal sorting and retrograde transport. In our previous study, we identified a novel role of Rab7l1 in phagosome maturation. However, its role in regulating macrophage innate-effector signaling and cytokine response is not clearly understood. In this study, we have demonstrated that upon treatment of Rab7l1-knocked-down (Rab7l1-KD) THP-1 macrophages with lipopolysaccharide (LPS) and Pam3CSK4 has led to higher induction levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) as compared to the control cells that received scrambled shRNA. Similar results were observed in Rab7l1-KD RAW 264.7 and Balb/c peritoneal macrophages. The phospho-ERK 1/2 (extracellular signal-regulated kinase 1/2) and phospho-p38 MAPK (mitogen-activated protein kinase) levels, known to be responsible for higher induction of TNF-α and IL-10 respectively, were higher in Rab7l1-KD THP-1 macrophages which also displayed higher nuclear translocation of p50/p65 nuclear factor kappa B (NF-κB) upon stimulation with LPS. Surface expression levels of toll-like receptor 2 (TLR2), TLR4 and CD14 receptors were higher in Rab7l1-KD THP-1 macrophages as compared to the control cells. However, intracellular levels of these receptors were lower in Rab7l1-KD THP-1 macrophages as compared to the control group. Together, our study suggests that Rab7l1 has a role in regulating MAPK signaling and cytokine effector responses in macrophages by regulating the surface expression of membrane receptors.

Abstract 13269: Effects of Nicotine-Containing Product Challenges on Interleukin-6 Pathway Expression in Human Pluripotent-Derived Arterial Endothelial Cells: The Clues Study

Background: Electronic nicotine delivery devices (ENDs) induce interleukin-6 (IL-6) pathway expression in airway epithelial cells, but their systemic effects on arterial endothelial cells (AECs) are unknown. We hypothesized differential expression (DE) of IL-6 pathway genes by human pluripotent-derived AECs exposed in vitro to participant serum drawn before and after use of ENDS by chronic vapers, use of combustible cigarettes by smokers, and no product use by controls. Methods: For this pilot study, we randomly selected 30 participants from the CLUES Study: 10 exclusive vapers (exhaled carbon monoxide [CO] <5 ppm, + urine NicCheck I), 10 exclusive smokers (CO >5 ppm, + urine), and 10 non-vaping/non-smoking controls (CO <5 ppm, negative urine). We generated purified populations of AECs (H9 CPTC C13 derivative human embryonic stem cell line) and exposed them for 6 hours to 50% concentrated participant serum drawn before and 15’ after a product use challenge. Total RNA was sequenced on a NextSeq 2000. The DESeq2 suite characterized DE with an a priori focus on genes in the IL-6 biosynthetic pathway gene ontology set. Z scores of normalized log2 gene expression values were determined and expressed as heat maps (Figure). Gene Set Variational Analysis (GSVA) was used to interrogate for enriched gene sets. P values were not adjusted for multiple hypothesis testing and are exploratory. Results: Vapers were a mean (SD) 28.9 (8.1) years old (30% female), smokers were 38.3 (11.6) years old (40% female), and controls were 33.6 (17.7) years old (50% female). Compared to controls, vapers had increased expression of the IL-6 pathway at baseline (p=0.004), but smokers did not (p=0.38). Following product use, vapers (p=0.0009) but not smokers (p=0.79) had increased expression of genes in the IL-6 biosynthetic pathway (Figure). Conclusion: Chronic vapers, but not smokers, may have increased expression of genes in the IL-6 pathway prior to product use with further increases afterwards.

Anti-diabetic effects of GLP1 analogs are mediated by thermogenic interleukin-6 signaling in adipocytes

Mechanisms underlying anti-diabetic effects of GLP1 analogs remain incompletely understood. We observed that in prediabetic humans exenatide treatment acutely induces interleukin-6 (IL-6) secretion by monocytes and IL-6 in systemic circulation. We hypothesized that GLP1 analogs signal through IL-6 in adipose tissue (AT) and used the mouse model to test if IL-6 receptor (IL-6R) signaling underlies the effects of the GLP1-IL-6 axis. We show that liraglutide transiently increases IL-6 in mouse circulation and IL-6R signaling in AT. Metronomic liraglutide treatment resulted in AT browning and thermogenesis linked with STAT3 activation. IL-6-blocking antibody treatment inhibited STAT3 activation in AT and suppressed liraglutide-induced increase in thermogenesis and glucose utilization. We show that adipose IL-6R knockout mice still display liraglutide-induced weight loss but lack thermogenic adipocyte browning and metabolism activation. We conclude that the anti-diabetic effects of GLP1 analogs are mediated by transient upregulation of IL-6, which activates canonical IL-6R signaling and thermogenesis.

Role of mucus-bacteria interactions in Enterotoxigenic Escherichia coli (ETEC) H10407 virulence and interplay with human microbiome

The intestinal mucus layer has a dual role in human health constituting a well-known microbial niche that supports gut microbiota maintenance but also acting as a physical barrier against enteric pathogens. Enterotoxigenic Escherichia coli (ETEC), the major agent responsible for traveler’s diarrhea, is able to bind and degrade intestinal mucins, representing an important but understudied virulent trait of the pathogen. Using a set of complementary in vitro approaches simulating the human digestive environment, this study aimed to describe how the mucus microenvironment could shape different aspects of the human ETEC strain H10407 pathophysiology, namely its survival, adhesion, virulence gene expression, interleukin-8 induction and interactions with human fecal microbiota. Using the TNO gastrointestinal model (TIM-1) simulating the physicochemical conditions of the human upper gastrointestinal (GI) tract, we reported that mucus secretion and physical surface sustained ETEC survival, probably by helping it to face GI stresses. When integrating the host part in Caco2/HT29-MTX co-culture model, we demonstrated that mucus secreting-cells favored ETEC adhesion and virulence gene expression, but did not impede ETEC Interleukin-8 (IL-8) induction. Furthermore, we proved that mucosal surface did not favor ETEC colonization in a complex gut microbial background simulated in batch fecal experiments. However, the mucus-specific microbiota was widely modified upon the ETEC challenge suggesting its role in the pathogen infectious cycle. Using multi-targeted in vitro approaches, this study supports the major role played by mucus in ETEC pathophysiology, opening avenues in the design of new treatment strategies.

Immunoglobulin A promotes IL-6 and IL-8 production, proliferation, and migration by the human bronchial smooth muscle cells

Immunoglobulin A (IgA) is important in biological defense, mainly in the mucosal area, and plays pathogenic roles in various diseases by activating both inflammatory and structural cells. The current study aimed to validate the effects of IgA on the human bronchial smooth muscle cell (BSMC), which plays a major role in airway inflammation and remodeling. Serum IgA induced interleukin (IL)-6 and IL-8 production at both mRNA and protein levels, and enhanced cell proliferation and migration by the BSMCs. The synthetic phenotype markers were regulated and the contractile phenotype markers were downregulated by serum IgA. Mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and nuclear factor-κB pathways were involved in IgA-induced IL-6 and IL-8 production. The BSMCs expressed transferrin receptor (TfR), and TfR siRNA transfection inhibited IL-6 and IL-8 production by serum IgA. In summary, serum IgA is a potent activator of the BSMCs at least partially via TfR.

Human Monocyte-Derived Dendritic Cells Are the Pharmacological Target of the Immunosuppressant Flavonoid Silibinin.

Silibinin, a natural polyphenolic flavonoid, is known to possess anti-inflammatory, anticancer, antioxidant, and immunomodulatory properties. However, the effects of Silibinin on the maturation and immunostimulatory functions of human dendritic cells (DC) remain to be elucidated. In this study, we have attempted to ascertain whether Silibinin influences the maturation, cytokine production, and antigen-presenting capacity of human monocyte-derived DC. We show that Silibinin significantly suppresses the upregulation of costimulatory and MHC molecules in LPS-stimulated mature DC and inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-12, IL-23, and TNF-α production. Furthermore, Silibinin impairs the proliferation response of the allogenic memory CD4 T lymphocytes elicited by LPS-matured DC and their Th1/Th17 profile. These findings demonstrate that Silibinin displays immunosuppressive activity by inhibiting the maturation and activation of human DC and support its potential application of adjuvant therapy in the treatment of autoimmune diseases.

Sub-toxic events induced by truck speed-facilitated PM2.5 and its counteraction by epigallocatechin-3-gallate in A549 human lung cells

To distinguish the influences of fuel type and truck speed on chemical composition and sub-toxic effects of particulates (PM2.5) from engine emissions, biomarkers—interleukin-6 (IL-6), cytochrome P450 (CYP) 1A1, heme oxygenase (HO)-1, and NADPH-quinone oxidoreductase (NQO)-1—were studied in A549 human lung cells. Fuel type and truck speed preferentially affected the quantity and ion/polycyclic aromatic hydrocarbon (PAH) composition of PM2.5, respectively. Under idling operation, phenanthrene was the most abundant PAH. At high speed, more than 50% of the PAHs had high molecular weight (HMW), of which benzo[a]pyrene (B[a]P), benzo[ghi]perylene (B[ghi]P), and indeno[1,2,3-cd]pyrene (I[cd]P) were the main PAHs. B[a]P, B[ghi]P, and I[cd]P caused potent induction of IL-6, CYP1A1, and NQO-1, whereas phenanthrene mildly induced CYP1A1. Based on the PAH-mediated induction, the predicted increases in biomarkers were positively correlated with the measured increases. HMW-PAHs contribute to the biomarker induction by PM2.5, at high speed, which was reduced by co-exposure to epigallocatechin-3-gallate.

SARS CoV-2 detected in neonatal stool remote from maternal COVID-19 during pregnancy.

BackgroundIn utero transmission of SARS coronavirus 2 (SARS-CoV-2) has not been fully investigated. We investigated whether newborns of mothers with COVID-19 during pregnancy might harbor SARS-CoV-2 in the gastrointestinal tract.MethodsThis cohort study investigated stool from 14 newborns born at 25–41 weeks admitted at delivery to our urban academic hospital whose mothers had COVID-19 during pregnancy. Eleven mothers had COVID-19 resolved more than 10 weeks before delivery. Newborn stool was evaluated for SARS-CoV-2 RNA, Spike protein, and induction of inflammatory cytokines interleukin-6 (IL-6) and interferon-γ (IFN-γ) in macrophages.ResultsDespite negative SARS CoV-2 nasal PCRs from all newborns, viral RNAs and Spike protein were detected in the stool of 11 out of 14 newborns as early as the first day of life and increased over time in 6. Stool homogenates from all 14 newborns elicited elevated inflammatory IL-6 and IFN-γ from macrophages. Most newborns were clinically well except for one death from gestational autoimmune liver disease and another who developed necrotizing enterocolitis.ConclusionsThese findings suggest in utero transmission of SARS-CoV-2 and possible persistent intestinal viral reservoirs in the newborns. Further investigation is required to understand the mechanisms and their clinical implications.Impact SARS-CoV-2 RNAs or Spike protein was detected in the stool of 11 out of 14 preterm newborns born to mothers with resolved COVID-19 weeks prior to delivery despite negative newborn nasal PCR swabs.These novel findings suggest risk of in utero SARS-CoV-2 transmission to the fetal intestine during gestation.The presence of SARS-CoV-2 RNAs and Spike protein in the intestines of newborns may potentially impact the development of the gut microbiome and the immune system; the long-term health impact on the preterm infants should be further investigated.

Early-Stage Identification and Avoidance of Antisense Oligonucleotides Causing Species-Specific Inflammatory Responses in Human Volunteer Peripheral Blood Mononuclear Cells.

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.

Neuroprotection against ischemic stroke requires a specific class of early responder T cells in mice.

Immunomodulation holds therapeutic promise against brain injuries, but leveraging this approach requires a precise understanding of mechanisms. We report that CD8+CD122+CD49dlo T regulatory-like cells (CD8+ TRLs) are among the earliest lymphocytes to infiltrate mouse brains after ischemic stroke and temper inflammation; they also confer neuroprotection. TRL depletion worsened stroke outcomes, an effect reversed by CD8+ TRL reconstitution. The CXCR3/CXCL10 axis served as the brain-homing mechanism for CD8+ TRLs. Upon brain entry, CD8+ TRLs were reprogrammed to upregulate leukemia inhibitory factor (LIF) receptor, epidermal growth factor–like transforming growth factor (ETGF), and interleukin 10 (IL-10). LIF/LIF receptor interactions induced ETGF and IL-10 production in CD8+ TRLs. While IL-10 induction was important for the antiinflammatory effects of CD8+ TRLs, ETGF provided direct neuroprotection. Poststroke intravenous transfer of CD8+ TRLs reduced infarction, promoting long-term neurological recovery in young males or aged mice of both sexes. Thus, these unique CD8+ TRLs serve as early responders to rally defenses against stroke, offering fresh perspectives for clinical translation.

Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity.

Many bacterial pathogens antagonize host defense responses by translocating effector proteins into cells. It remains an open question how those pathogens not encoding effectors counteract anti-bacterial immunity. Here, we show that Klebsiella pneumoniae exploits the evolutionary conserved innate protein SARM1 to regulate negatively MyD88- and TRIF-governed inflammation, and the activation of the MAP kinases ERK and JNK. SARM1 is required for Klebsiella induction of interleukin-10 (IL-10) by fine-tuning the p38-type I interferon (IFN) axis. SARM1 inhibits the activation of Klebsiella-induced absent in melanoma 2 inflammasome to limit IL-1β production, suppressing further inflammation. Klebsiella exploits type I IFNs to induce SARM1 in a capsule and lipopolysaccharide O-polysaccharide-dependent manner via the TLR4-TRAM-TRIF-IRF3-IFNAR1 pathway. Absence of SARM1 reduces the intracellular survival of K.pneumoniae in macrophages, whereas sarm1-deficient mice control the infection. Altogether, our results illustrate an anti-immunology strategy deployed by a human pathogen. SARM1 inhibition will show a beneficial effect to treat Klebsiella infections.

Oral intake of xanthohumol attenuates lipoteichoic acid-induced inflammatory response in human PBMCs

PurposeThe aim of the study was to determine if xanthohumol, a prenylated chalcone found in Hop (Humulus lupulus), has anti-inflammatory effects in healthy humans if applied in low doses achievable through dietary intake.MethodsIn a placebo-controlled single-blinded cross-over design study, 14 healthy young men and women either consumed a beverage containing 0.125 mg xanthohumol or a placebo. Peripheral blood mononuclear cells (PBMCs) were isolated before and 1 h after the intake of the beverages. Subsequently, PBMCs were stimulated with or without lipoteichoic acid (LTA) for 24 and 48 h. Concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6) and soluble cluster of differentiation (sCD14) protein were determined in cell culture supernatant. Furthermore, hTLR2 transfected HEK293 cells were stimulated with LTA in the presence or absence of xanthohumol and sCD14.ResultsThe stimulation of PBMCs with LTA for 24 and 48 h resulted in a significant induction of IL-1β, IL-6, and sCD14 protein release in PBMCs of both, fasted subjects and subjects after the ingestion of the placebo. In contrast, after ingesting xanthohumol, LTA-dependent induction of IL-1β, IL-6, and sCD14 protein release from PBMCs was not significantly higher than in unstimulated cells after 48 h. In hTLR2 transfected HEK293 cells xanthohumol significantly suppressed the LTA-dependent activation of cells, an effect attenuated when cells were co-incubated with sCD14.ConclusionThe results of our study suggest that an ingestion of low doses of xanthohumol can suppress the LTA-dependent stimulation of PBMCs through mechanisms involving the interaction of CD14 with TLR2. Study registered at ClinicalTrials.gov (NCT04847193, 22.03.2022).

Innate immune activation as cofactor in pemphigus disease manifestation.

Molecular mechanisms underlying auto-antibody-induced acantholysis in pemphigus vulgaris are subject of current research to date. To decipher the discrepancy between ubiquitous antibody binding to the epidermal desmosomes, but discontinuous disease manifestation, we were able to identify Ultraviolet A (UVA) as a cofactor for acantholysis. UVA induces interleukin (IL)-1 secretion in keratinocytes, mirroring innate immune system activation. In an in vitro keratinocyte dissociation assay increased fragmentation was observed when UVA was added to anti-Desmoglein 3 Immunoglobulins (anti-Dsg3 IgG). These results were confirmed in skin explants where UVA enhanced anti-Dsg3-mediated loss of epidermal adhesion. The UVA-mediated effect was blocked in vitro by the pan-caspase-inhibitor zVAD-fmk. Thus, we introduce UVA as a caspase-dependent exogenous cofactor for acantholysis which suggests that local innate immune responses largely contribute to overt clinical blister formation upon autoantibody binding to epidermal cells in pemphigus vulgaris.

Abstract 1318: Interleukin-1 promotes enhanced functional expression of dual oxidase 2 in human pancreatic cancer cells

Abstract Pancreatic inflammation contributes to increased risk of pancreatic adenocarcinoma (PDAC) development through the release of pro-inflammatory cytokines and generation of reactive oxygen species (ROS). Interleukin-1 (IL-1) is a major upstream pro-inflammatory cytokine secreted by malignant or microenvironmental cells that supports pancreatic tumorigenesis, invasiveness, and tumor heterogeneity. Previously, our laboratory has established that dual oxidase 2 (DUOX2), one of seven NADPH oxidase (NOX) family members, plays a critical role in H2O2-mediated host defense and chronic inflammation in the gastrointestinal tract. DUOX2 is upregulated and expressed along with its maturation factor, DUOXA2, in several human pancreatic cancer cell lines following exposure to various pro-inflammatory cytokines, including IFN-γ, IL-4, and IL-17A. Using a panel of PDAC cells lines, we report the induction of DUOX2/DUOXA2 mRNA and protein expression by both IL-1α and IL-1ß, two major agonists of IL-1. In BxPC-3, CFPAC-1 and Hs 766T (HTB-134) cells, prolonged treatment with IL-1α or IL-1ß results in transient activation of STAT1 and NF-kB signaling followed by prolonged induction of DUOX2/DUOXA2 mRNA and protein expression. Stimulation by IL-1α or IL-1ß results in a specific increase in the expression of DUOX2/DUOXA2; increased expression of other NOX family members was not observed. Activation of canonical IL-1 signaling in our PDAC cell lines was supported by the observation of IL-1-related upregulation of IL-8 (CXCL8) mRNA, a chemokine essential for neutrophil recruitment and a potent promoter of angiogenesis, in IL-1-treated PDAC cells. Moreover, when the anti-inflammatory compound anakinra (an IL-1R antagonist) was co-administered in vitro with IL-1, cytokine-induced DUOX2/DUOXA2 and CXCL8 induction was significantly suppressed. Finally, xenografts established with BxPC-3 cells demonstrated slower growth and significant downregulation of IL-1ß and DUOX2 when the immunosuppressive corticosteroid dexamethasone was administered. Given our previous findings that DUOX2 is highly expressed in the pancreas during episodes of chronic pancreatitis or pancreatic cancer, and contributes significantly to extracellular ROS formation, these studies suggest that IL-1 may play a role in promoting inflammation and tumorigenesis in pancreatic malignancies through upregulation of DUOX2. Citation Format: David J. Mallick, Yongzhong Wu, Mariam M. Konaté, Becky A. Diebold, Smitha Antony, Jennifer L. Meitzler, Guojian Jiang, Jiamo Lu, Krishnendu Roy, James H. Doroshow. Interleukin-1 promotes enhanced functional expression of dual oxidase 2 in human pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1318.

Abstract 3210: Oncostatin M modulates tumor-fibroblast crosstalk in pancreatic cancer without protecting against cachexia

Abstract Cancer cachexia is the involuntary loss of body weight, skeletal muscle, and adipose tissue associated with disease. Cachexia affects 85% of pancreatic cancer patients and patients suffer the greatest average weight loss among solid cancers. Inflammation is a key driver in cancer cachexia, specifically the Interleukin-6/GP130 family of cytokines that activate signaling of the JAK/STAT pathway. Among family members, Interleukin-6 (IL-6) and Leukemia Inhibitory Factor (LIF) promote cachexia and pancreatic cancer; however, less is known of the related Oncostatin M. Here we show cachexia and elevated circulating Osm coincide in the KrasG12D;Trp53R172H;Pdx1:Cre genetically engineered mouse model (KPC-GEMM) of pancreatic cancer and in mice bearing KPC cells implanted orthotopically. Osm expression was elevated in cardiac muscle and adipose tissue in KPC cachexia, while OSM receptor (Osmr) was elevated in cardiac and skeletal muscle and liver, indicating a systemic OSM circuit. Consistent with a feed-forward loop, KPC cells treated with OSM upregulated expression of OSMR. RNA-sequencing of OSM treated KPC and cancer-associated fibroblast (CAF) cell lines revealed activation of inflammation, fibrosis, and tumor progression pathways, including induction of IL-6. Functionally, exogenous OSM caused cardiac dysfunction, local muscle atrophy, fibrosis, and systemic bone loss in wildtype and Il6 null mice, suggesting OSM might mediate cachexia independent of IL-6. We hypothesized that deletion of OSM would be protective in cancer cachexia. Thus 14-week-old WT and Osm null mice were orthotopically implanted with KPC cells or underwent sham surgery; all groups were euthanized when one reached 5% fat mass. KPC tumors caused muscle weakness, body deconditioning and reduced activity as well as anemia and hematological inflammation. These findings were not genotype specific. There were also no differences in KPC-induced cardiac, skeletal muscle or fat wasting, nor in splenomegaly, organ wasting, or tumor size between WT and Osm null mice. Similarly, Osmr null mice showed no differences in KPC cachexia phenotypes versus wildtype mice. Nevertheless, tumors from Osm null mice showed alterations in the stromal compartment, including reduction in collagens I and III, decreased alpha smooth muscle actin staining, and altered mitochondrial metabolism and cellular composition by proteomics and RNAseq. Consistent with a role for OSM in modulating the stroma, excess OSM induced compaction of tumor cells and CAFs in 3D co-cultures. Together these data indicate that OSM signaling influences tumor-fibroblast crosstalk in the pancreatic cancer microenvironment but is not necessary for cachexia in the macroenvironment. Citation Format: Daenique H. Jengelley, Ashok Narasimhan, Joseph E. Rupert, Xiaoling Zhong, Andrew R. Young, Leonidas G. Koniaris, Teresa A. Zimmers. Oncostatin M modulates tumor-fibroblast crosstalk in pancreatic cancer without protecting against cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3210.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Activation of proteinase-activated receptor-2 (PAR-2) simultaneously induces interleukin 8 (IL-8) and suppresses monocyte chemoattractant protein-1 (MCP-1) in HaCaT cells

Allergic diseases are currently considered diseases of excessive type 2 inflammation created by orchestration between the innate and acquired immune systems. Since pattern recognition receptors (PRRs) are present in epidermal keratinocytes, it is noteworthy that aggravating factors of allergic diseases act directly on keratinocytes via PRRs. To investigate the relationship between the activation of PRRs and inflammation, we stimulated a keratinocyte cell line (HaCaT cells) with agonists against proteinase-activated receptor-2 (PAR-2), Toll-like receptor (TLR)2, and TLR4, alone or in combination, and we evaluated the changes in inflammatory cytokines and chemokines. Activation of TLR2 or TLR4 alone induced interleukin 6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) in an agonist concentration-dependent manner. Simultaneous activation of TLR2 and TLR4 induced IL-8 synergistically, MCP-1 in an additive trend, and IL-6 weakly but synergistically. PAR-2 activation of HaCaT cells induced IL-6 and IL-8 but suppressed MCP-1 in an agonist concentration-dependent manner. The enhancement of IL-8 and the suppression of MCP-1 by PAR-2 activation were both neutralized by the PAR-2 antagonist AZ3451, supporting the possibility that PAR-2 activation simultaneously induces the following opposing effects in inflammation: enhancement of IL-8 and suppression of MCP-1. The nuclear factor-κB (NF-κB) pathway inhibitor BAY 11-7082 neutralized the induction of IL-8 but not the suppression of MCP-1 by PAR-2 activation, indicating that PAR-2 activation induces activation of the NF-κB pathway, and that the suppression of MCP-1 by PAR-2 activation is not related to the NF-κB pathway.

Dual Effect of the Extract from the Fungus Coriolus versicolor on Lipopolysaccharide-Induced Cytokine Production in RAW 264.7 Macrophages Depending on the Lipopolysaccharide Concentration.

PurposeExtract from the fungus Coriolus versicolor (CV) is classified as an immunological response modifier. Previously, we have shown that this extract induces interleukin 6 (IL-6)-related extension of lipopolysaccharide (LPS)-induced fever. This study investigated the effect of CV extract on the production of pro-inflammatory cytokines and the expression of components of signal transduction pathways leading to the secretion of cytokines from RAW 264.7 macrophages stimulated with different doses of LPS.MethodsRAW 264.7 cells were stimulated with CV extract alone or co-treated with CV extract and LPS. The level of IL-6 and tumour necrosis factor α (TNF-α) in the culture media was measured using ELISA. Protein expression of Toll-like receptor (TLR) 4, phosphorylated IκB (p-IκB), CD14 glycoprotein and phospho-phosphatidylinositol 3-kinase (p-PI3K) was evaluated using Western blot. The effects of TLR4, nuclear factor κB (NF-κB) and p-PI3K on cytokine secretion were estimated using inhibitors: TAK-242, JSH-23 and LY294002.ResultsCV extract itself stimulates the secretion of IL-6 and TNF-α and increases the expression of TLR4, p-IκB and p-PI3K. The presence of CV extract during the treatment of cells with lower concentrations of LPS (10 and 100 ng/mL) increases the cytokine production. Co-stimulation of cells with CV extract and LPS at a higher dose (500 ng/mL) decreases the secretion of cytokines. This effect is related to the changes in the expression of TLR4, CD14 glycoprotein, p-IκB and p-PI3K.ConclusionThis is the first report showing that the CV extract-induced production of cytokines is mediated by the PI3K signalling pathway. This extract acts antagonistically or additively with LPS on the production of IL-6 and TNF-α, depending on the LPS concentration. Our results are helpful for illustrating the mechanisms for the immunostimulatory effect of CV extract in inflammatory processes.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

SUMMARYExternal and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

Mycobacterium avium-intracellulare complex promote release of pro-inflammatory enzymes matrix metalloproteinases by inducing neutrophil extracellular trap formation

The prevalence of and mortality from non-tuberculous mycobacteria (NTM) infections have been steadily increasing worldwide. Most NTM infections are caused by Mycobacterium avium-intracellulare complex (MAC). MAC can escape from killing by neutrophils, which are professional phagocytes. However, the involvement of neutrophils in the pathogenesis of MAC infection is poorly understood. The present study assessed the roles of neutrophil extracellular trap (NET) formation in neutrophil defense mechanisms against infection with MAC strains, including M. avium isolated from patients with severe or mild lung tissue destruction. Although all MAC induced NET formation, non-pathogenic mycobacteria (M. gordonae and M. smegmatis) slightly but not significantly induced NET formation. Peptidylarginine deiminase 4 (PAD4) inhibitor reduced MAC-induced NET formation but did not affect MAC escape from neutrophils. PAD4 inhibition attenuated the MAC-induced matrix metalloproteinase (MMP)-8 and 9 release to the levels of MMPs from non-pathogenic mycobacteria. MAC also induced interleukin (IL)-8 release by neutrophils, a process independent of MAC-induced NET formation. Taken together, these findings suggest that MAC induce NET formation, IL-8 release and NETs-dependent release of MMP-8 and -9 from neutrophils, leading to neutrophil accumulation and further inflammation, thereby enhancing the progression of infection in the lungs.