Secretion Of Chemokines Research Articles

LECs regulate neutrophil clearance through IL-17RC/CMTM4/NF-κB axis at sites of inflammation or infection

The lymphatic system plays a vital role in the regulation of tissue fluid balance and the immune response to inflammation or infection. The effects of lymphatic endothelial cells (LECs) on the regulation of neutrophil migration have not been well studied. In three murine models: imiquimod-induced skin inflammation, S. aureus-induced skin infection, and ligature-induced periodontitis, we show that numerous neutrophils migrate from inflamed or infected tissues to the draining lymph nodes via lymphatic vessels. Moreover, inflamed or infected tissues express a high level of IL-17A and TNF-α, simultaneously with a significant increase in the release of neutrophil attractors, including CXCL1, CXCL2, CXCL3, and CXCL5. Importantly, in vitro stimulation of LECs with IL-17A plus TNF-α synergistically promoted these chemokine secretion. Mechanistically, tetra-transmembrane protein CMTM4 directly binds to IL-17RC in LECs. IL-17A plus TNF-α stimulates CXC chemokine secretion by promoting NF-κB signaling. In contrast, knockdown of CMTM4 abrogates IL-17A plus TNF-α activated NF-κB signaling pathways. Lastly, the local administration of adeno-associated virus for CMTM4 in Prox1-CreERT2 mice, mediating LEC-specific overexpression of CMTM4, promotes the drainage of neutrophils by LECs and alleviates immune pathological responses. Thus, our findings reveal the vital role of LECs-mediated neutrophil attraction and clearance at sites of inflammation or infection.

Role of MNK/eIF4E pathway in CD4+ Th2 cytokine-induced lung fibrosis in chronic allergic asthma

Abstract Despite therapeutic advances in allergic asthma, the persistent challenge of airway remodeling still exists, necessitating further exploration. We aimed to investigate the influence of CD4+ Th2 cytokines on fibrosis associated with airway remodeling, with a focus on mesenchymal cell (MC) transformation. Using both human and mouse lung MCs, we investigated the pivotal role of MNK/eIF4E pathway in regulating fibrous protein expression driven by CD4+ Th2 cytokines. To assess this, we employed western blot and flow cytometry studies, while Luminex assays measured cytokine and chemokine secretion from activated lung MCs responding to Th2 cytokines. Treatment with Th2 cytokines significantly increased ATX expression in lung MCs, a response mitigated by the specific MNK1/2 inhibitor (eFT-508). Furthermore, this inhibitor not only abolished the phosphorylation of eIF4E but also reduced collagen levels within activated MCs induced by Th2 cytokines. Luminex assay results demonstrated a significant decrease in IL-6, CCL-2, and GM-CSF in the activated MCs upon inhibiting the MNK1/2 pathway. Additionally, eFT-508 exhibited a marked reduction in lymphocyte migration, utilizing the Boyden chamber transwell assay. Our study reveals that targeting the MNK/eIF4E pathway holds promise in mitigating airway remodeling associated with MCs fibrosis and provides valuable molecular insights for future therapeutic interventions related to fibrosis in the airway remodeling of allergic asthma.

Elucidating the Role of SYK in the Intestinal Epithelium

Background: Inflammatory Bowel Disease (IBD) involves chronic and recurring inflammation of the gastrointestinal tract. We identified several patients presenting with immune dysregulation and systemic inflammation, including inflammation of the gut. Analysis of Whole Exome Sequencing data from pediatric IBD patients has identified five rare protein coding Spleen Tyrosine Kinase (SYK) variants. Functional studies have demonstrated increased kinase activity implicating them in disease pathophysiology. While the role of SYK is well characterized in immune cells, it is not understood what role it plays in the intestinal epithelium. Aims: The primary aim of this project is to understand the role SYK plays in the intestinal epithelium, what molecular pathways it signals through, and how its dysregulation leads to intestinal disease. Methods: To understand SYKs role in the intestinal epithelium, a BioID screen was used to identify novel SYK interacting proteins. BioID was done with wild type SYK as well as the patient derived variants in HEK293 cells. BioID hits were validated using co-immunoprecipitation (CoIP) and proximity ligation assay (PLA). Further functional analysis was done in Caco2 cells, an intestinal epithelial cell model, with western blot, ELISA, and luciferase assays to assess the effect of SYK activity on downstream inflammatory pathways. Finally, we investigated the effect of small molecule kinase inhibitors to explore possible novel treatment options. Results: The BioID screen revealed several high confidence interactors, with dysregulated interactions in the patient variants. USP25, a ubiquitin specific peptidase that is widely expressed, including in the intestinal epithelium, was identified as one of the strongest hits based on peptide counts. The interaction was validated with CoIP and PLA, and further analysis showed SYK activation decreased USP25 protein levels by up to eight-fold. The secretion of the proinflammatory chemokine, IL-8, in response to lipopolysaccharide (LPS) was found to be almost twice as high in Caco2 cells with patient SYK variants relative to WT. Cells expressing the patient variants of SYK showed increased NFkB and AP1 levels, indicating increased inflammatory gene expression when stimulated with 100ng/mL LPS. NFkB was also active for longer in cells expressing the patient variant of SYK post stimulation, with WT cells returning to baseline in under half an hour, while the patient variant stayed in an inflamed state for 36 to 48 hours after LPS was removed. Conclusions: Increased SYK activity is associated with the degradation of USP25. USP25 is known to cleave ubiquitin off TNF receptor associated factors (TRAFs). TRAFs are activated downstream of innate immune receptors such as TLR4, and need to be ubiquitinated for activation. USP25, which is responsible for the deubiquitination of these TRAFs, acts as a negative regulator of these pathways. The hyperactive SYK variants degrade USP25 and impair the cells’ ability to switch off inflammatory signaling pathways, indicated by the cells elongated period in an inflamed state. This research is funded by grants from Canadian Institute for Health Research (CIHR) and the Leona M. and Harry B. Helmsley Charitable Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulation of neuroinflammation by interleukin-10 in mouse microglial cells

Abstract Microglia are resident macrophages of the central nervous system that are important for maintaining brain homeostasis. Persistent activation of microglia is associated with neuroinflammation in neurodegenerative disorders. Interest in immortalized microglia cell lines as cell culture models to study their biology and role in neuroinflammation has increased in the past decade, with mouse microglial BV-2 cells emerging as an in vitro model for neuronal inflammation similar to primary mouse microglial cells. We report here IL-10 pretreatment of BV-2 cells compared to primary microglia from CD-1 mice. A 45-plex Luminex immunoassay was used to measure IL-10 pretreatment attenuation of TNF-a, IL-6, CCL5/RANTES, and G-CSF secretion from cells treated with IFN-g and LPS. In addition, we demonstrate the effects of small molecule inhibitors MLN4924, tofacitinib (CP 690550), and Cardamonin on IL-10 pretreatment. MLN4924 inhibits the NFkB pathway and decreased secretion of multiple cytokines and chemokines, including TNF-a, IL-6, CCL2/MCP-1, CCL5, CCL3/MIP-1a, and CCL4/MIP-1b. Tofacitinib inhibits the Jak1/Tyk2-STAT3 pathway, decreasing STAT-3 TYR705 phosphorylation, but had little effect on IL-10 cytokine suppression. Interestingly, IL-10 pretreatment increased secretion of IL-1ra, an M2b microglia phenotype marker, and chemokines CCL2 and CCL3. Multiplex immunoassays are a valuable tool for investigating the mechanisms of neuroinflammation in primary and immortalized cell lines.

Deep pan-cancer analysis and multi-omics evidence reveal that ALG3 inhibits CD8+ T cell infiltration by suppressing chemokine secretion and is associated with 5-fluorouracil sensitivity

Backgroundα-1,3-mannosyltransferase (ALG3) holds significance as a key member within the mannosyltransferase family. Nevertheless, the exact function of ALG3 in cancer remains ambiguous. Consequently, the current research aimed to examine the function and potential mechanisms of ALG3 in various types of cancer. MethodsDeep pan-cancer analyses were conducted to investigate the expression patterns, prognostic value, genetic variations, single-cell omics, immunology, and drug responses associated with ALG3. Subsequently, in vitro experiments were executed to ascertain the biological role of ALG3 in breast cancer. Moreover, the link between ALG3 and CD8+ T cells was verified using immunofluorescence. Lastly, the association between ALG3 and chemokines was assessed using qRT-PCR and ELISA. ResultsDeep pan-cancer analysis demonstrated a heightened expression of ALG3 in the majority of tumors based on multi-omics evidence. ALG3 emerges as a diagnostic and prognostic biomarker across diverse cancer types. In addition, ALG3 participates in regulating the tumor immune microenvironment. Elevated levels of ALG3 were closely linked to the infiltration of bone marrow-derived suppressor cells (MDSC) and CD8+ T cells. According to in vitro experiments, ALG3 promotes proliferation and migration of breast cancer cells. Moreover, ALG3 inhibited CD8+ T cell infiltration by suppressing chemokine secretion. Finally, the inhibition of ALG3 enhanced the responsiveness of breast cancer cells to 5-fluorouracil treatment. ConclusionALG3 shows potential as both a prognostic indicator and immune infiltration biomarker across various types of cancer. Inhibition of ALG3 may represent a promising therapeutic strategy for tumor treatment.

Structural properties of immune complexes formed by viral antigens and specific antibodies shape the inflammatory response of macrophages

Data on the course of viral infections revealed severe inflammation as a consequence of antiviral immune response. Despite extensive research, there are insufficient data on the role of innate immune cells in promoting inflammation mediated by immune complexes (IC) of viral antigens and their specific antibodies. Recently, we demonstrated that antigens of human polyomaviruses (PyVs) induce an inflammatory response in macrophages. Here, we investigated macrophage activation by IC. We used primary murine macrophages as a cell model, virus-like particles (VLPs) of PyV capsid protein as antigens, and a collection of murine monoclonal antibodies (mAbs) of IgG1, IgG2a, IgG2b subclasses. The inflammatory response was investigated by analysing inflammatory chemokines and activation of NLRP3 inflammasome. We observed a diverse pattern of chemokine secretion in macrophages treated with different IC compared to VLPs alone. To link IC properties with cell activation status, we characterised the IC by advanced optical and acoustic techniques. Ellipsometry provided precise real-time kinetics of mAb-antigen interactions, while quartz crystal microbalance measurements showed changes in conformation and viscoelastic properties during IC formation. These results revealed differences in mAb-antigen interaction and mAb binding parameters of the investigated IC. We found that IC-mediated cell activation depends more on IC characteristics, including mAb affinity, than on mAb affinity for the activating Fc receptor. IC formed by the highest affinity mAb showed a significant enhancement of inflammasome activation. This may explain the hyperinflammation related to viral infection and vaccination. Our findings demonstrate that IC promote the viral antigen-induced inflammatory response depending on antibody properties.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

TRPV3 promotes sebocyte inflammation via transcriptional modulating TLR2 in acne

Acne is a common chronic inflammatory disease of the pilosebaceous unit. Transient receptor potential vanilloid 3 (TRPV3) is an ion channel that is involved in inflammatory dermatosis development. However, the involvement of TRPV3 in acne-related inflammation remains unclear. Here, we used acne-like mice and human sebocytes to examine the role of TRPV3 in the development of acne. We found that TRPV3 expression increased in the skin lesions of Propionibacterium acnes (P. acnes)-injected acne-like mice and the facial sebaceous glands (SGs) of acne patients. TRPV3 promoted inflammatory cytokines and chemokines secretion in human sebocytes and led to neutrophil infiltration surrounding the SGs in acne lesions, further exacerbating sebaceous inflammation and participating in acne development. Mechanistically, TRPV3 enhanced TLR2 level by promoting transcriptional factor phosphorylated-FOS-like antigen-1 (p-FOSL1) expression and its binding to the TLR2 promoter, leading to TLR2 upregulation and downstream NF-κB signaling activation. Genetic or pharmacological inhibition of TRPV3 both alleviated acne-like skin inflammation in mice via the TLR2-NF-κB axis. Thus, our study revealed the critical role of TRPV3 in sebaceous inflammation and indicated its potential as an acne therapeutic target.

Novel Fermentates Can Enhance Key Immune Responses Associated with Viral Immunity.

Fermented foods have long been known to have immunomodulatory capabilities, and fermentates derived from the lactic acid bacteria of dairy products can modulate the immune system. We have used skimmed milk powder to generate novel fermentates using Lb. helveticus strains SC234 and SC232 and we demonstrate here that these fermentates can enhance key immune mechanisms that are critical to the immune response to viruses. We show that our novel fermentates, SC234 and SC232, can positively impact on cytokine and chemokine secretion, nitric oxide (NO) production, cell surface marker expression, and phagocytosis in macrophage models. We demonstrate that the fermentates SC234 and SC232 increase the secretion of cytokines IL-1β, IL-6, TNF-α, IL-27, and IL-10; promote an M1 pro-inflammatory phenotype for viral immunity via NO induction; decrease chemokine expression of Monocyte Chemoattractant Protein (MCP); increase cell surface marker expression; and enhance phagocytosis in comparison to their starting material. These data suggest that these novel fermentates have potential as novel functional food ingredients for the treatment, management, and control of viral infection.

Exploration of the core pathway of inflammatory bowel disease complicated with metabolic fatty liver and two-sample Mendelian randomization study of the causal relationships behind the disease.

Inflammatory bowel disease (IBD) is often associated with complex extraintestinal manifestations. The incidence of nonalcoholic fatty liver disease (NAFLD) in IBD populations is increasing yearly. However, the mechanism of interaction between NAFLD and IBD is not clear. Consequently, this study aimed to explore the common genetic characteristics of IBD and NAFLD and identify potential therapeutic targets. Gene chip datasets for IBD and NAFLD were obtained from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was performed to identify modules in those datasets related to IBD and NAFLD. ClueGO was used for biological analysis of the shared genes between IBD and NAFLD. Based on the Human MicroRNA Disease Database (HMDD), microRNAs (miRNAs) common to NAFLD and IBD were obtained. Potential target genes for the miRNAs were predicted using the miRTarbase, miRDB, and TargetScan databases. Two-sample Mendelian randomization (MR) and two-way MR were used to explore the causal relationship between Interleukin-17 (IL-17) and the risk of IBD and NAFLD using data from GWAS retrieved from an open database. Through WGCNA, gene modules of interest were identified. GO enrichment analysis using ClueGO suggested that the abnormal secretion of chemokines may be a common pathophysiological feature of IBD and NAFLD, and that the IL-17-related pathway may be a common key pathway for the pathological changes that occur in IBD and NAFLD. The core differentially expressed genes (DEGs) in IBD and NAFLD were identified and included COL1A1, LUM, CCL22, CCL2, THBS2, COL1A2, MMP9, and CXCL8. Another cohort was used for validation. Finally, analysis of the miRNAs identified potential therapeutic targets. The MR results suggested that although there was no causal relationship between IBD and NAFLD, there were causal relationships between IL-17 and IBD and NAFLD. We established a comorbid model to explain the potential mechanism of IBD with NAFLD and identified the chemokine-related pathway mediated by cytokine IL-17 as the core pathway in IBD with NAFLD, in which miRNA also plays a role and thus provides potential therapeutic targets.

S100P facilitates LUAD progression via PKA/c-Jun-mediated tumor-associated macrophage recruitment and polarization

S100P, a member of the S100 calcium-binding protein family, is closely associated with abnormal proliferation, invasion, and metastasis of various cancers. However, its role in the lung adenocarcinoma (LUAD) tumor microenvironment (TME) remains unclear. In this study, we observed specific expression of S100P on tumor cells in LUAD patients through tissue immunofluorescence analysis. Furthermore, this expression was strongly correlated with the recruitment and polarization of tumor-associated macrophages (TAMs). Bioinformatics analysis revealed that high S100P expression is associated with poorer overall survival in LUAD patients. Subsequently, a subcutaneous mouse model demonstrated that S100P promotes recruitment and polarization of TAMs towards the M2 type. Finally, in vitro studies on LUAD cells revealed that S100P enhances the secretion of chemokines and polarizing factors by activating the PKA/c-Jun pathway, which is implicated in TAM recruitment and polarization towards the M2 phenotype. Moreover, inhibition of c-Jun expression impedes the ability of TAMs to infiltrate and polarize towards the M2 phenotype. In conclusion, our study demonstrates that S100P facilitates LUAD cells growth by recruiting M2 TAMs through PKA/c-Jun signaling, resulting in the production of various cytokines. Considering these findings, S100P holds promise as an important diagnostic marker and potential therapeutic target for LUAD.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. Epithelial activation and/or barrier effects upon exposure to 2-50 μg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 μg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

Wnt signaling regulates chemokine production and cell migration of circulating human monocytes

The β-catenin dependent canonical Wnt signaling pathway plays a crucial role in maintaining normal homeostasis. However, when dysregulated, Wnt signaling is closely associated with various pathological conditions, including inflammation and different types of cancer.Here, we show a new connection between the leukocyte inflammatory response and the Wnt signaling pathway. Specifically, we demonstrate that circulating human primary monocytes express distinct Wnt signaling components and are susceptible to stimulation by the classical Wnt ligand—Wnt-3a. Although this stimulation increased the levels of β-catenin protein, the expression of the classical Wnt-target genes was not affected. Intriguingly, treating circulating human monocytes with Wnt-3a induces the secretion of cytokines and chemokines, enhancing monocyte migration. Mechanistically, the enhanced monocyte migration in response to Wnt stimuli is mediated through CCL2, a strong monocyte-chemoattractant.To further explore the physiological relevance of these findings, we conducted ex-vivo experiments using blood samples of patients with rheumatic joint diseases (RJD) – conditions where monocytes are known to be dysfunctional. Wnt-3a generated a unique cytokine expression profile, which was significantly distinct from that observed in monocytes obtained from healthy donors.Thus, our results provide the first evidence that Wnt-3a may serve as a potent stimulator of monocyte-driven immune processes. These findings contribute to our understanding of inflammatory diseases and, more importantly, shed light on the role of a core signaling pathway in the circulation.

Regulatory role of Echinochrome A in cancer-associated fibroblast-mediated lung cancer cell migration.

Echinochrome A (Ech A), a marine biosubstance isolated from sea urchins, is a strong antioxidant, and its clinical form, histochrome, is being used to treat several diseases, such as ophthalmic, cardiovascular, and metabolic diseases. Cancer-associated fibroblasts (CAFs) are a component of the tumor stroma and induce phenotypes related to tumor malignancy, including epithelial-mesenchymal transition (EMT) and cancer stemness, through reciprocal interactions with cancer cells. Here, we investigated whether Ech A modulates the properties of CAFs and alleviates CAF-induced lung cancer cell migration. First, we observed that the expression levels of CAF markers, Vimentin and fibroblast-activating protein (FAP), were decreased in Ech A-treated CAF-like MRC5 cells. The mRNA transcriptome analysis revealed that in MRC5 cells, the expression of genes associated with cell migration was largely modulated after Ech A treatment. In particular, the expression and secretion of cytokine and chemokine, such as IL6 and CCL2, stimulating cancer cell metastasis was reduced through the inactivation of STAT3 and Akt in MRC5 cells treated with Ech A compared to untreated MRC5 cells. Moreover, while conditioned medium from MRC5 cells enhanced the migration of non-small cell lung cancer cells, conditioned medium from MRC5 cells treated with Ech A suppressed cancer cell migration. In conclusion, we suggest that Ech A might be a potent adjuvant that increases the efficacy of cancer treatments to mitigate lung cancer progression.

Role of formyl peptide receptor 2 in steatosis of L02 cells exposed to Mono-(2-ethylhexyl) phthalate.

Mono-(2-ethylhexyl) phthalate (MEHP) can accumulate in the liver and then lead to hepatic steatosis, while the underlying mechanism remains unclear. Inflammation plays an important role in the disorder of hepatic lipid metabolism. This study aims to clarify the role of the inflammatory response mediated by formyl peptide receptor 2 (FPR2) in steatosis of L02 cells exposed to MEHP. L02 cells were exposed to MEHP of different concentrations and different time. A steatosis model of L02 cells was induced with oleic acid and the cells were exposed to MEHP simultaneously. In addition, L02 cells were incubated with FPR2 antagonist and then exposed to MEHP. Lipid accumulation was determined by oil red O staining and extraction assay. The indicators related to lipid metabolism and inflammatory response were measured with appropriate kits. The relative expression levels of FPR2 and its ligand were determined by Western blot, and the interaction of them was detected by co-immunoprecipitation. As a result, MEHP exposure could promote the occurrence and progression of steatosis and the secretion of chemokines and inflammatory factors in L02 cells. MEHP could also affect the expression and activation of FPR2 and the secretion of FPR2 ligands. In addition, the promotion effect of MEHP on the secretion of total cholesterol and interleukin 1β in L02 cells could be significantly inhibited by the FPR2 antagonist. We concluded that FPR2 might affect the promotion effect of MEHP on steatosis of L02 cells by mediating inflammatory response.

Terminalia ferdinandiana Exell. extracts reduce pro-inflammatory cytokine and PGE2 secretion, decrease COX-2 expression and down-regulate cytosolic NF-κB levels

Based on their high antioxidant capacity and noteworthy phytochemistry, Terminalia ferdinandiana fruit and leaves have attracted considerable recent interest for their therapeutic potential. Whilst those studies have reported a variety of therapeutic properties for the fruit, the anti-inflammatory potential of T. ferdinandiana has been largely neglected and the leaves have been almost completely ignored. This study investigated the immune-modulatory and anti-inflammatory properties of T. ferdinandiana fruit and leaf extracts by evaluating their inhibition of multiple pro- and anti-inflammatory cytokines and chemokines secretion in lipopolysaccharide (LPS)-stimulated and unstimulated RAW 264.7 macrophages using multiplex bead immunoassays and ELISA assays. The methanolic extracts were particularly good immune-modulators, significantly inhibiting the secretion of all the cytokines and chemokines tested. Indeed, the methanolic extracts completely inhibited IL-10, IFN-γ, IL-1β, IL-6, MCP-1, and MIP-2a secretion, and almost completely inhibited the secretion of TNF-α. In addition, the methanolic T. ferdinandiana extracts also significantly inhibited cytosolic COX-2 levels (by 87–95%) and the synthesis of the PGE2 (by ~ 98%). In contrast, the methanolic extracts stimulated LTB4 secretion by ~ 60–90%, whilst the aqueous extracts significantly inhibited LTB4 secretion (by ~ 27% each). Exposure of RAW 264.7 cells to the methanolic T. ferdinandiana extracts also significantly down-regulated the cytosolic levels of NF-κB by 33–44%, indicating that the immune-modulatory and anti-inflammatory properties of the extracts may be regulated via a decrease in NF-κB transcription pathways. Taken together, these results demonstrate potent anti-inflammatory properties for the extracts and provide insights into their anti-inflammatory mechanisms.

Abstract LB062: ADT3_A potential first-in-class Vδ1-engager for in situ activation of Vδ1 γδ T cells and enhanced cytotoxicity towards solid tumors

Abstract Gamma delta (γδ) T cells are crucial in stress surveillance for infections and cancer. Studies have shown that tumour-infiltrating γδ T cells are a positive prognostic factor in cancer therapy. While γδ T cells share many characteristics with their αβ T cell counterpart, such as cytotoxic effector functions, they express a distinct TCR that is composed of a γ and a δ chain, belong to the HLA-class-I unrestricted immune cell subset with the ability to discriminate and specifically kill tumor cells via recognition of tumor stress ligands. Clinical trials using Vδ1 γδ T cell therapies have demonstrated promising results in Ph 1 trials, with early signs of favorable efficacy and safety profile.ADT3, is a novel bispecific engager that activates tissue-resident Vδ1+ T cells via (Epidermal Growth Factor Receptor) EGFR crosslinking on tumor cells. VD1 T cells isolated from skin biopsies, when activated, enhance the selective killing of EGFR positive tumor cells through innate mechanisms that recognize stress ligands that are otherwise absent on healthy cells. As a bispecific engager, ADT3 preserves high affinity binding to VD1 TCR through the Fab end of the molecule, and binding to two EGFR binding sites via the modified Fc domain. Crosslinking of VD1 and EGFR+ tumor cells with ADT3, mediates a series of events leading to proliferation of VD1 cells, upregulation of activation markers, CD107a degranulation and secretion of chemokines and cytokines (such as TNFa and IFNg) and tumor kill as determined by its potent cytotoxic activity in a T-cell dependent cellular cytotoxicity assay (TDCC). Apart from direct tumor kill, the secretion of such cytokines and chemokines via ADT3 activation are required for induction of immune orchestration of non-Vδ1 immune cells to enhance tumor kill. Preclinical data supports future clinical entry of ADT3, and IND enabling studies are in progress Citation Format: Joshua Freedman, Georgia Stevens, Julie Julie Hoong, Mihirban Tuna, Yaoyao Shi, Kathy J. Seidl, Katherine Galvin, Andrew Hutton, Antara Banerjee. ADT3_A potential first-in-class Vδ1-engager for in situ activation of Vδ1 γδ T cells and enhanced cytotoxicity towards solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB062.

The role of circulating neutrophils in the progression of kidney cancer

Introduction. Currently, the question of the role of neutrophils in the progression of kidney cancer remains relevant. Neutrophils are capable of exhibiting protumor properties through the secretion of cytokines, chemokines, and growth factors, which is determined by the expression of genes for these molecules. And the functional heterogeneity of neutrophils is characterized by differences in gene expression patterns.Aim. To assess the role of circulating neutrophils in the progression of kidney cancer.Materials and methods. In circulating neutrophils of patients with verified clear cell kidney cancer at stages I–III according to Tumor, Nodus and Metastasis (TNM) (n = 88) before surgical treatment and conditionally healthy donors (control group) (n = 20), the expression of NGAL genes was determined using quantitative reverse transcription polymerase chain reaction, MMP-13 and VEGF-A.Results. There was an increase in NGAL gene expression in circulating neutrophils (p = 0.05) at the initial stage and a decrease in it at advanced stages of kidney cancer (p = 0.03). High expression of the MMP-13 gene by circulating neutrophils was detected at all stages of kidney cancer relative to control values (at stage I p = 0.005; at stage II p = 0.003; at stage III p = 0.0008). A significant direct correlation was observed between the expression of the NGAL and MMP-13 genes in neutrophils at stage I kidney cancer (r = 0.696; p = 0.003). In the group of patients with kidney cancer, a direct correlation was found between the expression of the NGAL and VEGF-A genes (r = 0.322; p = 0.049). A multivariable Cox regression model for disease-free survival revealed the predictive value of VEGF-A and NGAL genes expression in circulating neutrophils. With an increase in the expression of the VEGF-A and NGAL genes in neutrophils by 1 unit, the risk of metastases increases by 0.80 (0.65–0.99; p = 0.043) and 1.42 (1.01–2.00; p = 0.046) times, respectively. The Kaplan–Meier analysis of disease-free survival in patients with kidney cancer showed the influence of NGAL expression in circulating neutrophils on progression-free time. In the group of patients with high NGAL expression, the median follow-up was 31.7 months, and in the group with low NGAL expression – more than 36 months (log-rank-test; p = 0.017).Conclusion. Thus, the data obtained suggest that circulating neutrophils play a leading role in the progression of kidney cancer. The level of expression of NGAL in circulating neutrophils can be used to predict the relapse-free period in patients with kidney cancer.

Neutrophil Membrane-Camouflaged Polyprodrug Nanomedicine for Inflammation Suppression in Ischemic Stroke Therapy.

Neuroinflammation has emerged as a major concern in ischemic stroke therapy because it exacebates neurological dysfunction and suppresses neurological recovery after ischemia/reperfusion. Fingolimod hydrochloride (FTY720) is an FDA-approved anti-inflammatory drug which exhibits potential neuroprotective effects in ischemic brain parenchyma. However, delivering a sufficient amount of FTY720 through the blood-brain barrier into brain lesions without inducing severe cardiovascular side effects remains challenging. Here, a neutrophil membrane-camouflaged polyprodrug nanomedicine that can migrate into ischemic brain tissues and in situ release FTY720 in response to elevated levels of reactive oxygen species. This nanomedicine delivers 15.2-fold more FTY720 into the ischemic brain and significantly reduces the risk of cardiotoxicity and infection compared with intravenously administered free drug. In addition, single-cell RNA-sequencing analysis identifies that the nanomedicine attenuates poststroke inflammation by reprogramming microglia toward anti-inflammatory phenotypes, which is realized via modulating Cebpb-regulated activation of NLRP3 inflammasomes and secretion of CXCL2 chemokine. This study offers new insights into the design and fabrication of polyprodrug nanomedicines for effective suppression of inflammation in ischemic stroke therapy.

The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.