Cytokine Expression In Cells Research Articles

5-Hydroxymethylfurfural Ameliorates Allergic Inflammation in HMC-1 Cells by Inactivating NF-κB and MAPK Signaling Pathways.

Allergic inflammation is the foundation of multiple allergic disorders, such as allergic rhinitis and asthma. Mast cells are effector cells that initiate inflammatory response. 5-hydroxymethylfurfural (5-HMF), a furfural compound, is the heat-processed product of various fruit, foods, drinks, as well as some Chinese herbal medicines. 5-HMF was previously reported to inhibit mast cell activation. Our study aimed to explore the functions of 5-HMF in both phorbol 12-mystate 13-acetate (PMA) plus calcium ionophore (A23187)-induced allergic inflammation in human mast cell line HMC-1 and ovalbumin (OVA)-induced asthma mouse models. HMC-1 cells were pretreated with 5-HMF and then stimulated by PMA+A23187. The cytotoxicity of 5-HMF on HMC-1 cells was evaluated by MTT assay. Histamine content in cell supernatants was measured by the o-phthaldialdehyde spectrofluorometric procedure. Intracellular calcium was determined using the fluorescent dye Fura-2AM. The production and expression of pro-inflammatory cytokines were detected by ELISA and RT-qPCR. Caspase-1 colorimetric assay was employed to examine the enzymatic activity of caspase-1. Asthma mouse models were induced by OVA sensitization. The bronchoalveolar lavage fluid (BALF) and blood samples were collected for the detection of total and differential cell count as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), OVA-immunoglobulin E (OVA-IgE), OVA-immunoglobulin G1 (OVA-IgG1), and pro-inflammatory cytokine levels. The left lung of mouse was dissected for histopathological examination by hematoxylin and eosin (H&E) staining. The protein expression of pro-caspase-1 and the phosphorylation of NF-κB and MAPK pathway-associated molecules were assessed by Western blotting. Our findings revealed that 5-HMF efficiently suppressed the PMA+A23187-induced enhancement in histamine production and intracellular calcium in HMC-1 cells. Pro-inflammatory cytokine production and expression in HMC-1 cells were elevated after PMA plus A23187 stimulation, which, however, were inhibited by pretreatment of 5-HMF. Additionally, 5-HMF suppressed the activity of caspase-1 and the phosphorylation of NF-κB and MAPK-associatedmolecules including p65 NF-κB, p38 MAPK, ERK, and JNK in HMC-1 cells. In vivo experiments demonstrated that 5-HMF treatment reduced the lung/body weight index and total and differential (macrophages, neutrophils, lymphocytes, and eosinophils) cell counts in BALF of asthmatic mice, but exerted no influence on serum AST and ALT levels. Besides, 5-HMF reduced serum OVA-IgE and OVA-IgG1 levels, mitigated lung inflammation, and inhibited the NF-κB and MAPK signaling pathways in asthma mouse models. 5-HMF mitigates allergic inflammation in asthma by inactivating caspase-1 and NF-κB and MAPK signaling pathways.

Macrophage extracellular traps promote tumor-like biologic behaviors of fibroblast-like synoviocytes through cGAS-mediated PI3K/Akt signaling pathway in patients with rheumatoid arthritis.

Rheumatoid arthritis is an autoimmune disease characterized by synovium hyperplasia and bone destruction. Macrophage extracellular traps are released from macrophages under various stimuli and may generate stable autoantigen-DNA complexes, as well as aggravate autoantibody generation and autoimmune responses. We aimed to investigate the role of macrophage extracellular traps on the biologic behaviors of rheumatoid arthritis fibroblast-like synoviocytes. Synovial tissues and fibroblast-like synoviocytes were obtained from patients with rheumatoid arthritis. Extracellular traps in synovium and synovial fluids were detected by immunofluorescence, immunohistochemistry, and SYTOX Green staining. Cell viability, migration, invasion, and cytokine expression of rheumatoid arthritis fibroblast-like synoviocytes were assessed by CCK-8, wound-healing assay, Transwell assays, and quantitative real-time polymerase chain reaction, respectively. RNA sequencing analysis was performed to explore the underlying mechanism, and Western blot was used to validate the active signaling pathways. We found that extracellular trap formation was abundant in rheumatoid arthritis and positively correlated to anti-CCP. Rheumatoid arthritis fibroblast-like synoviocytes stimulated with purified macrophage extracellular traps demonstrated the obvious promotion in tumor-like biologic behaviors. The DNA sensor cGAS in rheumatoid arthritis fibroblast-like synoviocytes was activated after macrophage extracellular trap stimuli. RNA sequencing revealed that differential genes were significantly enriched in the PI3K/Akt signaling pathway, and cGAS inhibitor RU.521 effectively reversed the promotion of tumor-like biologic behaviors in macrophage extracellular trap-treated rheumatoid arthritis fibroblast-like synoviocytes and downregulated the PI3K/Akt activation. In summary, our study demonstrates that macrophage extracellular traps promote the pathogenically biological behaviors of rheumatoid arthritis fibroblast-like synoviocytes through cGAS-mediated activation of the PI3K/Akt signaling pathway. These findings provide a novel insight into the pathogenesis of rheumatoid arthritis and the mechanisms of macrophages in modulating rheumatoid arthritis fibroblast-like synoviocyte tumor-like behaviors.

The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.

Study on the cytokines related to SARS-Cov-2 in testicular cells and the interaction network between cells based on scRNA-seq data.

Inflammatory cytokine storms (CS) in COVID-19 patients are associated with elevated levels of 13 specific cytokines, potentially impacting male fertility by causing testicular cell damage and disrupting the immune microenvironment. Some patients present with scrotal discomfort and orchitis. However, few studies have explored cytokine expression in testicular cells and their role in cell-to-cell communication. In this study, we integrated single-cell sequencing data sets of testicular cells, annotating 20 cell clusters using marker genes and the Human Cell Landscape database. We constructed cell pseudo-chronological trajectories, hub genes, and analyzed the cytokine interaction network between sperm cells using CellChat. Our findings identified 12 types of testicular cells, with four cytokines (IL8, CCL2, CCL3, and TNF) potentially involved in immune processes. Pseudo-chronological trajectory analysis indicated IL8 and CCL3's essential roles in testicular macrophages and endothelial cell development, affecting the immune microenvironment. We determined eight key cytokines (IL1, IL2, IL4, IL6, CCL, CSF3, TNF, and IFN-II) functions in cell interaction networks. Network analysis of exogenous cytokines directly acting on testicular cells showed IL2 potentially affecting all testicular cells, suggesting a vital role in cell communication. This research offers valuable insights into CSs effects on testicular cells and their potential impact on male fertility during COVID-19 infection.

Lymphocyte Expression of Intracellular Cytokines and Heat Shock Proteins in Peripheral Blood of Patients with Atopic Dermatitis.

The present study evaluates expression by activated CD4+ T helper1 (Th1) and T helper 2 (Th2) T lymphocytes of pro-inflammatory cytokines and cytoprotective heat shock proteins (HSPs) in peripheral blood of atopic dermatitis (AD) patients. This research represents preliminary work by the authors to identify correlates between critical immune parameters with the potential to serve as guidelines for the development of pharmacological strategies for altering these factors to promote the restoration of healthy immune profiles in persons afflicted with major atopic diseases. The major experimental strategy used in this research assessed immune activation by peripheral blood mononuclear cells (PBMC) from 21 AD patients and 12 age- and gender-matched healthy control subjects cultured with phorbol myristate acetate (PMA) and ionomycin (PMA/I), which are mutagenic immune activators, to induce expression of pro-inflammatory biomarkers in CD4+ T cells differentiated to express Th1 or Th2 cytokines and heme oxygenase-1 (HO-1) intracellularly (i). Evaluations were performed using an FC500 Beckman-Coulter flow cytometer. Elevated CD4+ T cell expression of cytokines, interleukin-4 (iIL-4), interleukin- 5 (iIL-5), interleukin-10 (iIL-10), interferon-gamma (iIFN-g), tumor necrosis factor-alpha (iTNF-α), were observed. Additionally, the heat shock proteins (HSP) iHO-1 and iHSP-70 were evaluated in cells from the blood of AD patients versus the control subjects. The present study demonstrated an elevated expression of both Th1 and Th2-associated cytokines in CD4+ T cells of AD patients, with a significant direct correlation between Th1 and Th2 cell populations, thus yielding insight into the immune features of the AD-associated systemic inflammatory profile. Finally, the observed increased iHO-1 and iHSP-70 expressions likely represent adaptive physiologic countermeasures to AD-associated inflammatory tissue damage, suggesting that HSP inducers are promising candidates for the management of atopic disorders.

Glutathione trisulfide prevents lipopolysaccharide-induced retinal inflammation via inhibition of proinflammatory cytokine production in glial cells

We aimed to investigate the impact of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammation in retinal glia. Inflammatory responses in mouse-derived glial cells and Wistar rat retinas were stimulated with administration of LPS. Cell survival and proinflammatory cytokine production were examined using the Calcein-AM assay, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Retinal microglia were visualized with immunohistochemistry for Iba1. Administration of LPS (10 µg/mL) or GSSSG (less than 100 µM) did not affect survival of cultured primary Müller cells and established microglial cells (BV-2). RT-qPCR and ELISA indicated that GSSSG inhibited LPS-induced gene upregulation and protein secretion of proinflammatory cytokines in these glial cells and rat retinas. GSSSG inhibited LPS-induced activation of TGF-β-activated kinase 1 (TAK1), which is an upstream kinase of NF-κB, in BV-2 cells. Finally, in vivo experiments indicated that intravitreal administration of GSSSG but not its relative glutathione disulfide (GSSG) inhibited LPS (500 ng)-induced accumulation of Iba1-immunopositive microglia in rat retinas. Taken together, GSSSG has the potential to prevent pathogenesis of inflammation-associated ocular diseases by inhibiting proinflammatory cytokine expression in retinal glial cells.

Tannerella forsythia scavenges Fusobacterium nucleatum secreted NOD2 stimulatory molecules to dampen oral epithelial cell inflammatory response.

The oral organism Tannerella forsythia is auxotrophic for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc). It survives in the oral cavity by scavenging MurNAc- and MurNAc-linked peptidoglycan fragments (muropeptides) secreted by co-habiting bacteria such as Fusobacterium nucleatum with which it forms synergistic biofilms. Muropeptides, MurNAc-l-Ala-d-isoGln (MDP, muramyl dipeptide) and d-γ-glutamyl-meso-DAP (iE-DAP dipeptide), are strong immunostimulatory molecules that activate nucleotide oligomerization domain (NOD)-like innate immune receptors and induce the expression of inflammatory cytokines and antimicrobial peptides. In this study, we utilized an in vitro T. forsythia-F. nucleatum co-culture model to determine if T. forsythia can selectively scavenge NOD ligands from the environment and impact NOD-mediated inflammation. The results showed that NOD-stimulatory molecules were secreted by F. nucleatum in the spent culture broth, which subsequently induced cytokine and antimicrobial peptide expression in oral epithelial cells. In the spent broth from T. forsythia-F. nucleatum co-cultures, the NOD-stimulatory activity was significantly reduced. These data indicated that F. nucleatum releases NOD2-stimulatory muropeptides in the environment, and T. forsythia can effectively scavenge the muropeptides released by co-habiting bacteria to dampen NOD-mediated host responses. This proof-of-principle study demonstrated that peptidoglycan scavenging by T. forsythia can impact the innate immunity of oral epithelium by dampening NOD activation.

Biodegradable nanoparticles targeting circulating immune cells reduce central and peripheral sensitization to alleviate neuropathic pain following spinal cord injury.

Neuropathic pain is a critical source of comorbidity following spinal cord injury (SCI) that can be exacerbated by immune-mediated pathologies in the central and peripheral nervous systems. In this article, we investigate whether drug-free, biodegradable, poly(lactide- co -glycolide) (PLG) nanoparticle treatment mitigates the development of post-SCI neuropathic pain in female mice. Our results show that acute treatment with PLG nanoparticles following thoracic SCI significantly reduces tactile and cold hypersensitivity scores in a durable fashion. Nanoparticles primarily reduce peripheral immune-mediated mechanisms of neuropathic pain, including neuropathic pain-associated gene transcript frequency, transient receptor potential ankyrin 1 nociceptor expression, and MCP-1 (CCL2) chemokine production in the subacute period after injury. Altered central neuropathic pain mechanisms during this period are limited to reduced innate immune cell cytokine expression. However, in the chronic phase of SCI, nanoparticle treatment induces changes in both central and peripheral neuropathic pain signaling, driving reductions in cytokine production and other immune-relevant markers. This research suggests that drug-free PLG nanoparticles reprogram peripheral proalgesic pathways subacutely after SCI to reduce neuropathic pain outcomes and improve chronic central pain signaling.

Sphingosine 1-phosphate elicits a ROS-mediated proinflammatory response in human endometrial stromal cells via ERK5 activation.

Endometriosis is a chronic gynecological disease affecting ~10% women in the reproductive age characterized by the growth of endometrial glands and stroma outside the uterine cavity. The inflammatory process has a key role in the initiation and progression of the disorder. Currently, there are no available early diagnostic tests and therapy relies exclusively on symptomatic drugs, so that elucidation of the complex molecular mechanisms involved in the pathogenesis of endometriosis is an unmet need. The signaling of the bioactive sphingolipid sphingosine 1-phosphate (S1P) is deeply dysregulated in endometriosis. S1P modulates a variety of fundamental cellular processes, including inflammation, neo-angiogenesis, and immune responses acting mainly as ligand of a family of G-protein-coupled receptors named S1P receptors (S1PR), S1P1-5 . Here, we demonstrated that the mitogen-activated protein kinase ERK5, that is expressed in endometriotic lesions as determined by quantitative PCR, is activated by S1P in human endometrial stromal cells. S1P-induced ERK5 activation was shown to be triggered by S1P1/3 receptors via a SFK/MEK5-dependent axis. S1P-induced ERK5 activation was, in turn, responsible for the increase of reactive oxygen species and proinflammatory cytokine expression in human endometrial stromal cells. The present findings indicate that the S1P signaling, via ERK5 activation, supports a proinflammatory response in the endometrium and establish the rationale for the exploitation of innovative therapeutic targets for endometriosis.

Ionizing Radiation Reduces Head and Neck Squamous Cell Carcinoma Cell Viability and Is Associated with Predictive Tumor-Specific T Cell Responses

Head and neck squamous cell carcinoma (HNSCC) is common and deadly, and there is a need for improved strategies to predict treatment responses. Ionizing radiation (IR) has been demonstrated to improve HNSCC outcomes, but its effects on immune responses are not well characterized. We determined the impact of IR on T cell immune responses ex vivo. Human and mouse HNSCC cells were exposed to IR ranging from 20 to 200 Gy to determine cell viability and the ability to stimulate T-cell-specific responses. Lymph node cells of LY2 and MOC2 tumor-bearing or non-tumor-bearing mice were re-stimulated with a tumor antigen derived from LY2 or MOC2 cells treated with 200 Gy IR, ultraviolet (UV) exposure, or freeze/thaw cycle treatments. T cell proliferation and cytokine production were compared to T cells restimulated with plate-bound CD3 and CD28 antibodies. Human and mouse HNSCC cells showed reduced viability in response to ionizing radiation in a dose-dependent manner, and induced expression of T cell chemotactic cytokines. Tumor antigens derived from IR-treated LY2 and MOC2 cells induced greater proliferation of lymph node cells from tumor-bearing mice and induced unique T cell cytokine expression profiles. Our results demonstrate that IR induces potent tumoral immune responses, and IR-generated tumor antigens can potentially serve as an indicator of antitumor immune responses to HNSCC in ex vivo T cell restimulation assays.

P-339 Activities and changes in the number of natural killer cells in endometriosis: systematic review and meta-analysis

Abstract Study question What are the differences in peripheral, uterine, and peritoneal NK (uNK, pNK, pfNK) cell counts/percentages, and activities in women with endometriosis compared controls Summary answer The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference in women with endometriosis compared controls What is known already NK cells play an important role in the pathogenesis of endometriosis Study design, size, duration This systematic review and meta-analysis included 36 experimental studies (case control and cross sectional). Narrative review is also conducted on the NK cell activity, cytokine expression, regulations and receptors. Participants/materials, setting, methods Women suffering with endometriosis confirmed with laparoscopy and/or pathology was the cases whereas, women having different pelvic pathology such as myoma, ovarian cyst, healthy woman and verified not having endometriosis with laparoscopy and/or pathology was considered as controls for this study. Databases (PubMed, Web of Science, Scopus, Google scholar, and EMBASE through OVID) were used to search for the available studies. RevMan 5.4 and STATA 14 were used to analyze the data. Main results and the role of chance The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference (standard mean difference SMD 0.13, 95%CI -0.35, 0.62; P = 0.59, I2 74%; total 393 women, 6 studies), (SMD 0.27, 95%CI -0.07, 0.61; P = 0.12, I2 80%; total 825 women, 11 studies), and (SMD 0.31, 95%CI -0.68, 01.29; P = 0.59, I2 94%; total 404 women,7 studies) in endometriosis patients compared with controls respectively. The pooled NK (uNK, pNK, pfNK) cell cytotoxicity/activity level is significantly higher in controls compared with women with endometriosis (MD 5.43, 95%CI 2.29, 8.57; P < 0.007, I2 50%; total 323 women, 7 studies). The NK cytotoxicity/activity level in early stages of endometriosis has no significant difference compared with advanced stages of endometriosis (MD 2.29, 95%CI –1.98-6.57; P = 0.29, I2 87%; total 263 women, 7 studies). There are variations in studies conducted in NK cell activities in endometriosis: broadly categorized as NK cell cytotoxicity, cytokine expression and NK cell regulation and receptors (Inhibition and activation). The cytotoxic activity of NK cells decreased in women with endometriosis and correlated with the severity of the disease, however the cytokine expression and inhibitor receptors are equivocal across studies. Limitations, reasons for caution We unable to conduct sub-group analysis to investigate the cytotoxic activity of NK cell in women with endometriosis and its stages in different samples such as pNK, uNK, and pfNK due to the lack of studies for the analysis. Wider implications of the findings This study revealed that the activity of NK cells in women with endometriosis is significantly lower than controls.Though the mean difference of NK cell count and percentage has no significant difference, the cytotoxicity activity of NK cell is higher in controls compared with women with endometriosis Trial registration number CRD42022384060

EFFECT OF LOW-LEVEL LASER ON CYTOKINE PRODUCTION BY MONOCYTES EXPOSED TO CISPLATIN AND STIMULATED WITH P. GINGIVALIS

Objective This study aimed to evaluate the effect of photobiomodulation (PBM) on monocytes exposed to cisplatin and P. gingivalis (Pg). Study Design Peripheral blood mononuclear cells from 10 healthy donors were exposed to cisplatin, irradiated with red laser (AlGaInP, 660nm, 0.028cm², 20mW, 0.71W/cm², 0.14J, 7s, 5J/cm²) and stimulated with Pg. Cell viability and cytokine expression by total monocytes and monocyte subpopulations were evaluated by flow cytometry. Results The treatments did not affect the viability of monocytes and TGF-β1 expression. In Pg-stimulated cells, cisplatin reduced the expression of IL-6, IL-8, and IL-10 in total and classical monocytes and increased IL-6 and TNF expression in the intermediate monocytes. In cisplatin exposed cells, PBM reduced the expression of IL-6 and IL-8 in total, classical, and intermediate monocytes, and IL-10 and TNF in total monocytes. In cisplatin and Pg-stimulated cells, PBM increased the expression of IL-6 and IL-8 in total and intermediate monocytes. Conclusion The red laser modulates the expression of cytokines by human monocytes treated with cisplatin, which may represent PBM mechanisms of action in the management of oral mucositis. Apoio: FAPEMIG APQ03601-16, FIP/PUC Minas 2020/24894-1S, and CAPES Code 001. This study aimed to evaluate the effect of photobiomodulation (PBM) on monocytes exposed to cisplatin and P. gingivalis (Pg). Peripheral blood mononuclear cells from 10 healthy donors were exposed to cisplatin, irradiated with red laser (AlGaInP, 660nm, 0.028cm², 20mW, 0.71W/cm², 0.14J, 7s, 5J/cm²) and stimulated with Pg. Cell viability and cytokine expression by total monocytes and monocyte subpopulations were evaluated by flow cytometry. The treatments did not affect the viability of monocytes and TGF-β1 expression. In Pg-stimulated cells, cisplatin reduced the expression of IL-6, IL-8, and IL-10 in total and classical monocytes and increased IL-6 and TNF expression in the intermediate monocytes. In cisplatin exposed cells, PBM reduced the expression of IL-6 and IL-8 in total, classical, and intermediate monocytes, and IL-10 and TNF in total monocytes. In cisplatin and Pg-stimulated cells, PBM increased the expression of IL-6 and IL-8 in total and intermediate monocytes. The red laser modulates the expression of cytokines by human monocytes treated with cisplatin, which may represent PBM mechanisms of action in the management of oral mucositis.

Magnolia kobus Extract Suppresses Porphyromonas gingivalis LPS-Induced Proinflammatory Cytokine and MMP Expression in HGF-1 Cells and Regulates Osteoclastogenesis in RANKL-Stimulated RAW264.7 Cells.

Clinical prevention is of utmost importance for the management of periodontal diseases. Periodontal disease starts with an inflammatory response in the gingival tissue, and results in alveolar bone destruction and subsequent tooth loss. This study aimed to confirm the anti-periodontitis effects of MKE. To confirm this, we studied its mechanism of action using qPCR and WB in LPS-treated HGF-1 cells and RANKL-induced osteoclasts. We found that MKE suppressed proinflammatory cytokine protein expression by inhibiting the TLR4/NF-κB pathway in LPS-PG-induced HGF-1 cells and blocking ECM degradation by regulating the expression of TIMPs and MMPs. We also confirmed that TRAP activity and multinucleated cell formation were reduced in RANKL-stimulated osteoclasts after exposure to MKE. These results were confirmed by inhibiting TRAF6/MAPK expression, which led to the suppression of NFATc1, CTSK, TRAP, and MMP expression at the gene and protein levels. Our results confirmed that MKE is a promising candidate for the management of periodontal disease based on its anti-inflammatory effects and inhibition of ECM degradation and osteoclastogenesis.

Single-cell transcriptomics suggest distinct upstream drivers of IL-17A/F in hidradenitis versus psoriasis

On the basis of the mounting evidence that type 17 T (T17) cells and increased IL-17 play a key role in driving hidradenitis suppurativa (HS) lesion development, biologic agents used previously in psoriasis that block signaling of IL-17A and/or IL-17F isoforms have been repurposed to treat HS. Our research aimed to characterize the transcriptome of HS T17 cells compared to the transcriptome of psoriasis T17 cells, along with their ligand-receptor interactions with neighborhood immune cell subsets. Single-cell data of 12,300 cutaneous immune cells from 8 deroofing surgical HS skin samples including dermal tunnels were compared to single-cell data of psoriasis skin (19,525 cells from 11 samples) and control skin (11,920 cells from 10 samples). All single-cell data were generated by the same protocol. HS T17 cells expressed lower levels of IL23R and higher levels of IL1R1 and IL17F compared to psoriasis T17 cells (P< .05). HS Treg cells expressed higher levels of IL1R1 and IL17F compared to psoriasis Treg cells (P< .05). Semimature dendritic cells were the major immune cell subsets expressing IL1B in HS, and IL-1β ligand-receptor interactions between semimature dendritic cells and T17 cells were increased in HS compared to psoriasis (P< .05). HS dermal tunnel keratinocytes expressed inflammatory cytokines (IL17C, IL1A, IL1B, and IL6) that differed from the HS epidermis keratinocytes (IL36G) (P< .05). IL6, which synergizes with IL1B to maintain cytokine expression in T17 cells, was mainly expressed by fibroblasts in HS, which also expressed IL11+ inflammatory fibroblast genes (IL11, IL24, IL6, and POSTN) involved in the paracrine IL-1/IL-6 loop. The IL-1β-T17 cell cytokine axis is likely a dominant pathway in HS with HS T17 cells activated by IL-1β signaling, unlike psoriasis T17 cells, which are activated by IL-23 signaling.

Research Highlights.

Research Highlights.

Dysregulation in IFN-γ signaling and response: the barricade to tumor immunotherapy.

Interferon-gamma (IFN-γ) has been identified as a crucial factor in determining the responsiveness to immunotherapy. Produced primarily by natural killer (NK) and T cells, IFN-γ promotes activation, maturation, proliferation, cytokine expression, and effector function in immune cells, while simultaneously inducing antigen presentation, growth arrest, and apoptosis in tumor cells. However, tumor cells can hijack the IFN-γ signaling pathway to mount IFN-γ resistance: rather than increasing antigenicity and succumbing to death, tumor cells acquire stemness characteristics and express immunosuppressive molecules to defend against antitumor immunity. In this review, we summarize the potential mechanisms of IFN-γ resistance occurring at two critical stages: disrupted signal transduction along the IFNG/IFNGR/JAK/STAT pathway, or preferential expression of specific interferon-stimulated genes (ISGs). Elucidating the molecular mechanisms through which tumor cells develop IFN-γ resistance help identify promising therapeutic targets to improve immunotherapy, with broad application value in conjugation with targeted, antibody or cellular therapies.

Unfolded Protein Response Factor ATF6 Augments T Helper Cell Responses and Promotes Mixed Granulocytic Airway Inflammation

Abstract The unfolded protein response (UPR) plays a critical role in many diseases, including airway inflammation. Several recent studies demonstrated a pathogenic role of Activating transcription factor 6a (ATF6a or ATF6), one of the essential arms of UPR, in airway structural cells. However, its role in T helper (TH) cells has not been well examined. In this study, we found that ATF6 was selectively induced by STAT6 and STAT3 in TH2 and TH17 cells, respectively. ATF6 upregulated UPR genes and promoted the differentiation and cytokine production of TH2 and TH17 cells. T cell-specific Atf6-deficiency impaired TH2 and TH17 responses in vitro and in vivo and attenuated mixed granulocytic experimental asthma. ATF6 inhibitor Ceapin A7 suppressed ATF6 downstream gene expression and TH cell cytokine expression in both murine and human memory CD4 +T cells. Administration of Ceapin A7 lessened both TH2 and TH17 responses in vivo, leading to alleviation of both airway neutrophilia and eosinophilia. Thus, our results demonstrate a novel role of ATF6 in TH2 and TH17 cell-driven mixed granulocytic airway disease, suggesting a novel option to combat steroid-resistant mixed and even T2-low endotypes of asthma through targeting ATF6. This work was supported in part by NIH grants HL148337 and AI142200 (X.O.Y.), and DK110439 and DK132643 (M.L.). We acknowledge the University of New Mexico Comprehensive Cancer Center Flow Cytometry Core supported by NIH CA118100 and the Autophagy, Inflammation, and Metabolism in Disease Center Inflammation and Metabolism Core supported by NIH GM121176

Differential silencing of STAT3 isoforms leads to changes in STAT3 activation.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in multiple fundamental biological processes and a key player in cancer development and progression. STAT3 is activated upon tyrosine phosphorylation and is constitutively active in various malignancies; therefore, the expression of pSTAT3 has been recognized as a predictor of poor survival. STAT3 encodes two alternatively-spliced STAT3 isoforms: the full-length STAT3α isoform and the truncated STAT3β isoform. These isoforms have been suggested as the reason for the occasionally observed opposing roles of STAT3 in cancer: an oncogene, on one hand, and a tumor suppressor on the other. To investigate their roles in aggressive breast cancer, we separately silenced each isoform and found that they affect each other's activation, impacting cell viability, cytokine expression, and migration. Silencing specific isoforms can lead to a more favorable balance of activated STAT3 proteins in the cell. Distinguishing between the two isoforms and their active forms is crucial for STAT3-related cancer diagnosis and therapy.

Abstract 6270: Combination therapy with selective SMARCA2 (BRM) degraders for treatment of SMARCA4 (BRG1)-deficient cancers

Abstract SMARCA2 (BRM) and SMARCA4 (BRG1) are mutually exclusive core catalytic subunits of the SWI/SNF complexes and use their ATPase domain to regulate the composition of nucleosomes. Between 4-5% of Non-Small Cell Lung Cancer (NSCLC) patients harbor SMARCA4 damaging mutations or SMARCA4 homo-deletions, resulting in loss of SMARCA4 protein expression. These SMARCA4-deleted cancer cells are predicted to be highly dependent on the paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type normal cells. Although the treatment of lung cancer has improved considerably in recent years with the development of immune checkpoint inhibitors (ICIs) and specific KRASG12C covalent inhibitors, KRASG12C mutations significantly co-occur in 15-17% of SMARCA4-deleted NSCLC patients and have also been shown to correlate with a worse clinical outcome. The co-occurrence of KRASG12C/SMARCA4-deletion mutations in NSCLC led us to investigate whether combining our selective SMARCA2 degraders with KRASG12C or other MAPK pathway inhibitors would demonstrate synergistic effects. To test this hypothesis, we treated SMARCA4 damaging/low expression KRASG12C mutant cell lines with our SMARCA2 degraders and several KRASG12C inhibitors, in addition to SHP2 and MEK inhibitors. Robust synergy was observed with these combinations, suggesting that targeting SMARCA2 together with agents that inhibit distinct nodes of the MAPK pathway in patients with SMARCA4-deleted cancer may be a promising therapeutic strategy. To further understand the mechanisms underpinning the synergy between our SMARCA2 degraders and KRASG12C/MAPK pathway inhibitors, we conducted RNA-seq and found unique transcriptional signatures in the synergistic combinations relative to those of either agent alone. We are currently testing if this in vitro synergy with our SMARCA2 degraders extends to in vivo combination efficacy in CDX and PDX models. Furthermore, early data suggested that SMARCA4 deletion may indicate poor outcomes of ICIs in lung cancer patients. Interestingly, our SMARCA2 degraders promote the antigen presentation pathway and induce pro-inflammatory cytokine expression in SMARCA4-deleted cancer cells. We are currently investigating combinations of our SMARCA2 degraders with ICIs using syngeneic mouse models and human ex-vivo approaches. In summary, our preclinical data suggest that potent and selective SMARCA2 targeted degraders may potentially improve patient outcomes when combined with therapeutic agents targeting the RAS/MAPK pathway and/or ICIs in SMARCA4-deleted cancers. The combination of SMARCA2 degraders with standard of care agents warrants further investigation as a potential novel, effective, and highly targeted combination approach. Citation Format: Michael Hulse, Margot Elkins, Jessica Burtell, Komali Vykuntam, Kris Vaddi, Andrew Combs, Koichi Ito, Peggy Scherle. Combination therapy with selective SMARCA2 (BRM) degraders for treatment of SMARCA4 (BRG1)-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6270.

Dynamics of inflammatory cytokine expression in bovine endometrial cells exposed to cow blood plasma small extracellular vesicles (sEV) may reflect high fertility

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30–200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein–Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.