Induction Of CCL2 Research Articles

Nuclear translocation of plasma membrane protein ADCY7 potentiates T cell-mediated antitumour immunity in HCC

BackgroundThe potency of T cell-mediated responses is a determinant of immunotherapy effectiveness in treating malignancies; however, the clinical efficacy of T-cell therapies has been limited in hepatocellular carcinoma (HCC) owing...

Hepatoprotective and Antioxidant Activities of Polyphenolic Extract of Pyrus communis Leaf in Carbon tetrachloride–Treated Albino Wistar Rats

Polyphenols are active plant compounds that are reportedly capable of eliminating or limiting the deleterious side effects of free radicals and consequently restoring the functional integrity of important organs such as the liver. This study investigated the liver-protecting and antioxidant activities of PEP.c (Polyphenol leaf extract of Pyrus communis) against carbon tetrachloride-induced hepatotoxicity in Wistar rats. Liver damage was induced via intraperitoneal (i.p.) injection of 1.5 mL/kg body weight (b.w.) of 50% carbon tetrachloride (CCl4) in olive oil on the 7th day of extract/drug administration. In-duction of CCl4 significantly (P < 0.05) increased the activities of serum gam-ma-glutamyl transferase (GGT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as a significant decrease (P < 0.05) in the serum level of total protein and the activity of superoxide dismutase and glutathione in the liver tissue with a concomitant increase in malondialdehyde (MDA) level. Oral administration of PEP.c (50, 100, and 250 mg/kg b.w.) for 7 days, significantly (P < 0.05) reduced the elevated serum levels of serum GGT, AST, ALT, ALP and increased the level of total protein as compared to CCl4-induced hepatotoxic-untreated group. However, administration of PEP.c significantly (P < 0.05) decreased concentration of malondialdehyde and increased the activity of the superoxide dismutase (SOD) as well as enhanced glutathione (GSH) level in the liver tissue. Results were compared to N-acetylcysteine, a known liver-protecting agent. Results from histopathological evaluation also supported the hepatoprotective-city of PEP.c in the CCl4-induced albino Wistar rats. The results of this study suggested that PEP.c can be used as a safe and alternative drug for the prevention and management of liver injury.

Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction.

Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models. Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone. Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1β and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models. Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

Sex-Specific Effects of THRβ Signaling on Metabolic Responses to High Fat Diet in Mice.

Thyroid hormone (TH) plays a crucial role in regulating the functions of both bone and adipose tissue. Given that TH exerts its cholesterol-lowering effects in hepatic tissue through the TH receptor-β (TRβ), we hypothesized that TRβ agonist therapy using MGL3196 (MGL) would be effective in treating increased adiposity and bone loss in response to a 12-week high-fat diet (HFD) in adult C57BL/6J mice. Transcriptional and serum profiling revealed that HFD-induced leptin promoted weight gain in both males and females, but MGL only suppressed leptin induction and weight gain in males. In vitro studies suggest that estrogen suppresses MGL activity in adipocytes, indicating that estrogen might interfere with MGL-TRβ function. Compared to systemic adiposity, HFD reduced bone mass in male but not female mice. Paradoxically, MGL treatment reversed macroscopic bone mineral density loss in appendicular bones, but micro-CT revealed that MGL exacerbated HFD-induced trabecular bone loss, and reduced bone strength. In studies on the mechanisms for HFD effects on bone, we found that HFD induced Rankl expression in male femurs that was blocked by MGL. By ex vivo assays, we found that RANKL indirectly represses osteoblast lineage allocation of osteoprogenitors by induction of inflammatory cytokines TNFα, IL-1β, and CCL2. Finally, we found that MGL functions in both systemic adiposity and bone by nongenomic TRβ signaling, as HFD-mediated phenotypes were not rescued in TRβ147F knockout mice with normal genomic but defective nongenomic TRβ signaling. Our findings demonstrate that the negative effects of HFD on body fat and bone phenotypes are impacted by MGL in a gender-specific manner.

Molecular mechanism of activation of human cardiac fibroblasts during Pseudomonas aeruginosa infection

Abstract The prevalence of cardiac dysfunction and its vast implications for human health highlight the need to investigate the mechanistic processes allowing its pathological manifestations. Studies from our lab revealed Pseudomonas aeruginosa (P.a.) infection causes severe cardiac inflammation and dysfunction, despite absence of the bacteria in the heart. However, the underlying mechanism is unknown. Thus, we hypothesize cardiac fibroblast activation by PAMPs enhances cytokine and chemokine release, driving cardiac inflammation. To test our hypothesis in vitro, we used human monocyte derived macrophages (hMDMs) and cardiac fibroblasts (HCFs). We harvested conditioned media (CM) from P.a. infected hMDMs and exposed to HCFs. RNA was extracted at different time points, next generation mRNA sequencing was performed and supernatants were harvested to determine inflammatory cytokine. Our data revealed exposing CM to HCFs upregulates cytokine and chemokine gene expression. Also, we found CCL2, TNF-α and IL-1β levels are high in supernatants harvested from HCFs. To further investigate mechanisms controlling CCL2 expression in HCFs, we identified JNK was involved in induction of CCL2 in HCF. In conclusion, activation of HCFs releases chemokines (CCL2) which recruit leukocytes into heart tissue, causing severe cardiac inflammation. Ongoing and future studies are needed to dive further into mechanisms of this pathway, showing its potential impact in driving cardiac inflammation in vivo.

Anlotinib enhanced CD8+ T cell infiltration via induction of CCL5 improves the efficacy of PD-1/PD-L1 blockade therapy in lung cancer

Non-small cell lung cancer (NSCLC) is a leading cause of mortality worldwide and requires effective treatment strategies. Recently, the development of a novel multiple-target tyrosine kinase inhibitor, anlotinib, has drawn increasing attention, especially it shows advantages when combined with PD-1/PD-L1 blockade. However, the mechanism by which anlotinib improves immunotherapy and remodeling of the tumor microenvironment remains unclear. In this study, we found that anlotinib combined with PD-1 blockade significantly inhibited tumor growth and reduced tumor weight in a lung cancer xenograft model compared to any single treatment. Both immunofluorescence and flow cytometry analyses revealed that anlotinib induced a CD8+ T cell dominated tumor microenvironment, which might account for its improved role in immunotherapy. Further investigations showed that CCL5-mediated CD8+ T cell recruitment plays a critical role in anlotinib and PD-1 blockade strategies. The depletion of CD8+ T cells abrogated this process. In conclusion, our findings showed that the combination of anlotinib and PD-1 blockade produced promising effects in the treatment of lung cancer, and that the induction of CCL5-mediced CD8+ T cell recruitment by anlotinib provided a novel mechanism of action.

Abstract 470: AR-induced miRNA124-3p.2 suppresses the metastasis-promoting factor CCL2

Abstract Objective: The standard treatment for advanced prostate cancer is androgen deprivation therapy that blocks androgen receptor (AR) signaling, but this therapy becomes ineffective within a few years, resulting in castration-resistant prostate cancer. We have previously shown that blockade of AR signaling induces CCL2 and activates cancer cells through an autocrine mechanism, but why AR blockade induces CCL2 secretion has not been elucidated. Micro RNAs (miRs) are endogenous non-coding RNAs that regulate the translation of various proteins. In this study, we investigated whether miRs are involved in CCL2 induction. Subjects and Methods: We used miRNA PCR Plate Array and Target Scan (https://www.targetscan.org) to search for miRs that are controlled by AR and bind to and repress the CCL2 gene. We knocked down the AR of human prostate cancer cell lines LNCaP and C4-2B with siRNA (siAR) and examined the expression of target miRs and CCL2. siAR-transfected LNCaP and C4-2B were transfected with target miR mimic and CCL2 expression was examined. siAR-transfected LNCaP, C4-2B, and C4-2B were transfected with target miR mimic and CCL2 expression was examined. siAR-transfected LNCaP and C4-2B were transfected with target miR mimic and CCL2 expression was examined. The target miR mimic was introduced into them, and the changes in migration ability were evaluated. Results: miR-124-3p.2 was extracted as a candidate from miRNA PCR Plate Array and Target Scan. miR-124-3p.2 expression was upregulated in both LNCaP and C4-2B when DHT was added and downregulated when CCL2 was added. In siAR LNCaP and C4-2B, the expression of CCL2 was decreased when miR-124-3p.2 mimic was introduced. Furthermore, in siAR-transfected LNCaP, C4-2B, the introduction of miR-124-3p.2 mimic suppressed the migration. Conclusion: AR suppresses CCL2 via miR124-3p.2. miR124-3p.2-mediated regulation of CCL2 may be a new therapeutic target for advanced prostate cancer. Citation Format: Shuhei Aoyama, Kouji Izumi, Atsushi Mizokami. AR-induced miRNA124-3p.2 suppresses the metastasis-promoting factor CCL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 470.

The cutaneous beta human papillomavirus type 8 E6 protein induces CCL2 through the CEBPα/miR-203/p63 pathway to support an inflammatory microenvironment in epidermodysplasia verruciformis skin lesions.

Human papillomavirus type 8 (HPV8), a cutaneous genus beta HPV type, has co-carcinogenic potential at sun-exposed sites in patients suffering from the inherited skin disease epidermodysplasia verruciformis (EV). We had previously shown that Langerhans cells responsible for epithelial immunosurveillance were strongly reduced at infected sites and that the HPV8 E7 protein interferes with the CCAAT/enhancer-binding protein (C/EBP)β to suppress the Langerhans cell chemokine CCL20. At the same time, however, we observed that EV lesions are heavily infiltrated with inflammatory immune cells, which is similar to the situation in HPV8 E6 transgenic mice. To identify critical inflammatory factors, we used a broad multiplex approach and found that the monocyte attracting chemokine CCL2 was significantly and strongly induced by HPV8 E6 but not E7-expressing HaCaT cells, which were used as a model for UV-damaged skin keratinocytes. Conditioned media from HPV8 E6-expressing keratinocytes enhanced CCL2-receptor (CCR2)-dependent monocyte recruitment in vitro, and macrophages predominated in the stroma but were also detected in the epidermal compartment of EV lesions in vivo. CCL2 induction by HPV8 E6 was even stronger than stimulation with the proinflammatory cytokine TNF-α, and both HPV8 E6 and TNF-α resulted in substantial suppression of the transcription factor C/EBPα. Using RNAi-mediated knockdown and overexpression approaches, we demonstrated a mechanistic role of the recently identified C/EBPα/miR-203/p63 pathway for HPV8 E6-mediated CCL2 induction at protein and transcriptional levels. Epithelial co-expression of p63 and CCL2 was confirmed in HPV8 E6-expressing organotypic air-liquid interface cultures and in lesional EV epidermis in vivo. In summary, our data demonstrate that HPV8 oncoproteins actively deregulate epidermal immune homeostasis through modulation of C/EBP factor-dependent pathways. While HPV8 E7 suppresses immunosurveillance required for viral persistence, the present study provides evidence that E6 involves the stemness-promoting factor p63 to support an inflammatory microenvironment that may fuel carcinogenesis in EV lesions.

Protective effect of Helianthus annuus seeds extract against CCl4-induced hepatocellular damage

Carbon tetrachloride (CCl4) is a hepatotoxin that causes toxicity in animals on its exposure. Hepatotoxins lead to hepatic demage that is treated by generating antioxidant effect. Synthetic and as well as natural drugs are available for this purpose but phyto-based herbal medicines got paramount importance against drug induced hepatotoxicity Current research was conducted to inspect the protective effect of aqueous extract of Helianthus annuus seeds pre-treatment on the carbon tetrachloride-induced hepatotoxicity in Balb C mice. Study covered the valuation of the enzymatic activity such as alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP) and Lactate dehydrogenase (LDH) and other biochemical components like bilirubin, total protein, catalase, glutathione (GSH), malondialdehyde bis-(dimethyl acetal) tetra ammonium (MDA). The induction of carbon tetrachloride (CCl4) caused rise in plasma ALAT, ASAT, ALP and LDH. Helianthus annuus seeds extract (HA extract) pre-treatment obliterated CCl4-induced deviations in the activities of these enzymes significantly. Bilirubin level increased whereas total protein contents decreased after the induction of CCl4 this effect was reversed by HA extract. Induction of CCl4 caused increased in MDA level while decrease in GSH and catalase. Helianthus annuus seeds extract also abolished these changes. Helianthus annuus seeds extract pre-treatment also prevented CCl4-induced changes in bilirubin and total protein contents. Carbon tetrachloride treatment resulted in huge hepatic damage. This was prevented by Helianthus annuus seeds extract. These results show that Helianthus annuus seeds extract pre-treatment prevented the mice from CCl4-induced hepatic damage, which visibly shows its defensive effects against hepatic damage.

Reprimo (RPRM) as a Potential Preventive and Therapeutic Target for Radiation-Induced Brain Injury via Multiple Mechanisms.

Patients receiving cranial radiotherapy for primary and metastatic brain tumors may experience radiation-induced brain injury (RIBI). Thus far, there has been a lack of effective preventive and therapeutic strategies for RIBI. Due to its complicated underlying pathogenic mechanisms, it is rather difficult to develop a single approach to target them simultaneously. We have recently reported that Reprimo (RPRM), a tumor suppressor gene, is a critical player in DNA damage repair, and RPRM deletion significantly confers radioresistance to mice. Herein, by using an RPRM knockout (KO) mouse model established in our laboratory, we found that RPRM deletion alleviated RIBI in mice via targeting its multiple underlying mechanisms. Specifically, RPRM knockout significantly reduced hippocampal DNA damage and apoptosis shortly after mice were exposed to whole-brain irradiation (WBI). For the late-delayed effect of WBI, RPRM knockout obviously ameliorated a radiation-induced decline in neurocognitive function and dramatically diminished WBI-induced neurogenesis inhibition. Moreover, RPRM KO mice exhibited a significantly lower level of acute and chronic inflammation response and microglial activation than wild-type (WT) mice post-WBI. Finally, we uncovered that RPRM knockout not only protected microglia against radiation-induced damage, thus preventing microglial activation, but also protected neurons and decreased the induction of CCL2 in neurons after irradiation, in turn attenuating the activation of microglial cells nearby through paracrine CCL2. Taken together, our results indicate that RPRM plays a crucial role in the occurrence of RIBI, suggesting that RPRM may serve as a novel potential target for the prevention and treatment of RIBI.

Ubiquitin-specific protease 34 in macrophages limits CD8 T cell-mediated onset of vitiligo in mice

As an autoimmune disorder, vitiligo is characterized by depigmented skin macules. CD8+T cells and macrophages enrichment promote the onset of vitiligo, while the role of macrophages to CD8+T is not well deciphered. To develop a mouse model of vitiligo with prominent epidermal depigmentation, Krt14-Kitl* transgenic mice containing an elevated number of melanocytes in the epidermis with membrane-bound Kit ligand (Kitl*) were adoptively transferred with premelanosome protein (PMEL) CD8+ T cells. On the other hand, Krt14-Kitl* mice were mated with ubiquitin-specific protease 34 (USP34)MKO mice to decipher the role of USP34 in vitiligo. Vitiligo scores and PMEL CD8+ T cell enrichment were detected with flow cytometry. Human peripheral blood mononuclear cells (PBMCs) or mice bone marrow-derived macrophages (BMDMs) were incubated with lipopolysaccharide (LPS), CpG, or co-incubated with KU-55933, an ataxia telangiectasia-mutated (ATM) inhibitor. Chemokine (C-C motif) ligand 2 (CCL2), Ccl5, and interleukin (Il)-12α expression was assayed with real-time PCR, and p-IKKα/β was assayed with Western blots. USP34 was up-regulated in the PBMCs of vitiligo patients and LPS-stimulated BMDMs. USP34 deficiency did not affect the differentiation of CD11b+F4/80+ macrophages in the bone marrow. Immunoprecipitation demonstrated the interaction between USP34 and ATM. USP34 deficiency or KU-55933 administration promoted the induction of Ccl2, Ccl5, Il12α, and p-IKKα/β in LPS or CpG stimulated BMDMs; KU-55933 administration could not affect the expression of the above molecules in USP34 deficient BMDMs. It further revealed that USP34 deficiency promoted the development of vitiligo with increased PMEL CD8+ T cell enrichment, which was not affected by KU-55933 administration. USP34 deficiency in macrophages promotes the onset of vitiligo with increased PMEL CD8+ T cell enrichment, and USP34/ATM complex can be considered as a therapy target.

Overcoming pancreatic cancer immune resistance by codelivery of CCR2 antagonist using a STING-activating gemcitabine-based nanocarrier

STING agonist has recently gained much attention for cancer treatment, but the therapeutic potential of STING agonist is hampered by STING-associated tumor immune resistance. In this work, guided by both bioinformatics and computer modeling, we rationally designed a “one stone hits two birds” nanoparticle-based strategy to simultaneously activate STING innate immune response while eliminating STING-associated immune resistance for the treatment of pancreatic ductal adenocarcinoma (PDAC). We discovered that the ultra-small sized micellar system based on gemcitabine-conjugated polymer (PGEM), which showed superior capacity of penetration in pancreatic tumor spheroid model and orthotopic tumor model, could serve as a novel “STING agonist”. The activation of STING signaling in dendritic cells (DCs) by PGEM increased both innate nature killer (NK) and adaptive anti-tumor T cell response. However, activation of STING signaling by PGEM in tumor cells also drove the induction of chemokines CCL2 and CCL7, resulting in immune resistance by recruiting tumor associated macrophage (TAM) and myeloid-derived suppressor cells (MDSCs). Through the combination of computer modeling and experimental screening, we developed a dual delivery modality by incorporating a CCR2 (the receptor shared by both CCL2 and CCL7) antagonist PF-6309 (PF) into PGEM micellar system. Our studies demonstrated that PGEM/PF formulation significantly reduced pancreatic tumor burden and induced potent anti-tumor immunity through reversing the CCL2/CCL7-mediated immunosuppression. Moreover, PGEM/PF sensitized PDAC tumors to anti-PD-1 therapy, leading to complete suppression/eradication of the tumors. Our work has shed light to the multi-faceted role of STING activation and provided a novel immunotherapy regimen to maximize the benefit of STING activation for PDAC treatment. In addition, this work paved a new way for bioinformatics and computer modeling-guided rational design of nanomedicine.

Individual Variabilities in Adipose Stem Cell Proliferation, Gene Expression and Responses to Lipopolysaccharide Stimulation.

Adipose stem cells (ASCs) are reported to play a role in normal physiology as well as in inflammation and disease. The objective of this work was to elucidate inter-individual differences in growth, gene expression and response to inflammatory stimuli in ASCs from different donors. Human ASC1 (male donor) and ASC2 (female donor) were purchased from Lonza (Walkersville, MD). Cell proliferation was determined by the sulforhodamine B assay. After time-dependent treatment of ASCs with or without bacterial lipopolysaccharide (LPS), marker gene mRNAs for proliferation, steroid hormones, and xenobiotic and immune pathways were determined using RT-PCR, and secreted cytokine levels in media were measured using the Bio-Plex cytokine assay kit. ASCs from both donors expressed androgen receptors but not estrogen receptors. ASC2 had a 2-fold higher proliferation rate and a 6-fold higher level of proliferation marker Ki67 mRNA than ASC1. ASC2 exhibited significantly greater fold induction of TNF-α and CCL2 by LPS compared to ASC1. TNF-α and GM-CSF protein levels were also significantly higher in the LPS-induced ASC2 media, but IL-6 secretion was higher in the LPS-induced ASC1 media. Our findings suggest that inter-individual variability and/or possible sex differences exist in ASCs, which may serve as a key determinant to inflammatory responses of ASCs.

Defining the impact of Chromobox 2 on the immune tumor microenvironment of high grade serous ovarian carcinoma (092)

Objectives: Chromobox 2 (CBX2), an epigenetic writer and subunit of Polycomb Repressor Complex 1 (PRC1), is associated with poor survival and chemoresistance in high-grade serous ovarian carcinoma (HGSOC). In prostate cancer, PRC1 drives an immunosuppressive tumor microenvironment (TME) via induction of CCL2 and recruitment of tumor-associated macrophages (TAMs). We sought to define the impact of genetic modulation of CBX2 on the immune tumor microenvironment (TME) in HGSOC. Methods: We performed lentiviral transduction of shCBX2 in HGSOC cells (OVCAR4). Knockdown was confirmed using immunoblot and mRNA expression via qPCR. RNA sequencing of shControl and shCBX2 cells was performed. Using an ID8 syngeneic mouse model (Trp53-/-, Brca2-/-), we inoculated five mice with either ID8 shControl, ID8 shCbx2/1, or ID8 shCbx2 and allowed tumors to develop. Tumors and ascites volume were measured. The NanoString Mouse Immune Oncology 360 panel was performed on both shControl and shCbx2 tumors to identify differential immune signatures. Multi-spectral immunohistochemistry (mIHC) was performed on 126 human-derived HGSOC tumors to define the relationship between CBX2 and macrophage infiltration. Using The Cancer Genome Atlas (TCGA) and the TIMERv2 platform, we evaluated correlations among CBX2, CCL2, and TAMs. Statistics were completed with ANOVA with Turkey correlation or Pearson, as indicated. A p-value of < 0.05 deemed significant. Conclusions: CBX2 remodels the TME of HGSOC to a more immunosuppressive state. Our data suggest this may be due to increased CCL2 and CXCL16-mediated macrophage activity in the tumor. Loss of CBX2 is associated with decreased levels of CCL2 and CXCL16, as well as inhibited tumor progression. CBX2 has the potential to serve as a novel therapeutic target and could optimize current immunotherapy. Objectives: Chromobox 2 (CBX2), an epigenetic writer and subunit of Polycomb Repressor Complex 1 (PRC1), is associated with poor survival and chemoresistance in high-grade serous ovarian carcinoma (HGSOC). In prostate cancer, PRC1 drives an immunosuppressive tumor microenvironment (TME) via induction of CCL2 and recruitment of tumor-associated macrophages (TAMs). We sought to define the impact of genetic modulation of CBX2 on the immune tumor microenvironment (TME) in HGSOC. Methods: We performed lentiviral transduction of shCBX2 in HGSOC cells (OVCAR4). Knockdown was confirmed using immunoblot and mRNA expression via qPCR. RNA sequencing of shControl and shCBX2 cells was performed. Using an ID8 syngeneic mouse model (Trp53-/-, Brca2-/-), we inoculated five mice with either ID8 shControl, ID8 shCbx2/1, or ID8 shCbx2 and allowed tumors to develop. Tumors and ascites volume were measured. The NanoString Mouse Immune Oncology 360 panel was performed on both shControl and shCbx2 tumors to identify differential immune signatures. Multi-spectral immunohistochemistry (mIHC) was performed on 126 human-derived HGSOC tumors to define the relationship between CBX2 and macrophage infiltration. Using The Cancer Genome Atlas (TCGA) and the TIMERv2 platform, we evaluated correlations among CBX2, CCL2, and TAMs. Statistics were completed with ANOVA with Turkey correlation or Pearson, as indicated. A p-value of < 0.05 deemed significant. Conclusions: CBX2 remodels the TME of HGSOC to a more immunosuppressive state. Our data suggest this may be due to increased CCL2 and CXCL16-mediated macrophage activity in the tumor. Loss of CBX2 is associated with decreased levels of CCL2 and CXCL16, as well as inhibited tumor progression. CBX2 has the potential to serve as a novel therapeutic target and could optimize current immunotherapy.

STAT3 regulates inflammatory cytokine production downstream of TNFR1 by inducing expression of TNFAIP3/A20.

Tumour Necrosis Factor (TNF) potently induces a transient inflammatory response that must be downregulated once any invasive stimulus has resolved. Yet, how TNF‐induced inflammation is shut down in normal cells is incompletely understood. The present study shows that STAT3 was activated in mouse embryo fibroblasts (MEFs) by treatment with TNF or an agonist antibody to TNFR1. STAT3 activation was inhibited by pharmacological inhibition of the Jak2 tyrosine kinase that associates with TNFR1. To identify STAT3 target genes, global transcriptome analysis by RNA sequencing was performed in wild‐type MEFs and MEFs from STAT3 knockout (STAT3KO) mice that were stimulated with TNF, and the results were validated at the protein level by using multiplex cytokine assays and immunoblotting. After TNF stimulation, STAT3KO MEFs showed greater gene and protein induction of the inflammatory chemokines Ccl2, Cxcl1 and Cxcl10 than WT MEFs. These observations show that, by activating STAT3, TNF selectively modulates expression of a cohort of chemokines that promote inflammation. The greater induction by TNF of chemokines in STAT3KO than WT MEFs suggested that TNF induced an inhibitory protein in WT MEFs. Consistent with this possibility, STAT3 activation by TNFR1 increased the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in WT MEFs but not in STAT3KO MEFs. Moreover, enforced expression of Tnfaip3/A20 in STAT3KO MEFs suppressed proinflammatory chemokine expression induced by TNF. Our observations identify Tnfaip3/A20 as a new downstream target for STAT3 which limits the induction of Ccl2, Cxcl1 and Cxcl10 and inflammation induced by TNF.

IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation.

BackgroundOverexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. Since both IFN-γ and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity.MethodsMonocytes (Human monocytic THP-1 cells) were stimulated with IFN-γ and LPS. CCL2 gene expression was determined by real-time RT-PCR. CCL2 protein was determined by ELISA. Signaling pathways were identified by using epigenetic inhibitors and STAT1 siRNA. Acetylation of H3K27 was analyzed by Western blotting. The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR.ResultsOur results show that the co-incubation of THP-1 monocytes with IFN-γ and LPS significantly enhanced the expression of CCL2, compared to treatment with IFN-γ or LPS alone. Similar results were obtained using primary monocytes and macrophages. Interestingly, IFN-γ priming was found to be more effective than LPS priming in inducing synergistic expression of CCL2. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. Mechanistically, we showed that IFN-γ priming induced acetylation of lysine 27 on histone 3 (H3K27ac) in THP-1 cells. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN-γ in this synergy.ConclusionOur findings suggest that IFN-γ, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

Anti-oxidative Effects of Butanol Seed Extract of Parkinsonia aculeata on Carbon Tetrachloride-Induced Liver Damage on Wistar Rats

Medicinal plants have protective effect because of the presence of several compounds which have different mechanism of action. This study sought to assess the anti-oxidative effects of butanol seed extract of Parkinsonia aculeata on carbon tetrachloride (CCl4)-induced liver damage on Wistar rats. The Wistar rats were put into five groups, each with six rats: Group A received a daily dosage of liquid paraffin (1ml/kg); Group B received 1ml/kg body weight of CCl4 (30% in liquid paraffin intraperitoneal); Group C, D, and E received the seed extracts at 100, 120, and 160 mg/kg every day for two weeks. Induction of CCl4 was three times a week for two weeks simultaneously with the extract to the last day. After sacrificed, the liver was harvested and homogenized, and used for further analyses. There was a significant increase (p&lt;0.05) in the levels of superoxide dismutase and catalase in all extract treated groups compared to positive control, except the catalase levels of group treated with 160mg/kg. Similar results was observed in vitamin C, vitamin E and glutathione in rats treated with 100 and 120mg/kg of the extract. The results of this study revealed that butanol seed extract from P. aculeata has antioxidant properties and can protect Wistar rats' livers from the damaging effects of CCl4.

Abstract 2043: The role of CCL20 in response to radiation in head and neck squamous cell carcinomas

Abstract Background: Radioresistance is responsible for most cases of tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC). Combined radiotherapy (RT) and immunotherapy (IT) treatment has improved survival for a minority of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To enhance the response of radioresistant tumors to radiation, an understanding of the factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs and their depletion improves tumor response to RT. In this study, we investigated how radiation modulates Treg infiltration through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity and that blocking CCL20 could lead to improved response to RT. Methods: Human (Cal27, SCC9) and mouse (MOC1, MOC2) cell lines with different immune phenotypes were irradiated at doses of 2Gy or 10Gy. Conditioned media (CM), RNA and protein were collected for the assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cell were were implanted into the right buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for the analysis of serum levels of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Results: Cal27 and MOC2 tumors had a gene signature associated with immunosuppression and Treg infiltration whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed microenvironment. In vitro, high-dose radiation significantly induced CCL20 in Cal27 and MOC2 cells relative to control. Multiplex analysis of chemokines from conditioned media samples showed that induction of CCL20 was associated with increased secretion of GM-CSF by MOC2 and Cal27 cells. In vivo, inhibition of CCL20 alone resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. The combination of RT and anti-CCL20 showed a significant decrease in tumor volume compared to RT alone. Conclusion: Our data suggests that high-dose radiation promotes the induction of CCL20 secretion by tumor cells which is responsible for attraction of Tregs. Inhibition of CCL20 secretion using anti-CCL20 antibodies can slow down tumor growth and enhance the response to radiation. Citation Format: Cleopatra Rutihinda, Ayman J. Oweida, Patrick Delage, Léanie Moreau, Safia Chelighem, Nour Elhouda Saidi. The role of CCL20 in response to radiation in head and neck squamous cell carcinomas [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 2043.

A Unique Cooperativity between Optineurin and CCL5 Triggers Early Cell Death during Herpes Simplex Virus-1 Infection of the Cornea

Abstract Induction of cell death is considered an effective strategy to control viral infections. Rapid killing of infected cells prevents propagation of viruses and thereby, effectively reduces the infection. Herpes Simplex Virus-1 (HSV-1) is known to cause ocular and nervous system infections. While studying an autophagy receptor protein, Optineurin (OPTN), we found evidence of a unique collaboration between OPTN and CCL5 to generate signal for early cell death in HSV-1 infected cells in the cornea. OPTN knockout mice tend to die due to rapid spread of HSV-1 infection to the brain causing encephalitis. Interestingly, the OPTN knockout animals also exhibit a marked reduction in CCL5 induction. Human transcriptomic profile dataset shows a strong positive correlation between CCL5 upregulation and OPTN expression during HSV infection. Our data shows that a well-coordinated activity of both, OPTN and CCL5, is needed to trigger an early cell death signal that mimics pyroptosis to prevent viral spread. Downregulation of CCL5 and/or OPTN causes interference with this signal and a corresponding delay in cell death. Our results provide new information on an anti-HSV1 chemokine signaling pathway that participates in innate immune control of HSV-1 spread and viral transmission.

IL-27 Induces CCL5 Production by T Lymphocytes, Which Contributes to Antitumor Activity.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this study, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent. In CD4+ T cells, IL-27-induced CCL5 production was primarily dependent on Stat1 activation, whereas in CD8+ T cells, Stat1 deficiency does not abrogate CCL5 induction. A chromatin immunoprecipitation assay revealed that in the CCL5 promoter region, both putative Stat3 binding sites exhibit significant binding to Stat3, whereas only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to an IL-27-mediated antitumor effect. This is signified by diminished tumor inhibition in anti-CCL5- and IL-27-treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to antitumor activity.