CCL2 Expression Research Articles

Intestinal Bacteroides drives glioma progression by regulating CD8+ T cell tumor infiltration.

The intestinal microbiota regulates normal brain physiology and the pathogenesis of several neurological disorders. While prior studies suggested that this regulation operates through immune cells, the underlying mechanisms remain unclear. Leveraging two well characterized murine models of low-grade glioma (LGG) occurring in the setting of the neurofibromatosis type 1 (NF1) cancer predisposition syndrome, we sought to determine the impact of the gut microbiome on optic glioma progression. Nf1-mutant mice genetically engineered to develop optic pathway gliomas (Nf1OPG mice) by 3 months of age were reared under germ-free (GF) conditions, treated with specific cocktails of antibiotics, or given fecal matter transplants (FMTs). Intestinal microbial species were identified by 16S genotyping. Neutralizing TGFβ antibodies were delivered systemically, while in vitro experiments used isolated murine microglia and T cells. Single cell RNA sequencing analysis was performed using established methods. Nf1 OPG mice raised in a GF environment or postnatally treated with vancomycin did not harbor optic gliomas or exhibit OPG-induced retinal nerve fiber layer thinning, which was reversed following conventionally raised mouse FMT or colonization with Bacteroides species. Moreover, this intestinal microbiota-regulated gliomagenesis was mediated by circulating TGFβ, such that systemic TGFβ neutralization reduced Nf1-OPG growth. TGFβ was shown to act on tumor-associated monocytes to induce Ccl3 expression and recruit CD8+ T cells necessary for glioma growth. Taken together, these findings establish, for the first time, a mechanistic relationship between Bacteroides in the intestinal microbiome and NF1-LGG pathobiology, suggesting both future predictive risk assessment strategies and therapeutic opportunities.

RNAi-based ALOX15B silencing augments keratinocyte inflammation in vitro via EGFR/STAT1/JAK1 signalling

Arachidonate 15-lipoxygenase type B (ALOX15B) peroxidises polyunsaturated fatty acids to their corresponding fatty acid hydroperoxides, which are subsequently reduced into hydroxy-fatty acids. A dysregulated abundance of these biological lipid mediators has been reported in the skin and blood of psoriatic compared to healthy individuals. RNAscope and immunohistochemistry revealed increased ALOX15B expression in lesional psoriasis samples. Using a cytokine cocktail containing IL-17A, interferon-gamma and tumour necrosis factor-alpha to produce a psoriasis-like phenotype, a role for ALOX15B in human epidermal keratinocyte inflammation was investigated. siRNA-mediated silencing of ALOX15B increased CCL2 expression and secretion. In addition to CCL2, secretion of CCL5 and CXCL10 were elevated in skin equivalents treated with lipoxygenase inhibitor ML351. Inhibition of the JAK1/STAT1 pathway reversed the enhanced CCL2 expression found with ALOX15B silencing. Previous studies have linked epidermal growth factor receptor (EGFR) inhibition with the upregulation of cytokines including CCL2, CCL5 and CXCL10. ALOX15B silencing reduced EGFR expression and inhibition of EGFR signalling potentiated the effect of ALOX15B silencing on increased CCL2, CCL5 and CXCL10 expression. Confirming previous findings, gene expression of cholesterol biosynthesis genes was reduced via reduced ERK phosphorylation. Reduced ERK phosphorylation was dependant on EGFR and NRF2 activation. Furthermore, plasma membrane lipids were investigated via confocal microscopy, revealing reduced cholesterol and lipid rafts. This study suggests a role for ALOX15B in keratinocyte inflammation through modulation of lipid peroxidation and the EGFR/JAK1/STAT1 signalling axis.

P1028 Personalised co-culture model of intestinal organoids and immune cells for evaluating biological therapy response in ulcerative colitis patients

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) affecting approximately 5 million people worldwide and is characterised by mucosal inflammation that may spread throughout the colon1. Existing therapies often have limited effectiveness or cause severe side effects2. Due to the absence of standardised criteria for selecting the most effective treatment for IBD, innovative preclinical drug testing systems are urgently needed. Methods The effect of upadacitinib (UPA) was tested in personalized co-cultures of colonic epithelial cells and macrophages derived from UC patients (n=12) and control (Con) individuals (n=11). Inflammation was induced with inflammatory cytokines TNF-α (10 ng/mL) and IFN-γ (10 ng/mL). Barrier integrity was measured by transepithelial electrical resistance (TEER). Expression of TNF-α, CCL5, and CXCL9 genes was evaluated using qRT-PCR. Data comparison was conducted using the Wilcoxon rank-sum test. A difference between the values was considered significant when p ≤ 0.05 or padj. ≤ 0.05. Results In cell cultures stimulated with inflammatory cytokines, barrier permeability increased (TEER decreased – Con: by 452.03 Ω·cm2, UC: 39.3 Ω·cm2), which partially recovered after removal of inflammatory factors (TEER increased – Con: by 65.91 Ω·cm2, UC – only 47.31 Ω·cm2). Epithelial integrity was restored after incubation with UPA (TEER increased – Con: by 116.82 Ω·cm2; UC: by 226.64 Ω·cm2). At the molecular level, overnight stimulation with TNF-α and IFN-γ increased expression of CXCL9 gene in epithelial cells (UC: fold change (FC) = 3064.49 padj. = 2.07 x 10-5; Con: FC = 664.44, padj. = 2.00 x 10-2) and macrophages (UC: FC = 140.47 padj. = 0.01; Con: FC = 314.43, padj. = 0.01) in both groups. Co-culture treatment with UPA showed an important trend of patient-specific response to therapeutic agent, which probably was the most important factor of non-significant values for observed changes in CCL5, CXCL9, TNF-α expression analysing summarized data. Conclusion To conclude, treatment with TNF-α and IFN-γ reduced epithelial barrier integrity and increased the expression of the inflammatory gene CXCL9 in both epithelial cells and macrophages. UPA tended to restore this disrupted barrier integrity. However, the effects of UPA on gene expression in both cell types showed variable impacts and effectiveness, revealing an individualized response to this biological agent.

Resolution of oncogene-induced senescence markers in HPV-infected cervical cancer tissue

BackgroundOncogene-Induced Senescence (OIS) is a form of senescence that occurs as a consequence of oncogenic overstimulation and possibly infection by oncogenic viruses. Whether senescence plays a role in the pathogenesis of cervical cancer (CC) is not well understood. Moreover, whether cervical epithelial cells that are part of the premalignant cervical intraepithelial neoplasia (CIN), exhibit markers of OIS in Human Papillomavirus (HPV)-infected tissue, has not been investigated.MethodsWe utilized a set of patient-derived premalignant and malignant tissue samples to investigate the protein (Ki67 and Lamin B1) and gene (TP53, IL1A, CCL2, and MMP9) expression of several OIS-associated biomarkers using immunohistochemistry (IHC) and qRT-PCR, respectively. Furthermore, we characterized the HPV status of all tissue samples.ResultsMost of the CC samples (34/37) were positive for HPV, mainly HPV-16 which was observed in 62.2% of the CC samples. Among CINs, HPV infection was found in 60.2% of the 32 samples with HPV-16 as the dominant genotype in 58.5% of the CINs. IHC analysis revealed a significant increase in the expression levels of both Ki67 and Lamin B1 proteins in CC tissue compared to CIN. On average, 93% of tumor cells were positive for Ki67 in comparison to only 25% of premalignant cells in CIN samples. Similarly, Lamin B1 expression was observed in 89% of tumor cells in malignant tissue on average, compared to 60% in CIN samples. Importantly, Lamin B1 expression was elevated in nonmalignant cervical tissue suggesting that its downregulation is more predominant in the premalignant state. Furthermore, RT-PCR revealed a significant decrease in the expression of TP53, IL1a, CCL2, and MMP9 markers in CC samples compared to CINs. Specifically, 84% of CC samples showed reduced TP53 expression, 90% showed reduced IL1a expression, 74% showed reduced CCL2 expression, and 76% showed reduced MMP9 expression when compared with their premalignant baseline. Infection of HPV was confirmed in 61% of the tumor tissues while only 25% of the CINs were positive for HPV.ConclusionThis work shall provide an opportunity to further examine the role of OIS in the process of HPV-driven CC development.

Effects of Production System With or Without Growth-Promoting Technologies on Growth and Blood Expression of (Cyto)Chemokines and Heat Shock and Tight Junction Proteins in Bos taurus and indicus Breeds During Summer Season.

Heat stress (HS) induced by global warming is a real welfare, productivity, and economic burden of cattle production. However, some cattle breeds have superior physiological adaptive traits to others, yet the underlying molecular mechanisms are not fully defined. The present study aimed, therefore, to determine the expression profile of stress-related molecular signatures in the blood of thermosensitive Angus (Bos taurus) and thermotolerant Brahman (Bos indicus) cattle breeds managed without (CON) or with growth-promoting technology (TRT) during the summer (April-October, 2023) season in Colorado, US. Body weight (BW) was significantly increased from April to October, and the amplitude was greater for the Angus compared to the Brahman breed. The TRT system slightly increased BW, mainly in the Angus breed. Molecular analyses showed that all tested genes were expressed in beef cattle blood. When comparing production systems, the expression of HSP1A1 was significantly upregulated, and HSP90 was downregulated in CON compared to TRT cattle. The expression of IL6, CCL20, and OCLN was induced by the CON system only in the Angus and not in the Brahman breed. At the breed level, Angus cattle exhibited greater expression of IL10, CCL20, and CLDN1 compared to their Brahman counterparts. There was a significant period by production system as well as period by breed interactions. The expression of HSP1A1 increased in both breeds during October. The expression of IL10, CXCL14, CXCR2, and CLDN1 was affected by the production systems in a period-dependent manner. However, the expression of IL6, CXCL14, CCL5, and CXCR2 was upregulated in Angus cattle in a period-sensitive manner. In summary, HSPs, (chemo)cytokines, and tight junction proteins are expressed in the whole blood of beef cattle, and their expression is regulated in a breed-, period-, and/or production system-dependent manner. This could open new vistas for future research to identify molecular signatures for non-invasive stress monitoring and/or marker-assisted genetic selection for robustness and resilience to HS.

5β-hydroxycostic acid from Laggera alata ameliorates sepsis-associated acute kidney injury through its anti-inflammatory and anti-ferroptosis effects via NF-κB and MAPK pathways.

The whole plant of Laggera alata is frequently utilize to remedy inflammatory diseases including nephritis as a traditional Chinese medicine. However, its active ingredients and mechanism of action against sepsis-associated acute kidney injury (SA-AKI) are unknown. This study aimed to identify active compounds from L. alata that inhibit renal inflammation and ameliorate SA-AKI, and to elucidate their mechanisms of action. The chemical constituents were separated from the ethyl acetate layer of L. alata methanol extract by column chromatography over silica gel, medium-pressure liquid chromatography and semipreparative high-performance liquid chromatography. Extensive spectroscopic techniques were applied to determine the chemical structures. The anti-inflammatory efficiency was measured by analyzing the NO production in RAW 264.7 cells. The levels of IL-6, IL-1β, CCL-2 and CCL-5 mRNA were determined by qRT-PCR. Cecal ligation and puncture (CLP) surgery is a frequently applied method to establish the mouse sepsis model. Sepsis was thus induced in mice via CLP. The effect in the treatment of SA-AKI was evaluated by H&E staining and ELISA detection. Western blotting was used to evaluate the protein levels involved in ferroptosis, NF-κB and MAPK signaling pathways. Twelve compounds were obtained from L.alata including four unreported sesquiterpenoids (1-4). Compound 5 exhibited the most significant inhibitory effect on NO production with the IC50 value of 6.034 μM and could restrain the mRNA expression of inflammatory factors IL-6, IL-1β, CCL-2 and CCL-5. The in vivo results demonstrated that compound 5 alleviated the renal injury by decreasing the serum IL-6, IL-1β, Cr, and BUN levels, reducing the kidney contents of Cys-C and KIM-1, and regulating the kidney levels of MDA, GSH, ferrous iron, GPX4, FTH1, and SLC7A11. Furthermore, Compound 5 also repressed the NF-κB and MAPK pathways in vitro and in vivo. This study revealed that compound 5 could ameliorate SA-AKI through exerting its anti-inflammatory and anti-ferroptosis effects via NF-κB and MAPK pathways. The current research supported the traditional use of L.alata in the treatment of renal diseases.

Lactate modulates microglial inflammatory responses through HIF-1α-mediated CCL7 signaling after cerebral ischemia in mice.

Lactate is a potent regulator of neuroinflammation. We recently demonstrated that lactate alleviated neuronal injury via HIF-1α-regulated microglial inflammation after oxygen-glucose deprivation (OGD). However, the underlying mechanisms and the effect of lactate on microglial responses after ischemic stroke remained unknown. Mouse acute cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion (MCAO). L-lactate (100mM, 2μl) was intracerebroventricularly administrated 30min after the reperfusion. Microglia responses were evidenced by the expression of multiple markers such as CD86, iNOS, arginase-1, CD206 and Ym1 in the peri-infarction 24h after MCAO using western blot analysis and quantitative real-time PCR. Inflammatory factors IL-6, TNF-α, TGF-β and IL-10, as well as NF-κB signaling were also detected. Infarct size and neuronal apoptosis in the peri-infarction at 24h, mice survival within 7days and long-term neurobehavioral function were evaluated. The involvement of HIF-1α in lactate-mediated microglial inflammation after MCAO was assessed using a HIF-1α inhibitor. Additionally, transcriptome analysis was used to identify the potential lactate targets in BV2 cells after OGD. The recombinant product of the identified CCL7 gene was used to verify its effect on cerebral ischemia-reperfusion injury in vivo. Lactate supplementation reduced infarction volume, neuronal apoptosis and neurological deficits. Lactate reduced the expression of CD86, iNOS, IL-6, TNF-α and elevated the expression of arginase-1, CD206, Ym1, TGF-β and IL-10 in the peri-infarction at 24h after reperfusion. Consistently, lactate inhibited the NF-κB signaling. Additionally, lactate upregulated HIF-1α in microglia 24h after reperfusion, while inhibition of HIF-1α reversed the effects of lactate on brain damage and neuroinflammation after cerebral ischemia. Furthermore, CCL7 was identified as the top down-regulated inflammatory gene induced by lactate in OGD-treated BV2 cells. It was also found high expression of CCL7 in the peri-infarction at 24h after reperfusion and lactate treatment inhibited CCL7 expression. However, HIF-1α inhibitor reversed the effect of lactate treatment on CCL7 expression. Finally, supplementation of recombinant CCL7 reversed the mitigated neuroinflammation and neuroprotective effect rendered by lactate treatment after MCAO. We concluded that treatment with lactate modulated the microglia inflammatory responses and alleviated cerebral ischemia injury. The inhibition of CCL7/NF-κB signaling by HIF-1α might be involved in the beneficial effect of lactate treatment.

Human leukocyte antigen DR alpha inhibits renal cell carcinoma progression by promoting the polarization of M2 macrophages to M1 via the NF-κB pathway

Human leukocyte antigen DR alpha (HLA-DRA) is recognized for its inhibitory effect on the progression of clear cell renal cell carcinoma (ccRCC); high HLA-DRA expression levels are positively correlated with improved prognosis in patients with ccRCC. In this study, we evaluated HLA-DRA expression in ccRCCs, its effects on tumor-associated macrophage recruitment, and the influence of polarization. Clinical cohort analyses revealed that elevated HLA-DRA expression in ccRCC cells was correlated with enhanced tumor infiltration by M1-type macrophages. In addition, ccRCC prognosis was predicted by combining HLA-DRA expression level analysis and the M1/M2 macrophage ratio. In vitro studies demonstrated that ccRCC cells with increased HLA-DRA expression promoted THP-1 cell migration and induced macrophage polarization toward the M1 phenotype. The effect was further substantiated in a mouse xenograft model in which an increase in M1 macrophages was observed. In addition, co-culturing macrophages with the supernatant from cells overexpressing HLA-DRA induced the expression of proteins associated with both M1 and M2 macrophage polarization. HLA-DRA was intricately linked to the expression and secretion of chemokines, including CCL2, CCL5, MIP-1ɑ, and CXCL-10. Moreover, the NF-κB pathway activation promoted polarization to M1 macrophages. This study shows that HLA-DRA and the M1/M2 ratio are indicators of favorable prognosis in patients with ccRCC. HLA-DRA promotes M1-like polarization by regulating NF-κB, which can be used as a therapeutic target to enhance anti-tumor immunity.

Integration of transcriptomics and metabolomics reveals the mechanism of Glycyrrhizae Radix Et Rhizoma extract inhibiting CCL5 in the treatment of acute pharyngitis.

Acute pharyngitis (AP) is a common condition marked by inflammation of the oropharynx, which can lead to severe throat swelling, breathing difficulties, and even suffocation, significantly impacting quality of life. Despite the beneficial anti-inflammatory activity of Glycyrrhizae Radix Et Rhizoma (GRER) and Isoliquiritigenin (ISL), their pharmacological mechanisms against AP remain unclear. This study explores the mechanisms by which GRER treats AP, utilizing both transcriptomics and metabolomics approaches. We identified the chemical components of GRER and those that enter the bloodstream using UPLC-MS/MS. Based on15 % ammonia-induced AP model, this study integrates transcriptomics and metabolomics to investigate the mechanism of GRER and ISL in the AP treatment. The results indicated that GRER has significantly protective and anti-inflammatory effects against AP. Our analysis identified 144 components of GRER in vitro and 17 components in vivo. Network pharmacology and quantitative analysis highlighted ISL as a key active ingredient responsible for GRER's anti-AP effects. Transcriptomics and metabolomics results indicate that GRER and its active ingredient ISL exert therapeutic effects on AP by inhibiting the expression of CCL5 in pharyngeal tissue, thereby downregulating the levels of pro-inflammatory metabolites malic acid and fumaric acid in the tricarboxylic acid (TCA) cycle pathway. The data in this article demonstrated that GRER and ISL has significantly anti-inflammatory effects and protective effects for AP by regulating CCL5 expression to reduce the levels of pro-inflammatory metabolites within TCA cycle pathway. It provides a scientific basis for prevention and treatment of AP.

Unveiling the interplay between hepatocyte SATB1 and innate immunity in autoimmune hepatitis

BackgroundInvestigating the function of SATB1 in hepatocytes is essential for developing therapeutic strategies for autoimmune hepatitis (AIH). Although SATB1 has been extensively studied in immune cells, its specific activity in hepatocytes within the context of AIH remains unclear. MethodsSATB1 expression in AIH hepatocytes was assessed by qRT-PCR, Western blotting, flow cytometry, and immunohistochemistry. In vivo modulation used RNA interference viruses and overexpression plasmids. SATB1′s proinflammatory effects were analyzed with protein microarray, immunohistochemistry, and flow cytometry. Chemotactic effects on RAW264.7 macrophages were tested in vitro, with mechanisms explored by dual-luciferase assays and CUT&RUN qPCR. Liver injury was evaluated by histopathology and serum biochemistry. ResultsSATB1 was significantly upregulated in hepatocytes of AIH patients and models, showing a stronger increase in hepatocytes than in CD45+ cells, and positively correlated with liver injury severity. In vivo RNAi-mediated SATB1 inhibition reduced liver inflammation, while SATB1 overexpression aggravated AIH progression. Both interference and overexpression experiments confirmed that SATB1 promotes liver injury by facilitating the infiltration of proinflammatory (Ly6Chigh) macrophage. In vitro, supernatant from SATB1-overexpressing hepatocytes enriched chemokine signaling pathways, leading to increased CCL2 expression and release, which attracted macrophages and drove their proinflammatory polarization. Mechanistically, SATB1 promoted CCL2 transcription by binding to its DNA and recruiting p300/CBP. ConclusionsThis study reveals that SATB1 is upregulated in hepatocytes in AIH. Elevated SATB1 levels in liver cells contribute to autoimmune hepatitis by increasing CCL2 expression, promoting the recruitment of inflammatory monocyte-derived macrophage, and reshaping the composition of the liver immune microenvironment.

Membrane palmitoylated protein MPP1 inhibits immune escape by regulating the USP12/ CCL5 axis in urothelial carcinoma.

The response rate to immunotherapy in patients with urothelial carcinoma remains limited. Studies have shown that membrane palmitoylated proteins (MPPs) play key roles in tumor progression. However, the mechanisms by which MPP1 regulates immune escape in urothelial carcinoma are not well understood. The TCGA and BEST databases were used to analyze the associations between the expression of members of the MPP family and the prognosis or immunotherapy sensitivity of urothelial carcinoma patients. MPP1 was identified due to its significant association with survival and immunotherapy sensitivity. MPP1 expression in urothelial carcinoma tissues and cell lines was examined. An MPP1 overexpression vector was used to transfect urothelial carcinoma cells. The functional assays included proliferation, migration, urothelial carcinoma cell-CD8+ T-cell coculture, CD8+ T-cell chemotaxis, and tumorigenesis in human immune reconstitution NOG mice (HuNOG). Bioinformatics, coimmunoprecipitation (CO-IP), mass spectrometry, quantitative real-time polymerase chain reaction (RT-qPCR), and western blotting were used to validate the activity of the MPP1/USP12/CCL5 cascade. Analysis of the BEST data revealed that, compared with other MPP family genes, MPP1 was more strongly associated with urothelial carcinoma prognosis and immunotherapy response. Low MPP1 expression was observed in urothelial carcinoma patients and was positively associated with better survival. MPP1 inhibited the proliferation and migration of urothelial carcinoma cells. Bioinformatics, in vitro coculture assays, and in vivo tumorigenesis experiments demonstrated that MPP1 promotes CCL5 production and CD8+ T-cell chemotaxis in the urothelial carcinoma tumor microenvironment (TME). Mechanistically, bioinformatics, mass spectrometry, co-IP, RT-qPCR, and western blot analyses indicated that MPP1 increases CCL5 expression by binding to and promoting USP12. MPP1 significantly inhibits urothelial carcinoma cell proliferation and immune escape via the MPP1/USP12/CCL5 cascade. MPP1 has the potential to serve as a biomarker for guiding immunotherapy in patients with urothelial carcinoma.

Identification of RN7SK LncRNA as a novel biomarker in Alzheimer’s disease using bioinformatics and expression analysis

Alzheimer’s disease (AD) is a degenerative illness that accounts for the common type of dementia among adults over the age of 65. Despite extensive studies on the pathogenesis of the disease, early diagnosis of AD is still debatable. In this research, we performed bioinformatics approaches on the AD-related E-MTAB 6094 dataset to uncover new potential biomarkers for AD diagnosis. To achieve this, we performed in-depth in silico assays, including differentially expressed genes analysis, weighted gene co-expression network analyses, module-trait association analyses, gene ontology and pathway enrichment analyses, and hub genes network analyses. Finally, the expression of the identified candidate genes was evaluated in AD patients PBMC samples by qRT-PCR. Through computational analyses, we found that RN7SK LncRNA and its co-expressed genes of TNF, TNFAIP3, CCLT3, and FLT3 are from key genes in AD development that are associated with inflammatory responses. Our experimental validation revealed that RN7SK LncRNA and TNF were substantially up-regulated in AD samples (P = 0.006 and P = 0.023, respectively). Whereas, TNFAIP3 expression was significantly decreased (P = 0.016). However, the expression of CCL3 and FLT3 did not differ significantly between two groups (P = 0.396 and P = 0.521, respectively). In conclusion, in this study a novel LncRNA associated with AD pathogenesis were identified, which may provide new diagnostic biomarker for AD.

LRP4 mutations promote tumour progression and resistance to anti-PD-1 therapy in recurrent hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) recurrence is a major factor limiting long-time survival and the cause of most deaths in patients with HCC. However, molecular characterisation and potential therapeutic targets of recurrent HCC remain mostly unknown. We performed whole-exome sequencing (WES) in 63 matched primary and recurrent HCC tumours and combined the data with whole-genome sequencing (WGS) results in 43 paired samples from our previous study. Sanger sequencing was used to identify all low-density lipoprotein receptor-related protein 4 (LRP4) coding exons in 203 additional patients with recurrent HCC. We identified LRP4 somatic mutations in 7.8% (24/309) of recurrent tumours and only 0.97% (3/309) of primary tumours (P<0.001). Prognosis after the second liver resection was poorer in patients with an LRP4 mutation. Biofunctional investigations demonstrated that inactivating LRP4 mutations promoted tumour progression and immunosuppression. Mechanistically, mutated LRP4 reduced intratumoural conventional type 1 dendritic cell and CD8+ T cell infiltration by repressing CCL4 expression and secretion through activation of β-catenin signalling, resulting in resistance to anti-programmed cell death protein-1 (PD-1) therapy. Patients with recurrent HCC carrying an LRP4 mutation did not benefit from anti-PD-1 treatment after their second resection surgery. A β-catenin inhibitor reversed LRP4-induced resistance to anti-PD-1 therapy in humanised tumour-bearing mice. Our results identified novel LRP4 mutations important in recurrent HCC. Inactivating LRP4 mutations were associated with resistance to anti-PD-1 therapy and could be useful biomarkers for precision therapy in patients with recurrent HCC.

Lung immune incompetency after mild peritoneal sepsis and its partial restoration by type 1 interferon: a mouse model study

BackgroundSepsis is commonly associated with acute respiratory distress syndrome (ARDS). Although the exaggerated inflammation may damage intact lung tissues, a percentage of patients with ARDS are reportedly immunocompromised, with worse outcomes. Herein, using a murine sepsis model, time-course immune reprogramming after sepsis was evaluated to explore whether the host is immunocompromised. Leukocyte kinetics in the lung tissue were evaluated in a male C57/BL6 mouse model of mild peritoneal sepsis induced by cecal ligation and puncture, with the survival rate exceeds 90%. Lung immune reactivity was evaluated by intratracheal instillation of lipopolysaccharide (LPS; 30 µg). Furthermore, the effect of interferon (IFN)-β in vivo and ex vivo was evaluated.ResultsFour days after sepsis, the lung water content remained high, even among mice in clinical recovery. While monocytes and neutrophils gradually accumulated in the lung interstitium, the inflammatory cytokine/chemokine expression levels in the lungs continued to decline. Intratracheal LPS instillation induced more leukocyte trafficking and protein leakage into the alveoli in the septic lung, indicating more severe lung injury. However, LPS stimulation-associated mRNA expression of tnf, il6, ccl2, and cxcl1 was suppressed. Intra-alveolar expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and keratinocyte-derived cytokine (KC) was also suppressed. Monocytes isolated from the lung tissue showed an impaired response in il6, ccl2, and cxcl1 to LPS. Systemic IFN-β restored the above impaired regulator function of monocytes, as did coculturing these cells from lung tissue with IFN-β.ConclusionsHistologically accelerated inflammation and paradoxically suppressed immunological regulator signaling were observed in the early recovery phase of sepsis. This observation may provide a model for the immunologically irresponsive state that occurs in some patients with sepsis. Systemic IFN-β partly restored the post-septic immunocompromised state, indicating its therapeutic potential for the immunosuppressive state seen in some patients with sepsis/ARDS.

Potent phytoceuticals cocktail exhibits anti-inflammatory and antioxidant activity on LPS-triggered RAW264.7 macrophages in vitro.

Chronic inflammatory conditions, which include respiratory diseases and other ailments, are characterized by persistent inflammation and oxidative stress, and represent a significant health burden, often inadequately managed by current therapies which include conventional inhaled bronchodilators and oral or inhaled corticosteroids in the case of respiratory disorders. The present study explores the potential of Vedicinals®9 Advanced, a polyherbal formulation, to mitigate LPS-induced inflammation and oxidative stress in RAW264.7 mouse macrophages. The cells were pre-treated with Vedicinals®9 Advanced, followed by exposure to LPS to induce an inflammatory response. Key experimental outcomes were assessed, including nitric oxide (NO) and reactive oxygen species (ROS) production, as well as the expression of inflammatory and oxidative stress-related genes and proteins. Vedicinals®9 Advanced significantly reduced LPS-induced NO and ROS production, indicating strong anti-inflammatory and antioxidant properties. Additionally, the formulation downregulated the LPS-upregulated mRNA expression of pro-inflammatory cytokines, such as TNF-α and CXCL1, and oxidative stress markers, including GSTP1 and NQO1. Furthermore, Vedicinals®9 Advanced downregulated the LPS-induced protein expression of the chemokines CCL2 and CCL6, the LPS co-receptor, CD14, and the pro-inflammatory cytokines G-CSF and IL-1β. These findings highlight the potential of Vedicinals®9 Advanced as a therapeutic option for managing CRDs and other inflammatory conditions. The formulation's ability to simultaneously target inflammation and oxidative stress suggests it may offer advantages over existing treatments, with potential for broader application in inflammatory diseases.

Identification and phenotypic characterization of neoantigen-specific cytotoxic CD4+ T cells in endometrial cancer.

Tumor-reactive CD4+ T cells often accumulate in the tumor microenvironment (TME) in human cancer, but their functions and roles in antitumor responses remain elusive. Here, we investigated the immunopeptidome of HLA class II-positive (HLA-II+) endometrial cancer with an inflamed TME using a proteogenomic approach. We identified HLA-II neoantigens, one of which induced polyclonal CD4+ tumor-infiltrating lymphocyte (TIL) responses. We then experimentally demonstrated that neoantigen-specific CD4+ TILs lyse target cells in an HLA-II-dependent manner. Single cell transcriptomic analysis of the TME coupled with T-cell receptor (TCR) sequencing revealed the presence of CD4+ T-cell clusters characterized by CXCL13 expression. The CXCL13+ clusters contained two subclusters with distinct cytotoxic gene expression patterns. The identified neoantigen-specific CD4+ T cells were found exclusively in one of the CXCL13+ subclusters characterized by granzyme B and CCL5 expression. These results demonstrate the involvement of tumor-reactive CD4+ T cells with cytotoxic function in immune surveillance of endometrial cancer and reveal their transcriptomic signature.

Leptin/LPS-treated dendritic cells reduce the expression of genes involved in tumor tissue metastasis and angiogenesis in an animal model of breast cancer.

Leptin, an immune-regulating protein, enhances the maturation of dendritic cells (DCs). We previously demonstrated that leptin and lipopolysaccharide (LPS) promote the expression of co-stimulatory molecules on the surface of DCs. Leptin/LPS-treated DCs increased T cell responses against 4T1 breast cancer in mice. Therefore, in the present study, we investigate the effects of a DC vaccine treated with leptin and LPS on the genes involved in tumor metastasis, angiogenesis, and related cytokines in a mouse model of breast cancer. Tumor induction was achieved through subcutaneous injection of 4T1 cells into syngeneic mice. On days 12 and 19, the mouse groups received the DC vaccine treated with leptin and a combination of leptin and LPS. After sacrificing the mice on day 26, the levels of IL-6 and IL-33 in the serum were assayed using the ELISA technique, and the expression levels of the VEGF, CCL2, MMP9, and CCL5 genes in the tumors were measured by Real-Time PCR. Compared to untreated tumor-bearing mice, the leptin-treated mature DC (mDC) group exhibited a significant reduction in the expression of MMP9 (0.33-fold, p = 0.01) and CCL5 (0.81-fold, p = 0.02). The leptin-LPS-treated mDC group showed decreased expression of genes involved in metastasis and tumor growth, including VEGF (0.72-fold, p = 0.03), MMP9 (0.26-fold, p = 0.001), and CCL5 (0.3-fold, p = 0.006), indicating more efficient prevention of metastasis. The CCL2 gene expression levels in both treatment groups showed a slight decreasing trend, but these changes were not statistically significant. The leptin-treated mDC group reduced IL-6 production by approximately 16% (p = 0.02), while treatment with the leptin-LPS-treated mDC significantly decreased IL-6 production by approximately 22% (p = 0.01) and increased IL-33 production by approximately 42% (p = 0.03). The findings of the present study indicate that the leptin-LPS-treated mDC vaccine group reduced the expression of genes and cytokines involved in metastasis and angiogenesis, demonstrating greater efficacy compared to the leptin-treated mDC vaccine group.

In vitro culture of leukemic cells in collagen scaffolds and carboxymethyl cellulose-polyethylene glycol gel.

Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene (MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).

FC04 Dynamic KC-neutrophil-fibroblast communication network centred on IL-36/TNFSF15 responses characterizes inflammatory responses in generalized pustular psoriasis

Abstract Generalized pustular psoriasis (GPP) is a severe psoriatic subtype characterized by epidermal neutrophil infiltration, often occurring in acute, potentially life-threatening episodes. However, the characterization of the immune microenvironment in GPP lesions compared with plaque psoriasis remains largely unknown. Here, we used single-cell RNA profiling to interrogate the transcriptomes of ∼60 000 single cells from GPP lesional skin (n = 13) and healthy adult skin (n = 4), combined with spatial RNA sequencing. GPP was associated with major shifts in multiple cell populations, including, in particular, increased frequency of myeloid immune cells and supraspinous keratinocytes. We identified a unique myeloid-derived neutrophil subset characterized by the absence of CASP8. CASP8− neutrophils were accompanied by high expression of inflammatory pathway genes, including RIPK1, NF-KB1, IL1B, CXCL1, and CXCL8 in a stable disease state and were found at low levels in an inactive state in healthy skin. Spatial mapping of these genes illustrated the neutrophil transition from pre-inflammation to a pro-inflammation state through casting off CASP8. In silico and in vitro co-culture studies demonstrated that the communication network between IL36G+ spinous KCs and neutrophils is enlarged in the GPP lesions, with TNFSF15 (TL1A) released from neutrophils exaggerating the inflammatory crosstalk. While there was a slight increase in Th17 cells, there was greater prominence of CD8+ cytotoxic T cells highlighted by upregulated GZMB, CCL5, NKG7, and PRF1 expression, hinting at a cytotoxicity tendency rather than inflammation response in GPP lesions. Notably, a subset of S100A9+ fibroblasts in GPP was observed to contribute to the amplification of the GPP immune network through the transition to a pro-inflammatory state, connected by ligand–receptor interactions with other spatially proximate immune cells, including Th17 cells, CD8+ Tc cells, neutrophils, and IL1B+ macrophages. In sum, these data provide an in-depth view of immune cell participation and highlight the role of neutrophil–KC–fibroblast crosstalk in GPP pathogenesis.

Unveiling Key Biomarkers and Mechanisms in Septic Cardiomyopathy: A Comprehensive Transcriptome Analysis.

Septic cardiomyopathy (SCM) is a significant global public health concern characterized by substantial morbidity and mortality, which has not been improved for decades due to lack of early diagnosis and effective therapies. This study aimed to identify hub biomarkers in SCM and explore their potential mechanisms. We utilized the GSE53007 and GSE207363 datasets for transcriptome analysis of normal and SCM mice. Hub biomarkers were identified through a protein-protein interaction (PPI) network and validated using LPS-treated C57/BL6 mice. Functional enrichment analysis was performed to uncover relevant signaling pathways, while single-cell RNA sequencing was used to examine key genes and regulatory mechanisms associated with SCM. A total of 374 differentially expressed genes (DEGs) were identified, with 268 genes up-regulated and 106 genes down-regulated. Functional enrichment highlighted chemokine activity and receptor binding, with KEGG pathways revealing significant involvement of the TNF and IL-7 signaling pathways. Deterioration of cardiac function, elevated inflammatory markers such as IL-1β, IL-6, and increased cardiac injury biomarkers such as cTnI indicated the successful establishment of our SCM model. Subsequently, qPCR was conducted to validate the expression of the top 10 genes, through which we identified Cd40, Tlr2, Cxcl10, Ccl5, Cxcl1, Cd14, Gbp2, Ifit2, and Vegfa as key biomarkers. Single-cell sequencing indicated increased neutrophil and macrophage populations, with decreased B cells and cardiomyocytes. Additionally, transcription regulators Irf1 and Stat1 were found to potentially regulate the expression of Gbp2, Cxcl10, Ccl5, and Cd40, linking SCM to immune response, ferroptosis, pyroptosis, cuproptosis, and m6A RNA methylation modification. This study identified nine hub biomarkers and two transcription regulators associated with SCM. Exploring the connections between SCM and immunity, ferroptosis, pyroptosis, cuproptosis, and m6A RNA methylation might provide insights into the underlying mechanisms. These findings enhanced our understanding of SCM's underlying mechanisms and might pave the way for novel therapeutic strategies to improve clinical outcomes.