The bioinfomatics analysis of the M1 macrophage-related gene CXCL9 signature in cervical cancer.

BACKGROUND: The expression and function of coexpression genes of M1 macrophage in cervical cancer have not been identified. And the CXCL9-expressing tumour-associated macrophage has been poorly reported in cervical cancer. METHODS: To clarify the regulatory gene network of M1 macrophage in cervical cancer, we downloaded gene expression profiles of cervical cancer patients in TCGA database to identify M1 macrophage coexpression genes. Then we constructed the protein-protein interaction networks by STRING database and performed functional enrichment analysis to investigate the biological effects of the coexpression genes. Next, we used multiple bioinformatics databases and experiments to overall investigate coexpression gene CXCL9, including western blot assay and immunohistochemistry assay, GeneMANIA, Kaplan-Meier Plotter, Xenashiny, TISCH2, ACLBI, HPA, TISIDB, GSCA and cBioPortal databases. RESULTS: There were 77 positive coexpression genes and 5 negative coexpression genes in M1 macrophage. The coexpression genes in M1 macrophage participated in the production and function of chemokines and chemokine receptors. Especially, CXCL9 was positively correlated with M1 macrophage infiltration levels in cervical cancer. CXCL9 expression would significantly decrease and high CXCL9 levels were linked to good prognosis in the cervical cancer tumour patients, it manifestly expressed in blood immune cells, and was positively related to immune checkpoints. CXCL9 amplification was the most common type of mutation. The CXCL9 gene interaction network could regulate immune-related signalling pathways, and CXCL9 amplification was the most common mutation type in cervical cancer. Meanwhile, CXCL9 may had clinical significance for the drug response in cervical cancer, possibly mediating resistance to chemotherapy and targeted drug therapy. CONCLUSION: Our findings may provide new insight into the M1 macrophage coexpression gene network and molecular mechanisms in cervical cancer, and indicated that M1 macrophage association gene CXCL9 may serve as a good prognostic gene and a potential therapeutic target for cervical cancer therapies.

Cervical cancer is a common gynaecological malignancy, investigating the precise gene expression regulation of M1 macrophage is crucial for understanding the changes in the immune microenvironment of cervical cancer. In our study, a total of 82 coexpression genes with M1 macrophages were identified, and these genes were involved in the production and biological processes of chemokines and chemokine receptors. Especially, the chemokine CXCL9 was positively correlated with M1 macrophage infiltration levels in cervical cancer. CXCL9 as a protective factor, it manifestly expressed in blood immune cells, and was positively related to immune checkpoints. CXCL9 amplification was the most common type of mutation. And CXCL9 expression could have an effect on the sensitivity of some chemicals or targeted drugs against cervical cancer. These findings may provide new insight into the M1 macrophage coexpression gene network and molecular mechanisms, and shed light on the role of CXCL9 in cervical cancer.

CXCL9, IL2RB, and SPP1, potential diagnostic biomarkers in the co-morbidity pattern of atherosclerosis and non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a hepatocyte inflammation based on hepatocellular steatosis, yet there is no effective drug treatment. Atherosclerosis (AS) is caused by lipid deposition in the endothelium, which can lead to various cardiovascular diseases. NASH and AS share common risk factors, and NASH can also elevate the risk of AS, causing a higher morbidity and mortality rate for atherosclerotic heart disease. Therefore, timely detection and diagnosis of NASH and AS are particularly important. In this study, differential gene expression analysis and weighted gene co-expression network analysis were performed on the AS (GSE100927) and NASH (GSE89632) datasets to obtain common crosstalk genes, respectively. Then, candidate Hub genes were screened using four topological algorithms and externally validated in the GSE43292 and GSE63067 datasets to obtain Hub genes. Furthermore, immune infiltration analysis and gene set variation analysis were performed on the Hub genes to explore the underlying mechanisms. The DGIbd database was used to screen candidate drugs for AS and NASH. Finally, a NASH model was constructed using free fatty acid-induced human L02 cells, an AS model was constructed using lipopolysaccharide-induced HUVECs, and a co-morbidity model was constructed using L02 cells and HUVECs to verify Hub gene expression. The result showed that a total of 113 genes common to both AS and NASH were identified as crosstalk genes, and enrichment analysis indicated that these genes were mainly involved in the regulation of immune and metabolism-related pathways. 28 candidate Hub genes were screened according to four topological algorithms, and CXCL9, IL2RB, and SPP1 were identified as Hub genes after in vitro experiments and external dataset validation. The ROC curves and SVM modeling demonstrated the good diagnostic efficacy of these three Hub genes. In addition, the Hub genes are strongly associated with immune cell infiltration, especially macrophages and γ-δ T cell infiltration. Finally, five potential therapeutic drugs were identified. has-miR-185 and hsa-miR-335 were closely related to AS and NASH. This study demonstrates that CXCL9, IL2RB, and SPP1 may serve as potential biomarkers for the diagnosis of the co-morbidity patterns of AS and NASH and as potential targets for drug therapy.

Identification of immune- and oxidative stress-related signature genes as potential targets for mRNA vaccines for pancreatic cancer patients.

Adenocarcinoma of the pancreas (PAAD) is one of the deadliest malignant tumors, and messenger ribonucleic acid vaccines, which constitute the latest generation of vaccine technology, are expected to lead to new ideas for the treatment of pancreatic cancer. The Cancer Genome Atlas-PAAD and Genotype-Tissue Expression data were merged and analyzed. Weighted gene coexpression network analysis was used to identify gene modules associated with tumor mutational burden among the genes related to both immunity and oxidative stress. Differentially expressed immune-related oxidative stress genes were screened via univariate Cox regression analysis, and these genes were analyzed via nonnegative matrix factorization. After immune infiltration analysis, least absolute shrinkage and selection operator regression combined with Cox regression was used to construct the model, and the usefulness of the model was predicted based on the receiver operating characteristic curve and decision curve analysis curves after model construction. Finally, metabolic pathway enrichment was analyzed using gene set enrichment analysis combined with Kyoto Encyclopedia of Genes and Genomes and gene ontology biological process analyses. This model consisting of the ERAP2, mesenchymal-epithelial transition factor (MET), CXCL9, and angiotensinogen (AGT) genes can be used to help predict the prognosis of pancreatic cancer patients more accurately than existing models. ERAP2 is involved in immune activation and is important in cancer immune evasion. MET binds to hepatocyte growth factor, leading to the dimerization and phosphorylation of c-MET. This activates various signaling pathways, including MAPK and PI3K, to regulate the proliferation, invasion, and migration of cancer cells. CXCL9 overexpression is associated with a poor patient prognosis and reduces the number of CD8 + cytotoxic T lymphocytes in the PAAD tumor microenvironment. AGT is cleaved by the renin enzyme to produce angiotensin 1, and AGT-converting enzyme cleaves angiotensin 1 to produce angiotensin 2. Exposure to AGT-converting enzyme inhibitors after pancreatic cancer diagnosis is associated with improved survival. The 4 genes identified in the present study - ERAP2, MET, CXCL9, and AGT - are expected to serve as targets for messenger ribonucleic acid vaccine development and need to be further investigated in depth.

Age-related dysregulation of CXCL9/10 in monocytes is linked to impaired innate immune responses in a mouse model of Staphylococcus aureus osteomyelitis.

BACKGROUND: Age-associated impairments in innate immunity are believed to be a causative factor responsible for severe pathogenesis of Staphylococcus aureus (S. aureus) infection in the bone tissue. However, the basis for age-associated decline in innate immune response upon S. aureus infection remains poorly understood. RESULTS: Our transcriptional data (GEO: GSE166522) from a mouse model of S. aureus osteomyelitis show up-regulated CXCL9 and CXCL10 (CXCL9/10), which is further confirmed in vitro and in vivo by the present study. Notably, monocytes are a main source for CXCL9/10 production in bone marrow upon S. aureus challenge, but this response declines in middle-aged mice. Interestingly, conditional medium of bone marrow monocytes from middle-aged mice has a strikingly decreased effect on bactericidal functions of neutrophils and macrophages compares with that from young mice. We further show that activation of CXCL9/10-CXCR3 axis between monocytes and macrophages/neutrophils promotes the bactericidal function of the cells, whereas blocking the axis impairs such function. Importantly, treatment with either exogenous CXCL9 or CXCL10 in a middle-aged mice model enhances, while pharmacological inhibition of CXCR3 in young mice model impairs, bacterial clearance and bone marrow structure. CONCLUSIONS: These findings demonstrate that bone marrow monocytes act as a critical promotor of innate immune response via the CXLCL9/10-CXCR3 axis upon S. aureus infection, and that the increased susceptibility to S. aureus infection in skeleton in an aged host may be largely attributable to the declined induction of CXCR9/10 in monocytes.

Evaluation of significantly changed chemokine factors of idiopathic granulomatous mastitis in non-puerperal patients.

Idiopathic granulomatous mastitis (IGM), a recurrent inflammation disease of the non-lactating breast, has had an increasing clinical morbidity rate in recent years, and its complicated symptoms and unclear etiology make it challenging to treat. This rare benign inflammatory breast disease, centered on the lobules, represents the most challenging type of non-puerperal mastitis (NPM), also known as non-lactating mastitis. In this study, patients diagnosed with IGM (M, n = 23) were recruited as cases, and patients with benign control breast disease (C, n = 17) were enrolled as controls. Cytokine microarray detection measured and analyzed the differentially expressed cytokine factors between IGM and control patients. Then, we verified the mRNA and protein expression levels of the significantly changed cytokine factors using Q-RT-PCR, ELISA, western blot, and IHC experiments. The cytokine factor expression levels significantly changed compared to the control group. We observed a significant increase between IGM and control patients in cytokine factors expression, such as interleukin-1ß (IL-1ß), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1ß, tumor necrosis factor receptor 2 (TNF RII). Then, we verified the expression of these top five dysregulated factors in both mRNA and protein levels. Our results demonstrated the cytokine map in IGM and indicated that several cytokines, especially chemokines, were associated with and significantly dysregulated in IGM tissues compared to the control group. The chemokine factors involved might be essential in developing and treating IGM. These findings would be helpful for a better understanding of IGM and offer valuable insights for devising novel diagnostic and therapeutic strategies.

Augmentation of tumor expression of HLA-DR, CXCL9, and CXCL10 may improve olfactory neuroblastoma immunotherapeutic responses.

BACKGROUND: Olfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches. METHODS: Multispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed. RESULTS: A significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration. CONCLUSION: These data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.

Exploring shared molecular signatures and regulatory mechanisms in nonalcoholic steatohepatitis and inflammatory bowel disease using integrative bioinformatics analysis.

The co-existence of inflammatory bowel disease (IBD) and non-alcoholic steatohepatitis (NASH) has raised interest in identifying shared molecular mechanisms and potential therapeutic targets. However, the relationship between these two diseases remains unclear and effective medical treatments are still lacking. Through the bioinformatics analysis in this study, 116 shared differentially expressed genes (SDEGs) were identified between IBD and NASH datasets. GO and KEGG pathway analyses revealed significant involvement of SDEGs in apoptotic processes, cell death, defense response, cytokine and chemokine activity, and signaling pathways. Furthermore, weighted gene co-expression network analysis (WGCNA) identified five shared signature genes associated specifically with IBD and NASH, they were CXCL9, GIMAP2, ADAMTS5, GRAP, and PRF1. These five genes represented potential diagnostic biomarkers for distinguishing patients with diseases from healthy individuals by using two classifier algorithms and were positively related to autophagy, ferroptosis, angiogenesis, and immune checkpoint factors in the two diseases. Additionally, single-cell analysis of IBD and NASH samples highlighted the expression of regulatory genes in various immune cell subtypes, emphasizing their significance in disease pathogenesis. Our work elucidated the shared signature genes and regulatory mechanisms of IBD and NASH, which could provide new potential therapies for patients with IBD and NASH.

Diagnostic accuracy and cellular origin of pleural fluid CXCR3 ligands for tuberculous pleural effusion.

BACKGROUND: Pleural biomarkers represent potential diagnostic tools for tuberculous pleural effusion (TPE) due to their advantages of low cost, short turnaround time, and less invasiveness. This study evaluated the diagnostic accuracy of two CXCR3 ligands, C-X-C motif chemokine ligand 9 (CXCL9) and CXCL11, for TPE. In addition, we investigated the cellular origins and biological roles of CXCL9 and CXCL11 in the development of TPE. METHODS: This double-blind study prospectively enrolled patients with undiagnosed pleural effusion from two centers (Hohhot and Changshu) in China. Pleural fluid on admission was obtained and levels of CXCL9 and CXCL11 were measured by an enzyme-linked immunosorbent assay (ELISA). The receiver operating characteristic (ROC) curve and the decision curve analysis (DCA) were used to evaluate their diagnostic accuracy and net benefit, respectively. THP-1 cell-derived macrophages were treated with Bacillus Calmette-Guérin (BCG), and quantitative real-time PCR (qRT-PCR) and ELISA were used to determine the mRNA and protein levels of CXCL9 and CXCL11. The chemoattractant activities of CXCL9 and CXCL11 for T helper (Th) cells were analyzed by a transwell assay. RESULTS: One hundred and fifty-three (20 TPEs and 133 non-TPEs) patients were enrolled in the Hohhot Center, and 58 (13 TPEs and 45 non-TPEs) were enrolled in the Changshu Center. In both centers, we observed increased CXCL9 and CXCL11 in TPE patients. The areas under the ROC curves (AUCs) of pleural CXCL9 and CXCL11 in the Hohhot Center were 0.70 (95 % CI: 0.55-0.85) and 0.68 (95 % CI: 0.52-0.84), respectively. In the Changshu Center, the AUCs of CXCL9 and CXCL11 were 0.96 (95 % CI: 0.92-1.00) and 0.97 (95 % CI: 0.94-1.00), respectively. The AUCs of CXCL9 and CXCL11 decreased with the advancement of age. The decision curves of CXCL9 and CXCL11 showed net benefits in both centers. CXCL9 and CXCL11 were upregulated in BCG-treated macrophages. Pleural fluid from TPE and conditioned medium from BCG-treated macrophages were chemotactic for Th cells. Anti-CXCL9 or CXCL11 neutralizing antibodies could partly block the chemotactic activity. CONCLUSIONS: Pleural CXCL9 and CXCL11 are potential diagnostic markers for TPE, but their diagnostic accuracy is compromised in elderly patients. CXCL9 and CXCL11 can promote the migration of peripheral Th cells, thus representing a therapeutic target for the treatment of TPE.

Microwave ablation combined with PD-L1 blockade synergistically promotes Cxcl9-mediated antitumor immunity.

Although microwave ablation (MWA) is an important curative therapy in colorectal cancer liver metastasis, recurrence still occurs clinically. Our previous studies have shown that the expression of programmed cell death 1 ligand 1 (PD-L1) is upregulated following MWA, suggesting that MWA combined with anti-PD-L1 treatment can serve as a promising clinical therapeutic strategy against cancer. Using MWA-treated preclinical mice models, MWA combined with αPD-L1 treatment decreased tumor growth and prolonged overall survival (OS). Furthermore, through flow cytometry and single-cell RNA sequencing analysis, we determined that the MWA plus αPD-L1 therapy significantly suppressed CD8+ T cell exhaustion and enhanced their effector function. A significant increase in γ-interferon (IFN-γ) stimulated transcription factors, specifically Irf8, was observed. This enhancement facilitated the polarization of tumor-associated macrophages (TAM1s and TAM2s) through the nuclear factor-κB/JAK-STAT1 signaling pathway. Furthermore, the combination therapy stimulated the production of CXC motif chemokine ligand (CXCL9) by TAM1s and tumor cells, potentially increasing the chemotaxis of CD8 T cells and Th1 cells. Knocking out Cxcl9 in MC38 tumor cells or using CXCL9 blockade enhanced tumor growth of untreated tumors and shortened OS. Taken together, our study showed that blocking the IFN-γ-Cxcl9-CD8+ T axis promoted tumor progression and discovered a potential involvement of IRF8-regulated TAMs in preventing T cell exhaustion. Collectively, we identified that the combination of MWA with anti-PD-L1 treatment holds promise as a therapeutic strategy to rejuvenate the immune response against tumors. This merits further exploration in clinical studies.

RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

The glycosaminoglycan-binding chemokine fragment CXCL9(74-103) reduces inflammation and tissue damage in mouse models of coronavirus infection.

Introduction: Pulmonary diseases represent a significant burden to patients and the healthcare system and are one of the leading causes of mortality worldwide. Particularly, the COVID-19 pandemic has had a profound global impact, affecting public health, economies, and daily life. While the peak of the crisis has subsided, the global number of reported COVID-19 cases remains significantly high, according to medical agencies around the world. Furthermore, despite the success of vaccines in reducing the number of deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains a gap in the treatment of the disease, especially in addressing uncontrolled inflammation. The massive recruitment of leukocytes to lung tissue and alveoli is a hallmark factor in COVID-19, being essential for effectively responding to the pulmonary insult but also linked to inflammation and lung damage. In this context, mice models are a crucial tool, offering valuable insights into both the pathogenesis of the disease and potential therapeutic approaches. Methods: Here, we investigated the anti-inflammatory effect of the glycosaminoglycan (GAG)-binding chemokine fragment CXCL9(74-103), a molecule that potentially decreases neutrophil transmigration by competing with chemokines for GAG-binding sites, in two models of pneumonia caused by coronavirus infection. Results: In a murine model of betacoronavirus MHV-3 infection, the treatment with CXCL9(74-103) decreased the accumulation of total leukocytes, mainly neutrophils, to the alveolar space and improved several parameters of lung dysfunction 3 days after infection. Additionally, this treatment also reduced the lung damage. In the SARS-CoV-2 model in K18-hACE2-mice, CXCL9(74-103) significantly improved the clinical manifestations of the disease, reducing pulmonary damage and decreasing viral titers in the lungs. Discussion: These findings indicate that CXCL9(74-103) resulted in highly favorable outcomes in controlling pneumonia caused by coronavirus, as it effectively diminishes the clinical consequences of the infections and reduces both local and systemic inflammation.

HIF-1α Mediates Immunosuppression and Chemoresistance in Colorectal Cancer by Inhibiting CXCL9, -10 and -11.

Uncertainty exists regarding the mechanisms by which hypoxia-inducible factors (HIFs) control CD8+T-cell migration into tumor microenvironments. Here, we found that HIF-1α knockdown or overexpression resulted in increased or decreased CXCL9, -10, and -11 expression in vitro, respectively. Gene Set Variation Analysis revealed that elevated HIF-1α levels correlated with a poor prognosis, severe pathological stage, and an absence of CD8+ T cells in the tumor microenvironment in colorectal cancer (CRC) patients. HIF-1α was inversely associated with pathways beneficial to anti-tumor immunotherapy and cytokine/chemokine function. In vivo, inhibiting HIF-1α or its upstream regulator BIRC2 significantly suppressed tumor growth and promoted CD8+ T-cell infiltration. CXCR3 neutralizing antibodies reversed these effects, implicating the involvement of CXCL9, -10, and -11/CXCR3 axis. The presence of HIF-1α weakened the upregulation of CXCL9, -10, and -11 by bleomycin and doxorubicin. Combining HIF-1α inhibition with bleomycin promoted CD8+ T-cell infiltration and tumor suppression in vivo. Moreover, doxorubicin could upregulate CXCL9, -10 and -11 by suppressing HIF-1α. Our findings highlight the potential of HIF-1α inhibition to improve CRC microenvironments and increase chemotherapy sensitivity.

Pharmacological Polarization of Tumor-Associated Macrophages Toward a CXCL9 Antitumor Phenotype.

Tumor-associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM-specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small-molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM-avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM-specific states, an insight a novel therapeutic anticancer approach is used to discover.

Increased type 1 inflammation in gynecologic cervicovaginal samples in patients with APS-1.

BACKGROUND: Inborn errors of immunity offer important insights into mucosal immunity. In autoimmune polyendocrine syndrome type-1 (APS-1), chronic mucocutaneous candidiasis has been ascribed to neutralizing IL-17 autoantibodies. Recent evidence implicates excessive T-cell IFN-γ secretion and ensuing epithelial barrier disruption in predisposition to candidiasis, but these results remain to be replicated. Whether IL-17 paucity, increased type I inflammation, or their combination underlies susceptibility to chronic mucocutaneus candidiasis in APS-1 is debated. OBJECTIVE: Our aim was to characterize the immunologic features in the cervicovaginal mucosa of females with APS-1. METHODS: Vaginal fluid was collected with a flocked swab from 17 females with APS-1 and 18 controls, and cytokine composition was analyzed using Luminex (Luminex Corporation, Austin, Tex). Cervical cell samples were obtained with a cervix brush from 6 patients and 6 healthy controls and subjected to transcriptome analysis. RESULTS: The vaginal fluid samples from patients with APS-1 had IFN-γ concentrations comparable to those of the controls (2.6 vs 2.4 pg/mL) but high concentrations of the TH1 chemokines CXCL9 and CXCL10 (1094 vs 110 pg/mL [P < .001] and 4033 vs 273 pg/mL [P = .001], respectively), whereas the IL-17 levels in the samples from the 2 groups were comparable (28 vs 8.8 pg/mL). RNA sequencing of the cervical cells revealed upregulation of pathways related to mucosal inflammation and cell death in the patients with APS-1. CONCLUSION: Excessive TH1 cell response appears to underlie disruption of the mucosal immune responses in the genital tract of patients with APS-1 and may contribute to susceptibility to candidiasis in the genital tract as well.

Intratumoral delivery of a highly active form of XCL1 enhances antitumor CTL responses through recruitment of CXCL9-expressing conventional type-1 dendritic cells.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

Quantifying dynamic pro-inflammatory gene expression and heterogeneity in single macrophage cells.

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics-that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFNγ) stimulation. We found that IRF1 responds to low concentration or short duration IFNγ stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFNγ stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression.

CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Differential Anti-Tumor Effects of IFN-Inducible Chemokines CXCL9, CXCL10, and CXCL11 on a Mouse Squamous Cell Carcinoma Cell Line.

Chemokines are a group of cytokines involved in the mobilization of leukocytes, which play a role in host defense and a variety of pathological conditions, including cancer. Interferon (IFN)-inducible chemokines C-X-C motif ligand 9 (CXCL), CXCL10, and CXCL11 are anti-tumor chemokines; however, the differential anti-tumor effects of IFN-inducible chemokines are not completely understood. In this study, we investigated the anti-tumor effects of IFN-inducible chemokines by transferring chemokine expression vectors into a mouse squamous cell carcinoma cell line, SCCVII, to generate a cell line stably expressing chemokines and transplanted it into nude mice. The results showed that CXCL9- and CXCL11-expressing cells markedly inhibited tumor growth, whereas CXCL10-expressing cells did not inhibit growth. The NH2-terminal amino acid sequence of mouse CXCL10 contains a cleavage sequence by dipeptidyl peptidase 4 (DPP4), an enzyme that cleaves the peptide chain of chemokines. IHC staining indicated DPP4 expression in the stromal tissue, suggesting CXCL10 inactivation. These results suggest that the anti-tumor effects of IFN-inducible chemokines are affected by the expression of chemokine-cleaving enzymes in tumor tissues.

HDAC inhibitor HPTA initiates anti-tumor response by CXCL9/10-recruited CXCR3+CD4+T cells against PAHs carcinogenicity.

Polycyclic aromatic hydrocarbons (PAHs) exposure in food is closely associated with the occurrence and development of breast cancer, which may attribute to altered immunotoxicity and immune regulation. Currently, cancer immunotherapy aims to promote tumor-specific T cell responses, especially CD4+T helper cells (Th) for anti-tumor immunity. The histone deacetylase inhibitors (HDACis) are found to exert an anti-tumor effect by reshaping the tumor immune microenvironment, but the immune regulatory mechanism of HDACis in PAHs-induced breast tumor remains elusive. Here, using established breast cancer models induced by 7,12-dimethylbenz[a]anthracene (DMBA), a potent carcinogenic agent of PAH, the novel HDACi, 2-hexyl-4-pentylene acid (HPTA) exhibited anti-tumor effect by activating T lymphocytes immune function. HPTA recruited CXCR3+CD4+T cells into chemokines CXCL9/10-enriched tumor sites, and the increased secretion of CXCL9/10 was regulated by the NF-κB-mediated pathway. Furthermore, HPTA promoted Th1 differentiation and assisted cytotoxic CD8+T cells in the elimination of breast cancer cells. These findings support the proposition of HPTA as a potential therapeutic in the treatment of PAHs-induced carcinogenicity.

CXCL9 Links Skin Inflammation and Fibrosis through CXCR3-Dependent Upregulation of Col1a1 in Fibroblasts.

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.