Chronic obstructive pulmonary disease (COPD) was the 3rd leading cause of death in 2012 worldwide. It is particularly severe in the elderly, who are at risk of death by coughing, mucous hypersecretion, and finally breathlessness. Recently, anti-COPD drug development has increased, and many animal screening systems have been studied. Tobacco smoke animal models are the best known animal screening system, but have several preparation requirements, such as a tobacco smoke generator and a separate facility to prevent smoke release. Accordingly, we evaluated the properties of a lipopolysaccharide (LPS) murine model for COPD screening and the effect of the time elapsed from 0 to 72 hr after LPS intranasal instillation on various biomarkers of COPD severity, such as WBC and neutrophils in bronchoalveolar fluid (BALF), IgE in serum, histopathology in the lung, and cytokines (IL-8, TNF-α, IFN-γ, and TGF-β) and chemokines (CCL-2, CXCL1, CXCL9, CXCL10, and CXCL11) in the respiratory system. Although from 48 hr after LPS treatment several factors which could be evaluated as biomarkers for COPD establishment such as WBC and neutrophil in BALF, IgE in serum, cytokines (IL-8, TNF-α, and IFN-γ), and chemokines (CCL-2, CXCL1, CXCL9, CXCL10, and CXCL11) increased at 72 hr the increment of important factors for COPD establishment such as IgE, fibrosis in the lung, and cytokines (IL-8, TNF-α, and IFN-γ) was more clear. Based on our results, we concluded that the optimal time after LPS intranasal instillation is 72 hr.

BACKGROUND

Inhibition of the PD-L1/PD-1 immune checkpoint axis represents one of the most promising approaches of immunotherapy for various cancer types. However, immune checkpoint inhibition is successful only in subpopulations of patients emphasizing the need for powerful biomarkers that adequately reflect the complex interaction between the tumor and the immune system. Recently, recurrent copy number gains (CNG) in chromosome 9p involving PD-L1 were detected in many cancer types including lung cancer, melanoma, bladder cancer, head and neck cancer, cervical cancer, soft tissue sarcoma, prostate cancer, gastric cancer, ovarian cancer, and triple-negative breast cancer.

METHODS

Here, we applied functional genomics to analyze global mRNA expression changes associated with chromosome 9p gains. Using the TCGA data set, we identified a list of 75 genes that were strongly up-regulated in tumors with chromosome 9p gains across many cancer types.

RESULTS

As expected, the gene set was enriched for chromosome 9p and in particular chromosome 9p24 (36 genes and 23 genes). Furthermore, we found enrichment of two expression programs derived from genes within and beyond 9p: one implicated in cell cycle regulation (22 genes) and the other implicated in modulation of the immune system (16 genes). Among these were specific cytokines and chemokines, e.g. CCL4, CCL8, CXCL10, CXCL11, other immunoregulatory genes such as IFN-G and IDO1 as well as highly expressed proliferation-related kinases and genes including PLK1, TTK, MELK and CDC20 that represent potential drug targets.

CONCLUSIONS

Collectively, these data shed light on mechanisms of immune escape and stimulation of proliferation in cancer with PD-L1 CNG and highlight additional vulnerabilities that may be therapeutically exploitable.

Chronic Obstructive Pulmonary Disease (COPD) is considered to be a progressive disease characterized by chronic inflammation and irreversible airflow obstruction, mainly caused by tobacco smoking. Based on World Health Organization data, COPD will be the fourth cause of mortality in 2020, after vascular, cardiac and cerebral diseases and cancer. To date no therapy retards or suppresses progression of COPD. The chronic inflammatory process caused by tobacco smoking promotes structural changes predominantly in the small airways (less than 2mm). Macrophages, neutrophils and T cells are thought to be important key players, as well as structural cells like fibroblasts, epithelial and smooth muscle cells. The interaction between macrophages and lymphocytes, especially CD8+, has been implicated in the pathogenesis of COPD. Chemokines such as CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC and CCL5/ RANTES have been described as possibly responsible for recruitment of T cells and blood monocytes increasing the number of macrophages and CD8+ T cells in the lung of COPD patients. The study of the influx of mononuclear cells to the lung is very important not only to promote a better understanding of the COPD physiopathology but also to help identify new targets for treatment. Based on this new evidence, the study of several mediator antagonists that can block the recruitment of inflammatory cells to the lung have been tested in COPD.

OBJECTIVE

To determine the changes in the airway inflammation-related cytokine/chemokine profiles after exposure to cigarette smoke (CS) and smoking cessation (SC).

METHODS

A total of 18 male C57BL/6 mice were equally divided into three groups: CS group, SC group, and normal control group. The airway resistance, lung morphology, and collagen deposition around airways were determined. HE staining and Masson trichrome staining were used for histopathological analysis. The inflammatory cells in bronchoalveolar lavage fluid (BALF) were assessed. The inflammation-associated cytokines were determined using real-time PCR and immunohistochemistry. Expressions of CXCR3 ligands including the CXCL9, CXCL10, CXCL11 and other cytokines in lung tissue and BALF were also analyzed.

RESULTS

The airway resistance significantly increased in both CS group and SC group when compared with the normal control group. Lung pathological scores in both CS group and SC group were also higher than that in the normal control group, while there was no significant difference between the CS group and SC group. Inflammatory cells including the neutrophils, macrophages, and lymphocytes also increased in both the CS group and SC group at both mRNA and protein levels. The mRNA levels of CXCL9, CXCL10, MMP9, and MMP12 were significantly higher in CS group and SC group than those in the normal control group (all P<0.05). The protein expression levels of CXCL9, CXCL10, CXCL11, MMP2, MMP9, MMP12, and TGF-Β1 were significantly higher in CS group and SC group than those in the normal control group (all P<0.05). Compared with the normal control group,the concentrations of CXCL9, CXCL10, CXCL11, IL-8, and TGF-Β1 in the BALF supernatants of the CS group and SC group significantly increased (P<0.05); in addition, the IL-6 and TNF-Α concentrations also increased in the CS group (both P<0.05).

CONCLUSIONS

CS exposure triggers inflammatory cell flux and accumulation in the lung parenchyma and BALF. As a consequence, the inflammatory cytokines increase dramatically. After CS, the cytokines/chemokines can decrease, but is still higher than in non-smokers.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and follow-up of personalized cancer therapy, including immunotherapy. Fine needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FNA samples from patients with breast cancer (BC; n=25) and benign lesions (n=32). We demonstrate that the FNA BC samples could be divided into two main clusters, characterized by differences in expression levels of the estrogen receptor (ER) and the proliferation marker Ki67. This clustering corresponded to some extent to established BC subtypes. Our analysis also revealed several proteins whose expression levels differed between BC and benign lesions (e.g. CA9, GZMB, IL6, VEGFA, CXCL11, PDL1 and PCD1), as well as several chemokines correlating with ER and Ki67 status (e.g. CCL4, CCL8, CCL20, CXCL8, CXCL9 and CXCL17). Finally, we also identified three signatures that could predict Ki67 status, ER status and tumor grade, respectively, based on a small subset of proteins, which was dominated by chemokines. To our knowledge, expression profiles of CCL13 in benign lesions and BC have not previously been described but were shown herein to correlate with proliferation (p=0.00095), suggesting a role in advanced BC. Given the broad functional range of the proteins analyzed, immune-related proteins were overrepresented among the observed alterations. Our pilot study supports the emerging role of chemokines in BC progression. Due to the minimally traumatic sampling and clinically important molecular information for therapeutic decisions, this methodology is promising for future immunoscoring and monitoring of treatment efficacy in BC.

It has been suggested that CXCR3 is important for nociception. Our experiments were conducted to evaluate involvement of CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11/CCL21) in neuropathic pain. Our studies give new evidence that intrathecal administration of each CXCR3 ligand induces pain-like behaviour in naive mice that occurs shortly after injection due to its location of neurons, which is confirmed by immunofluorescent staining. Moreover, intrathecal administrations of CXCL9, CXCL10, CCL21 neutralizing antibodies diminished pain-related behaviour. RT-PCR/Western blot analysis unprecedentedly showed spinal elevated levels of CXCR3 after chronic constriction injury of the sciatic nerve in rats in parallel with different time-course changes of its endogenous ligands. Initially, on day 2 we observed spinal increased levels of CXCL10 and CXCL11 indicating that these chemokines have important roles in triggering neuropathy. Then, on day 7, we observed increased levels of CXCL4, CXCL9, CXCL10. Interestingly, changes in CXCL9 level persisted until day 28, suggesting that these chemokines are responsible for long-term, persistent neuropathy. Additionally, in DRG the CXCL4, CXCL9 were elevated. The results obtained from primary glial cultures, suggests that all CXCR3 ligands can be produced in microglia, but also, except for CXCL4, in astrocytes. We provide the first evidence that in neuropathy chronic intrathecal administration of CXCR3 antagonist, (±)-NBI-74330, attenuates hypersensitivity with concomitant occurrence of microglial and some of CXCR3 ligands activation observed in the spinal cord and/or DRG level. This paper underlies the significance of CXCR3 in neuropathic pain and shows therapeutic potential of its blockade for enhancement of morphine analgesia as the major novelty of this work.

Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period in pigs.

OBJECTIVE

Impaired angiogenesis is linked to adipose tissue (AT) dysfunction, inflammation, and insulin resistance in human obesity. Chemokine (C-X-C motif) receptor. (CXCR3) ligands are important regulators of angiogenesis in different disease contexts such as cancer; however, their role in human morbid obesity is unknown. We investigated the role of the CXCR3 axis in AT angiogenesis in morbidly obese patients.

METHODS

The study group comprised 50 morbidly obese patients (mean age 44 ± 1 years, body mass index 44 ± 1 kg/m2) who had undergone laparoscopic Roux-Y-gastric bypass surgery, and 25 age-matched non-obese control subjects. We measured the circulating levels of the CXCR3 ligands monokine induced by interferon-γ (MIG/CXCL9), interferon-γ inducible protein 10 (IP-10/CXCL10), and interferon-γ-inducible T-cell alpha chemoattractant (I-TAC/CXCL11) in all studied subjects. Additionally, the expression of CXCR3 ligands was analyzed in paired biopsies of subcutaneous and visceral AT obtained during the laparoscopic procedure in morbidly obese patients. Additionally, we explored the functional role of CXCR3 ligands on angiogenesis in AT from morbidly obese patients using an ex vivo assay.

RESULTS

Plasma levels of CXCL10 and CXCL11 were significantly higher in morbidly obese patients than in controls (p < 0.01). In ex vivo assays, angiogenic growth was markedly lower in visceral AT than in subcutaneous AT (p < 0.05), which was related to significant tissue upregulation of CXCL10, CXCL11 and CXCR3 (p < 0.05). CXCL10 or CXCL11 inhibited AT angiogenesis (p < 0.05), and blockade of CXCR3 function significantly increased capillary sprouting in visceral fat deposits (p < 0.05). Western blot analysis showed that the p38 mitogen-activated protein kinase signaling pathway was implicated in the angiostatic effects of CXCR3 in AT.

CONCLUSIONS

CXCL10 and CXCL11 may play. deleterious role in obesity as potential inhibitors of AT angiogenesis. Accordingly, pharmacological blockade of CXCR3 could represent. therapy to prevent AT dysfunction in obesity.

H2O2 is an early danger cue required for innate immune cell recruitment to wounds. To date, little is known about whether H2O2 is required for the migration of human adaptive immune cells to sites of inflammation. However, oxidative stress is known to impair T cell activity, induce actin stiffness, and inhibit cell polarization. In this study, we show that low oxidative concentrations of H2O2 also impede chemokinesis and chemotaxis of previously activated human T cells to CXCL11, but not CXCL10 or CXCL12. We show that this deficiency in migration is due to a reduction in inflammatory chemokine receptor CXCR3 surface expression and cellular activation of lipid phosphatase SHIP-1. We demonstrate that H2O2 acts through an Src kinase to activate a negative regulator of PI3K signaling, SHIP-1 via phosphorylation, providing a molecular mechanism for H2O2-induced chemotaxis deficiency. We hypothesize that although H2O2 serves as an early recruitment trigger for innate immune cells, it appears to operate as an inhibitor of T lymphocyte immune adaptive responses that are not required until later in the repair process.

Pancreatic cancer (PC) is a malignant tumor with a poor prognosis and high mortality. However, the biological role of miR-548t-5p in PC has not been reported. In this study, we found that miR-548t-5p expression was significantly decreased in PC tissues compared with adjacent tissues, and that low miR-548t-5p expression was associated with malignant PC behavior. In addition, high miR-548t-5p expression inhibited the proliferation, migration, and invasion of PC cell lines. Regarding the molecular mechanism, the luciferase reporter gene, chromatin immunoprecipitation (ChIP), and functional recovery assays revealed that YY1 binds to the miR-548t-5p promoter and positively regulates the expression and function of miR-548t-5p. miR-548t-5p also directly regulates CXCL11 to inhibit its expression. A high level of CXCL11 was associated with worse Tumor Node Metastasis (TNM) staging in patients with PC, enhancing proliferation and metastasis in PC cells. Our study shows that the YY1/miR-548t-5p/CXCL11 axis plays an important role in PC and provides a new potential candidate for the treatment of PC.

BACKGROUND

Ingenol mebutat (IM)-gel is effective for the topical treatment of epithelial tumors, including actinic keratoses (AKs) or anogenital warts (AGW). AK patients treated with IM develop intensified inflammatory reactions on sights of prior clinical visible or palpable AKs as compared to the surrounding actinically damaged skin, suggesting the induction of a tumor cell-directed inflammation. AGW patients treated with IM develop even stronger inflammatory reactions with large erosions, suggesting a directed inflammatory response against HPV-infected keratinocytes. Of note, even widespread erosions heal very fast without any superinfections. Here, we set out to elucidate underlying molecular and cellular mechanisms of these clinical observations.

METHODS

The effects of IM (10-9-10-5 M) on the expression and translation of a comprehensive set of chemokines (CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCL14, CCL2, CCL5, CCL20, CCL27) and antimicrobial peptides (AMP) (HBD1, HBD2, HBD3, LL37, RNase7) were analyzed in primary human epithelial keratinocytes (HEK) and a set of epithelial cancer cell lines by RT-qPCR and ELISA in vitro. To study the possible effects of different concentrations of IM on migratory, respectively wound healing responses, an in vitro scratch assay was conducted on HEK.

RESULTS

Ingenol mebutat significantly and dose-dependently induced the expression of proinflammatory chemokines (CXCL8, CCL2) and AMP (RNase7, HBD3) in HEK and epithelial cancer cell lines. A significantly stronger induction of CXCL8 and CCL2 was observed in our tested tumor cells as compared to HEK. We did not observe any significant effect of IM on HEK migration, respectively wound healing responses in vitro for any tested concentration (10-9, 10-8, 10-6 M) except 10-7 M, which induced a significant inhibition.

CONCLUSIONS

Our data suggest that tumor cells are more susceptible to IM as compared to differentiated HEK. This is evident by a stronger IM-mediated induction of proinflammatory chemokines in tumor cells, which may result in a tumor cell-directed inflammatory response and rapid tumor destruction. In addition, IM induces AMP in keratinocytes and seems not to severely interfere with keratinocyte migration, which contributes to a fast and uncomplicated wound healing. Surprising is a selective inhibition of keratinocyte migration by IM at the concentration of 10-7 M pointing to very dose depending biological effects, induced by IM.

Plasma cytokines are biomarkers of disease extent and mycobacterial burden in pulmonary tuberculosis (PTB). Whether chemokines can perform the same role in PTB is not known. We examined the plasma levels of chemokines in individuals with PTB, latent TB (LTB) or healthy controls (HC) and their association with disease severity and mycobacterial burdens in PTB. We also examined the chemokines in PTB individuals at the end of anti-tuberculous chemotherapy (ATT). PTB individuals exhibited significantly higher levels of CCL1, CCL3, CXCL1, CXCL2, CXCL9 and CXCL10 in comparison to LTB and/or HC individuals. PTB individuals with bilateral or cavitary disease displayed significantly elevated levels of CCL1, CCL3, CXCL1, CXCL10 and CXCL11 compared to those with unilateral or non-cavitary disease and also exhibited a significant positive relationship with bacterial burdens. In addition, PTB individuals with slower culture conversion displayed significantly elevated levels of CCL1, CCL3, CXCL1 and CXCL9 at the time of PTB diagnosis and prior to ATT. Finally, the chemokines were significantly reduced following successful ATT. Our data demonstrate that PTB is associated with elevated levels of chemokines, which are partially reversed followed chemotherapy. Our data demonstrate that chemokines are markers of disease severity, predicting increased bacterial burden and delayed culture conversion in PTB.

Innate lymphoid cells play an important role in the early effector cytokine-mediated response. In Wistar Kyoto rats, CD8⁺ non-T lymphocytes (CD8⁺Lym) infiltrate into glomeruli during the development of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. Here, we examined the profiles and roles of CD8⁺Lym in anti-GBM glomerulonephritis. The regulation of CD8⁺Lym by peroxisome proliferator-activated receptor (PPAR)-α in anti-GBM glomerulonephritis was also evaluated. Glomerular infiltrating CD8⁺Lym were lineage-negative cells that showed markedly high expression of IFN-γ and T-bet mRNAs but not Eomes, indicating these cells are group 1 innate lymphoid cells. In anti-GBM glomerulonephritis, the glomerular mRNAs of innate lymphoid cell-related cytokines (IFN-γ and TNF-α) and chemokines (CXCL9, CXCL10, and CXCL11) are significantly increased. Treatment with a PPARα agonist ameliorated renal injury, with reduced expression of these mRNAs. In vitro, enhanced IFN-γ production from innate lymphoid cells upon IL-12 and IL-18 stimulation was reduced by the PPARα agonist. Moreover, CXCL9 mRNA in glomerular endothelial cells and CXCL9, CXCL10, and CXCL11 mRNAs in podocytes and macrophages were upregulated by IFN-γ, whereas the PPARα agonist downregulated their expression. We also detected the infiltration of innate lymphoid cells into glomeruli in human anti-GBM glomerulonephritis. Thus, innate lymphoid cells are involved in the progression of anti-GBM glomerulonephritis and regulated directly or indirectly by PPARα. Our findings suggest that innate lymphoid cells could serve as novel therapeutic targets for anti-GBM glomerulonephritis.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 induces severe infection, and it is responsible for a worldwide disease outbreak starting in late 2019. Currently, there are no effective medications against coronavirus. In the present study, we utilized a holistic bioinformatics approach to study gene signatures of SARS-CoV- and SARS-CoV-2-infected Calu-3 lung adenocarcinoma cells. Through the Gene Ontology platform, we determined that several cytokine genes were up-regulated after SARS-CoV-2 infection, including TNF, IL6, CSF2, IFNL1, IL-17C, CXCL10, and CXCL11. Differentially regulated pathways were detected by the Kyoto Encyclopedia of Genes and Genomes, gene ontology, and Hallmark platform, including chemokines, cytokines, cytokine receptors, cytokine metabolism, inflammation, immune responses, and cellular responses to the virus. A Venn diagram was utilized to illustrate common overlapping genes from SARS-CoV- and SARS-CoV-2-infected datasets. An Ingenuity pathway analysis discovered an enrichment of tumor necrosis factor- (TNF-) and interleukin (IL)-17-related signaling in a gene set enrichment analysis. Downstream networks were predicted by the Database for Annotation, Visualization, and Integrated Discovery platform also revealed that TNF and TNF receptor 2 signaling elicited leukocyte recruitment, activation, and survival of host cells after coronavirus infection. Our discovery provides essential evidence for transcript regulation and downstream signaling of SARS-CoV and SARS-CoV-2 infection.

Background: Screening for donor-specific antibodies (DSA) has limited diagnostic value in patients with late antibody-mediated rejection (ABMR). Here, we evaluated whether biomarkers reflecting microcirculation inflammation or tissue injury-as an adjunct to DSA detection-are able to improve non-invasive ABMR monitoring. Methods: Upon prospective cross-sectional antibody screening of 741 long-term kidney transplant recipients with a silent clinical course, 86 DSA-positive patients were identified and biopsied. Serum and urine levels of E-selectin/CD62E, vascular cell adhesion molecule 1 (VCAM-1), granzyme B, hepatocyte growth factor (HGF), C-C motif chemokine ligand (CCL)3, CCL4, C-X-C motif chemokine ligand (CXCL)9, CXCL10, and CXCL11 in DSA-positive recipients were investigated applying multiplexed bead-based immunoassays. Results: Diagnosis of ABMR (50 patients) was associated with significantly higher levels of CXCL9 and CXCL10 in blood and urine and of HGF in blood. Overall, urinary CXCL9 had the highest diagnostic accuracy for ABMR (area under the receiver operating characteristic curve: 0.77; accuracy: 80%) and its combined evaluation with the mean fluorescence intensity of the immunodominant DSA (DSAmax MFI) revealed a net reclassification improvement of 73% compared to DSAmax MFI alone. Conclusions: Our results suggest urinary CXCL9 testing, combined with DSA analysis, as a valuable non-invasive tool to uncover clinically silent ABMR late after transplantation.

HSV-2 infection-induced CXCR3 ligands are important for the recruitment of virus-specific CD8⁺ T cells, but their impact on CD4⁺ T cell trafficking remains to be further determined. Given that recruitment of CD4⁺ T cells to infection areas may be one of the mechanisms that account for HSV-2 infection-mediated enhancement of HIV-1 sexual transmission, here we investigated the functionality of HSV-2 infection-induced CXCR3 ligands CXCL9, CXCL10, and CXCL11 in vivo and in vitro, and determined the viral components responsive for such induction and the underlying mechanisms. We first found that the expression of CXCR3 ligands CXCL9, CXCL10, and CXCL11 was increased in mice following vaginal challenge with HSV-2, while CXCL9 played a predominant role in the recruitment of CD4⁺ T cells to the vaginal foci of infected mice. HSV-2 infection also induced the production of CXCL9, CXCL10, and CXCL11 in human cervical epithelial cells. Of note, although HSV-2 induced the expression of all the three CXCR3 ligands, the induced CXCL9 appeared to play a predominant role in promoting CD4⁺ T cell migration, reflecting that the concentrations of CXCL10 and CXCL11 required for CD4⁺ T cell migration are higher than that of CXCL9. We further revealed that, ICP4, an immediate-early protein of HSV-2, is crucial in promoting CXCR3 ligand expression through the activation of p38 MAPK pathway. Mechanistically, ICP4 binds to corresponding promoters of CXCR3 ligands via interacting with the TATA binding protein (TBP), resulting in the transcriptional activation of the corresponding promoters. Taken together, our study highlights HSV-2 ICP4 as a vital viral protein in promoting CXCR3 ligand expression and CXCL9 as the key induced chemokine in mediating CD4⁺ T cell migration. Findings in this study have shed light on HSV-2 induced leukocyte recruitment which may be important for understanding HSV-2 infection-enhanced HIV-1 sexual transmission and the development of intervention strategies.

The contractile to synthetic phenotypic switching of vascular smooth muscle cells (VSMCs) in response to fibroblast growth factor (FGF) has been previously described. However, the role of the inflammatory response induced by FGF signaling in VSMCs and its occurrence in atherosclerosis remains unclear. In the present study, FGF signaling promoted a contractile to secretory phenotypic transition in VSMCs. VSMCs (primary human aortic smooth muscle cells) treated with FGF exhibited a decrease in the protein expression levels of factors involved in contractility and the secretion of various chemokines was increased, as assessed by reverse transcription‑quantitative PCR and ELISA. Additionally, inhibition of FGF signaling by silencing FGF receptor substrate 2 (FRS2) decreased the protein expression levels of various chemokines. Furthermore, VSMCs in the medial layers of arteries from apolipoprotein E‑deficient mice and human atherosclerotic samples exhibited an increase in FGF signaling that was identified to be associated with an increase in the protein expression levels of pro‑inflammatory molecules, including C‑C motif chemokine ligand 2, C‑X‑C motif chemokine ligand (CXCL) 9, CXCL10 and CXCL11, compared with wild‑type mice and healthy control samples, respectively. The present results suggested that FGF signaling induced dedifferentiation of contractile VSMCs and the transition to a secretory phenotype, which may be involved in the progression of atherosclerosis. Collectively, the present results suggested that the FGF signaling pathway may represent a novel target for the treatment of atherosclerosis.

The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses. Although unable to trigger G protein activation, both ligands activate G protein-independent ACKR3 responses and prompt arrestin recruitment. This offers a model to specifically study ligand-specific receptor conformations leading to G protein-independent signaling and to functional parameters such as receptor transport and chemokine degradation. We here show chemokine specificity in arrestin recruitment, by different effects of single amino acid substitutions in ACKR3 on arrestin in response to CXCL12 or CXCL11. Chemokine specificity in receptor transport was also observed, as CXCL11 induced faster receptor internalization, slower recycling, and longer intracellular sojourn of ACKR3 than CXCL12. Internalization and recycling rates of the ACKR3 R1423.50A substitution in response to each chemokine were similar; however, ACKR3 R1423.50A degraded only CXCL12 and not CXCL11. This suggests that ligand-specific intracellular receptor transport is required for chemokine degradation. Remarkably, the failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine. This suggests the involvement of another, yet unidentified, ACKR3 effector in scavenging. In summary, our study correlates ACKR3 ligand-specific conformational transitions with chemokine-dependent receptor transport dynamics and points toward unexpected ligand specificity in the mechanisms of chemokine degradation.

Papillary thyroid cancer (PTC) is the most prevalent endocrine neoplasia. The increased incidence of PTC in patients with thyroiditis and the frequent immune infiltrate found in PTC suggest that inflammation might be a risk factor for PTC development. The CXCR3-ligand system is involved in thyroid inflammation and CXCR3 has been found upregulated in many tumors, suggesting its pro-tumorigenic role under the inflammatory microenvironment. CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) trigger antagonistic responses partly due to the presence of two splice variants, CXCR3A and CXCR3B. Whereas CXCR3A promotes cell proliferation, CXCR3B induces apoptosis. However, the relation between CXCR3 variant expression with chronic inflammation and PTC development remains unknown. Here, we characterized the expression pattern of CXCR3 variants and their ligands in benign tumors and PTC. We found that CXCR3A and CXCL10 mRNA levels were increased in non-metastatic PTC when compared to non-neoplastic tissue. This increment was also observed in a PTC epithelial cell line (TPC-1). Although elevated protein levels of both isoforms were detected in benign and malignant tumors, the CXCR3A expression remained greater than CXCR3B and promoted proliferation in Nthy-ori-3-1 cells. In non-metastatic PTC, inflammation was conditioning for the CXCR3 ligands increased availability. Consistently, CXCL10 was strongly induced by interferon gamma in normal and tumor thyrocytes. Our results suggest that persistent inflammation upregulates CXCL10 expression favoring tumor development via enhanced CXCR3A-CXCL10 signaling. These findings may help to further understand the contribution of inflammation as a risk factor in PTC development and set the basis for potential therapeutic studies.

Long-term exposure to traffic-related pollutants can lead to a variety of respiratory diseases, including inflammation, asthma, and lung cancer; however, the underlying biological mechanisms are not fully understood. We focused on the effects of exposure to different air pollutants on the expression of genes associated with inflammatory immune responses, allergic reactions and asthma, and lung cancer. In order to understand the cellular responses induced by exposure to different traffic-related pollutants, we performed PCR array to evaluate the mRNA expression of genes associated with inflammatory immune responses, allergic reactions and asthma, and lung cancer in the lungs of mice exposed to three different environments, including the laboratory (clean air), and polluted parking garages in Foshan and Guangzhou for four weeks. Cytokines (IFN-γ, IL-4, and IL-17A) were analyzed by Flow cytometry; the morphological structures were detected by Haematoxylin and eosin (H&E) staining. Our results revealed that the main pollutant in Guangzhou was PM2.5, the main pollutants in Foshan were gaseous pollutants including CO, NOx and SO2. IFN-γ was significantly lower, and IL-4, and IL-17A were significantly higher in mice in the Guangzhou and Foshan groups compared with laboratory group. The morphological structures were damaged in Guangzhou and Foshan groups. In addition, we found that exposure to traffic-related pollutants triggered the expression of inflammatory genes (Cxcl11 and Tnfs4), allergy and asthma genes (Clca3 and Prg2), and lung cancer genes (Agr2, Col11a1, and Sostdc1). As such, our results demonstrate that persistent exposure to traffic-related pollutants may elevate the incidence of immune disorders and asthma, and may be as a risk factor for lung cancer.

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44⁺CD24^- phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.

Loss-of-function mutations of the breast cancer type 1 susceptibility protein (BRCA1) are associated with breast (BC) and ovarian cancer (OC). To identify gene signatures regulated by epigenetic mechanisms in OC cells carrying BRCA1 mutations, we assessed cellular responses to epigenome modifiers and performed genome-wide RNA- and chromatin immunoprecipitation-sequencing in isogenic OC cells UWB1.289 (carrying a BRCA1 mutation, BRCA1-null) and UWB1.289 transduced with wild-type BRCA1 (BRCA1+). Increased sensitivity to histone deacetylase inhibitors (HDACi) was observed in BRCA1-null vs. BRCA1+ cells. Gene expression profiles of BRCA1-null vs. BRCA1+ cells and treated with HDACi were integrated with chromatin mapping of histone H3 lysine 9 or 27 acetylation. Gene networks activated in BRCA1-null vs. BRCA1 + OC cells related to cellular movement, cellular development, cellular growth and proliferation, and activated upstream regulators included TGFβ1, TNF, and IFN-γ. The IFN-γ pathway was altered by HDACi in BRCA1+ vs. BRCA1-null cells, and in BRCA1-mutated/or low vs. BRCA1-normal OC tumors profiled in the TCGA. Key IFN-γ-induced genes upregulated at baseline in BRCA1-null vs. BRCA1+OC and BC cells included CXCL10, CXCL11, and IFI16. Increased localization of STAT1 in the promoters of these genes occurred in BRCA1-null OC cells, resulting in diminished responses to IFN-γ or to STAT1 knockdown. The IFN-γ signature was associated with improved survival among OC patients profiled in the TCGA. In all, our results support that changes affecting IFN-γ responses are associated with inactivating BRCA1 mutations in OC. This signature may contribute to altered responses to anti-tumor immunity in BRCA1-mutated cells or tumors.