Parabacteroides distasonis-Derived Outer Membrane Vesicles Enhance Antitumor Immunity Against Colon Tumors by Modulating CXCL10 and CD8+ T Cells.

Purpose: Given the potent immunostimulatory effects of bacterial outer membrane vesicles (OMVs) and the significant anti-colon tumor properties of Parabacteroides distasonis (Pd), this study aimed to elucidate the role and potential mechanisms of Pd-derived OMVs (Pd-OMVs) against colon cancer. Methods: This study isolated and purified Pd-OMVs from Pd cultures and assessed their characteristics. The effects of Pd-OMVs on CT26 cell uptake, proliferation, and invasion were investigated in vitro. In vivo, a CT26 colon tumor model was used to investigate the anti-colon tumor effects and underlying mechanisms of Pd-OMVs. Finally, we evaluated the biosafety of Pd-OMVs. Results: Purified Pd-OMVs had a uniform cup-shaped structure with an average size of 165.5 nm and a zeta potential of approximately -9.56 mV, and their proteins were associated with pathways related to immunity and apoptosis. In vitro experiments demonstrated that CT26 cells internalized the Pd-OMVs, resulting in a significant decrease in their proliferation and invasion abilities. Further in vivo studies confirmed the accumulation of Pd-OMVs in tumor tissues, which significantly inhibited the growth of colon tumors. Mechanistically, Pd-OMVs increased the expression of CXCL10, promoting infiltration of CD8+ T cells into tumor tissues and expression of pro-inflammatory factors TNF-α, IL-1ß, and IL-6. Notably, Pd-OMVs demonstrated a high level of biosafety. Conclusion: This paper elucidates that Pd-OMVs can exert significant anti-colon tumor effects by upregulating the expression of the chemokine CXCL10, thereby increasing the infiltration of CD8+ T cells into tumors and enhancing antitumor immune responses. This suggests that Pd-OMVs may be developed as a novel nanoscale potent immunostimulant with great potential for application in tumor immunotherapy. As well as developed as a novel nano-delivery carrier for combination with other antitumor drugs.

CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Coinfection with respiratory syncytial virus and rhinovirus increases IFN-λ1 and CXCL10 expression in human primary bronchial epithelial cells.

Acute respiratory tract infection (ARTI) is common in all age groups, especially in children and the elderly. About 85% of children who present with bronchiolitis are infected with respiratory syncytial virus (RSV); however, nearly one-third are coinfected with another respiratory virus, such as human rhinovirus (HRV). Therefore, it is necessary to explore the immune response to coinfection to better understand the molecular and cellular pathways involving virus-virus interactions that might be modulated by innate immunity and additional host cell response mechanisms. This study aims to investigate the host innate immune response against RSV-HRV coinfection compared with monoinfection. Human primary bronchial/tracheal epithelial cells (HPECs) were infected with RSV, HRV, or coinfected with both viruses, and the infected cells were collected at 48 and 72 hours. Gene expression profiles of IL-6, CCL5, TNF-α, IFN-ß, IFN-λ1, CXCL10, IL-10, IL-13, IRF3, and IRF7 were investigated using real-time quantitative PCR, which revealed that RSV-infected cells exhibited increased expression of IL-10, whereas HRV infection increased the expression of CXCL10, IL-10, and CCL5. IFN-λ1 and CXCL10 expression was significantly different between the coinfection and monoinfection groups. In conclusion, our study revealed that two important cytokines, IFN-λ1 and CXCL10, exhibited increased expression during coinfection.

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.

Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10.

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Distinct T cell signatures are associated with Staphylococcus aureus skin infection in pediatric atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin condition with a childhood prevalence of up to 25%. Microbial dysbiosis is characteristic of AD, with Staphylococcus aureus the most frequent pathogen associated with disease flares and increasingly implicated in disease pathogenesis. Therapeutics to mitigate the effects of S. aureus have had limited efficacy and S. aureus-associated temporal disease flares are synonymous with AD. An alternative approach is an anti-S. aureus vaccine, tailored to AD. Experimental vaccines have highlighted the importance of T cells in conferring protective anti-S. aureus responses; however, correlates of T cell immunity against S. aureus in AD have not been identified. We identify a systemic and cutaneous immunological signature associated with S. aureus skin infection (ADS.aureus) in a pediatric AD cohort, using a combined Bayesian multinomial analysis. ADS.aureus was most highly associated with elevated cutaneous chemokines IP10 and TARC, which preferentially direct Th1 and Th2 cells to skin. Systemic CD4+ and CD8+ T cells, except for Th2 cells, were suppressed in ADS.aureus, particularly circulating Th1, memory IL-10+ T cells, and skin-homing memory Th17 cells. Systemic γδ T cell expansion in ADS.aureus was also observed. This study suggests that augmentation of protective T cell subsets is a potential therapeutic strategy in the management of S. aureus in AD.

CXCL10 and IL15 co-expressing chimeric antigen receptor T cells enhance anti-tumor effects in gastric cancer by increasing cytotoxic effector cell accumulation and survival.

Chimeric antigen receptor (CAR) T cells have demonstrated outstanding therapeutic success in hematological malignancies. Yet, their efficacy against solid tumors remains constrained due to inadequate infiltration of cytotoxic T and CAR-T cells in the tumor microenvironment (TME), a factor correlated with poor prognosis in patients with solid tumors. To overcome this limitation, we engineered CAR-T cells to secrete CXCL10 and IL15 (10 × 15 CAR-T), which sustain T cell viability and enhance their recruitment, thereby amplifying the long-term cytotoxic capacity of CAR-T cells in vitro. In a xenograft model employing NUGC4-T21 cells, mice receiving 10 × 15 CAR-T cells showed superior tumor reduction and extended survival rates compared to those treated with second-generation CAR-T cells. Histopathological evaluations indicated a pronounced increase in cytotoxic T cell accumulation in the TME post 10 × 15 CAR-T cell treatment. Therefore, the synergistic secretion of CXCL10 and IL15 in these CAR-T cells enhances T cell recruitment and adaptability within tumor tissues, improving tumor control. This approach may offer a promising strategy for advancing CAR-T therapies in the treatment of solid tumors.

CXCR3+ effector regulatory T cells associate with disease tolerance during lower respiratory pneumovirus infection.

Lifestyle factors like poor maternal diet or antibiotic exposure disrupt early life microbiome assembly in infants, increasing the risk of severe lower respiratory infections (sLRI). Our prior studies in mice indicated that a maternal low-fibre diet (LFD) exacerbates LRI severity in infants by impairing recruitment of plasmacytoid dendritic cells (pDC) and consequently attenuating expansion of lung regulatory T (Treg) cells during pneumonia virus of mice (PVM) infection. Here, we investigated whether maternal dietary fibre intake influences Treg cell phenotypes in the mediastinal lymph nodes (mLN) and lungs of PVM-infected neonatal mice. Using high dimensional flow cytometry, we identified distinct clusters of regulatory T cells (Treg cells), which differed between lungs and mLN during infection, with notably greater effector Treg cell accumulation in the lungs. Compared to high-fibre diet (HFD)-reared pups, frequencies of various effector Treg cell subsets were decreased in the lungs of LFD-reared pups. Particularly, recruitment of chemokine receptor 3 (CXCR3+) expressing Treg cells was attenuated in LFD-reared pups, correlating with lower lung expression of CXCL9 and CXCL10 chemokines. The recruitment of this subset in response to PVM infection was similarly impaired in pDC depleted mice or following anti-CXCR3 treatment, increasing immunopathology in the lungs. In summary, PVM infection leads to the sequential recruitment and expansion of distinct Treg cell subsets to the lungs and mLN. The attenuated recruitment of the CXCR3+ subset in LFD-reared pups increases LRI severity, suggesting that strategies to enhance pDCs or CXCL9/CXCL10 expression will lower immune-mediated pathogenesis.

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.

CSF1R blockade slows progression of cerebral hemorrhage by reducing microglial proliferation and increasing infiltration of CD8 + CD122+ T cells into the brain.

Microglia play a pivotal role in the neuroinflammatory response after brain injury, and their proliferation is dependent on colony-stimulating factors. In the present study, we investigated the effect of inhibiting microglia proliferation on neurological damage post intracerebral hemorrhage (ICH) in a mouse model, an aspect that has never been studied before. Using a colony-stimulating factor-1 receptor antagonist (GW2580), we observed that inhibition of microglia proliferation significantly ameliorated neurobehavioral deficits, attenuated cerebral edema, and reduced hematoma volume after ICH. This intervention was associated with a decrease in pro-inflammatory factors in microglia and an increased infiltration of peripheral regulatory CD8 + CD122+ T cells into the injured brain tissue. The CXCR3/CXCL10 axis is the mechanism of brain homing of regulatory CD8 + CD122+ T cells, and the high expression of IL-10 is the hallmark of their synergistic anti-inflammatory effect with microglia. And activated astrocytes around the insult site are a prominent source of CXCL10. Thus, inhibition of microglial proliferation offers a new perspective for clinical translation. The cross-talk between multiple cells involved in the regulation of the inflammatory response highlights the comprehensive nature of neuroimmunomodulation.

Staphylococcus aureus acquires resistance to glycopeptide antibiotic vancomycin via CXCL10.

BACKGROUND: Glycopeptide antibiotic vancomycin is a bactericidal antibiotic available for the infection to Staphylococcus aureus (SA), however, SA has a strong adaptive capacity and thereby acquires resistance to vancomycin. This study aims to illuminate the possible molecular mechanism of vancomycin resistance of SA based on the 16S rRNA sequencing data and microarray profiling data. METHODS: 16S rRNA sequencing data of control samples and urinary tract infection samples were retrieved from the EMBL-EBI (European Molecular Biology Laboratory - European Bioinformatics Institute) database. Correlation of gut flora and clinical indicators was evaluated. The possible targets regulated by SA were predicted by microarray profiling and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. CXCL10 gene knockout and overexpression were introduced to evaluate the effect of CXCL10 on the virulence of SA and the resistance to vancomycin. SA strains were co-cultured with urethral epithelial cells in vitro. The presence of SA virulence factors was detected using PCR. Biofilm formation of SA strains was assessed using the microtiter plate method. Furthermore, the antibiotic sensitivity of SA strains was evaluated through vancomycin testing. RESULTS: Gut flora and its species abundance had significant difference between urinary tract infection and control samples. SA was significantly differentially expressed in urinary tract infection samples. Resistance of SA to vancomycin mainly linked to the D-alanine metabolism pathway. SA may participate in the occurrence of urinary tract infection by upregulating CXCL10. In addition, CXCL10 mainly affected the SA resistance to vancomycin through the TLR signaling pathway. In vitro experimental results further confirmed that the overexpression of CXCL10 in SA increased SA virulence and decreased its susceptibility to vancomycin. In vitro experimental validation demonstrated that the knockout of CXCL10 in urethral epithelial cells enhanced the sensitivity of Staphylococcus aureus (SA) to vancomycin. CONCLUSION: SA upregulates the expression of CXCL10 in urethral epithelial cells, thereby activating the TLR signaling pathway and promoting resistance to glycopeptide antibiotics in SA.

Radiotherapy Enhances Metastasis Through Immune Suppression by Inducing PD-L1 and MDSC in Distal Sites.

PURPOSE: Radiotherapy (RT) is a widely employed anticancer treatment. Emerging evidence suggests that RT can elicit both tumor-inhibiting and tumor-promoting immune effects. The purpose of this study is to investigate immune suppressive factors of radiotherapy. EXPERIMENTAL DESIGN: We used a heterologous two-tumor model in which adaptive concomitant immunity was eliminated. RESULTS: Through analysis of PD-L1 expression and myeloid-derived suppressor cells (MDSC) frequencies using patient peripheral blood mononuclear cells and murine two-tumor and metastasis models, we report that local irradiation can induce a systemic increase in MDSC, as well as PD-L1 expression on dendritic cells and myeloid cells, and thereby increase the potential for metastatic dissemination in distal, nonirradiated tissue. In a mouse model using two distinct tumors, we found that PD-L1 induction by ionizing radiation was dependent on elevated chemokine CXCL10 signaling. Inhibiting PD-L1 or MDSC can potentially abrogate RT-induced metastasis and improve clinical outcomes for patients receiving RT. CONCLUSIONS: Blockade of PD-L1/CXCL10 axis or MDSC infiltration during irradiation can enhance abscopal tumor control and reduce metastasis.

Characterizing of intra-amniotic inflammatory changes associated with chronic inflammation in the placenta marked by elevated amniotic fluid interferon gamma-induced protein 10 (IP-10) in pregnancies complicated by preterm prelabor rupture of membranes.

OBJECTIVES: This study aimed to determine the occurrence of intra-amniotic inflammatory changes associated with chronic inflammation in the placenta, marked by elevated levels of interferon gamma-induced protein 10 (IP-10) (≥2200 pg/mL) in the amniotic fluid of women with preterm prelabor rupture of membranes (PPROM). Specifically, the study investigated whether these intra-amniotic inflammatory changes were more common in women with microbial invasion of amniotic cavity (MIAC) and intra-amniotic inflammation (IAI), as indicated by increased amniotic fluid interleukin (IL)-6 concentration (≥3000 pg/mL). STUDY DESIGN: A cohort of 114 women with singleton pregnancies complicated by PPROM between 24+0 and 36+6 weeks of gestation were included. Amniotic fluid samples were obtained via amniocentesis upon admission. MIAC diagnosis involved aerobic and anaerobic cultures, as well as polymerase chain reaction (PCR) analysis of the amniotic fluid. Immunoassay tests and enzyme-linked immunosorbent assay (ELISA) were used to determine IL-6 and IP-10 concentrations, respectively. RESULTS: Among the participants, 19.3 % and 15.8 % had MIAC and IAI, respectively. The occurrence of intra-amniotic inflammatory changes associated with chronic inflammation in the placenta was similar between women with and without MIAC (25 % vs. 40.9 %, p = 0.136, adjusted p = 0.213). The rate of intra-amniotic inflammatory changes associated with chronic inflammation in the placenta was significantly higher in women with IAI compared to those without, after adjusting for gestational age at sampling (55.6 % vs. 22.9 %, p = 0.005, adjusted p = 0.011). CONCLUSION: This study revealed comparable rates of intra-amniotic inflammatory changes associated with chronic inflammation in the placenta in women with and without MIAC, but a higher prevalence of intra-amniotic inflammatory changes associated with chronic inflammation in the placenta in women with IAI. These findings suggest involvement of chronic inflammation even in women with PPROM with acute intra-amniotic inflammation.

Induction of SK-MEL-28 Invasion by Brain Cortical Cell-Conditioned Medium Through CXCL10 Signaling.

Melanoma, an infrequent yet significant variant of skin cancer, emerges as a primary cause of brain metastasis among various malignancies. Despite recognizing the involvement of inflammatory molecules, particularly chemokines, in shaping the metastatic microenvironment, the intricate cellular signaling mechanisms underlying cerebral metastasis remain elusive. In our pursuit to unravel the role of cytokines in melanoma metastasis, we devised a protocol utilizing mixed cerebral cortical cells and SK-MEL-28 melanoma cell lines. Contrary to expectations, we observed no discernible morphological change in melanoma cells exposed to a cerebral conditioned medium (CM). However, a substantial increase in both migration and proliferation was quantitatively noted. Profiling the chemokine secretion by melanoma in response to the cerebral CM unveiled the pivotal role of interferon gamma-induced protein 10 (CXCL10), inhibiting the secretion of interleukin 8 (CXCL8). Furthermore, through a transwell assay, we demonstrated that knockdown CXCL10 led to a significant decrease in the migration of the SK-MEL-28 cell line. In conclusion, our findings suggest that a cerebral CM induces melanoma cell migration, while modulating the secretion of CXCL10 and CXCL8 in the context of brain metastases. These insights advance our understanding of the underlying mechanisms in melanoma cerebral metastasis, paving the way for further exploration and targeted therapeutic interventions.

LAMP1 controls CXCL10-CXCR3 axis mediated inflammatory regulation of macrophage polarization during inflammatory stimulation.

Increased expression of CXCL10 and its receptor CXCR3 represents an inflammatory response in cells and tissues. Macrophage polarization and autophagy are major functions in inflammatory macrophages; however, the cellular functions of the CXCL10-CXCR3 axis in macrophages are not well understood. Here, we examined the role of CXCL10-CXCR3-axis-regulated autophagy in macrophage polarization. First, in non-inflammatory macrophages, whereas CXCL10 promotes M2 polarization and inhibits M1 polarization, CXCR3 antagonist AMG487 induces the opposite macrophage polarization. Next, CXCL10 promotes the expression of autophagy proteins (Atg5-Atg12 complex, p62, LC3-II, and LAMP1) and AMG487 inhibits their expression. Knockdown of LAMP1 by short interfering RNA switches the CXCL10-induced polarization from M2 to M1 in non-inflammatory macrophages. Furthermore, in inflammatory macrophages stimulated by poly(I:C), CXCL10 induces M1 polarization and AMG487 induces M2 polarization in association with a decrease in LAMP1. Finally, AMG487 alleviates lung injury after poly(I:C) treatment in mice. In conclusion, CXCL10-CXCR3 axis differentially directs macrophage polarization in inflammatory and non-inflammatory states, and autophagy protein LAMP1 acts as the switch controlling the direction of macrophage polarization by CXCL10-CXCR3.

Homocysteine modulates CXCL10/CXCR3 axis activity to induce endothelial dysfunction.

Elevated homocysteine (Hcy) levels have been linked to the development of cardiovascular diseases, notably endothelial dysfunction, a critical precursor to atherosclerosis. In this extensive investigation, we explore the intricate pathways through which Hcy influences endothelial dysfunction, with particular attention to the CXCL10/CXCR3 axis. Employing a dual approach encompassing both in vitro and in vivo models, we scrutinize the repercussions of Hcy exposure on endothelial functionality. Our results reveal that Hcy significantly impairs crucial endothelial processes, including cell migration, proliferation, and tube formation. Concomitantly, Hcy upregulates the expression of adhesion molecules, exacerbating endothelial dysfunction. In a murine hyperhomocysteinemia (HHcy) model, we observed a parallel increase in plasma Hcy levels and adverse vascular effects. Moreover, our study unraveled a pivotal role of the CXCL10/CXCR3 axis in Hcy-induced endothelial dysfunction. Hcy exposure led to the upregulation of CXCL10 and CXCR3, both in vitro and in HHcy mice. Importantly, the blockade of this axis, achieved through specific antibodies or NBI-74330, mitigated the detrimental effects of Hcy on endothelial function. In conclusion, our findings illuminated the central role of the CXCL10/CXCR3 axis in mediating Hcy-induced endothelial dysfunction, providing valuable insights for potential therapeutic strategies in managing HHcy-related cardiovascular diseases.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

BACKGROUND: Interferon-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. METHODS: Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cell migration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. RESULTS: Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. CONCLUSION: Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1ß(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

CXCL10 upregulation in radiation-exposed human peripheral blood mononuclear cells as a candidate biomarker for rapid triage after radiation exposure.

PURPOSE: In case of a nuclear accident, individuals with high-dose radiation exposure (>1-2 Gy) should be rapidly identified. While ferredoxin reductase (FDXR) was recently suggested as a radiation-responsive gene, the use of a single gene biomarker limits radiation dose assessment. To overcome this limitation, we sought to identify reliable radiation-responsive gene biomarkers. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from mice after total body irradiation, and gene expression was analyzed using a microarray approach to identify radiation-responsive genes. RESULTS: In light of the essential role of the immune response following radiation exposure, we selected several immune-related candidate genes upregulated by radiation exposure in both mouse and human PBMCs. In particular, the expression of ACOD1 and CXCL10 increased in a radiation dose-dependent manner, while remaining unchanged following lipopolysaccharide (LPS) stimulation in human PBMCs. The expression of both genes was further evaluated in the blood of cancer patients before and after radiotherapy. CXCL10 expression exhibited a distinct increase after radiotherapy and was positively correlated with FDXR expression. CONCLUSIONS: CXCL10 expression in irradiated PBMCs represents a potential biomarker for radiation exposure.