Ligand-specific conformational transitions and intracellular transport are required for atypical chemokine receptor 3-mediated chemokine scavenging.

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.

The combination of CXCL9, CXCL10 and CXCL11 levels during primary HIV infection predicts HIV disease progression.

BACKGROUND: Chemokines are small chemotactic cytokines involved in inflammation, cell migration, and immune regulation in both physiological and pathological contexts. Here, we investigated the profile of chemokines during primary HIV infection (PHI). METHODS: Fifty-four participants with blood samples before and during HIV infection and clinical information available were selected from an HIV-negative man who have sex with men (MSM) prospective cohort. Thirty chemokines and 10 cytokines were measured pre- and post-HIV infection in the same individuals using a Bio-Plex Pro™ Human Chemokine Panel. RESULTS: Levels of 18 chemokines/cytokines changed significantly during PHI relative to pre-HIV infection levels; 14 were up-regulated and 4 down-regulated. Among them, CXCL9, CXCL10, and CXCL11 were the most prominently raised. Levels of CXCL9 and CXCL10 were much higher in the high-set point group (log viral load (lgVL) ≥ 4.5) than those in the low-set point group (lgVL < 4.5) and levels of CXCL9, CXCL10, and CXCL11 were higher in the low-CD4+ T-cell count group (CD4+ T-cell count ≥ 500). A formula to predict HIV disease progression using a combination panel comprising CXCL9, CXCL10, and CXCL11 was developed, where risk score = 0.007 × CXCL9 + 0.004 × CXCL10 - 0.033 × CXCL11 - 1.724, with risk score values higher than the cutoff threshold (0.5211) indicating more rapid HIV disease progression. CONCLUSIONS: A panel of plasma CXCL9, CXCL10, and CXCL11 measured during primary HIV-1 infection could predict long-term HIV disease prognosis in an MSM group and has potential as a novel biomarker in the clinic.

Elevated specific peripheral cytokines found in major depressive disorder patients with childhood trauma exposure: a cytokine antibody array analysis.

Taking into consideration the previous evidence of revealing the relationship of early life adversity, major depressive disorder (MDD), and stress-linked immunological changes, we recruited 22 MDD patients with childhood trauma exposures (CTE), 21 MDD patients without CTE, and 22 healthy controls without CTE, and then utilized a novel cytokine antibody array methodology to detect potential biomarkers underlying MDD in 120 peripheral cytokines and to evaluate the effect of CTE on cytokine changes in MDD patients. Although 13 cytokines were identified with highly significant differences in expressions between MDD patients and normal controls, this relationship was significantly attenuated and no longer significant after consideration of the effect of CTE in MDD patients. Depressed individuals with CTE (TD patients) were more likely to have higher peripheral levels of those cytokines. Severity of depression was associated with plasma levels of certain increased cytokines; meanwhile, the increased cytokines led to a proper separation of TD patients from normal controls during clustering analyses. Our research outcomes add great strength to the relationship between depression and cytokine changes and suggest that childhood trauma may play a vital role in the co-appearance of cytokine changes and depression.

Functional Analysis of CXCR3 Splicing Variants and Their Ligands Using NanoBiT-Based Molecular Interaction Assays.

CXCR3 regulates leukocyte trafficking, maturation, and various pathophysiological conditions. Alternative splicing generates three CXCR3 isoforms in humans. Previous studies investigated the roles of CXCR3 isoforms, and some biochemical data are not correlated with biological relevance analyses. RT-PCR analyses indicate that most cells express all three splicing variants, suggesting that they may mutually affect the chemokine binding and cellular responses of other splicing variants. Here, we performed an integrative analysis of the functional relations among CXCR3 splicing variants and their chemokine-dependent signaling using NanoBiT live cell protein interaction assays. The results indicated that the CXCR3 N-terminal region affected cell surface expression levels and ligand-dependent activation. CXCR3A was efficiently expressed in the plasma membrane and responded to I-TAC, IP-10, and MIG chemokines. By contrast, CXCR3B had low plasma membrane expression and mediated I-TAC-stimulated cellular responses. CXCR3Alt was rarely expressed on the cell surface and did not mediate any cell responses to the tested chemokines; however, CXCR3Alt negatively affected the plasma membrane expression of CXCR3A and CXCR3B and their chemokine-stimulated cellular responses. Jurkat cells express endogenous CXCR3, and exogenous CXCR3A expression enhanced chemotactic activity in response to I-TAC, IP-10, and MIG. By contrast, exogenous expression of CXCR3B and CXCR3Alt eliminated or reduced the CXCR3A-induced chemotactic activity. The PF-4 chemokine did not activate any CXCR3-mediated cellular responses. NanoBiT technology are useful to integrative studies of CXCR3-mediated cell signaling, and expand our knowledge of the cellular responses mediated by molecular interactions among the splicing variants, including cell surface expression, ligand-dependent receptor activation, and chemotaxis.

Evaluation of I-TAC as a potential early plasma marker to differentiate between critical and non-critical COVID-19.

The COVID-19 pandemic has led to significant global health and economic consequences. There is an unmet need to define a molecular fingerprint of severity of the disease that may guide an early, rational and directed intervention preventing severe illness. We collected plasma from patients with moderate (nine cases), severe (22 cases) and critical (five cases) COVID-19 within three days of hospitalization (approximately one week after symptom onset) and used a cytokine antibody array to screen the 105 cytokines included in the array. We found that I-TAC, IP-10, ST2 and IL-1ra were significantly upregulated in patients with critical disease as compared to the non-critical (moderate and severe combined). ELISA further quantified I-TAC levels as 590.24±410.89, 645.35±517.59 and 1613.53±1010.59 pg/ml in moderate, severe and critical groups, respectively. Statistical analysis showed that I-TAC levels were significantly higher in patients with critical disease when compared with moderate (p = 0.04), severe (p = 0.03) or the combined non-critical (p = 0.02) group. Although limited by the low sample numbers, this study may suggest a role of I-TAC as a potential early marker to discriminate between critical and non-critical COVID-19 cases. Such knowledge is urgently needed for appropriate allocation of resources and to serve as a platform for future research towards early interventions that could mitigate disease severity and save lives.

The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

The chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, ß-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving ß-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the ß-arrestin-bias and the atypical nature of CXCR3-B.

Study of CXCL9-11 gene polymorphisms in liver fibrosis among patients with chronic hepatitis C.

Several factors are associated with the progression of chronic hepatitis C: comorbidities, lifestyle, and pathogenic factors, including immune response, apoptosis and heredity. Single nucleotide polymorphisms (SNPs) in the PNPLA3 and TM6SF2 genes are more widely studied genetic risk factors, while CXCL9-11 chemokines produced by hepatocytes in the process of infection are less well studied. Our aim was to evaluate the influence of CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 in liver fibrosis when analysed together with PNPLA3 rs738409 and TM6SF2 rs58542926. The study included 219 patients with chronic hepatitis C. SNP genotyping was performed by real-time PCR. Univariate and multivariate analyses were used to detect the association between SNPs and advanced fibrosis in a recessive genetic model. All SNPs had a minimum allele frequency >5%, and CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 were in high linkage disequilibrium (D' ≥ 0.84). In the multivariate analysis, we observed that male gender (P = 0.000), older age (P = 0.025), moderate to intense inflammatory activity (P = 0.002), moderate to accentuated hepatic steatosis (P = 0.026) and the CT genotype of the TM6SF2 rs58542926 SNP (P = 0.014) presented significant associations with advanced fibrosis. Overall, the CXCL9 rs10336, CXCL10 rs3921, CXCL11 rs4619915 and PNPLA3 rs738409 SNPs did not influence liver fibrosis among patients with chronic hepatitis C.

Sarcoidosis and the Th1 chemokine MIG.

Sarcoidosis is a systemic inflammatory disease, affecting any organ, and that can be discovered by accident in approximately 5% of cases. High levels of the type-1 helper (Th1)-dependent chemokine, monokine induced by interferon (IFN)-γ (MIG)/chemokine (C-X-C motif) ligand (CXCL)9, and its receptor CXCR3 have been reported in bronchoalveolar lavage and biopsy samples of patients with sarcoidosis. These elevated levels are related with the amount of CD4+ lymphocytes and total lymphocytes. Alveolar macrophages resulted stained positive for all CXCR3 ligands and produced elevated levels of these chemokines. It has been shown that the epithelioid and giant cells of the sarcoid lungs were stained positive for MIG, IFN-inducible T-cell α chemoattractant (I-TAC) and IFN-γ-inducible protein 10 (IP-10), suggesting that MIG plays an important role in the accumulation of Th1 lymphocytes in sarcoid lungs. In addition, serum levels of MIG were related with the severity of the disease, and a correlation between the serial measurements of MIG and the clinical course of the disease was shown, indicating MIG as a potentially useful biomarker of sarcoidosis and its severity.

The Role of CXCR3 and Associated Chemokines in the Development of Atherosclerosis and During Myocardial Infarction.

The chemokine receptor CXCR3 and associated CXC chemokines have been extensively investigated in several inflammatory and autoimmune diseases as well as in tumor development. Recent studies have indicated the role of these chemokines also in cardiovascular diseases. We aimed to present current knowledge regarding the role of CXCR3-binding chemokines in the pathogenesis of atherosclerosis and during acute myocardial infarction.

Alpha chemokines in Crohn's disease.

Many studies have shown that chemokine (C-X-C motif) receptor (CXCR)3 and its ligand chemokines, as monokine induced by interferon (IFN)-γ (MIG), interferon-γ inducible protein (IP-10) and IFN-inducible T cell-α chemoattractant (I-TAC), are strongly overexpressed both in the intestinal mucosa of mice with experimental colitis, and in patients with Crohn's disease (CD) in lymphocytes, in macrophages and in epithelial cells. IFN-γ induces CXCR3 and its chemokines expression in epithelial intestinal cells; these chemokines are important for the recruitment of granulocytes and mononuclear cells, thus for the maintenance of inflammation in CD. Serum IP-10 levels might reflect CD disease activity, and it may be a marker for the responsiveness of patients to treatments. However other studies are needed to document the use of IP-10 in clinical setting. Attempts are currently underway to inhibit CXCR3 or its chemokines in CD as a possible therapy of CD.

Th1 cytokines and chemokines in primary biliary cirrhosis.

T- helper 1 (Th1) cytokines and chemokines in primary biliary cirrhosis (PBC) has been investigated in several studies. The involvement of (C-X-C motif) receptor 3 (CXCR3) and its ligands in the pathogenesis of PBC was studied in autoimmune cholangitis animal models suggesting that CXCR3 chemokines contribute to the development of PBC. In humans with PBC, interferon (IFN)γ-induced protein 10 (IP-10) and chemokine (C-X-C motif) ligand 9 (MIG) expressions, and CXCR3-positive cells were present in the portal areas of diseased livers. MIG and IP-10 were positively associated with the severity of liver fibrosis. Circulating IP-10 and MIG levels, and CXCR3-expressing cells, in PBC were increased significantly compared to controls and appeared to increase with disease progression. Furthermore, a significant reduction of these chemokines in PBC patients' serum after ursodeoxycholic acid (UDCA) treatment has been shown.

The CXCR3 chemokines in inflammatory myopathies.

The α-chemokines (C-X-C motif) ligand 9 (CXCL9) and interferon γ-induced protein 10 (IP-10) are expressed in idiopathic inflammatory myopathies muscle. Abundant expression of IP-10 was observed on macrophages and T cells surrounding and invading non-necrotic muscle fibers in polymyositis and sporadic inclusion body myositis and in T cells in perimysial infiltrates of dermatomyositis. IP-10 was also localized to blood vessel endothelial cells in all inflammatory and normal muscle tissues. Serum IP-10 is high in patients with inflammatory myopathies. Human skeletal muscle cells might actively self-promote muscular inflammation by eliciting IP-10 secretion, under the influence of cytokines [Interferon (IFN-γ), Tumor Necrosis Factor (TNF-α)], which can amplify T helper (Th)1 cell tissue infiltration in vivo. It has been shown that drugs able to block the IP-10/chemokine (C-X-C motif) receptor 3 (CXCR3) axis can suppress inflammation in muscle.

Interaction of the chemokines I-TAC (CXCL11) and SDF-1 (CXCL12) in the regulation of tumor angiogenesis of colorectal cancer.

The chemokine CXCL12 has a decisive role in tumor progression by mediating pro-angiogenic and pro-metastatic effects through its receptor CXCR4. The CXCL12 pathway is connected with another chemokine, CXCL11, through its second receptor CXCR7. CXCL11 also binds to the CXCR3 receptor. CXCL11 function in tumor angiogenesis is likely receptor dependent because CXCR3 predominantly mediates angiostatic signals whereas CXCR7 mediated signaling is rather angiogenic. We therefore studied the interaction of CXCL12 and CXCL11 in an in vivo model of colorectal cancer metastasis. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of syngeneic BALB/c mice. The animals received either peritumoral application of CXCL11 or intraperitoneal injections with neutralizing antibodies against CXCL11, CXCL12 or both. Tumor growth characteristics, angiogenesis, cell migration, invasive tumor growth, tumor cell proliferation and apoptosis were studied by intravital fluorescence microscopy and immunohistochemistry during an observation period of 14 days. Local exposure to CXCL11 significantly stimulated tumor growth compared to controls and enhanced invasive growth characteristics without affecting tumor angiogenesis and tumor cell migration. Neither CXCL11 nor CXCL12-blockade had a significant impact on tumor growth and angiogenesis, whereas the combined neutralization of CXCL11 and CXCL12 almost completely abrogated tumor vessel formation. As a consequence, tumor growth and invasive growth characteristics were reduced compared to the other groups. The results of the present study underline the interaction of CXCL12 and CXCL11 during tumor angiogenesis. The combined blockade of both signaling pathways may provide an interesting anti-angiogenic approach for anti-tumor therapy.

Tumor stroma-derived factors skew monocyte to dendritic cell differentiation toward a suppressive CD14+ PD-L1+ phenotype in prostate cancer.

Tumor-associated stromal myofibroblasts are essential for the progression and metastatic spread of solid tumors. Corresponding myeloid cell infiltration into primary tumors is a negative prognostic factor in some malignancies. The aim of this study was to define the exact role of stromal myofibroblasts and stromal factors in early prostate carcinoma (PCa) regulating monocyte infiltration and differentiation into dendritic cells (DCs). Epithelial and stromal primary cultures were generated from PCa biopsies and their purity confirmed. Stromal cells produced significantly more of the (C-C) motif chemokine ligand 2 (CCL2), interleukin 6 (IL-6) and transforming growth factor ß (TGFß) than epithelial cells. Monocyte chemoattraction was predominantly due to stromal-derived factors, mainly CCL2. DCs generated in the presence of stromal (but not epithelial) factors upregulated CD209, but failed to downregulate the monocyte marker CD14 in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Monocytes exposed to stromal factors did not produce detectable amounts of IL-10, however, upon lipopolysaccharide stimulation, stromal factor generated dendritic cells (sDC) produced significantly more IL-10 and less IL-12 than their conventional DC counterparts. sDC failed to cross-present tumor-antigen to CD8+ T cells and suppressed T-cell proliferation. Most importantly, sDC expressed significantly elevated levels of programmed cell death ligand-1 (PD-L1) in a primarily STAT3 and IL-6-dependent manner. In parallel with our findings in vitro, tumor-infiltrating CD14+ cells in situ were found to express both PD-L1 and CD209, and a higher percentage of tumor-associated CD3+ T cells expressed programmed cell death-1 (PD-1) molecules compared to T cells in blood. These results demonstrate a hitherto undescribed, fundamental contribution of tumor-associated stromal myofibroblasts to the development of an immunosuppressive microenvironment in early PCa.

MIG (CXCL9), IP-10 (CXCL10) and I-TAC (CXCL11) concentrations after nasal allergen challenge in patients with allergic rhinitis.

INTRODUCTION: The role of monokine induced by interferon-γ (IFN-γ, MIG/CXCL9), IFN-γ-inducible protein (IP-10/CXCL10), and IFN-inducible T cell α chemoattractant (I-TAC/CXCL11) in allergic inflammation has not been explored in detail in vivo. The aim of the study was to examine the changes in concentrations of MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in nasal lavages collected from healthy and allergic subjects during nasal allergen challenge. MATERIAL AND METHODS: Subjects allergic to grass pollen and healthy controls were included. Nasal allergen challenge preceded by placebo administration was performed outside the pollen season. Nasal lavages were collected before and 30 min after application of the placebo and 30 min after allergen administration. Concentrations of chemokines were determined using ELISA. RESULTS: We observed significantly higher concentrations of IP-10/CXCL10 in allergic patients compared to the healthy subjects before (354.49 ±329.24 vs. 164.62 ±175.94 pg/ml; p = 0.036), 30 min after placebo (420.3 ±421.28 vs. 246.88 ±353.24 pg/ml; p = 0.021) and 30 min after allergen administration (403.28 ±359.29 vs. 162.68 ±148.69 pg/ml; p = 0.025). IP-10/CXCL10 levels did not change 30 min after allergen provocation. In contrast, MIG/CXCL9 levels were similar in both groups before and after placebo. However, a significant rise in MIG/CXCL9 concentration was noted in allergic patients 30 min after the allergen (138.88 ±109.59 vs. 395.8 ±301.2 pg/ml; p = 0.00026). I-TAC/CXCL11 concentrations increased after placebo as well as the allergen in both groups. CONCLUSIONS: IP-10/CXCL10 concentrations are elevated in nasal lavages from allergic patients and this chemokine may play a role in chronic allergic inflammation. MIG/CXCL9 levels increase rapidly after allergen application, which may suggest its role in the early allergic response. Results on I-TAC/CXCL11 concentrations remain inconclusive.

Altered Th1/Th2 commitment contributes to lung senescence in CXCR3-deficient mice.

Aging is an inevitable process associated with immune imbalance, which is characterized by a progressive functional decline in major organs, including lung. However, effects of altered Th1/Th2 commitment on lung senescence are largely unknown. To examine effects of altered Th1/Th2 balance on lung aging, we measured proportions of Th1 and Th2 cells and expression of cytokines, chemokines, collagen deposition and other relevant physiological and pathological parameters in 2- and 20-months-old (mo) CXCR3-deficient (CXCR3(-/-)) C57BL/6J mice compared with wild-type (WT) mice. There was a significant weight-loss observed in 20-mo CXCR3(-/-) mice compared with the same aged WT group. Although lung function and structure changed with age in both groups, central airway resistance (Rn), tissue elastance (H) and damping (G) were significantly lower in 20-mo CXCR3(-/-) mice than those of WT mice. In contrast, the whole lung volume (V(L)), the mean linear intercept length of alveolar (L(m)), and the total lung collagen content were significantly elevated in 20-mo CXCR3(-/-) mice. With aging, the lungs of WT mice had typical Th1-type status (increased population of Th1 cells and concentrations of cytokine IFN-γ and CXCR3 ligands) while CXCR3(-/-) mice showed Th2-type polarization (decreased proportion of Th1 cells and concentrations of CXCR3 ligands but increased level of IL-4). Our data suggest that Immunosenescence is associated with lung aging, and that altered Th1/Th2 imbalance favors Th2 predominance in CXCR3(-/-) mice, which contributes to the process of accelerated lung aging in this model.