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

황종익,Huong Thi Nguyen,Sunghoon Hurh,Lan Phuong Nguyen,Thai Uy Nguyen,Hee-Kyung Park,Jae Young Seong,Cheol Soon Lee,Byung-Joo Ham 한국분자세포생물학회 2023 Molecules and cells Vol.46 No.5

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.

Time-dependent Expression of CXCR3 Ligands and IFN-γ in Tuberculosis Infection

( Wou Young Chung ),( Hye Eun Byeon ),( Kwang Joo Park ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

Interferon (IFN)-γ and CXCR3 ligands play important roles in host defence against tuberculosis (TB) infection. We previously reported the significant increased of CXCR3 ligands levels in TB antigen-stimulated blood of TB patients compared to latent TB infection (LTBI) as well as healthy controls. Venous blood of the control group (n = 12), LTBI patient group (n = 12), and active TB patients (n = 12) was drawn and stimulated with TB-specific antigens. After 0, 1, 2, 8, and 24 h of incubation, lymphocytes and monocytes were isolated. The mRNA expression levels IFN-γ and CXCR3 ligands (CXCL9, CXCL10, amd CXCL11) were evaluated using real time RT-PCR, and their concentrations in supernatant were measured using ELISA. Also, mRNA expression of CXCR3, CCR3, and TNF-α was evaluated. The Results showed that time-dependent increases of lymphocyte mRNA levels of CXCR3 ligands were more prominent than those of IFN-γ in TB patients. Protein level measurements of them in supernatant showed the similar Results. The protein concentrations and mRNA levels of CXCR3 ligands in TB patients were significantly higher in TB patients than those in LTBI and the controls, while those of IFN-γ levels were not different among groups. The ratio of lymphocyte mRNA levels of CXCR9, CXCL10, CXCL11, and IFN-γ at 24 h in TB patients relative to LTBI patients were 2.4, 2.0, 3.1, and 1.5, respectively. In conclusion, transcription activation levels of CXCR3 ligands were more prominent than those of IFN-γ in TB patients. The measurement of transcription levels of CXCR3 ligands could differentiate TB patients and LTBI patients.

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Sung Hee Yoon,Sun Ok Yun,Jung Yong Park,Hee Yeun Won,Eun-Kyung Kim,Hyun-Jung Sohn,Hyun-Il Cho,김태규 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.3

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-γ, TNF-α, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-α and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-γ secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+ CCR4- CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy. Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-γ, TNF-α, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-α and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-γ secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+ CCR4- CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.

Homology Modeling of Chemokine Receptor CXCR3: A Novel Therapeutic Target against Inflammatory Diseases

M, Shalini,Madhavan, Thirumurthy The Basic Science Institute Chosun University 2015 조선자연과학논문집 Vol.8 No.3

CXCR3 is a C-X-C chemokine receptor type 3 also known as GPR9 and CD183. CXCR3 is a G-Protein coupled chemokine receptor which interacts with three endogenous interferon inducible chemokine's (CXCL9, CXCL10 and CXCL11) and is proved to play a vital role in the Th1 inflammatory responses. CXCR3 has been implicated to be associated with various disease conditions like inflammatory diseases, autoimmune diseases, type I diabetes and acute cardiac allograft rejection. Therefore CXCR3 receptor is found to be an attractive therapeutic target for the treatment of inflammatory diseases. Inorder to decipher the biological function of a CXCR3, 3D structure is of much important but the crystal structure for CXCR3 has not yet been resolved. Hence, in the current study Homology modeling of CXCR3 was performed against various templates and validated using different parameters to suggest the best model for CXCR3. The reported best model can be used for further studies such as docking to identify the important binding site residues.

Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse <i>Hippocampus abdominalis</i>: Molecular perspectives and immune defensive role upon pathogenic stress

Priyathilaka, Thanthrige Thiunuwan,Oh, Minyoung,Bathige, S.D.N.K.,De Zoysa, Mahanama,Lee, Jehee Elsevier 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.65 No.-

<P><B>Abstract</B></P> <P>CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse <I>CXCR3</I> and <I>CXCR4</I> (designated as <I>HaCXCR3</I> and <I>HaCXCR4</I>) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. <I>HaCXCR3</I> and <I>HaCXCR4</I> encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that <I>HaCXCR3</I> and <I>HaCXCR4</I> were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney <I>HaCXCR3</I> and <I>HaCXCR4</I> mRNA expressions were differently modulated when challenged with <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Full length coding sequences of CXC chemokine receptor 3 and 4 from Seahorse (HaCXCR3 and HaCXCR4) were identified. </LI> <LI> The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors. </LI> <LI> The <I>HaCXCR3</I> and <I>HaCXCR4</I> mRNA was significantly up-regulated upon live pathogens and PAMPs in blood and kidney. </LI> </UL> </P>

Neuroprotective effect of miR‑410‑3p against sevoflurane anesthesia‑induced cognitive dysfunction in rats through PI3K/Akt signaling pathway via targeting C–X–C motif chemokine receptor 5

Rui Su,Ping Sun,Dianhong Zhang,Wei Xiao,Chun Feng,Liang Zhong 한국유전학회 2019 Genes & Genomics Vol.41 No.10

Background Postoperative cognitive dysfunction (POCD) is a neurodegenerative disorder with impairment of cognition. Sevoflurane anesthesia has been found to lead to CD and microRNAs (miRNAs) were reported to affect cognitive function. This study investigates the neuroprotective effect against sevoflurane anesthesia-induced CD. Methods HE staining was used to detect the pathological change of hippocampal neuron. Morris water maze test was used to analyze latency time, platform crossing and swimming speed. Quantitative real-time PCR (qRT-PCR) and western blotting were performed to examine the mRNA and protein expression of miR-410-3p, IL-6, TNF-α, IL-1β and C–X–C motif chemokine receptor 5 (CXCR5). Dual-luciferase reporter assay was used to detect the relationship between miR-410-3p and CXCR5. Results MiR-410-3p was downregulated in sevoflurane anesthesia-induced rats and cells and act as a suppressor in sevoflurane anesthesia-induced hippocampal neuron apoptosis and inflammation. Furthermore, miR-410-3p was identified to bind with CXCR5. Further analysis showed that CXCR5 expression was increased by sevoflurane treatment, whereas was repressed by miR-410-3p overexpression. Moreover, miR-410-3p could inhibit sevoflurane anesthesia-induced hippocampal neuron apoptosis by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Conclusion These data indicated that miR-410-3p exhibited its neuroprotective effect on sevoflurane anesthesia-induced CD by targeting CXCR5 via PI3K/Akt signaling pathway. Our study may potentially provide a new light on the pathogenesis and therapeutic method for sevoflurane anesthesia-induced CD.

여포성 종양에서 CXC Chemokine Receptor 4와 Galectin-3의 면역조직화학적 발현

박지은,김부경,구상건,박요한,최영식,김영옥 대한갑상선학회 2012 International Journal of Thyroidology Vol.5 No.1

Background and Objectives: Follicular tumors can present a difficult diagnostic challenge for cytological evaluation and ultrasound findings. Therefore, new methods which could help distinguish follicular adenoma from follicular carcinoma simply and accurately are greatly desired. This study investigated the usefulness of immnunohistochemical expression of CXC chemokine receptor 4 (CXCR4) and galectin-3 as marker of differentiated thyroid carcinomas. Materials and Methods: Expression of CXCR4 and galectin-3 were examined immunohistochemically in the 60 paraffin embedded tissues which were already diagnosed as follicular adenoma (n=20), follicullar carcinoma (n=20), and papillary carcinoma (n=20) of thyroid. Results: Galatin-3 was expressed significantly high in follicular carcinoma than follicular adenoma (p=0.022). CXCR4 was also expressed significantly high in follicular carcinoma than follicular adenoma (p=0.027). The sensitivity of CXCR4 and galectin-3 was 70% and 80% and specificity, 65% and 60% for differential diagnosis of follicular tumors. Conclusion: An immunohistochemical panel, including galatin-3 and CXCR4, could be useful in the differential diagnosis between follicular adenoma from follicular carcinoma.

F-7 폐결핵 환자의 단순혈액 및 항원자극혈액에서 CXCR3 ligands 측정의 유용성 비교

박광주,정우영,정윤정,이규성,박주헌,신승수 대한결핵 및 호흡기학회 2017 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.124 No.-

배경: 결핵에서 Th1 세포면역에 관여하는 CXCR3 ligands의 임상적 유용성이 보고된 바 있다. 방법: 활동성 폐결핵 환자 (n = 65) 및 건강대조군 (n = 64)의 단순혈액 (혈청) 및 결핵항원자극혈액에서 interferon (IFN)-γ 및 CXCR3 ligands인 CXCL9과 CXCL11의 농도를 ELISA 법으로 측정하였다. 결과: 1. 폐결핵 환자의 혈청 CXCR3 ligands는 대조군보다 높았으나 IFN-γ는 차이가 없었다. 항원자극혈액에서는 세 표지자 모두 환자군에서 높았다. 2. 폐결핵과 대조군의 감별을 위한 AUC는 혈청 CXCL9, CXCL11, IFN-γ에서 각각 0.94, 0.90, 0.48이었고 항원자극혈액에서는 각각 0.97, 0.99, 0.93이었다. 3. 폐결핵과 IGRA양성 대조군과의 감별을 위한 AUC는 혈청 CXCL9, CXCL11, IFN-γ에서 각각 0.93, 0.88, 0.39였고 항원자극혈액에서는 각각 0.91, 0.96, 0.81이었다. 4. 결핵과 대조군의 감별을 위해 혈청 CXCL9와 항원자극혈액 CXCL11 농도를 합산하여 구한 AUC는 0.99였다. 5. 결핵과 대조군의 감별에서 항원자극혈액 CXCL11은 민감도 96.9%, 특이도 93.7%였고 혈청 CXCL9 양성 또는 항원자극혈액 CXCL11이 양성일 때 결핵으로 진단한 조합은 민감도 100%, 특이도 78.1%였다. 결론: 폐결핵에서 단순혈액 CXCR3 ligands 측정은 결핵항원자극혈액을 이용한 진단법에 보완적 역할을 하며, 특히 IGRA 양성 대조군과 감별에서 유용한 표지자로 나타났다.

A common intronic variant of <i>CXCR3</i> is functionally associated with gene expression levels and the polymorphic immune cell responses to stimuli

Choi, Jung-Won,Park, Choon-Sik,Hwang, Minyoung,Nam, Hye-Young,Chang, Hun Soo,Park, Seong Gyu,Han, Bok-Ghee,Kimm, Kuchan,Kim, Hyung Lae,Oh, Bermseok,Kim, Yeonjung Elsevier 2008 The Journal of allergy and clinical immunology Vol.122 No.6

<P><B>Background</B></P><P>CXCR3 is a chemokine receptor that plays important roles in mediating chemotactic signals and modulating the activation of lymphocytes. We have previously conducted a case-control study by using a candidate gene approach to investigate the association of <I>CXCR3</I> polymorphisms with the risk of asthma. Results from the epidemiologic study showed that a common nucleotide variant in the <I>CXCR3</I> intron <I>(rs2280964G>A)</I> was associated with disease susceptibility (1006 cases and 384 control subjects; odds ratio, 0.81; 95% CI, 0.69-0.94; <I>P</I> = .007).</P><P><B>Objective</B></P><P>The aim of our study was to evaluate the epidemiologic study and provide functional evidence for the association of <I>rs2280964G>A</I> with asthma by investigating the effects of intronic variant on chemokine-mediated phenotypes of human-derived T cells.</P><P><B>Methods</B></P><P>We used cell line–based <I>in vitro</I> and human primary T cell–based <I>ex vivo</I> studies to examine the functional consequences of the intronic polymorphism, focusing on the regulation of gene expression, splicing, and immune responsiveness toward activating signals.</P><P><B>Results</B></P><P>We present functional evidence indicating that the <I>rs2280964A</I> allele significantly correlates with decreased <I>CXCR3</I> gene expression, which would lead to variation in immune cell responses to chemokine-cytokine signals <I>in vitro</I> and <I>ex vivo</I> that includes a decrease in chemotactic activity.</P><P><B>Conclusion</B></P><P>These findings, in conjunction with those of our previous epidemiologic studies, might implicate a functional link between a common nucleotide variant of a chemokine receptor gene, <I>CXCR3</I>, and a cause for a complex-trait disease, asthma.</P>

Pathogenic roles of CXCL10 signaling through CXCR3 and TLR4 in macrophages and T cells: relevance for arthritis

Lee, Jong-Ho,Kim, Bongjun,Jin, Won Jong,Kim, Hong-Hee,Ha, Hyunil,Lee, Zang Hee BioMed Central 2017 Arthritis research & therapy Vol.19 No.-

<P><B>Background</B></P><P>Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by uncontrolled joint inflammation and destruction of bone and cartilage. We previously reported that C-X-C motif chemokine 10 (CXCL10; also called IP-10) has important roles in joint inflammation and bone destruction in arthritis. However, the specific mechanisms by which CXCL10 regulates the recruitment of inflammatory cells and the production of osteoclastogenic cytokines in RA progression are not fully understood.</P><P><B>Methods</B></P><P>Bone marrow-derived macrophages and CD4<SUP>+</SUP> T cells were isolated from wild-type (WT), <I>Cxcl10</I><SUP>–/–</SUP>, and <I>Cxcr3</I><SUP>–/–</SUP> mice. CXCL10-induced migration was performed using a Boyden chamber, and CXCL10-stimulated production of osteoclastogenic cytokines was measured by quantitative real-time PCR and ELISA. Collagen antibody-induced arthritis (CAIA) was induced by administration of collagen type II antibodies and lipopolysaccharide to the mice. Clinical scores were analyzed and hind paws were collected for high-resolution micro-CT, and histomorphometry. Serum was used to assess bone turnover and levels of osteoclastogenic cytokines.</P><P><B>Results</B></P><P>CXCL10 increased the migration of inflammatory cells through C-X-C chemokine receptor 3 (CXCR3)-mediated, but not toll-like receptor 4 (TLR4)-mediated, ERK activation. Interestingly, both receptors CXCR3 and TLR4 were simultaneously required for CXCL10-stimulated production of osteoclastogenic cytokines in CD4<SUP>+</SUP> T cells. Furthermore, calcineurin-dependent NFATc1 activation was essential for CXCL10-induced RANKL expression. In vivo, F4/80<SUP>+</SUP> macrophages and CD4<SUP>+</SUP> T cells robustly infiltrated into synovium of WT mice with CAIA but were significantly reduced in both <I>Cxcl10</I><SUP><I>–/–</I></SUP> and <I>Cxcr3</I><SUP><I>–/–</I></SUP> mice. Serum concentrations of osteoclastogenic cytokines and bone destruction were also reduced in the knockout mice, leading to attenuated progression of arthritis.</P><P><B>Conclusion</B></P><P>These findings highlight the importance of CXCL10 signaling in the pathogenesis of RA and provide previously unidentified details of the mechanisms by which CXCL10 promotes the development of arthritis.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s13075-017-1353-6) contains supplementary material, which is available to authorized users.</P>