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CC chemokine receptors (CCRs), the numbers of the G protein-coupled receptor (GPCR) superfamily, had crucial roles in treating infection, inflammation, and tissue damage by binding to their ligands. In this study, five fish-specific CCRs, namely CCR4La, CCR4Lc, CCR12a1, CCR12a2, and CCR12b, were identified in largemouth bass (Micropterus salmoides). The correction of nomenclatures of these CCRs were confirmed by phylogenetic analysis, structural analysis and genomic synteny analysis. Following 1 × 10 6  CFU/mL and 1 × 10 7  CFU/mL Edwardsiella piscicida infection, these five CCRs were significantly induced in spleen of largemouth bass, indicating their important roles in the immune response against bacterial infection. Selection pressure analysis revealed that CCR4La, CCR4Lc, CCR12a1, and CCR12a2 underwent negative selection pressure, whereas CCR12b experienced positive selection pressure. Robust selection site detection methods identified that positive selected sites of CCR4La, CCR4Lc, CCR12a1, and CCR12a2 mainly distributed in their extracellular regions, which involved in ligand binding and pathogen interaction. Similarly, the positive selected sites of CCR12b were also located in its extracellular regions. The accuracy of the pressure selected sites were also validated by molecular docking analysis. The potential ligands for these five CCRs were identified by molecular docking analysis, with finding that CCL3 and CCL5 might be the ligands of largemouth bass CCR4La/Lc, and CCL5, CCL8, CCL7, CCL13 and CCL26 might be that of largemouth bass CCR12a1/a2/b. Our results provided basis for elucidating the functions of chemokine-receptor complex in largemouth bass., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Inflammatory bowel diseases (IBDs) involve chronic inflammation of the gastrointestinal tract, where effector CD4 + T-cells play a central role. Thereby, the recruitment of T-cells into the colonic mucosa represents a key process in IBD. We recently found that CCR9 and DRD5 might form a heteromeric complex on the T-cell surface. The increase in CCL25 production and the reduction in dopamine levels associated with colonic inflammation represent a dual signal stimulating the CCR9:DRD5 heteromer, which promotes the recruitment of CD4 + T-cells into the colonic lamina propria. Here, we aimed to analyse the molecular requirements involved in the heteromer assembly as well as to determine the underlying cellular mechanisms involved in the colonic tropism given by the stimulation of the CCR9:DRD5 complex. The results show that dual stimulation of the CCR9:DRD5 heteromer potentiates the phosphorylation of the myosin light chain 2 (MLC2) and the migration speed in confined microchannels. Accordingly, disrupting the CCR9:DRD5 assembly induced a sharp reduction in the pMLC2 in vitro, decreased the migratory speed in confined microchannels, and dampened the recruitment of CD4 + T-cells into the inflamed colonic mucosa. Furthermore, in silico analysis confirmed that the interface of interaction of CCR9:DRD5 is formed by the transmembrane segments 5 and 6 from each protomer. Our findings demonstrated that the CCR9:DRD5 heteromeric complex plays a fundamental role in the migration of CD4 + T-cells into the colonic mucosa upon inflammation. Thereby, the present study encourages the design of strategies for disassembling the formation of the CCR9:DRD5 as a therapeutic opportunity to treat IBD.

A substantial body of literature, including our own, points to a connection between hepatitis B virus (HBV) infection and the development of drug resistance in hepatocellular carcinoma (HCC), particularly against sorafenib. However, the influence of HBV on resistance to regorafenib, another therapeutic agent, has been less studied. In this study, we used the GEO database (GSE87630) and clinical samples to demonstrate that C-C motif chemokine receptor 9 (CCR9) was highly expressed in HBV-related HCC and predicted poor overall survival. Its overexpression correlated with HBsAg-positive HCC patients. Both univariate and multivariable Cox regression analysis elucidated CCR9 was an independent risk factor for poor overall survival in HCC patients. Our in vitro findings further revealed that HBV structural proteins, small HBV surface antigen (SHBs), triggered an upregulation of CCR9. Functional assays showed that SHBs enhanced HCC cell proliferation, migration, and invasion, increased ABCB1 and ABCC1 expression, and promoted regorafenib resistance via CCR9. Intriguingly, overexpression of HBV plasmid and an AAV-HBV mouse model both exhibited a significant elevation in global N6-methyladenosine (m6A) levels. Further investigations revealed that SHBs elevated these m6A levels, upregulated CCR9 and stabilized CCR9 mRNA through KIAA1429-mediated m6A modification, with sites 1373 and 1496 on CCR9 mRNA being critical for modification. In conclusion, SHBs promoted HCC progression and regorafenib resistance via KIAA1429-mediated m6A modification of CCR9. Our findings suggested that CCR9 could be a potential prognostic biomarker and a valuable molecular therapeutic target of regorafenib resistance in HBV-related HCC., (© 2024 Wiley Periodicals LLC.)

Introduction: Restoring immune tolerance is a promising area of therapy for autoimmune diseases. One method that helps restore immunological tolerance is the approach using tolerogenic dendritic cells (tolDCs). In our study, we analyzed the effectiveness of using dendritic cells transfected with DNA constructs encoding IL-10, type II collagen, and CCR9 to induce immune tolerance in an experimental model of arthritis., Methods: Dendritic cell cultures were obtained from bone marrow cells of Balb/c mice. Dendritic cells (DCs) cultures were transfected with pmaxCCR9, pmaxIL-10, and pmaxCollagen type II by electroporation. The phenotype and functions of DCs were studied using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Migration of electroporated DCs was assessed in vitro . Induction of antigen-collagen induced arthritis (ACIA) was carried out according to the protocol in Balb/c mice. DCs were then administered to ACIA mice. The development of arthritis was monitored by measuring paw swelling with a caliper at different time points. The immunological changes were assessed by analyzing the content of antibodies to type II collagen using enzyme immunoassay. Additionally, a histological examination of the joint tissue was conducted, followed by data analysis., The Results Are as Follows: DCs were obtained, characterized by reduced expression of CD80, CD86, and H-2Db (MHC class I), increased expression of CCR9, as well as producing IL-10 and having migratory activity to thymus cells. Transfected DCs induced T-regulatory cells (T-reg) and increased the intracellular content of IL-10 and TGF-β in CD4 + T cells in their co-culture, and also suppressed their proliferative activity in response to antigen. The administration of tolDCs transfected with DNA constructs encoding type II collagen, IL-10, and CCR9 to mice with ACIA demonstrated a reduction in paw swelling, a reduction in the level of antibodies to type II collagen, and a regression of histological changes., Conclusion: The study presents an approach by which DCs transfected with DNA constructs encoding epitopes of type II collagen, IL-10 and CCR9 promote the development of antigen-specific tolerance, control inflammation and reduce the severity of experimental arthritis through the studied mechanisms: induction of T-reg, IL-10, TGF-β., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fisher, Kurilin, Bulygin, Shevchenko, Philippova, Taranov, Ivleva, Maksyutov and Sennikov.)

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance., (©2023 American Association for Cancer Research.)

Our previous studies demonstrated that CCR9 plays an important role in several aspects of T-cell acute lymphoblastic leukemia progression and that CCR9 is a potential therapeutic target. However, the underlying mechanism that regulates CCR9 expression remains incompletely understood. In this study, bioinformatics analysis and validation in clinical samples revealed the lncRNA15691 to be positively correlated with CCR9 mRNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9high T-cell acute lymphoblastic leukemia cell lines. LncRNA15691, a previously uncharacterized lncRNA, was found to be located in both the cytoplasm and the nucleus via fluorescence in situ hybridization assay. In addition, lncRNA15691 upregulated the expression of CCR9 and was involved in T-cell acute lymphoblastic leukemia cell invasion. In vivo experiments showed that lncRNA15691 promoted leukemia cell homing/infiltration into the bone marrow, blood, and spleen, whereas the CCR9 ligand, CCL25, augmented the extramedullary infiltration of CCR9low leukemia cells overexpressing lncRNA15691 into blood, spleen, and liver. Subsequently, RNA protein pull-down assays, coupled with liquid chromatography-tandem mass spectrometry, were used to uncover potential lncRNA15691-interacting proteins, which were then validated by RNA immunoprecipitation. These mechanistic studies revealed that lncRNA15691 upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA. Collectively, our study revealed a novel mechanism of regulating CCR9 expression and implicated lncRNA15691 as a potential novel biomarker for T-cell acute lymphoblastic leukemia infiltration., Competing Interests: Conflict of interest The authors declared that they have no conflicts of interest to this work., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chemokine (C-C motif) receptor-like 2 (CCRL2), is a seven transmembrane receptor closely related to the chemokine receptors CCR1, CCR2, CCR3, and CCR5. Nevertheless, CCRL2 is unable to activate conventional G-protein dependent signaling and to induce cell directional migration. The only commonly accepted CCRL2 ligand is the nonchemokine chemotactic protein chemerin (RARRES2). The chemerin binding to CCLR2 does induce leukocyte chemotaxis, yet, genetic targeting of CCRL2 was shown to modulate the inflammatory response in different experimental models. This mechanism was shown to be crucial for lung dendritic cell migration, neutrophil recruitment, and Natural Killer cell-dependent immune surveillance in lung cancer. To gain more insight in the interactions involved in the CCRL2-chemerin, the binding complexes were generated by protein-protein docking, then submitted to accelerated molecular dynamics. The obtained trajectories were inspected by principal component analyses followed by kernel density estimation to identify the ligand-receptor regions most frequently involved in the binding. To conclude, the reported analyses led to the identification of the putative hot-spot residues involved in CCRL2-chemerin binding., (© 2022 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Background: CC chemokine receptor 9 (CCR9), an organ-specific chemokine receptor, interacts with its exclusive ligand CCL25 to promote tumor proliferation and metastasis. However, the effect of CCR9 on salivary adenoid cystic carcinoma (SACC) malignant behavior remains unknown. This study aimed to investigate the specific molecular mechanism by which CCR9/CCL25 modulates malignant progression in SACC., Methods: Immunohistochemistry staining and RT-qPCR analyses were performed to detect the correlation of CCR9 expression and tumor progression-associated markers in SACC. In vitro , SACC cell proliferation and apoptosis were evaluated using Cell Counting Kit-8 and colon formation, and cell migration and invasion were detected by wound healing and transwell assays. Vercirnon was used as an inhibitor of CCR9, and LY294002 was used as an inhibitor of the PI3K/AKT pathway in this study. Western blot and RT-qPCR assays were carried out to measure the downstream factors of the interaction of CCL25 and CCR9. The effect of CCL25 on the development of SACC in vivo was examined by a xenograft tumor model in nude mice following CCL25, Vercirnon and LY294002 treatment., Results: CCR9 was highly expressed in SACC compared with adjacent salivary gland tissues, and its level was associated with tumor proliferation and metastases. CCL25 enhanced cell proliferation, migration, and invasion through its interaction with CCR9 and exerted an antiapoptotic effect on SACC cells. Targeting CCR9 via Vercirnon significantly reduced the phosphorylation level of AKT induced by CCL25. CCL25/CCR9 could activate its downstream factors through the PI3K/AKT signaling pathway, such as cyclin D1, BCL2 and SLUG, thus promoting SACC cell proliferation, antiapoptosis, invasion and metastasis. The in vivo data from the xenograft mouse models further proved that CCL25 administration promoted malignant tumor progression by activating the PI3K/AKT pathway., Conclusion: The interaction of CCL25 and CCR9 promotes tumor growth and metastasis in SACC by activating the PI3K/AKT signaling pathway, offering a promising strategy for SACC treatment., Competing Interests: The authors declare that they have no competing interests., (© 2022 Chai et al.)

The chemokine receptor CCR9 equips T cells with the ability to respond to CCL25, a chemokine that is highly expressed in the thymus and the small intestine (SI). Notably, CCR9 is mostly expressed on CD8 but not on CD4 lineage T cells, thus imposing distinct tissue tropism on CD4 and CD8 T cells. The molecular basis and the consequences for such a dichotomy, however, have not been fully examined and explained. Here, we demonstrate that the forced expression of CCR9 interferes with the tissue trafficking and differentiation of CD4 T cells in SI intraepithelial tissues. While CCR9 overexpression did not alter CD4 T cell generation in the thymus, the forced expression of CCR9 was detrimental for the proper tissue distribution of CD4 T cells in the periphery, and strikingly also for their terminal differentiation in the gut epithelium. Specifically, the differentiation of SI epithelial CD4 T cells into immunoregulatory CD4 + CD8αα + T cells was impaired by overexpression of CCR9 and conversely increased by the genetic deletion of CCR9. Collectively, our results reveal a previously unappreciated role for CCR9 in the tissue homeostasis and effector function of CD4 T cells in the gut., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Altering T cell trafficking to mucosal regions can enhance immune responses towards pathogenic infections and cancers at these sites, leading to better outcomes. All- trans -retinoic acid (ATRA) promotes T cell migration to mucosal surfaces by inducing transcription of the mucosal-homing receptors CCR9 and α4β7 via binding to retinoic acid receptors (RARs), which heterodimerize with retinoid X receptors (RXRs) to function. However, the unstable nature and toxicity of ATRA limit its use as a widespread treatment modality for mucosal diseases. Therefore, identifying alternatives that could reduce or eliminate the use of ATRA are needed. Rexinoids are synthetically derived compounds structurally similar to ATRA. Originally named for their ability to bind RXRs, rexinoids can enhance RAR-mediated gene transcription. Furthermore, rexinoids are more stable than ATRA and possess an improved safety profile, making them attractive candidates for use in clinical settings. Here we show that select novel rexinoids act as ATRA mimics, as they cause increased CCR9 and α4β7 expression and enhanced migration to the CCR9 ligand, CCL25 in vitro , even in the absence of ATRA. Conversely, other rexinoids act synergistically with ATRA, as culturing cells with suboptimal doses of both compounds resulted in CCR9 expression and migration to CCL25. Overall, our findings show that rexinoids can be used independently or synergistically with ATRA to promote mucosal homing of T cells in vitro , and lends support for the prospective clinical use of these compounds in immunotherapeutic approaches for pathogenic infections or cancers at mucosal surfaces., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Manhas, Marshall, Wagner, Jurutka, Mancenido, Debray and Blattman.)

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in T H 2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations., (© 2021. The Author(s).)

Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, eleven CC chemokine receptors (CCRs) and seven CXC chemokine receptors (CXCRs) were identified from turbot genome. Phylogenetic and syntenic analyses were performed to annotate these genes, indicating the closest relationship between the turbot chemokine receptors and their counterparts of Japanese flounders (Paralichthys olivaceus). Evolutionary analyses revealed that the tandem duplications of CCR8 and CXCR3, the whole genome duplications of CCR6, CCR9, CCR12, and CXCR4, and the teleost-specific CCR12 led to the expansion of turbot chemokine receptors. In addition, turbot chemokine receptors were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in spleen, gill, and head kidney. Moreover, most turbot chemokine receptors were significantly differentially expressed in spleen and gill after Aeromonas salmonicida infection, and exhibited general down-regulations at early time points and then gradually up-regulated. Finally, protein-protein interaction network (PPI) analyses indicated that chemokine receptors interacted with a few immune-related genes such as interleukins, Grk genes, CD genes, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokine receptors in turbots., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vibrio cholerae is a bacterial pathogen which causes the severe acute diarrheal disease cholera. Given that a symptomatic incident of cholera can lead to long term protection, a thorough understanding of the immune response to this pathogen is needed to identify parameters critical to the generation and durability of immunity. To approach this, we utilized a live attenuated cholera vaccine to model the response to V. cholerae infection in 12 naïve subjects. We found that this live attenuated vaccine induced durable vibriocidal antibody titers that were maintained at least one year after vaccination. Similar to what we previously reported in infected patients from Bangladesh, we found that vaccination induced plasmablast responses were primarily specific to the two immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). Interestingly, the magnitude of the early plasmablast response at day 7 predicted the serological outcome of vaccination at day 30. However, this correlation was no longer present at later timepoints. The acute responses displayed preferential immunoglobulin isotype usage, with LPS specific cells being largely IgM or IgA producing, while cholera toxin responses were predominantly IgG. Finally, CCR9 was highly expressed on vaccine induced plasmablasts, especially on IgM and IgA producing cells, suggesting a role in migration to the gastrointestinal tract. Collectively, these findings demonstrate that the use of a live attenuated cholera vaccine is an effective tool to examine the primary and long-term immune response following V. cholerae exposure. Additionally, it provides insight into the phenotype and specificity of the cells which likely return to and mediate immunity at the intestinal mucosa. A thorough understanding of these properties both in peripheral blood and in the intestinal mucosae will inform future vaccine development against both cholera and other mucosal pathogens. Trial Registration: NCT03251495., Competing Interests: The authors have declared that no competing interests exist.

The coronavirus disease 2019 (COVID-19) is an infectious disease that mainly affects the host respiratory system with ~ 80% asymptomatic or mild cases and ~ 5% severe cases. Recent genome-wide association studies (GWAS) have identified several genetic loci associated with the severe COVID-19 symptoms. Delineating the genetic variants and genes is important for better understanding its biological mechanisms. We implemented integrative approaches, including transcriptome-wide association studies (TWAS), colocalization analysis, and functional element prediction analysis, to interpret the genetic risks using two independent GWAS datasets in lung and immune cells. To understand the context-specific molecular alteration, we further performed deep learning-based single-cell transcriptomic analyses on a bronchoalveolar lavage fluid (BALF) dataset from moderate and severe COVID-19 patients. We discovered and replicated the genetically regulated expression of CXCR6 and CCR9 genes. These two genes have a protective effect on lung, and a risk effect on whole blood, respectively. The colocalization analysis of GWAS and cis-expression quantitative trait loci highlighted the regulatory effect on CXCR6 expression in lung and immune cells. In the lung-resident memory CD8 + T (T RM ) cells, we found a 2.24-fold decrease of cell proportion among CD8 + T cells and lower expression of CXCR6 in the severe patients than moderate patients. Pro-inflammatory transcriptional programs were highlighted in the T RM cellular trajectory from moderate to severe patients. CXCR6 from the 3p21.31 locus is associated with severe COVID-19. CXCR6 tends to have a lower expression in lung T RM cells of severe patients, which aligns with the protective effect of CXCR6 from TWAS analysis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: HIV infection promotes the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, tissue fibrosis and disease progression. Early antiretroviral treatment (ART) upon HIV infection improves CD4 count and decreases immune activation. However, Treg dynamics and their epigenetic regulation following early ART initiation remain understudied., Methods: Treg subsets were characterized by flow cytometry in 103 individuals, including untreated HIV-infected participants in acute and chronic phases, ART-treated in early infection, elite controllers (ECs), immunological controllers (ICs), and HIV-uninfected controls. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq technology., Findings: Total Treg frequency increased overtime during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CD39, and LAP(TGF-β1)), which remained unchanged following early ART. Expression of gut migration markers (CCR9, Integrin-β7) by Tregs was elevated during untreated HIV infection, while they declined with the duration of ART but not upon early ART initiation. Notably, gut-homing Tregs expressing LAP(TGF-β1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-β1) + Tregs overtime were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene. Remarkably, LAP(TGF-β1)-expressing Tregs in ECs were significantly higher than in uninfected subjects, while the markers of Treg activation and gut migration were not different., Interpretation: Early ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and HIV disease progression., Funding: This study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and in part by the AIDS and Infectious Diseases Network of the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S)., Competing Interests: Declaration of Competing Interest We have no competing of interest to declare., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Breast cancer is the most severe malignant tumor in women. Chemokines and their receptors appear to be implicated in tumorigenesis and metastatic pattern. Also the scavenger atypical chemokine receptors are emerging as crucial regulators for the availability of chemokines. Therefore the aim of the present study is to evaluate the expression of CCR7, ACKR4 and their ligand; CCL21 in human breast cancer., Main Methods: In this study, RT-PCR was done to detect the expression of CCR7 and ACKR4 in 50 non-metastatic and 30 metastatic breast cancer tissue. Also CCL21 level in the serum of study group was detected by ELISA. The expression of all markers is compared to 80 control healthy individual., Key Findings: Our results revealed the increase in expression of CCR7 and CCL21 level in metastatic group compared to non-metastatic and control groups while ACKR4 expression is significantly increased in breast tissues of non-metastatic patients compared to both control and metastatic groups. Also there was significant positive correlation between CCR7 expression and CCL21 level in cancer patients and significant negative correlation between ACKR4 and both CCR-7 and CCL21 in both non-metastatic and metastatic cancer groups., Significance: Thus, it might be elucidating that ACKR4 and CCR7 could be a novel target for tumor therapy as targeting the chemokine-receptors axis might represent a powerful tool to prevent infiltration and metastasis and consequently improve cancer prognosis and treatment., (Copyright © 2021. Published by Elsevier Inc.)

Introduction: Most of the typical chemokine receptors (CKRs) have been identified as coreceptors for a variety of human and simian immunodeficiency viruses (HIVs and SIVs). This study evaluated CCRL2 to examine if it was an HIV/SIV coreceptor., Methods: The Human glioma cell line, NP-2, is normally resistant to infection by HIV and SIV. The cell was transduced with amplified cluster of differentiation 4 (CD4) as a receptor and CCR5, CXCR4 and CCRL2 as coreceptor candidates to produce NP-2/CD4/coreceptor cells (). The cells were infected with multiplicity of infection (MOI) 1.0. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa staining. Proviral DNA was detected by polymerase chain reaction (PCR), and reverse transcriptase (RT) activity was measured., Results: IFA detected viral antigens of the primary isolates, HIV-1HAN2 and HIV-2MIR in infected NP-2/CD4/CCRL2 cells, indicated CCRL2 as a functional coreceptor. IFA results were confirmed by the detection of proviral DNA and measurement of RT-activity in the spent cell supernatants. Additionally, MGC was detected in HIV-2MIR-infected NP-2/CD4/CCCRL2 cells. HIV-2MIR were found more potent users of CCRL2 than HIV-1HAN2. Moreover, GWAS studies, gene ontology and cell signaling pathways of the HIV-associated genes show interaction of CCRL2 with HIV/SIV envelope protein., Conclusions: In vitro experiments showed CCRL2 to function as a newly identified coreceptor for primary HIV-2 isolates conveniently. The findings contribute additional insights into HIV/SIV transmission and pathogenesis. However, its in vivo relevance still needs to be evaluated. Confirming in vivo relevance, ligands of CCRL2 can be investigated as potential targets for HIV entry-inhibitor drugs., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Macrophages are the key regulator of T-cell responses depending on their activation state. C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical receptor originally cloned from LPS-activated macrophages, has recently been shown to regulate immune responses under several inflammatory conditions. However, whether CCRL2 influences macrophage function and regulates tumor immunity remains unknown. Here, we found that tumoral CCRL2 expression is a predictive indicator of robust antitumor T-cell responses in human cancers. CCRL2 is selectively expressed in tumor-associated macrophages (TAM) with immunostimulatory phenotype in humans and mice. Conditioned media from tumor cells could induce CCRL2 expression in macrophages primarily via TLR4, which is negated by immunosuppressive factors. Ccrl2 -/- mice exhibit accelerated melanoma growth and impaired antitumor immunity characterized by significant reductions in immunostimulatory macrophages and T-cell responses in tumor. Depletion of CD8 + T cells or macrophages eliminates the difference in tumor growth between WT and Ccrl2 -/- mice. Moreover, CCRL2 deficiency impairs immunogenic activation of macrophages, resulting in attenuated antitumor T-cell responses and aggravated tumor growth in a coinjection tumor model. Mechanically, CCRL2 interacts with TLR4 on the cell surface to retain membrane TLR4 expression and further enhance its downstream Myd88-NF-κB inflammatory signaling in macrophages. Similarly, Tlr4 -/- mice exhibit reduced CCRL2 expression in TAM and accelerated melanoma growth. Collectively, our study reveals a functional role of CCRL2 in activating immunostimulatory macrophages, thereby potentiating antitumor T-cell response and tumor rejection, and suggests CCLR2 as a potential biomarker candidate and therapeutic target for cancer immunotherapy., Competing Interests: The authors declare no competing interest.

Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism. To systematically characterize these effects, we analysed G protein- and β-arrestin-mediated signal transduction resulting from stimulation of these receptors by each of their cognate chemokine ligands within the same cellular background. Although the chemokines did not elicit ligand-biased agonism, the three receptors exhibited different arrays of signaling outcomes. Stimulation of CCR4 by either CC chemokine ligand 17 (CCL17) or CCL22 induced β-arrestin recruitment but not G protein-mediated signaling, suggesting that CCR4 has the potential to act as a scavenger receptor. At CCR7, both CCL19 and CCL21 stimulated G protein signaling and β-arrestin recruitment, with CCL19 consistently displaying higher potency. At CCR10, CCL27 and CCL28(4-108) stimulated both G protein signaling and β-arrestin recruitment, whereas CCL28(1-108) was inactive, suggesting that CCL28(4-108) is the biologically relevant form of this chemokine. These comparisons emphasize the intrinsic abilities of different receptors to couple with different downstream signaling pathways. Comparison of these results with previous studies indicates that differential agonism at these receptors may be highly dependent on the cellular context.

Fibrotic scarring is tightly linked to the development of heart failure in patients with post-myocardial infarction (MI). Atypical chemokine receptor 4 (ACKR4) can eliminate chemokines, such as C-C chemokine ligand 21 (CCL21), which is independently associated with heart failure mortality. However, the role of ACKR4 in the heart during MI is unrevealed. This study aimed to determine whether ACKR4 modulates cardiac remodeling following MI and to illuminate the potential molecular mechanisms. The expression of ACKR4 was upregulated in the border/infarct area, and ACKR4 was predominantly expressed in cardiac fibroblasts (CFs). Knockout of ACKR4 protected against adverse ventricular remodeling in mice post-MI. These protective effects of ACKR4 deficiency were independent of dendritic cell immune response but could be attributed to downregulated CF-derived IL-6, affecting CF proliferation and endothelial cell (EC) functions, which consequently inhibited cardiac fibrosis. ACKR4 promoted IL-6 generation and proliferation of CFs. Besides, ACKR4 induced endothelial-to-mesenchymal transition (EndMT) in ECs through IL-6 paracrine effect. The p38 MAPK/NF-κB signaling pathway was involved in ACKR4 facilitated IL-6 generation. Moreover, ACKR4 overexpression in vivo via AAV9 carrying a periostin promoter aggravated heart functional impairment post-MI, which was abolished by IL-6 neutralizing antibody. Therefore, our study established a novel link between ACKR4 and IL-6 post-MI, indicating that ACKR4 may be a novel therapeutic target to ameliorate cardiac remodeling., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sex differences in human placenta exist from early pregnancy to term, however, it is unclear whether these differences are driven solely by sex chromosome complement or are subject to differential sex hormonal regulation. Here, we survey the human chorionic villus (CV) transcriptome for sex-linked signatures from 11 to 16 gestational weeks, corresponding to the first window of increasing testis-derived androgen production in male fetuses. Illumina HiSeq RNA sequencing was performed on Lexogen Quantseq 3' libraries derived from CV biopsies (n = 11 females, n = 12 males). Differential expression (DE) was performed to identify sex-linked transcriptional signatures, followed by chromosome mapping, pathway analysis, predicted protein interaction, and post-hoc linear regressions to identify transcripts that trend over time. We observe 322 transcripts DE between male and female CV from 11 to 16 weeks, with 22 transcripts logFC > 1. Contrary to our predictions, the difference between male and female expression of DE autosomal genes was more pronounced at the earlier gestational ages. In females, we found selective upregulation of extracellular matrix components, along with a number of X-linked genes. In males, DE transcripts centered on chromosome 19, with mitochondrial, immune, and pregnancy maintenance-related transcripts upregulated. Among the highest differentially expressed autosomal genes were CCRL2, LGALS13, and LGALS14, which are known to regulate immune cell interactions. Our results provide insight into sex-linked gene expression in late first and early second trimester developing human placenta and lay the groundwork to understand the mechanistic origins of sex differences in prenatal development.

Aims/hypothesis: Accumulation of adipose tissue macrophages is considered pivotal in the development of obesity-associated inflammation and insulin resistance. In addition, recent studies suggest an involvement of the intestine as the primary organ in inducing hyperglycaemia and insulin resistance. We have reported that the C-C motif chemokine receptor (CCR) CCR9 is associated with intestinal immunity and has a pathogenic role in various liver diseases. However, its contribution to type 2 diabetes is unknown. In the current study, we aimed to clarify the involvement of CCR9 in the pathology of type 2 diabetes and the potential underlying mechanisms., Methods: To elucidate how CCR9 affects the development of metabolic phenotypes, we examined the impact of CCR9 deficiency on the pathogenesis of type 2 diabetes using male C57BL/6J (wild-type [WT]) and CCR9-deficient (CCR9 knockout [KO]) mice fed a 60% high-fat diet (HFD) for 12 weeks., Results: WT and Ccr9KO mice fed an HFD exhibited a comparable weight gain; however, glucose tolerance and insulin resistance were significantly improved in Ccr9KO mice. Moreover, visceral adipose tissue (VAT) and the liver of Ccr9KO mice presented with less inflammation and increased expression of glucose metabolism-related genes than WT mice. Ccr9 and Ccl25 expression were specifically higher in the small intestine but was not altered by HFD feeding and type 2 diabetes development. Accumulation of IFN-γ-producing CD4 + T lymphocytes and increased intestinal permeability in the small intestine was observed in WT mice following HFD feeding, but these changes were suppressed in HFD-fed Ccr9KO mice. Adoptive transfer of gut-tropic CCR9-expressing T lymphocytes partially reversed the favourable glucose tolerance found in Ccr9KO mice via exacerbated inflammation in the small intestine and VAT., Conclusions/interpretation: CCR9 plays a central role in the pathogenesis of type 2 diabetes by inducing an inflammatory shift in the small intestine. Our findings support CCR9 as a new therapeutic target for type 2 diabetes via the gut-VAT-liver axis.

Background & Aims: The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH., Methods: Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9 -/- mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH., Results: Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9 -/- mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9 + CD11b + inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9 -/- mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine., Conclusions: These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH., Lay Summary: Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Abnormal crosstalk between gut immune and the liver was involved in nonalcoholic steatohepatitis (NASH). Mice with methionine choline-deficient (MCD) diet-induced NASH presented an imbalance of pro-(IL-6 and IFN-γ) and anti-inflammatory cytokines (IL-10) in the intestine. We also clarified that the ratio of CD4 + T cells and found that the NASH mesenteric lymph node (MLN) presents decreased numbers of CD4 + Th17 cells but increased numbers of CD4 + CD8 + FoxP3 + regulatory T cells (Tregs). Furthermore, the intestinal immune imbalance in NASH was attributed to impaired gut chemokine receptor 9 (CCR9)/chemokine ligand 25 (CCL25) signalling, which is a crucial pathway for immune cell homing in the gut. We also demonstrated that CD4 + CCR9 + T cell homing was dependent on CCL25 and that the numbers and migration abilities of CD4 + CCR9 + T cells were reduced in NASH. Interestingly, the analysis of dendritic cell (DC) subsets showed that the numbers and retinal dehydrogenase (RALDH) activity of CD103 + CD11b + DCs were decreased and that the ability of these cells to upregulate CD4 + T cell CCR9 expression was damaged in NASH. Taken together, impaired intestinal CCR9/CCL25 signalling induced by CD103 + CD11b + DC dysfunction contributes to the gut immune imbalance observed in NASH., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Facilitating resolution of inflammation using atypical chemokine receptors (ACKR) as an anticancer strategy is considered but requires a deeper understanding of receptor role in carcinogenesis. We aimed at transcriptional analysis (RTqPCR) of ACKR2 and ACKR4 expression in colorectal adenoma-adenocarcinoma sequence in paired normal-neoplastic tissues from 96 polyps and 51 cancers. On average, ACKR2 was downregulated in neoplastic as compared to non-affected tissue in polyp (by 2.7-fold) and cancer (by 3.1-fold) patients. The maximal downregulation (by 8.2-fold) was observed in adenomas with the highest potential for malignancy and was gradually lessening through cancer stages I-IV, owing to increased receptor expression in tumors. On average, ACKR4 was significantly downregulated solely in adenocarcinomas (by 1.5-fold), less so in patients with lymph node metastasis, owing to a gradual decrease in ACKR4 expression among N0-N1-N2 cancers in non-affected tissue without changes in tumors. In adenomas, ACKR4 downregulation in neoplastic tissue increased with increasing potential for malignancy and contribution of villous growth pattern. ACKR4 expression increased in non-affected tissue with a concomitant decrease in pathological mucosa. In conclusion, the changes in ACKRs expression occur already in precancerous colorectal lesions, culminating in the adenomas with the highest potential for malignancy. Therefore, chemoprevention by manipulating ACKRs' expression is worth exploration.

GWAS analysis of severe Covid patients implicates a major locus on chromosome 3. The corresponding 50 kb segment appears to originate from Neanderthal/Sapiens crossings, raising interesting evolutionary questions., (© 2020 médecine/sciences – Inserm.)

Background: Vitamin A (VA) stores are low in early infancy and may impair development of the immune system., Objective: This study determined if neonatal VA supplementation (VAS) affects the following: 1) development of regulatory T (Treg) cells; 2) chemokine receptor 9 (CCR9) expression, which directs mucosal targeting of immune cells; and 3) systemic endotoxin exposure as indicated by changed plasma concentrations of soluble CD14 (sCD14). Secondarily, VA status, growth, and systemic inflammation were investigated., Methods: In total, 306 Bangladeshi infants were randomly assigned to receive 50,000 IU VA or placebo (PL) within 48 h of birth, and immune function was assessed at 6 wk, 15 wk, and 2 y. Primary outcomes included the following: 1) peripheral blood Treg cells; 2) percentage of Treg, T, and B cells expressing CCR9; and 3) plasma sCD14. Secondary outcomes included the following: 4) VA status measured using the modified relative dose-response (MRDR) test and plasma retinol; 5) infant growth; and 6) plasma C-reactive protein (CRP). Statistical analysis identified group differences and interactions with sex and birthweight., Results: VAS increased (P = 0.004) the percentage of CCR9+ Treg cells (13.2 ± 1.37%) relative to PL (9.17 ± 1.15%) in children below the median birthweight but had the opposite effect (P = 0.04) in those with higher birthweight (VA, 9.13 ± 0.89; PL, 12.1 ± 1.31%) at 6 and 15 wk (values are combined mean ± SE). VAS decreased (P = 0.003) plasma sCD14 (1.56 ± 0.025 mg/L) relative to PL (1.67 ± 0.032 mg/L) and decreased (P = 0.034) the prevalence of VA deficiency (2.3%) relative to PL (9.2%) at 2 y., Conclusions: Neonatal VAS enhanced mucosal targeting of Treg cells in low-birthweight infants. The decreased systemic exposure to endotoxin and improved VA status at 2 y may have been due to VA-mediated improvements in gut development resulting in improved barrier function and nutrient absorption. This trial was registered at clinicaltrials.gov as NCT01583972 and NCT02027610., (Published by Oxford University Press on behalf of the American Society for Nutrition 2020.)

The majority of genetically modified C57BL/6 mice contain congenic passenger DNA around the targeted gene locus as they were generated from 129-derived embryonic stem cells (ESCs) with subsequent backcrossing to the C57BL/6 genetic background. When studying the role of atypical chemokine receptor 4 (ACKR4) in the immune system, we realized that the two available Ackr4-deficient mouse strains (Ackr4 -/- and Ackr4 GFP/GFP ) show profoundly different phenotypes: Compared to wild-type and Ackr4 GFP/GFP mice, Ackr4 -/- mice show a strong accumulation of plasma blasts in mesenteric lymph node and spleen as well as increased B cell proliferation after in vitro activation. This phenotype was maintained after further backcrossing to C57BL/6 mice and was even present in heterozygous Ackr4 +/- animals, suggesting that a gene variant on the targeted chromosome might cause this phenotype. Exome sequencing revealed that a region of approximately 20 Mbp around the Ackr4 locus on chromosome 9 still originates from the 129 background based on high variant density observed. In activated Ackr4 -/- and Ackr4 GFP/GFP B cells, transcripts of genes around the Ackr4 locus were equally deregulated compared to C57BL/6 B cells, whereas increased expression of IL-6 was selectively observed in B cells of Ackr4 -/- mice. Because the gene encoding for IL-6 is placed on chromosome 5 these findings suggest that passenger DNA around the Ackr4 locus has an indirect effect on B cell activation and IL-6 production. Results of the present study should not only lead to the reinterpretation of data from earlier studies using Ackr4 -/- mice but should remind the scientific community about the limitations of mouse models using mice created by gene-targeting of nonsyngeneic ESCs., (©2019 Society for Leukocyte Biology.)

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit β-arrestin1 and β-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit β-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4., (© 2019 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Chemokines are essential for guiding cell migration. Atypical chemokine receptors (ACKRs) contribute to the cell migration process by binding, internalizing and degrading local chemokines, which enables the formation of confined gradients. ACKRs are heptahelical membrane spanning molecules structurally related to G-protein coupled receptors (GPCRs), but seem to be unable to signal through G-proteins upon ligand binding. ACKR4 internalizes the chemokines CCL19, CCL21, and CCL25 and is best known for shaping functional CCL21 gradients. Ligand binding to ACKR4 has been shown to recruit β-arrestins that has led to the assumption that chemokine scavenging relies on β-arrestin-mediated ACKR4 trafficking, a common internalization route taken by class A GPCRs. Here, we show that CCL19, CCL21, and CCL25 readily recruited β-arrestin1 and β-arrestin2 to human ACKR4, but found no evidence for β-arrestin-dependent or independent ACKR4-mediated activation of the kinases Erk1/2, Akt, or Src. However, we demonstrate that β-arrestins interacted with ACKR4 in the steady-state and contributed to the spontaneous trafficking of the receptor in the absence of chemokines. Deleting the C-terminus of ACKR4 not only interfered with the interaction of β-arrestins, but also with the uptake of fluorescently labeled cognate chemokines. We identify the GPCR kinase GRK3, and to a lesser extent GRK2, but not GRK4, GRK5, and GRK6, to be recruited to chemokine-stimulated ACKR4. We show that GRK3 recruitment proceded the recruitment of β-arrestins upon ACKR4 engagement and that GRK2/3 inhibition partially interfered with steady-state interaction and chemokine-driven recruitment of β-arrestins to ACKR4. Overexpressing β-arrestin2 accelerated the uptake of fluorescently labeled CCL19, indicating that β-arrestins contribute to the chemokine scavenging activity of ACKR4. By contrast, cells lacking β-arrestins were still capable to take up fluorescently labeled CCL19 demonstrating that β-arrestins are dispensable for chemokine scavenging by ACKR4., (Copyright © 2020 Matti, Salnikov, Artinger, D'Agostino, Kindinger, Uguccioni, Thelen and Legler.)

The chemokines CCL21 and CCL19, through binding of their cognate receptor CCR7, orchestrate lymph node homing of dendritic cells and naïve T cells. CCL21 differs from CCL19 via an unstructured 32 residue C-terminal domain. Previously described roles for the CCL21 C-terminus include GAG-binding, spatial localization to lymphatic vessels, and autoinhibitory modulation of CCR7-mediated chemotaxis. While truncation of the C-terminal tail induced chemical shift changes in the folded chemokine domain, the structural basis for its influence on CCL21 function remains largely unexplored. CCL21 concentration-dependent NMR chemical shifts revealed weak, nonphysiological self-association that mimics the truncation of the C-terminal tail. We generated a series of C-terminal truncation variants to dissect the C-terminus influence on CCL21 structure and receptor activation. Using NMR spectroscopy, we found that CCL21 residues 80-90 mediate contacts with the chemokine domain. In cell-based assays for CCR7 and ACKR4 activation, we also found that residues 92-100 reduced CCL21 potency in calcium flux, cAMP inhibition, and β-arrestin recruitment. Taken together, these structure-function studies support a model wherein intramolecular interactions with specific residues of the flexible C-terminus stabilize a less active monomer conformation of the CCL21. We speculate that the autoinhibitory intramolecular contacts between the C-terminal tail and chemokine body are disrupted by GAG binding and/or interactions with the CCR7 receptor to ensure optimal functionality.

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs., Competing Interests: The authors have declared that no competing interests exist.

Aim : CC chemokine receptor 9 (CCR9) interacts with its exclusive ligand CCL25, resulting in promoting tumor progression and metastasis. However, the effect and mechanisms of CCR9 on lung adenocarcinoma distant metastasis remain largely unknown. To preliminary clarify the underlying mechanisms, we investigate the correlation between CCR9 and ALDH1A1 + cancer stem cells (CSCs), as well as the effect of CCR9 on the migration and invasion of CSCs. Methods : Immunohistochemistry was performed to detect the expression of CCR9 in lung adenocarcinoma tissues. The correlations of CCR9 with distant metastasis and overall survival were investigated. Serial paraffin-embedded tissue blocks were used to detect ALDH1A1 + CSCs expression. The correlations between CCR9 expression and ALDH1A1 + CSCs were evaluated. We further studied the effect of CCR9/CCL25 on the migration and invasion of CSCs using transwell assays. Results : There were positive correlations between CCR9 expression and distant metastasis, as well as poor overall survival. Patients with high CCR9 expression were more likely to develop distant metastasis and demonstrated poorer overall survival than patients with low CCR9 expression. In addition, there was positive correlation between the expression of CCR9 and ALDH1A1 in the same tumor microenvironment. ALDH high CSCs demonstrated enhanced expression of CCR9 than ALDH low cells. Further transwell assays demonstrated that the numbers of CSCs migrated or invaded in response to CCL25 were more than that without CCL25 stimulation. Additional application of anti-CCR9 antibody reversed the CCL25-induced migration and invasion of CSCs. Conclusions : In summary, our study demonstrated that CCR9/CCL25 promoted the migration and invasion of CSCs, which might contribute to distant metastasis and poor overall survival. Our findings provided evidence that CCR9/CCL25 could be used as novel therapeutic targets for lung adenocarcinoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

CCR9 + T cells have an increased potential to be activated and therefore may mediate strong antitumor responses. Here, we found, however, that CCL25, the only chemokine for CCR9 + cells, is not expressed in human or murine triple-negative breast cancers (TNBCs), raising a hypothesis that intratumoral delivery of CCL25 may enhance antitumor immunotherapy in TNBCs. We first determined whether this approach can enhance CD47-targeted immunotherapy using a tumor acidity-responsive nanoparticle delivery system (NP-siCD47/CCL25) to sequentially release CCL25 protein and CD47 small interfering RNA in tumor. NP-siCD47/CCL25 significantly increased infiltration of CCR9 + CD8 + T cells and down-regulated CD47 expression in tumor, resulting in inhibition of tumor growth and metastasis through a T cell-dependent immunity. Furthermore, the antitumor effect of NP-siCD47/CCL25 was synergistically enhanced when used in combination with programmed cell death protein-1/programmed death ligand-1 blockades. This study offers a strategy to enhance immunotherapy by promoting CCR9 + CD8 + T cell tumor infiltration., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

β-carotene is a robust modulator of mucosal barriers, and it can amplify the immunoglobulin A (IgA) response via the retinoic acid (RA)-mediated pathway. We investigated the influence of β-carotene on intestinal barriers in layer-type cockerels. In this study, β-carotene has a positive influence on growth performance and intestinal morphology. β-carotene remarkably enhanced serum secretory immunoglobulin A (sIgA) levels, jejunal mucosal sIgA, and IgA concentrations. β-Carotene significantly enhanced mRNA expression levels of IgA, CC chemokine receptor-9 (CCR9), polymeric immunoglobulin receptor (pIgR), and retinoic acid receptor α (RARα) in the ileal tissues and pIgR in the jejunal tissues. β-Carotene improves mRNA expression of intestinal barrier-related proteins including: mucin-2 (MUC-2), zonula occludens-2 (ZO-2), occludins (OCLN), and zonula occludens-1 (ZO-1) in the ileal tissues. Moreover, β-carotene decreased the levels of Escherichia coli and elevates the levels of Lactobacillus. The results indicate that β-carotene can promote growth performance and contribute to the gradual development of intestinal barriers in Hyline Brown chicks. This study enriches our knowledge about the effects of β-carotene on intestinal barrier and highlights a theoretical basis of β-carotene application in the poultry industry., (© 2020 Japanese Society of Animal Science.)

Obesity impacts over 30% of the United States population, resulting in a wide array of complications. Included among these is the deterioration of the intestinal barrier, which has been implicated in type 2 diabetes and susceptibility to bacterial transepithelial migration. The intestinal epithelium is maintained by αβ and γδ intraepithelial T lymphocytes, which migrate along the epithelia, support epithelial homeostasis, and protect from infection. In this study, we investigate how obesity impacts intraepithelial lymphocyte (IEL) persistence and function in intestinal homeostasis and repair. Mice were fed a high-fat diet to induce obesity and to study immunomodulation in the intestine. There is a striking reduction in αβ and γδ IEL persistence as obesity progresses with a different mechanism in αβ versus γδ IEL populations. CD4 + and CD4 + CD8 + αβ intraepithelial T lymphocytes exhibit reduced homeostatic proliferation in obesity, whereas both αβ and γδ IELs downregulate CD103 and CCR9. The reduction in intraepithelial T lymphocytes occurs within 7 wk of high-fat diet administration and is not dependent on chronic inflammation via TNF-α. Young mice administered a high-fat diet upon weaning exhibit the most dramatic phenotype, showing that childhood obesity has consequences on intestinal IEL seeding. Together, this dysfunction in the intestinal epithelium renders obese mice more susceptible to dextran sulfate sodium-induced colitis. Diet-induced weight loss restores IEL number and CD103/CCR9 expression and improves outcome in colitis. Together, these data confirm that obesity has immunomodulatory consequences in intestinal tissues that can be improved with weight loss., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Chimpanzees, humans' closest relatives, are in danger of extinction. Aside from direct human impacts such as hunting and habitat destruction, a key threat is transmissible disease. As humans continue to encroach upon their habitats, which shrink in size and grow in density, the risk of inter-population and cross-species viral transmission increases, a point dramatically made in the reverse with the global HIV/AIDS pandemic. Inhabiting central Africa, the four subspecies of chimpanzees differ in demographic history and geographical range, and are likely differentially adapted to their particular local environments. To quantitatively explore genetic adaptation, we investigated the genic enrichment for SNPs highly differentiated between chimpanzee subspecies. Previous analyses of such patterns in human populations exhibited limited evidence of adaptation. In contrast, chimpanzees show evidence of recent positive selection, with differences among subspecies. Specifically, we observe strong evidence of recent selection in eastern chimpanzees, with highly differentiated SNPs being uniquely enriched in genic sites in a way that is expected under recent adaptation but not under neutral evolution or background selection. These sites are enriched for genes involved in immune responses to pathogens, and for genes inferred to differentiate the immune response to infection by simian immunodeficiency virus (SIV) in natural vs. non-natural host species. Conversely, central chimpanzees exhibit an enrichment of signatures of positive selection only at cytokine receptors, due to selective sweeps in CCR3, CCR9 and CXCR6 -paralogs of CCR5 and CXCR4, the two major receptors utilized by HIV to enter human cells. Thus, our results suggest that positive selection has contributed to the genetic and phenotypic differentiation of chimpanzee subspecies, and that viruses likely play a predominate role in this differentiation, with SIV being a likely selective agent. Interestingly, our results suggest that SIV has elicited distinctive adaptive responses in these two chimpanzee subspecies., Competing Interests: The authors have declared that no competing interests exist.

The present study aimed to determine the clinical significance and potential molecular mechanisms of C‑C motif chemokine receptor (CCR) genes in patients with early‑stage pancreatic ductal adenocarcinoma (PDAC). The transcriptomic, survival and clinical data of 112 patients with early‑stage PDAC who underwent pancreaticoduodenectomy were obtained from The Cancer Genome Atlas. The prognostic values of the CCR genes involved in early‑stage PDAC were evaluated using Kaplan‑Meier analysis and the multivariate Cox proportional risk regression model, and the potential molecular mechanisms were determined using bioinformatics tools. The identified CCRs closely interacted with each other at both the gene and protein levels. High expression levels of CCR5 [adjusted P=0.012; adjusted hazard ration (HR)=0.478, 95% confidence interval (CI)=0.269‑0.852], CCR6 (adjusted P=0.026; adjusted HR=0.527, 95% CI=0.299‑0.927) and CCR9 (adjusted P=0.001; adjusted HR=0.374, 95% CI=0.209‑0.670) were significantly associated with longer overall survival times in patients with early‑stage PDAC. The contribution of CCR5, CCR6 and CCR9 to the outcome of early‑stage PDAC was also demonstrated. Combined survival analysis of CCR5, CCR6 and CCR9 suggested that patients with high expression levels of these CCRs exhibited the most favorable outcomes. A prognostic signature was constructed in terms of the expression level of CC5, CCR6 and CCR9, and time‑dependent receiver operating characteristic curves indicated that this signature was able to effectively predict the outcome of patients with early‑stage PDAC. The potential molecular mechanisms of CCR5, CC6 and CCR9 in PDAC include its intersection of the P53, nuclear factor (NF)‑κB, generic transcription, mitogen‑activated protein kinase and STAT signaling pathways. Collectively, this highlights that CCR5, CCR6 and CCR9 are potential prognostic biomarkers for early‑stage PDAC.

Ikzf1 is a Krüppel-like zinc-finger transcription factor that plays indispensable roles in T and B cell development. Although the function of Ikzf1 has been studied extensively, the molecular mechanism underlying T lymphopoiesis remains incompletely defined during the embryonic stage. Here we report that the genetic ablation of ikzf1 in mutant zebrafish resulted in abrogated embryonic T lymphopoiesis. This was ascribed to impaired thymic migration, proliferation, and differentiation of hematopoietic stem/progenitor cells (HSPCs). Ccr9a and Irf4a, two indispensable factors in T lymphopoiesis, were the direct targets of Ikzf1 and were absent in the ikzf1 mutants. Genetic deletion of either ccr9a or irf4a in the corresponding mutant embryos led to obvious T cell development deficiency, which was mainly caused by disrupted thymic migration of HSPCs. Restoration of ccr9 a in ikzf1 mutants obviously promoted HSPC thymus homing. However, the HSPCs then failed to differentiate into T cells. Additional replenishment of irf4a efficiently induced HSPC proliferation and T cell differentiation. Our findings further demonstrate that Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 and provide new insight into the genetic network of T lymphocyte development., (© 2019 Huang et al.)

CCRL2 is a nonsignaling seven-transmembrane domain receptor. CCRL2 binds chemerin, a protein that promotes chemotaxis of leukocytes, including macrophages and natural killer (NK) cells. In addition, CCRL2 controls the inflammatory response in different pathologic settings, such as hypersensitivity, inflammatory arthritis, and experimental autoimmune encephalitis. Here, we investigated the role of CCRL2 in the regulation of lung cancer-related inflammation. The genetic deletion of Ccrl2 promoted tumor progression in urethane-induced and in Kras G12D/+ / p53 LoxP lung tumor mouse models. Similarly, a Kras -mutant lung tumor displayed enhanced growth in Ccrl2 -deficient mice. This phenotype was associated with a reduced inflammatory infiltrate characterized by the impaired recruitment of several leukocyte populations including NK cells. Bone marrow chimeras showed that CCRL2 expression by the nonhematopoietic cell compartment was responsible for the increased tumor formation observed in Kras -mutant Ccrl2 -deficient mice. In human and mouse lungs, CCRL2 was expressed by a fraction of CD31 + endothelial cells, where it could control NK infiltration. Elevated CCRL2 expression in biopsies from human lung adenocarcinoma positively correlated with clinical outcome. These results provide evidence for a crucial role of CCRL2 in shaping an anti-lung tumor immune response., (©2019 American Association for Cancer Research.)

Chemerin is a multifunctional adipokine with established roles in inflammation, adipogenesis and glucose homeostasis. Increasing evidence suggest an important function of chemerin in cancer. Chemerin's main cellular receptors, chemokine-like receptor 1 (CMKLR1), G-protein coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) are expressed in most normal and tumor tissues. Chemerin's role in cancer is considered controversial, since it is able to exert both anti-tumoral and tumor-promoting effects, which are mediated by different mechanisms like recruiting innate immune defenses or activation of endothelial angiogenesis. For this review article, original research articles on the role of chemerin and its receptors in cancer were considered, which are listed in the PubMed database. Additionally, we included meta-analyses of publicly accessible DNA microarray data to elucidate the association of expression of chemerin and its receptors in tumor tissues with patients' survival.

The mechanism underlying the progression of relapsing-remitting multiple sclerosis to secondary progressive multiple sclerosis (SPMS), characterized by accumulating fixed disability, is yet to be fully understood. Although alterations in the gut microbiota have recently been highlighted in multiple sclerosis pathogenesis, the mechanism linking the altered gut environment with the remote CNS pathology remains unclear. Here, we analyse human CD4+ memory T cells expressing the gut-homing chemokine receptor CCR9 and found a reduced frequency of CCR9+ memory T cells in the peripheral blood of patients with SPMS relative to healthy controls. The reduction in the proportion of CCR9+ cells among CD4+ memory T cells (%CCR9) in SPMS did not correlate with age, disease duration or expanded disability status scale score, although %CCR9 decreased linearly with age in healthy controls. During the clinical relapse of both, relapsing-remitting multiple sclerosis and neuromyelitis optica, a high proportion of cells expressing the lymphocyte activating 3 gene (LAG3) was detected among CCR9+ memory T cells isolated from the CSF, similar to that observed for mouse regulatory intraepithelial lymphocytes. In healthy individuals, CCR9+ memory T cells expressed higher levels of CCR6, a CNS-homing chemokine receptor, and exhibited a regulatory profile characterized by both the expression of C-MAF and the production of IL-4 and IL-10. However, in CCR9+ memory T cells, the expression of RORγt was specifically upregulated, and the production of IL-17A and IFNγ was high in patients with SPMS, indicating a loss of regulatory function. The evaluation of other cytokines supported the finding that CCR9+ memory T cells acquire a more inflammatory profile in SPMS, reporting similar aspects to CCR9+ memory T cells of the elderly healthy controls. CCR9+ memory T cell frequency decreased in germ-free mice, whereas antibiotic treatment increased their number in specific pathogen-free conditions. Here, we also demonstrate that CCR9+ memory T cells preferentially infiltrate into the inflamed CNS resulting from the initial phase and that they express LAG3 in the late phase in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. Antibiotic treatment reduced experimental autoimmune encephalomyelitis symptoms and was accompanied by an increase in CCR9+ memory T cells in the peripheral blood. Antibodies against mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), which is capable of blocking cell migration to the gut, also ameliorated experimental autoimmune encephalomyelitis. Overall, we postulate that the alterations in CCR9+ memory T cells observed, caused by either the gut microbiota changes or ageing, may lead to the development of SPMS., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Analyzing phylogenetic relationships using mathematical methods has always been of importance in bioinformatics. Quantitative research may interpret the raw biological data in a precise way. Multiple Sequence Alignment (MSA) is used frequently to analyze biological evolutions, but is very time-consuming. When the scale of data is large, alignment methods cannot finish calculation in reasonable time. Therefore, we present a new method using moments of cumulative Fourier power spectrum in clustering the DNA sequences. Each sequence is translated into a vector in Euclidean space. Distances between the vectors can reflect the relationships between sequences. The mapping between the spectra and moment vector is one-to-one, which means that no information is lost in the power spectra during the calculation. We cluster and classify several datasets including Influenza A, primates, and human rhinovirus (HRV) datasets to build up the phylogenetic trees. Results show that the new proposed cumulative Fourier power spectrum is much faster and more accurately than MSA and another alignment-free method known as k-mer. The research provides us new insights in the study of phylogeny, evolution, and efficient DNA comparison algorithms for large genomes. The computer programs of the cumulative Fourier power spectrum are available at GitHub (https://github.com/YaulabTsinghua/cumulative-Fourier-power-spectrum)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Atypical chemokine receptors (ACKRs) are expressed by discrete populations of stromal cells at specific anatomical locations where they control leukocyte migration by scavenging or transporting chemokines. ACKR4 is an atypical receptor for CCL19, CCL21, and CCL25. In skin, ACKR4 plays indispensable roles in regulating CCR7-dependent APC migration, and there is a paucity of migratory APCs in the skin-draining lymph nodes of Ackr4 -deficient mice under steady-state and inflammatory conditions. This is caused by loss of ACKR4-mediated CCL19/21 scavenging by keratinocytes and lymphatic endothelial cells. In contrast, we show in this study that Ackr4 deficiency does not affect dendritic cell abundance in the small intestine and mesenteric lymph nodes, at steady state or after R848-induced mobilization. Moreover, Ackr4 expression is largely restricted to mesenchymal cells in the intestine, where it identifies a previously uncharacterized population of fibroblasts residing exclusively in the submucosa. Compared with related Ackr4 - mesenchymal cells, these Ackr4 + fibroblasts have elevated expression of genes encoding endothelial cell regulators and lie in close proximity to submucosal blood and lymphatic vessels. We also provide evidence that Ackr4 + fibroblasts form physical interactions with lymphatic endothelial cells, and engage in molecular interactions with these cells via the VEGFD/VEGFR3 and CCL21/ACKR4 pathways. Thus, intestinal submucosal fibroblasts in mice are a distinct population of intestinal mesenchymal cells that can be identified by their expression of Ackr4 and have transcriptional and anatomical properties that strongly suggest roles in endothelial cell regulation., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration., Competing Interests: DM, JS, ER No competing interests declared, LS Reviewing editor, eLife, (© 2018, Malhotra et al.)

Multipotent hematopoietic progenitors must acquire thymus-homing capacity to initiate T lymphocyte development. Despite its importance, the transcriptional program underlying this process remains elusive. Cbfβ forms transcription factor complexes with Runx proteins, and here we show that Cbfβ2, encoded by an RNA splice variant of the Cbfb gene, is essential for extrathymic differentiation of T cell progenitors. Furthermore, Cbfβ2 endows extrathymic progenitors with thymus-homing capacity by inducing expression of the principal thymus-homing receptor, Ccr9. This occurs via direct binding of Cbfβ2 to cell type-specific enhancers, as is observed in Rorγt induction during differentiation of lymphoid tissue inducer cells by activation of an intronic enhancer. As in mice, an alternative splicing event in zebrafish generates a Cbfβ2-specific mRNA, important for ccr9 expression. Thus, despite phylogenetically and ontogenetically variable sites of origin of T cell progenitors, their robust thymus-homing capacity is ensured by an evolutionarily conserved mechanism emerging from functional diversification of Runx transcription factor complexes by acquisition of a novel splice variant., (© 2018 Tenno et al.)

Objective: To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn's disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody., Methods: In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, β7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing., Results: A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [-87% to -98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for β7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for β7+ effector memory T cells [placebo, 8%; PF-00547659, 84-138%] and β7+ naïve T cells [8%; 13-50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in β7+ T cells., Conclusions: Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes., (Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Neanderthals contributed genetic material to modern humans via multiple admixture events. Initial admixture events presumably occurred in Western Asia shortly after humans migrated out of Africa. Despite being a focal point of admixture, earlier studies indicate lower Neanderthal introgression rates in some Western Asian populations as compared with other Eurasian populations. To better understand the genome-wide and phenotypic impact of Neanderthal introgression in the region, we sequenced whole genomes of nine present-day Europeans, Africans, and the Western Asian Druze at high depth, and analyzed available whole genome data from various other populations, including 16 genomes from present-day Turkey. Our results confirmed previous observations that contemporary Western Asian populations, on an average, have lower levels of Neanderthal-introgressed DNA relative to other Eurasian populations. Modern Western Asians also show comparatively high variability in Neanderthal ancestry, which may be attributed to the complex demographic history of the region. We further replicated the previously described depletion of putatively functional sequences among Neanderthal-introgressed haplotypes. Still, we find dozens of common Neanderthal-introgressed haplotypes in the Turkish sample associated with human phenotypes, including anthropometric and metabolic traits, as well as the immune response. One of these haplotypes is unusually long and harbors variants that affect the expression of members of the CCR gene family and are associated with celiac disease. Overall, our results paint a complex first picture of the genomic impact of Neanderthal introgression in the Western Asian populations., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)