Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions.

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.

Sustained Secretion of CCL21 via an Implantable Cell Reservoir Hydrogel Enhances the Systemic Antitumor Effect of Radiotherapy.

The combination of radiotherapy (RT) and immunotherapy shows promise in improving the clinical treatment of solid tumors; however, it faces challenges of low response rates and systemic toxicity. Herein, an implantable alginate/collagen hydrogel encapsulating C-C motif ligand 21 (CCL21)-expressing dendritic cells (CCL21-DCs@gel) was developed to potentiate the systemic antitumor effects of RT. The hydrogel functioned as a suitable reservoir for in vivo culture and proliferation of CCL21-DCs, thereby enabling sustained CCL21 release. The local CCL21 gradient induced by CCL21-DCs@gel significantly enhanced the efficacy of RT in suppressing primary tumor growth and inhibiting distant metastasis across several mouse models. Furthermore, the combination of RT with CCL21-DCs@gel provided complete prophylactic protection to mice. Mechanistic investigations revealed that CCL21-DCs@gel potentiated RT by promoting tumor lymphangiogenesis and attracting immune cell infiltration into the tumor. Collectively, these results suggest that CCL21-DCs@gel is a promising adjunct to RT for effectively eradicating tumors and preventing tumor recurrence.

The medulla controls effector primed γδT-cell development in the adult mouse thymus.

γδT cells are produced in the thymus throughout life and provide immunity at epithelial-rich sites. Unlike conventional αßT cells, γδT-cell development involves intrathymic acquisition of effector function, with priming for either IL17 or IFN-γ production occurring during embryonic or adult life, respectively. How the thymus controls effector-primed γδT-cell generation in adulthood is poorly understood. Here, we distinguished de novo γδT cells from those undergoing thymus recirculation and/or retention using Rag2GFP mice alongside markers of maturation/effector priming including CD24, CD25, CD73, and IFN-γ, the latter by crossing with IFN-γYFP GREAT mice. We categorize newly developing γδT-cells into an ordered sequence where CD25+ CD73- IFN-γYFP- precursors are followed sequentially by CD25- CD73+ IFN-γYFP- intermediates and CD25- CD73+ IFN-γYFP+ effectors. To determine intrathymic requirements controlling this sequence, we examined γδT-cell development in Relb-/- thymus grafts that lack medullary microenvironments. Interestingly, medulla deficiency did not alter CD25+ γδT-cell precursor generation, but significantly impaired development of effector primed stages. This impact on γδT-cell priming was mirrored in plt/plt mice lacking the medullary chemoattractants CCL19 and CCL21, and also Ccl21a-/- but not Ccl19-/- mice. Collectively, we identify the medulla as an important site for effector priming during adult γδT-cell development and demonstrate a specific role for the medullary epithelial product CCL21 in this process.

Chemokine CCL21 determines immunotherapy response in hepatocellular carcinoma by affecting neutrophil polarization.

BACKGROUND: The efficacy of immune checkpoint inhibitors (ICIs) in hepatocellular carcinoma (HCC) is poor and great heterogeneity among individuals. Chemokines are highly correlated with tumor immune response. Here, we aimed to identify an effective chemokine for predicting the efficacy of immunotherapy in HCC. METHODS: Chemokine C-C motif ligand 21 (CCL21) was screened by transcriptomic analysis in tumor tissues from HCC patients with different responses to ICIs. The least absolute shrinkage and selection operator (LASSO) regression analysis was conducted to construct a predictive nomogram. Neutrophils in vitro and HCC subcutaneous tumor model in vivo were applied to explore the role of CCL21 on the tumor microenvironment (TME) of HCC. RESULTS: Transcriptome analysis showed that CCL21 level was much higher in HCC patients with response to immunotherapy. The predictive nomogram was constructed and validated as a classifier. CCL21 could inhibit N2 neutrophil polarization by suppressing the activation of nuclear factor kappa B (NF-κB) pathway. In addition, CCL21 enhanced the therapeutic efficacy of ICIs. CONCLUSION: CCL21 may serve as a predictive biomarker for immunotherapy response in HCC patients. High levels of CCL21 in TME inhibit immunosuppressive polarization of neutrophils. CCL21 in combination with ICIs may offer a novel therapeutic strategy for HCC.

CCL21 and CLDN11 Are Key Driving Factors of Lymph Node Metastasis in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Understanding the molecular mechanisms of GC metastasis is crucial for improving patient survival outcomes. METHODS: RNA sequencing and analysis were performed on tissue samples from primary and lymph node metastatic lesions of gastric cancer. Differential gene analysis and functional pathway analysis were conducted. Immune infiltrating environment and protein expression levels were evaluated using immunohistochemistry. Cell experiments were conducted to investigate the role of CCL21 in GC metastasis. RESULTS: ACTG2, CNN1, DES, MUC6, and PGC were significantly upregulated in primary tumor cells, while CCL21, MS4A1, CR2, CLDN11, and FDCSP were significantly upregulated in metastatic tumor cells. Functional pathway analysis revealed enrichment in pathways related to immune response. CLDN11 and CCL21 were found to play important roles in promoting gastric cancer metastasis. Cell experiments confirmed the role of CCL21 in promoting GC cell growth and metastasis. CCL21 is highly expressed in GC tissues and binds to CCR7, leading to upregulation of CLDN11. This results in GC-lymph node metastasis and abnormal activation of immune cells (B cells and CD4+ T cells). CONCLUSION: Inhibition of CCL21 and CLDN11 proteins may be a promising strategy for treating GC and preventing lymph node metastasis. These findings provide specific molecular markers for early lymph node metastases of GC, which can aid in developing treatment strategies and predicting patient prognosis.

CCL21-Ser expression in melanoma cells recruits CCR7+ naïve T cells to tumor tissues and promotes tumor growth.

CCL21-Ser, a chemokine encoded by the Ccl21a gene, is constitutively expressed in the thymic epithelial cells and stromal cells of secondary lymphoid organs. It regulates immune cell migration and survival through its receptor CCR7. Herein, using CCL21-Ser-expressing melanoma cells and the Ccl21a-deficient mice, we demonstrated the functional role of cancer cell-derived CCL21-Ser in melanoma growth in vivo. The B16-F10 tumor growth was significantly decreased in Ccl21a-deficient mice compared with that in wild-type mice, indicating that host-derived CCL21-Ser contributes to melanoma proliferation in vivo. In Ccl21a-deficient mice, tumor growth of melanoma cells expressing CCL21-Ser was significantly enhanced, suggesting that CCL21-Ser from melanoma cells promotes tumor growth in the absence of host-derived CCL21-Ser. The increase in tumor growth was associated with an increase in the CCR7+ CD62L+ T cell frequency in the tumor tissue but was inversely correlated with Treg frequency, suggesting that naïve T cells primarily promote tumor growth. Adoptive transfer experiments demonstrated that naïve T cells are preferentially recruited from the blood into tumors with melanoma cell-derived CCL21-Ser expression. These results suggest that CCL21-Ser from melanoma cells promotes the infiltration of CCR7+ naïve T cells into the tumor tissues and creates a tumor microenvironment favorable for melanoma growth.

ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development.

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal-ventral patterning during early embryogenesis in Xenopus.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

iNKT cells with high PLZF expression are recruited into the lung via CCL21-CCR7 signaling to facilitate the development of asthma tolerance in mice.

Establishment of immune tolerance is crucial to protect humans against asthma. Promyelocytic leukemia zinc finger (PLZF) is an emerging suppressor of inflammatory responses. CCL21-CCR7 signaling mediates tolerance development. However, whether PLZF and CCL21-CCR7 are required for the development of asthma tolerance is unknown. Here, we found that Zbtb16 (coding PLZF) and Ccl21 were upregulated in OVA-induced asthma tolerance (OT) lungs by RNA-seq. PLZF physically interacted with GATA3 and its expression was higher in GATA3+ Th2 cells and ILC2s in OT lungs. Zbtb16-knockdown in lymphocytes promoted the differentiation of CD3e+ CD4+ T cells, particularly those producing IL-4 and IL-5. Moreover, iNKT cells with high expression of PLZF were recruited into the lungs via draining lymph nodes during tolerance. Blockade of CCL21-CCR7 signaling in OT mice decreased the PLZF+  cell population, abolished CCR7-induced PLZF+ iNKT recruitment to the lungs, enhanced Th2responses and exacerbated lung pathology. In OT mice, respiratory syncytial virus (RSV) infection impeded PLZF+  cell and CCR7+ PLZF+ iNKT cellrecruitment to the lungs and increased airway resistance. Collectively, these results indicate that PLZF could interact with GATA3 and restrain differentiation of IL-4- and IL-5-producing T cells, iNKT cells with high PLZF expression are recruited to the lungs via CCL21-CCR7 signaling to facilitate the development of asthma tolerance.

Metabolic reprogramming of macrophages instigates CCL21-induced arthritis.

This study was designed to delineate the functional significance of CCL21 in metabolic reprogramming in experimental arthritis and differentiated rheumatoid arthritis (RA) macrophages (MΦs). To characterize the influence of CCL21 on immunometabolism, its mechanism of action was elucidated by dysregulating glucose uptake in preclinical arthritis and RA MΦs. In CCL21 arthritic joints, the glycolytic intermediates hypoxia-inducible factor 1α (HIF1α), cMYC and GLUT1 were overexpressed compared with oxidative regulators estrogen-related receptor γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1)-α. Interestingly, 2-deoxy-D-glucose (2-DG) therapy mitigated CCL21-induced arthritis by restraining the number of joint F4/80+ iNOS+ MΦs without impacting F4/80+ Arginase+ MΦs. Similar to the preclinical findings, blockade of glycolysis negated CCL21-polarized CD14+ CD86+ GLUT+ MΦ frequency; however, CD14+ CD206+ GLUT+ MΦs were not implicated in this process. In CCL21-induced arthritis and differentiated RA MΦs, the inflammatory imprint was uniquely intercepted by 2-DG via interleukin-6 (IL-6) downregulation. Despite the more expansive inflammatory response of CCL21 in the arthritic joints relative to the differentiated RA MΦs, 2-DG was ineffective in joint tumor necrosis factor-α, IL-1ß, CCL2 and CCL5 enrichment. By contrast, disruption of glycolysis markedly impaired CCL21-induced HIF1α and cMYC signaling in arthritic mice. Notably, in RA MΦs, glycolysis interception was directed toward dysregulating CCL21-enhanced HIF1α transcription. Nonetheless, in concurrence with the diminished IL-6 levels, CCL21 differentiation of CD14+ CD86+ GLUT1+ MΦs was reversed by glycolysis and HIIF1α inhibition. Moreover, in the CCL21 experimental arthritis or differentiated RA MΦs, the malfunctioning metabolic machinery was accompanied by impaired oxidative phosphorylation because of reduced PGC1α or peroxisome proliferator-activated receptor-γ expression. CCL21 reconfigures naïve myeloid cells into glycolytic RA CD14+ CD86+ GLUT+ IL-6high HIF1αhigh MΦs. Therefore, inhibiting the CCL21/CCR7 pathway may provide a promising therapeutic strategy.

Drainage of senescent astrocytes from brain via meningeal lymphatic routes.

Recent progress on the central lymphatic system has greatly increased our understanding of how the brain maintains its own waste homeostasis. Here, we showed that perivascular spaces and meningeal lymphatic vessels form a functional route for clearance of senescent astrocytes from the aging brain. Blocking meningeal lymphatic drainage by ligation of the deep cervical lymph nodes impaired clearance of senescent astrocytes from brain parenchyma, subsequently increasing neuroinflammation in aged mice. By contrast, enhancing meningeal lymphatic vessel diameter by a recombinant adeno-associated virus encoding mouse vascular endothelial growth factor-C (VEGF-C) improved clearance of senescent astrocytes and mitigated neuroinflammation. Mechanistically, VEGF-C was highly expressed in senescent astrocytes, contributing themselves to migrate across lymphatic vessels along C-C motif chemokine ligand 21 (CCL21) gradient by interacting with VEGF receptor 3. Moreover, intra-cisternal injection of antibody against CCL21 hampered senescent astrocytes into the lymphatic vessels and exacerbated short memory defects of aged mice. Together, these findings reveal a new perspective for the meningeal lymphatics in the removal of senescent astrocytes, thus offering a valuable target for therapeutic intervention.

Trichinella spiralis nurse cell formation is regulated via CCR7+ dendritic cells.

The chemokine receptor CCR7 is a well-established homing receptor for dendritic cells (DCs) and T-cells. Interaction with the CCL19 and CCL21 ligands promotes priming of immune responses in lymphoid tissues; however, the mechanism underlying CCR7-induced immune responses against helminth parasite infection remains unknown. Thus, we examined the role of CCR7 in generating protective immune responses against intracellular Trichinella spiralis infection. The results showed significantly increased CCR7, CCL19 and CCL21 expression in the muscle tissue compared to that in the intestinal tissue in T. spiralis-infected mice. The CCR7-expressing DC population increased in the mesenteric and peripheral lymph nodes (PLNs) during T. spiralis infection. Notably, the number of CCR7-expressing cells in PLNs increased by more than 30% at 28 days post-infection; however, this increase was significantly inhibited in CCR7-blocked mice treated with CCR7-specific antibodies. T helper 2 (Th2)-and regulatory T (Treg )-related cytokine levels were also reduced by CCR7-specific antibody treatment. CCR7-blocked mice lost their resistance to T. spiralis infection in the muscle phase but not in the intestinal phase. Furthermore, fewer eosinophils around the nurse cells and reduced total and T. spiralis-specific IgE in the serum were observed in CCR7-blocked mice compared to those infected with only T. spiralis. CCR7 blockade led to the T. spiralis infection-induced suppression of Th2- and Treg -related cytokine production in vitro. These results suggest that CCR7 in DCs might play an essential role in host defence mechanisms against T. spiralis infection, particularly in the muscle stage of the infection, by accelerating Th2 and Treg cell responses.

Selective Boosting of CCR7-Acting Chemokines; Short Peptides Boost Chemokines with Short Basic Tails, Longer Peptides Boost Chemokines with Long Basic Tails.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 regulate the lymph node homing of dendritic cells and naïve T-cells and the following induction of a motile DC-T cell priming state. Although CCL19 and CCL21 bind CCR7 with similar affinities, CCL21 is a weak agonist compared to CCL19. Using a chimeric chemokine, CCL19CCL21N-term|C-term, harboring the N-terminus and the C-terminus of CCL21 attached to the core domain of CCL19, we show that these parts of CCL21 act in a synergistic manner to lower ligand potency and determine the way CCL21 engages with CCR7. We have published that a naturally occurring basic C-terminal fragment of CCL21 (C21TP) boosts the signaling of both CCL19 and CCL21. Boosting occurs as a direct consequence of C21TP binding to the CCR7 N-terminus, which seems to free chemokines with basic C-termini from an unfavorable interaction with negatively charged posttranslational modifications in CCR7. Here, we confirm this using a CCL19-variant lacking the basic C-terminus. This variant displays a 22-fold higher potency at CCR7 compared to WT CCL19 and is highly unaffected by the presence of C21TP. WT CCL19 has a short basic C-terminus, CCL21 a longer one. Here, we propose a way to differentially boost CCL19 and CCL21 activity as short and long versions of C21TP boost CCL19 activity, whereas only a long C21TP version can boost chemokines with a full-length CCL21 C-terminus.

Distinct Chemokine Receptor Expression Profiles in De Novo DLBCL, Transformed Follicular Lymphoma, Richter's Trans-Formed DLBCL and Germinal Center B-Cells.

Chemokine receptors and their ligands have been identified as playing an important role in the development of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and Richter syndrome (RS). Our aim was to investigate the different expression profiles in de novo DLBCL, transformed follicular lymphoma (tFL), and RS. Here, we profiled the mRNA expression levels of 18 chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, CX3CR1 and XCR1) using RQ-PCR, as well as immunohistochemistry of seven chemokine receptors (CCR1, CCR4-CCR8 and CXCR2) in RS, de novo DLBCL, and tFL biopsy-derived tissues. Tonsil-derived germinal center B-cells (GC-B) served as non-neoplastic controls. The chemokine receptor expression profiles of de novo DLBCL and tFL substantially differed from those of GC-B, with at least 5-fold higher expression of 15 out of the 18 investigated chemokine receptors (CCR1-CCR9, CXCR1, CXCR2, CXCR6, CXCR7, CX3CR1 and XCR1) in these lymphoma subtypes. Interestingly, the de novo DLBCL and tFL exhibited at least 22-fold higher expression of CCR1, CCR5, CCR8, and CXCR6 compared with RS, whereas no significant difference in chemokine receptor expression profile was detected when comparing de novo DLBCL with tFL. Furthermore, in de novo DLBCL and tFLs, a high expression of CCR7 was associated with a poor overall survival in our study cohort, as well as in an independent patient cohort. Our data indicate that the chemokine receptor expression profile of RS differs substantially from that of de novo DLBCL and tFL. Thus, these multiple dysregulated chemokine receptors could represent novel clinical markers as diagnostic and prognostic tools. Moreover, this study highlights the relevance of chemokine signaling crosstalk in the tumor microenvironment of aggressive lymphomas.

Chemokine C-C motif ligand 21 synergized with programmed death-ligand 1 blockade restrains tumor growth.

Programmed death-ligand 1 (PD-L1) blockade has revolutionized the prognosis of several cancers, but shows a weak effect on pancreatic cancer (PC) due to poor effective immune infiltration. Chemokine C-C motif ligand 21 (CCL21), a chemokine promoting T cell immunity by recruiting and colocalizing dendritic cells (DCs) and T cells, serves as a potential antitumor agent in many cancers. However, its antitumor response and mechanism combined with PD-L1 blockade in PC remain unclear. In our study, we found CCL21 played an important role in leukocyte chemotaxis, inflammatory response, and positive regulation of PI3K-AKT signaling in PC using Metascape and gene set enrichment analysis. The CCL21 level was verified to be positively correlated with infiltration of CD8+ T cells by the CIBERSORT algorithm, but no significant difference in survival was observed in either The Cancer Genome Atlas or the International Cancer Genome Consortium cohort when stratified by CCL21 expression. Additionally, we found the growth rate of allograft tumors was reduced and T cell infiltration was increased, but tumor PD-L1 abundance elevated simultaneously in the CCL21-overexpressed tumors. Then, CCL21 was further verified to increase tumor PD-L1 level through the AKT-glycogen synthase kinase-3ß axis in human PC cells, which partly impaired the antitumor T cell immunity. Finally, the combination of CCL21 and PD-L1 blockade showed superior synergistic tumor suppression in vitro and in vivo. Together, our findings suggested that CCL21 in combination with PD-L1 blockade might be an efficient and promising option for the treatment of PC.

Urinary small extracellular vesicles derived CCL21 mRNA as biomarker linked with pathogenesis for diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. METHODS: Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. RESULTS: The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. CONCLUSIONS: Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

A formulated poly (I:C)/CCL21 as an effective mucosal adjuvant for gamma-irradiated influenza vaccine.

BACKGROUND: Several studies on gamma-irradiated influenza A virus (γ-Flu) have revealed its superior efficacy for inducing homologous and heterologous virus-specific immunity. However, many inactivated vaccines, notably in nasal delivery, require adjuvants to increase the quality and magnitude of vaccine responses. METHODS: To illustrate the impacts of co-administration of the gamma-irradiated H1N1 vaccine with poly (I:C) and recombinant murine CCL21, either alone or in combination with each other, as adjuvants on the vaccine potency, mice were inoculated intranasally 3 times at one-week interval with γ-Flu alone or with any of the three adjuvant combinations and then challenged with a high lethal dose (10 LD50) of A/PR/8/34 (H1N1) influenza virus. Virus-specific humoral, mucosal, and cell-mediated immunity, as well as cytokine profiles in the spleen (IFN-γ, IL-12, and IL-4), and in the lung homogenates (IL-6 and IL-10) were measured by ELISA. The proliferative response of restimulated splenocytes was also determined by MTT assay. RESULTS: The findings showed that the co-delivery of the γ-Flu vaccine and CCL21 or Poly (I:C) significantly increased the vaccine immunogenicity compared to the non-adjuvanted vaccine, associated with more potent protection following challenge infection. However, the mice given a combination of CCL21 with poly (I:C) had strong antibody- and cell-mediated immunity, which were considerably higher than responses of mice receiving the γ-Flu vaccine with each adjuvant separately. This combination also reduced inflammatory mediator levels (notably IL-10) in lung homogenate samples. CONCLUSIONS: The results indicate that adjuvantation with the CCL21 and poly (I:C) can successfully induce vigorous vaccine-mediated protection, suggesting a robust propensity for CCL21 plus poly (I:C) as a potent mucosal adjuvant.

CCL21/CCR7 signaling in macrophages promotes joint inflammation and Th17-mediated osteoclast formation in rheumatoid arthritis.

In rheumatoid arthritis (RA), synovial tissue abundantly expresses CCL21, a chemokine strongly associated with RA susceptibility. In this study, we aimed to characterize the functional significance of CCL21/CCR7 signaling in different phases of RA pathogenesis. We determined that CCR7 is a hallmark of RA M1 synovial fluid (SF) macrophages, and its expression in RA monocytes and in vitro differentiated macrophages is closely associated with disease activity score (DAS28). In early stages of RA, monocytes infiltrate the synovial tissue. However, blockade of SF CCL21 or CCR7 prevents RA SF-mediated monocyte migration. CCR7 expression in the newly migrated macrophages can be accentuated by LPS and IFNγ and suppressed by IL-4 treatment. We also uncovered that CCL21 stimulation increases the number of M1-polarized macrophages (CD14+CD86+), resulting in elevated transcription of IL-6 and IL-23. These CCL21-induced M1 cytokines differentiate naïve T cells to Th17 cells, without affecting Th1 cell polarization. In the erosive stages of disease, CCL21 potentiates RA osteoclastogenesis through M1-driven Th17 polarization. Disruption of this intricate crosstalk, by blocking IL-6, IL-23, or IL-17 function, impairs the osteoclastogenic capacity of CCL21. Consistent with our in vitro findings, we establish that arthritis mediated by CCL21 expands the joint inflammation to bone erosion by connecting the differentiation of M1 macrophages with Th17 cells. Disease progression is further exacerbated by CCL21-induced neovascularization. We conclude that CCL21 is an attractive novel target for RA therapy, as blockade of its function may abrogate erosive arthritis modulated by M1 macrophages and Th17 cell crosstalk.

Fucoxanthin Prevents Pancreatic Tumorigenesis in C57BL/6J Mice That Received Allogenic and Orthotopic Transplants of Cancer Cells.

Fucoxanthin (Fx) is a marine carotenoid with anti-inflammatory and anti-cancer properties in various animal models of carcinogenesis. However, there is currently no information on the effects of Fx in animal models of pancreatic cancer. We investigated the chemopreventive effects of Fx in C57BL/6J mice that received allogenic and orthotopic transplantations of cancer cells (KMPC44) derived from a pancreatic cancer murine model (Ptf1aCre/+; LSL-krasG12D/+). Using microarray, immunofluorescence, western blot, and siRNA analyses, alterations in cancer-related genes and protein expression were evaluated in pancreatic tumors of Fx-administered mice. Fx administration prevented the adenocarcinoma (ADC) development of pancreatic and parietal peritoneum tissues in a pancreatic cancer murine model, but not the incidence of ADC. Gene and protein expressions showed that the suppression of chemokine (C-C motif) ligand 21 (CCL21)/chemokine receptor 7 (CCR7) axis, its downstream of Rho A, B- and T-lymphocyte attenuator (BTLA), N-cadherin, αSMA, pFAK(Tyr397), and pPaxillin(Tyr31) were significantly suppressed in the pancreatic tumors of mice treated with Fx. In addition, Ccr7 knockdown significantly attenuated the growth of KMPC44 cells. These results suggest that Fx is a promising candidate for pancreatic cancer chemoprevention that mediates the suppression of the CCL21/CCR7 axis, BTLA, tumor microenvironment, epithelial mesenchymal transition, and adhesion.

A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions.

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and metastasize to lymphoid organs. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. We established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6Dy649P1 and CCL21-S6Dy649P1 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit ß-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6Dy649P1 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations.