Re-analysis of single cell and spatial transcriptomics data reveals B cell landscape in gastric cancer microenvironment and its potential crosstalk with tumor cells for clinical prognosis.

BACKGROUND: At present, immunotherapy has become a powerful treatment for advanced gastric cancer (AGC), but not all patients can benefit from it. According to the latest research, the impact of B cell subpopulations on the immune microenvironment of gastric cancer (GC) is unknown. Exploring whether the interaction between B cells and tumor cells in GC affects the effectiveness of immunotherapy has attracted our interest. METHODS: This study involved the re-analysis of single-cell RNA (scRNA) and spatial transcriptomics (ST) data from publicly available datasets. The focus was on investigating the subpopulations and differentiation trajectories of B cells in the gastric cancer (GC) tumor immune microenvironment (TIME). Spatial transcriptomics (ST) and multiple immunofluorescence (mIF) revealed a clear co-localization pattern between B cells and tumor cells. Multiple immunotherapy datasets were collected to identify unique immunotherapy biomarkers. The unique immunotherapeutic potential of targeting CCL28 was validated through a mouse gastric cancer model. In addition, flow cytometry revealed changes in the tumor immune microenvironment targeting CCL28. RESULTS: The re-analysis of ST data from multiple cancer types revealed a co-localization pattern between B cells and tumor cells. A significant number of IgA plasma cells were identified in the GC TIME. Five different tumor-infiltrating B cell subpopulations and two unique B cell differentiation trajectories were characterized, along with seven GC-related states. By analyzing the communication between GC cells and B cells, it was further discovered that tumor cells can influence and recruit plasma cells through CCL28-CCR10 signaling. Additionally, there was a crosstalk between GC cells and B cells. Finally, we identified the LAMA/CD44 signaling axis as a potential prognostic marker for immunotherapy through a large amount of immunotherapy data. We also validated through various animal tumor models that targeting CCL28 can significantly promote CD8+T cell infiltration and function in the TME by regulating B cell and plasma cell functions, and has the ability to synergize immunotherapy. CONCLUSION: The co-localization and crosstalk between GC cells and B cells significantly affect the efficacy of immunotherapy, and inhibiting the CCL28-CCR10 signal axis is a potential immunotherapy target for GC. Meanwhile, LAMA/CD44 pair may be a potential adverse indicator for immunotherapy and tumor prognosis.

CCL28 promotes progression of hepatocellular carcinoma through PDGFD-regulated MMP9 and VEGFA pathways.

Hepatocellular carcinoma (HCC) remains a major global health concern with limited therapeutic options and poor prognosis. Chemokines have emerged as critical regulators in the progression and metastasis of HCC. This study aims to investigate the mechanisms involved in CCL28-promoted progression of HCC and provide novel therapeutic targets for HCC treatment. Relationship between CCL28 expression and HCC progression were investigated by bioinformatic analysis and immunohistochemical staining assays. CCK-8, Transwell, and colony formation assay were conducted to explore the impact of CCL28 on the growth, migration and invasion of HCC cells. Quantitative real-time PCR and western blotting assays were performed to learn potential molecular mechanisms underlying the transformation of HCC driven by CCL28. The results showed that there was a direct link between increased CCL28 levels and the advancement of HCC, leading to a worse outcome. CCL28 significantly augmented malignant transformation of HCC cells, containing proliferation, migration, invasion, and clonogenicity, via activation of PDGFD-regulated MMP9 and VEGFA pathways. CCL28 emerges as a pivotal contributor to HCC tumorigenesis, propelling HCC development through the PDGFD signaling pathway. Our findings unveil potential therapeutic targets for HCC treatment.

Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

BACKGROUND: It has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure. METHOD: Overexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC-MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter. RESULTS: Our study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells. CONCLUSION: We reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.

CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments.

The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.

Analysis of the mucosal chemokines CCL28, CXCL14, and CXCL17 in dry eye disease: An in vitro and clinical investigation.

Mucosal chemokines have antimicrobial properties and play an important role in mucosal immunity. However, little is known about their expression on the ocular surface. This study aimed to analyze the expression of the mucosal chemokines CCL28, CXCL14 and CXCL17 in corneal and conjunctival epithelial cells under in vitro dry eye (DE) conditions, and in conjunctival samples from healthy subjects and DE patients. Human corneal epithelial cells (HCE) and immortalized human conjunctival epithelial cells (IM-HConEpiC) were incubated under hyperosmolar (400-500 mOsM) or inflammatory (TNF-α 25 ng/mL) conditions for 6 h and 24 h to measure CCL28, CXCL14, and CXCL17 gene expression by RT-PCR and their secretion by immunobead-based analysis (CCL28, CXCL14) and ELISA (CXCL17). Additionally, twenty-seven DE patients and 13 healthy subjects were included in this study. DE-related questionnaires (OSDI, mSIDEQ and NRS) evaluated symptomatology. Ocular surface integrity was assessed using vital staining. Tactile sensitivity was measured with Cochet-Bonnet esthesiometer, and mechanic and thermal (heat and cold) sensitivity using Belmonte's non-contact esthesiometer. Subbasal nerve plexus and dendritic cell density were analyzed by in vivo confocal microscopy. Conjunctival cells from participants were collected by impression cytology to measure mucosal chemokines gene expression by RT-PCR. Our results showed that HCE and IM-HConEpiC cells increased CCL28, CXCL14, and CXCL17 secretion under hyperosmolar conditions. The gene expression of CCL28 was significantly upregulated in conjunctival samples from DE patients. CCL28 expression correlated positively with symptomatology, corneal staining, heat sensitivity threshold, and dendritic cell density. CXCL14 expression correlated positively with age, ocular pain, conjunctival staining, tactile sensitivity, and image reflectivity. CXCL17 expression correlated positively with corneal staining. These results suggest that corneal and conjunctival epithelial cells could be a source of CCL28, CXCL14, and CXCL17 on the ocular surface and that CCL28 might be involved in DE pathogenesis.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

BACKGROUND/AIM: The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. MATERIALS AND METHODS: We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. RESULTS: CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. CONCLUSION: This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Transcriptionally activates CCL28 expression to inhibit M2 polarization of macrophages and prevent immune escape in colorectal cancer cells.

OBJECTIVE: This study aimed to investigate the potential molecular mechanism of SPDEF in immune evasion of colorectal cancer (CRC) and examine its impact on macrophage M2 polarization using the TCGA and GEO databases. METHODS: By combining TCGA and GEO databases, differential gene expression between CRC samples and standard tissue samples was analyzed to screen for immune-related genes (IRGs) associated with the prognosis of CRC patients. A predictive risk model was constructed based on 18 key IRGs, which were then validated using the GEO dataset. The relationship between transcription factors and IRGs was further explored to investigate their regulatory network in CRC. In vivo and in vitro experiments were carried out to validate these regulatory relationships and explore the function of SPDEF and CCL28 in CRC. RESULTS: Twelve key IRGs associated with clinical and pathological characteristics of CRC patients were identified. Among them, CCL28 significantly impacted macrophage infiltration in CRC cells and may be a critical factor in immune evasion. In both in vitro and in vivo experiments, overexpression of SPDEF upregulated CCL28 expression, thereby suppressing M2 polarization of macrophages and inhibiting CRC cell proliferation and tumor growth. Notably, interference with CCL28 could reverse the effect of SPDEF overexpression. CONCLUSION: SPDEF can suppress immune evasion of CRC cells by activating CCL28, which is achieved through the modulation of M2 polarization of macrophages. This provides a new research direction and potential therapeutic target for immunotherapy in CRC.

LncRNA H19 aggravates primary graft dysfunction after lung transplantation via KLF5-mediated activation of CCL28.

The present study aims to elucidate the possible involvement of H19 in primary graft dysfunction (PGD) following lung transplantation (LT) and the underlying mechanism. The transcriptome data were obtained through high-throughput sequencing analysis, and the differential long noncoding RNAs and messenger RNAs were screened for coexpression analysis. The interaction among H19, KLF5 and CCL28 was analyzed. A hypoxia-induced human pulmonary microvascular endothelial cell injury model was established, in which H19 was knocked down to elucidate its effect on the lung function, inflammatory response, and cell apoptosis. An orthotopic left LT model was constructed for in vivo mechanistic validation. High-throughput transcriptome sequencing analysis revealed the involvement of the H19/KLF5/CCL28 signaling axis in PGD. Silencing of H19 reduced inflammatory response and thus improved PGD. CCL28 secreted by human pulmonary microvascular endothelial cells after LT recruited neutrophils and macrophages. Mechanistic investigations indicated that H19 augmented the expression of CCL28 by binding to the transcription factor KLF5. Abundant expression of CCL28 reversed the alleviating effect of H19 silencing on PGD. In conclusion, the results point out that H19 exerts a promoting effect on PGD through increasing KLF5 expression and the subsequent CCL28 expression. Our study provides a novel insight into the mechanism of action of H19.

High Frequencies of Antiviral Effector Memory TEM Cells and Memory B Cells Mobilized into Herpes Infected Vaginal Mucosa Associated With Protection Against Genital Herpes.

Vaginal mucosa-resident anti-viral effector memory B- and T cells appeared to play a crucial role in protection against genital herpes. However, how to mobilize such protective immune cells into the vaginal tissue close to infected epithelial cells remains to be determined. In the present study, we investigate whether and how, CCL28, a major mucosal-associated chemokine, mobilizes effector memory B- and T cells in leading to protecting mucosal surfaces from herpes infection and disease. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). We found the presence of significant frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10 receptor, in herpes-infected asymptomatic (ASYMP) women compared to symptomatic (SYMP) women. A significant amount of the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP B6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected asymptomatic mice. In contrast, compared to wild-type (WT) B6 mice, the CCL28 knockout (CCL28(-/-)) mice: (i) Appeared more susceptible to intravaginal infection and re-infection with HSV-2; (ii) Exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+ CD62L- CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. The results imply a critical role of the CCL28/CCR10 chemokine axis in the mobilization of anti-viral memory B and T cells within the VM to protect against genital herpes infection and disease.

Fragment-based drug discovery of small molecule ligands for the human chemokine CCL28.

The mucosal chemokine CCL28 is a promising target for immunotherapy drug development due to its elevated expression level in epithelial cells and critical role in creating and maintaining an immunosuppressive tumor microenvironment. Using sulfotyrosine as a probe, NMR chemical shift mapping identified a potential receptor-binding hotspot on the human CCL28 surface. CCL28 was screened against 2,678 commercially available chemical fragments by 2D NMR, yielding thirteen verified hits. Computational docking predicted that two fragments could occupy adjoining subsites within the sulfotyrosine recognition cleft. Dual NMR titrations confirmed their ability to bind CCL28 simultaneously, thereby validating an initial fragment pair for linking and merging strategies to design high-potency CCL28 inhibitors.

Neutralization of excessive CCL28 improves wound healing in diabetic mice.

Introduction: Chronic, non-healing skin wounds such as diabetic foot ulcers (DFUs) are common in patients with type 2 diabetes mellitus (T2DM) and often result in limb amputation and even death. However, mechanisms by which T2DM and inflammation negatively impact skin wound healing remains poorly understood. Here we investigate a mechanism by which an excessive level of chemokine CCL28, through its receptor CCR10, impairs wound healing in patients and mice with T2DM. Methods & Results: Firstly, a higher level of CCL28 was observed in skin and plasma in both patients with T2DM, and in obesity-induced type 2 diabetic db/db mice. Compared with WT mice, adipose tissue from db/db mice released 50% more CCL28, as well as 2- to 3-fold more IL-1ß, IL-6, and TNF-α, and less VEGF, as determined by ELISA measurements. Secondly, overexpression of CCL28 with adenovirus (Adv-CCL28) caused elevation of proinflammatory cytokines as well as CCR10 expression and also reduced eNOS expression in the dorsal skin of WT mice as compared with control Adv. Thirdly, topical application of neutralizing anti-CCL28 Ab dose-dependently accelerated wound closure and eNOS expression, and decreased IL-6 level, with an optimal dose of 1 µg/wound. In addition, mRNA levels of eNOS and anti-inflammatory cytokine IL-4 were increased as shown by real-time RT-PCR. The interaction between eNOS and CCR10 was significantly reduced in diabetic mouse wounds following application of the optimal dose of anti-CCL28 Ab, and eNOS expression increased. Finally, enhanced VEGF production and increased subdermal vessel density as indicated by CD31 immunostaining were also observed with anti-CCL28 Ab. Discussion: Taken together, topical application of neutralizing anti-CCL28 Ab improved dorsal skin wound healing by reducing CCR10 activation and inflammation in part by preventing eNOS downregulation, increasing VEGF production, and restoring angiogenesis. These results indicate anti-CCL28 Ab has significant potential as a therapeutic strategy for treatment of chronic non-healing diabetic skin wounds such as DFUs.

Detection of CCL25 and the correlation between CCL25, CCL28, IL-7, and TSLP in human breast milk.

In this study, CCL25, a chemokine that contributes to the immunological function of the thymus and intestines, was detected in human breast milk (HBM) for the first time. We then focused on the correlations of CCL25 with CCL28, TSLP, and IL-7, which were predicted to interact with CCL25 in HBM. We also compared their levels between primiparous and multiparous women. A total of 53 parturient women were recruited. Their HBM was collected during 0-5 days and at 1 month after parturition and the CCL25, CCL28, IL-7, and TSLP levels in the HBM were analyzed using ELISA. The results showed that CCL25 and TSLP levels were significantly higher in colostrum than in mature milk. Moreover, CCL28 and IL-7 levels in colostrum showed a positive correlation. These results indicate that CCL28 and IL-7 in colostrum might interact positively with each other when produced in the mammary glands during lactation. The findings also suggest that the level of parity has no effect on their levels in HBM. In conclusion, our results clarify that CCL25 is present in HBM and that the concentrations of CCL25 and TSLP are higher in colostrum than in mature milk. Moreover, the production of CCL28 and IL-7 might be closely correlated in human colostrum.

[Role of the CCL28-CCR10 pathway in monocyte migration in rheumatoid arthritis].

OBJECTIVE: To examine the expression of chemokine receptor CCR10 on monocytes/macrophages in the joints of patients with rheumatoid arthritis (RA), and to investigate the role of chemokine CCL28 and its receptor CCR10 in the migration of RA monocytes and its mechanism. METHODS: The expression of CCR10 in synovial tissues from 8 RA patients, 4 osteoarthritis (OA) patients, and 4 normal controls was analyzed by immunohistochemistry, and cell staining was scored on a 0-5 scales. Flow cytometry was used to measure the percentage of CCR10 positive cells in CD14+ monocytes from peripheral blood of 26 RA patients and 20 healthy controls, as well as from synovial fluid of 15 RA patients. The chemotactic migration of monocytes from RA patients and healthy controls in response to CCL28 was evaluated using an in vitro Transwell system. Western blotting was conducted to assess phosphorylation of the extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) pathways in RA monocytes upon CCL28 treatment. RESULTS: CCR10 was predominantly expressed in RA synovial lining cells and sublining macrophages, endothelial cells, and lymphocytes. CCR10 expression was significantly increased on lining cells and sublining macrophages in RA synovial tissue compared with OA and normal synovial tissue (both P < 0.01). The patients with RA had markedly elevated expression of CCR10 on peripheral blood CD14+ monocytes compared with the healthy controls [(15.6±3.0)% vs. (7.7±3.8)%, P < 0.01]. CCR10 expression on synovial fluid monocytes from the RA patients was (32.0±15.0)%, which was significantly higher than that on RA peripheral blood monocytes (P < 0.01). In vitro, CCL28 caused significant migration of CD14+ monocytes from peripheral blood of the RA patients and the healthy controls at concentrations ranging from 10-100 µg/L (all P < 0.01). The presence of neutralizing antibody to CCR10 greatly suppressed CCL28-driven chemotaxis of RA monocytes (P < 0.01). Stimulation of RA monocytes with CCL28 induced a remarkable increase in phosphorylation of ERK and Akt (both P < 0.05). ERK inhibitor (U0126) and phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) strongly reduced the migration of RA monocytes in response to CCL28 (both P < 0.01). CONCLUSION: RA patients had increased CCR10 expression on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages. CCL28 ligation to CCR10 promoted RA monocyte migration through activation of the ERK and PI3K/Akt signaling pathways. The CCL28-CCR10 pathway could participate in monocyte recruitment into RA joints, thereby contributing to synovial inflammation and bone destruction.

Persistent Depletion of Neuroprotective Factors Accompanies Neuroinflammatory, Neurodegenerative, and Vascular Remodeling Spectra in Serum Three Months after Non-Emergent Cardiac Surgery.

We hypothesized that the persistent depletion of neuroprotective markers accompanies neuroinflammation and neurodegeneration in patients after cardiac surgery. A total of 158 patients underwent elective heart surgery with their blood collected before surgery (tbaseline) and 24 h (t24hr), seven days (t7d), and three months (t3m) post-surgery. The patients' serum was measured for markers of neurodegeneration (τau, τaup181-183, amyloid ß1-40/ß2-42, and S100), atypical neurodegeneration (KLK6 and NRGN), neuro-injury (neurofilament light/heavy, UC-HL, and GFAP), neuroinflammation (YKL-40 and TDP-43), peripheral nerve damage (NCAM-1), neuroprotection (apoE4, BDNF, fetuin, and clusterin), and vascular smoldering inflammation (C-reactive protein, CCL-28 IL-6, and IL-8). The mortality at 28 days, incidence of cerebrovascular accidents (CVA), and functional status were followed for three months. The levels of amyloid ß1-40/ß1-42 and NF-L were significantly elevated at all time points. The levels of τau, S100, KLK6, NRGN, and NCAM-1 were significantly elevated at 24 h. A cluster analysis demonstrated groupings around amyloids, KLK6, and NCAM-1. YKL-40, but not TDP-43, was significantly elevated across all time points. BDNF, apoE4, fetuin, and clusterin levels were significantly diminished long-term. IL-6 and IL-8 levles returned to baseline at t3m. The levels of CRP, CCL-28, and Hsp-70 remained elevated. At 3 months, 8.2% of the patients experienced a stroke, with transfusion volume being a significant variable. Cardiac-surgery patients exhibited persistent peripheral and neuronal inflammation, blood vessel remodeling, and the depletion of neuroprotective factors 3 months post-procedure.

Roles of CCR10/CCL27-CCL28 axis in tumour development: mechanisms, diagnostic and therapeutic approaches, and perspectives.

Cancer is now one of the major causes of death across the globe. The imbalance of cytokine and chemokine secretion has been reported to be involved in cancer development. Meanwhile, CC chemokines have received considerable interest in cancer research. CCR10, as the latest identified CC chemokine receptor (CCR), has been implicated in the recruitment and infiltration of immune cells, especially lymphocytes, into epithelia such as skin via ligation to two ligands, CCL27 and CCL28. Other than homoeostatic function, several mechanisms have been shown to dysregulate CCR10/CCL27-CCL28 expression in the tumour microenvironment. As such, these receptors and ligands mediate T-cell trafficking in the tumour microenvironment. Depending on the types of lymphocytes recruited, CCR10/CCL27-CCL28 interaction has been shown to play conflicting roles in cancer development. If they were T helper and cytotoxic T cells and natural killer cells, the role of this axis would be tumour-suppressive. In contrast, if CCR10/CCL27-CCL28 recruited regulatory T cells, cancer-associated fibroblasts or myeloid-derived suppressor cells, it would lead to tumour progression. In addition to the trafficking of lymphocytes and immune cells, CCR10 also leads to the migration of tumour cells or endothelial cells (called angiogenesis and lymphangiogenesis) to promote tumour metastasis. Furthermore, CCR10 signalling triggers tumour-promoting signalling such as PI3K/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase, resulting in tumour cell growth. Since CCR10/CCL27-CCL28 is dysregulated in the tumour tissues, it is suggested that analysis and measurement of them might predict tumour development. Finally, it is hoped using therapeutic approaches based on this axis might increase our knowledge to overcome tumour progression.

CCL28 Enhances HSV-2 gB-Specific Th1-Polarized Immune Responses against Lethal Vaginal Challenge in Mice.

Plasmid DNA (pDNA) represents a promising "genetic vaccine platform" capable of overcoming major histocompatibility complex barriers. We previously demonstrated that low-to-moderate doses of mucosae-associated epithelial chemokine (MEC or CCL28) as an immunomodulatory adjuvant can trigger effective and long-lasting systemic and mucosal HSV-2 gD-specific immune responses, whereas mice immunized with gD in combination with high-dose CCL28 showed toxicity and lost their immunoprotective effects after lethal HSV-2 challenge. The exact causes underlying high-dose, CCL28-induced lesions remain unknown. In an intramuscularly immunized mouse model, we investigated the immune-enhancement mechanisms of low-dose CCL28 as a molecular adjuvant combined with the relatively weak immunogen HSV-2 gB. Compared with the plasmid gB antigen group, we found that a low-dose of plasmid CCL28 (pCCL28) codelivered with pgB induced increased levels of gB-specific serum IgG and vaginal fluid IgA, serum neutralizing antibodies (NAb), Th1-polarized IgG2a, and cytokine IL-2 (>5-fold). Furthermore, low-dose pCCL28 codelivery with pgB enhanced CCL28/CCR10-axis responsive CCR10− plus CCR10+ B-cell (~1.2-fold) and DC pools (~4-fold) in the spleen, CCR10− plus CCR10+ T-cell pools (~2-fold) in mesenteric lymph nodes (MLNs), and the levels of IgA-ASCs in colorectal mucosal tissues, leading to an improved protective effect against a lethal dose of HSV-2 challenge. Findings in this study provide a basis for the development of CCL28-adjuvant vaccines against viral mucosal infections.

Protective effects of bicistronic DNA vaccines of herpes simplex virus type 2 glycoprotein D and adjuvant CCL28 on mice / 中华微生物学和免疫学杂志

Objective:To study the protective effects of bicistronic DNA vaccines carrying herpes simplex virus type 2 glycoprotein D (HSV-2 gD) and adjuvant CCL28 sequences that were connected by internal ribosome entry site (IRES) sequence in mouse model.Methods:The recombinant DNA vaccines, pgD-IRES-CCL28 and pCCL28-IRES-gD, encoding HSV-2 gD and adjuvant CCL28 were constructed with IRES sequence. After verified by sequencing, they were intramuscularly injected twice into BALB/c mice. Serum samples and vaginal lavage fluids were collected regularly. Splenocytes, mesenteric lymph node cells and rectal mucosa tissues were separated and collected. The titers of antigen-specific antibodies in immunized mice were analyzed with end-point ELISA. In vitro neutralization assay was used to measure neutralizing antibody titers in serum and vaginal lavage fluid after vaccination and virus challenge. CCL28-responsive immune cells in splenocytes, mesenteric lymph node cells and rectal tissues were detected by chemotaxis experiment and immunohistochemical staining. The protective effects of the bicistronic DNA vaccines were evaluated by fluorescent quantitative PCR, weighing and disease severity assessment. Humoral and cellular immune responses induced by the bicistronic DNA vaccines and their efficacy in immunoprotection were analyzed by comparing with pgD+ pCCL28 group. Results:IgG titers in serum samples and IgA antibody titers in vaginal lavage fluids of mice immunized with pCCL28-IRES-gD were similar to those in pgD+ pCCL28 group. The neutralizing ability of antibodies, the number of rectal mucosal IgA+ plasma cells and CCL28-responsive immune cells in mucosal tissues were increased in pCCL28-IRES-gD group. Serum neutralizing antibodies were not produced immediately in the mice challenged with HSV-2, but no weight loss, disease symptoms or death was observed. However, pgD+ pcDNA3.1 and pgD-IRES-CCL28 were ineffective against HSV-2 infection in mice.Conclusions:The recombinant bicistronic DNA vaccine of pCCL28-IRES-gD could induce stronger mucosal immune response in mice and provide better protective effects.

CCL28 Downregulation Attenuates Pancreatic Cancer Progression Through Tumor Cell-Intrinsic and -Extrinsic Mechanisms.

C-C motif chemokine ligand 28 (CCL28) has been reported to be pro-tumoral in several cancer types. However, the role of CCL28 in pancreatic ductal adenocarcinoma (PDAC) progression remains unclear. CCL28 mRNA expression in tumors from PDAC patients was found to be elevated as compared to normal pancreas. CCL28 expression was also negatively correlated with overall survival (OS) in pancreatic cancer patients. Our in vitro experiments showed that CCL28 knockdown impairs the proliferation of mouse pancreatic cancer cell line PAN02. Moreover, in both immunocompetent syngeneic mice and immunodeficient NOD-SCID mice, CCL28 deficiency significantly attenuated the growth of subcutaneous PAN02 tumors. In syngeneic mouse model, CCL28 downregulation remodeled the pancreatic tumor microenvironment by suppressing the infiltration of both regulatory T (Treg) cells, myeloid-derived suppressor cells, and activated pancreatic stellate cells, and upregulating the expression of lymphocyte cytotoxic proteins including perforin and granzyme B. In conclusion, our work demonstrates that CCL28 is a potential target for pancreatic cancer treatment and CCL28 blockade could inhibit tumor growth through both tumor-cell-intrinsic and extrinsic mechanisms.

Construction of ceRNA regulatory network in mice with Echinococcosis-induced allergic reactions.

BACKGROUND: Cystic echinococcosis (CE) is a common chronic zoonotic parasitic disease infected with the Echinococcus granulosus. This study aimed to construct the competitive endogenous RNA (ceRNA) network and investigate the mechanism of mice in echinococcosis sensitization. METHODS: The animal model of echinococcosis was established in mice, and the RNA sequencing was then performed using cysts. The differentially expressed RNAs (DERNAs: DEmRNAs, DEmiRNAs, and DElncRNAs) were screened between anaphylactic mice or non-anaphylactic mice and controls, respectively. The interactions of two DERNAs groups were identified and the ceRNA network was constructed. Moreover, the potential biological functions and pathways of the DEmRNAs were explored by enrichment analyses. Finally, the qRT-PCR and the western blot were performed to validate the expression levels of key RNAs and proteins. RESULTS: A total of 285 common DEmRNAs, 157 common DElncRNAs and 4 common DEmiRNAs were observed. CeRNA network contained 3 DElncRNAs, 4 DEmiRNAs, and 27 DEmRNAs corporately. Enrichment results revealed that the functions of DEmRNAs focus on biological functions and pathways that specifically interact with the immune inflammatory response. In addition, the expression of 1700099I09Rik, let-7a-5p, Ccl28 and IL-13 was validated by RT-qPCR and western blot. CONCLUSION: In this study, ceRNA network associated with CE sensitization in mice was constructed. The DEmRNAs in this network may be key clues for the immune mechanism of CE sensitization.