C-C Motif) Ligand 2 Research Articles

Silver nanoparticles combat Salmonella Typhimurium: Suppressing intracellular infection and activating dendritic cells

Salmonella Typhimurium (ST) can hide inside cells, avoid antibiotic therapy and being killed by host’s immune system to cause persistent infection in humans and animals. Metal nanoparticles are regarded as an alternative to overcome the above limitations, silver nanoparticles especially have been applied in combating drug-resistant bacteria. However, the therapeutic effects of silver nanoparticles against intracellular infection and their impacts on host immunity remain an area of further investigation. In this work, we synthesized Ganoderma extract-capped silver nanoparticles (Ag@Ge) and explored the therapeutic potential and immune adjuvant effects of Ag@Ge against intracellular ST. Firstly, Ag@Ge had a small particle size of 35.52±7.46 nm, good stability, and biocompatibility. Then, Ag@Ge effectively entered RAW 264.7 cells, suppressed intracellular ST infection. Furthermore, Ag@Ge activated mouse dendritic cells (DCs) in vitro, evidenced by increased phenotypic markers (CD80/CD86/CD40/major compatibility complex II (MHCII)) expression and cytokine and chemokine (interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL-2), and chemokine (C-C motif) receptor-7 (CCR-7)) transcription. More notably, the combination of Ag@Ge with inactivated ST recruited intestinal DCs to mitigate ST infection in mice, evidenced by decreased body weight loss and bacterial loads in the tissues (liver, jejunum, and colon), and improved platelets count. The above findings indicate that Ag@Ge has the potential as an alternative nano-antibiotic against intracellular ST infection.

Ubiquitin-specific protease 34 in macrophages limits CD8 T cell-mediated onset of vitiligo in mice

As an autoimmune disorder, vitiligo is characterized by depigmented skin macules. CD8+T cells and macrophages enrichment promote the onset of vitiligo, while the role of macrophages to CD8+T is not well deciphered. To develop a mouse model of vitiligo with prominent epidermal depigmentation, Krt14-Kitl* transgenic mice containing an elevated number of melanocytes in the epidermis with membrane-bound Kit ligand (Kitl*) were adoptively transferred with premelanosome protein (PMEL) CD8+ T cells. On the other hand, Krt14-Kitl* mice were mated with ubiquitin-specific protease 34 (USP34)MKO mice to decipher the role of USP34 in vitiligo. Vitiligo scores and PMEL CD8+ T cell enrichment were detected with flow cytometry. Human peripheral blood mononuclear cells (PBMCs) or mice bone marrow-derived macrophages (BMDMs) were incubated with lipopolysaccharide (LPS), CpG, or co-incubated with KU-55933, an ataxia telangiectasia-mutated (ATM) inhibitor. Chemokine (C-C motif) ligand 2 (CCL2), Ccl5, and interleukin (Il)-12α expression was assayed with real-time PCR, and p-IKKα/β was assayed with Western blots. USP34 was up-regulated in the PBMCs of vitiligo patients and LPS-stimulated BMDMs. USP34 deficiency did not affect the differentiation of CD11b+F4/80+ macrophages in the bone marrow. Immunoprecipitation demonstrated the interaction between USP34 and ATM. USP34 deficiency or KU-55933 administration promoted the induction of Ccl2, Ccl5, Il12α, and p-IKKα/β in LPS or CpG stimulated BMDMs; KU-55933 administration could not affect the expression of the above molecules in USP34 deficient BMDMs. It further revealed that USP34 deficiency promoted the development of vitiligo with increased PMEL CD8+ T cell enrichment, which was not affected by KU-55933 administration. USP34 deficiency in macrophages promotes the onset of vitiligo with increased PMEL CD8+ T cell enrichment, and USP34/ATM complex can be considered as a therapy target.

Tumor microenvironment signaling and therapeutics in cancer progression.

Tumor development and metastasis are facilitated by the complex interactions between cancer cells and their microenvironment, which comprises stromal cells and extracellular matrix (ECM) components, among other factors. Stromal cells can adopt new phenotypes to promote tumor cell invasion. A deep understanding of the signaling pathways involved in cell-to-cell and cell-to-ECM interactions is needed to design effective intervention strategies that might interrupt these interactions. In this review, we describe the tumor microenvironment (TME) components and associated therapeutics. We discuss the clinical advances in the prevalent and newly discovered signaling pathways in the TME, the immune checkpoints and immunosuppressive chemokines, and currently used inhibitors targeting these pathways. These include both intrinsic and non-autonomous tumor cell signaling pathways in the TME: protein kinase C (PKC) signaling, Notch, and transforming growth factor (TGF-β) signaling, Endoplasmic Reticulum (ER) stress response, lactate signaling, Metabolic reprogramming, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and Siglec signaling pathways. We also discuss the recent advances in Programmed Cell Death Protein 1 (PD-1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), T-cell immunoglobulin mucin-3 (TIM-3) and Lymphocyte Activating Gene 3 (LAG3) immune checkpoint inhibitors along with the C-C chemokine receptor 4 (CCR4)- C-C class chemokines 22 (CCL22)/ and 17 (CCL17), C-C chemokine receptor type 2 (CCR2)- chemokine (C-C motif) ligand 2 (CCL2), C-C chemokine receptor type 5 (CCR5)- chemokine (C-C motif) ligand 3 (CCL3) chemokine signaling axis in the TME. In addition, this review provides a holistic understanding of the TME as we discuss the three-dimensional and microfluidic models of the TME, which are believed to recapitulate the original characteristics of the patient tumor and hence may be used as a platform to study new mechanisms and screen for various anti-cancer therapies. We further discuss the systemic influences of gut microbiota in TME reprogramming and treatment response. Overall, this review provides a comprehensive analysis of the diverse and most critical signaling pathways in the TME, highlighting the associated newest and critical preclinical and clinical studies along with their underlying biology. We highlight the importance of the most recent technologies of microfluidics and lab-on-chip models for TME research and also present an overview of extrinsic factors, such as the inhabitant human microbiome, which have the potential to modulate TME biology and drug responses.

Epigenetic regulation of chemokine (CC-motif) ligand 2 in inflammatory diseases.

Appropriate responses to inflammation are conducive to pathogen elimination and tissue repair, while uncontrolled inflammatory reactions are likely to result in the damage of tissues. Chemokine (CC-motif) Ligand 2 (CCL2) is the main chemokine and activator of monocytes, macrophages, and neutrophils. CCL2 played a key role in amplifying and accelerating the inflammatory cascade and is closely related to chronic non-controllable inflammation (cirrhosis, neuropathic pain, insulin resistance, atherosclerosis, deforming arthritis, ischemic injury, cancer, etc.). The crucial regulatory roles of CCL2 may provide potential targets for the treatment of inflammatory diseases. Therefore, we presented a review of the regulatory mechanisms of CCL2. Gene expression is largely affected by the state of chromatin. Different epigenetic modifications, including DNA methylation, post-translational modification of histones, histone variants, ATP-dependent chromatin remodelling, and non-coding RNA, could affect the 'open' or 'closed' state of DNA, and then significantly affect the expression of target genes. Since most epigenetic modifications are proven to be reversible, targeting the epigenetic mechanisms of CCL2 is expected to be a promising therapeutic strategy for inflammatory diseases. This review focuses on the epigenetic regulation of CCL2 in inflammatory diseases.

Chemokine (C-C motif) Ligand 2 Is Regulated Through the EGFR/Src Pathway in HER2-positive Breast Cancer Cells.

Chemokine (C-C motif) ligand 2 (CCL2) influences growth and metastasis and is associated with poor prognosis in various cancers. However, the regulatory mechanism of CCL2 induction by human epidermal growth factor receptor 2 (HER2) is not fully understood in breast cancer. Thus, we investigated how CCL2 expression is regulated in HER2-positive (HER2+) breast cancer. A human cytokine array was performed to investigate the differential expression of cytokines by HER2 overexpression. Quantitative reverse transcription PCR, enzyme-linked immunosorbent assay and western blot were performed to detect the levels of mRNA and protein expression. Cell cycle and proliferation were analyzed by flow cytometry. Cell invasion was analyzed by Boyden chamber assay. Our results showed that HER2 overexpression augmented CCL2 expression. Epidermal growth factor receptor (EGFR) and Src activities were increased in the HER2-overexpressed breast cancer cells. Interestingly, HER2-induced CCL2 expression could not be down-regulated by trastuzumab, while neratinib or saracatinib led to a decrease in the expression of CCL2 in HER2+ breast cancer cells. CCL2 expression is regulated through the EGFR/Src-dependent signaling in HER2+ breast cancer.

T Helper 17–Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis

Background: Infiltration of cluster of differentiation (CD) 3+ (CD3+) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. Hypothesis: An imbalance of the ratio of regulatory T cells and T helper 17 cells would be associated with disease progression in posttraumatic osteoarthritis, suggesting opportunities for immunomodulatory therapy. Study Design: Descriptive laboratory study. Methods: Synovial fluid was aspirated from the joints of equine clinical patients undergoing arthroscopic surgery for posttraumatic osteoarthritis resulting from intra-articular fragmentation. Joints were classified as having mild or moderate posttraumatic osteoarthritis. Synovial fluid was also obtained from nonoperated horses with normal cartilage. Peripheral blood was obtained from horses with normal cartilage and those with mild and moderate posttraumatic osteoarthritis. Synovial fluid and peripheral blood cells were analyzed by flow cytometry, and native synovial fluid was analyzed by enzyme-linked immunosorbent assay. Results: CD3+ T cells represented 81% of lymphocytes in synovial fluid, which increased in animals with moderate posttraumatic osteoarthritis to 88.3% (P = .02). CD14+ macrophages were doubled in those with moderate posttraumatic osteoarthritis compared with mild posttraumatic osteoarthritis and controls (P < .001). Less than 5% of CD3+ T cells found within the joint were forkhead box P3 protein+ (Foxp3+) regulatory T cells, but a 4- to 8-times higher percentage of nonoperated and mild posttraumatic osteoarthritis joint regulatory T cells secreted interleukin (IL)–10 than peripheral blood Tregs (P < .005). T regulatory-1 cells that secreted IL-10 but did not express Foxp3 accounted for approximately 5% of CD3+ T cells in all joints. T helper 17 cells and Th17-like regulatory T cells were increased in those with moderate posttraumatic osteoarthritis (P < .0001) compared with mild and nonoperated patients. IL-10, IL-17A, IL-6, chemokine (C-C motif) ligand (CCL) 2 (CCL2), and CCL5 concentrations detected by enzyme-linked immunosorbent assay in synovial fluid were not different between groups. Conclusions: An imbalance of the ratio of regulatory T cells and T helper 17 cells and an increase in T helper 17 cell–like regulatory T cells in synovial fluid from joints with more severe disease provide novel insights into immunological mechanisms that are associated with posttraumatic osteoarthritis progression and pathogenesis. Clinical Relevance: Early and targeted use of immunotherapeutics in the mitigation of posttraumatic osteoarthritis may improve patient clinical outcomes.

Potential Mechanisms of Perillae folium Against COVID-19: A Network Pharmacology Approach.

In China, Perillae folium is widely used to treat colds, especially in the early stages of cold; the effect of taking P. folium is readily noticeable at that time. The active compounds and targets of P. folium were screened from Traditional Chinese Medicine Systems Pharmacology, Chinese Pharmacopoeia, and UniProt. Targets related to the initiation and progression of 2019 Coronavirus Disease (COVID-19) were retrieved from Online Mendelian Inheritance in Man and GeneCards. The potential therapeutic targets of P. folium on COVID-19 were the cross targets between them. Enrichment analysis of Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were conducted by using the Database for Annotation, Visualization and Integrated Discovery website. Molecular docking between key compounds and core targets was performed with AutoDock. The effects of P. folium extract and rosmarinic acid on inflammatory cytokines were tested by a cellular inflammatory model. The "Perillae folium-compound-target-COVID-19" network contained 11 kinds of compounds and 33 matching targets. There were 261 items in the GO functions (P < .05) and 67 items linked to the KEGG signaling pathways (P < .05). Luteolin and rosmarinic acid were key compounds of P. folium. Their docking with the core targets mitogen-activated protein kinase 1 (MAPK1) and chemokine (C-C motif) ligand 2 (CCL2), respectively, showed that they had good affinity with each other. Cell experiments demonstrated that P. folium extract had inhibitory effects on interleukin-6 and tumor necrosis factor (TNF)-α in cells, and was better than rosmarinic acid. Luteolin, rosmarinic acid, and other individual active compounds in P. folium, which may participate in PI3K-Akt, TNF, Jak-STAT, COVID-19, and other multisignaling pathways through multiple targets such as MAPK1 and CCL2, and play a therapeutic role in COVID-19.

Oncolytic adenovirus-mediated expression of CCL5 and IL12 facilitates CA9-targeting CAR-T therapy against renal cell carcinoma

Chimeric antigen receptor T-cell (CAR-T) is particularly prominent in hematological but not in solid tumors, mainly based on the complex tumor immune microenvironment. Oncolytic virus (OVs) is an emerging adjuvant therapy method. OVs may prime tumor lesions to induce anti-tumor immune response, thereby enhancing CAR-T cells functionality and possibly increasing response rates. Here, we combined CAR-T cells targeting carbonic anhydrase 9 (CA9) and an oncolytic adenovirus (OAV) carrying chemokine (C-C motif) ligand 5 (CCL5), cytokine interleukin-12 (IL12) to explore the anti-tumor effects of this combination strategy. The data showed that Ad5-ZD55-hCCL5-hIL12 could infect and replicate in renal cancer cell lines and induced a moderate inhibition of xenografted tumor in nude mice. IL12 mediated by Ad5-ZD55-hCCL5-hIL12 promoted the phosphorylation of Stat4 in CAR-T cells, induced CAR-T cells to secrete more IFN-γ. We also found that Ad5-ZD55-hCCL5-hIL-12 combined with CA9-CAR-T cells significantly increased the infiltration of CAR-T cells in tumor mass, prolonged the survival of the mice and restrained tumor growth in immunodeficient mice. Ad5-ZD55-mCCL5-mIL-12 could also increase CD45+CD3+T cell infiltration and prolong mice survival in immunocompetent mice. These results provided feasibility for the combination of oncolytic adenovirus and CAR-T cells, which demonstrated the sufficient potential and prospects of CAR-T for the treatment of solid tumors.

HUCMSC-derived Exosomes Suppress the Titanium Particles-induced Osteolysis in Mice through Inhibiting CCL2 and CCL3.

Wear particles induce inflammation and the further osteolysis around the prosthesis, has been proven to be the main cause of aseptic hip joint loosening. In this research, we aimed to clarify whether human umbilical cord mesenchymal stem cells (HUCMSCs) could inhibit the titanium particles-induced osteolysis and shed light upon its mechanism. The expression of chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5) were examinjed in clinical specimens of aseptic hip prosthesis loosening patients. Local injection of lentivirus that knocked down CCL2 or CCL3 in a cranial osteolysis mice model were used to exam the effect of CCL2 and CCL3 on titanium particles-induced osteolysis in vivo. Transwell assay was used to examine the effect of CCL2 and CCL3 on titanium particles-induced activation of macrophage in vitro. Furthermore, the therapeutic effect of HUCMSCs, and exosomes from HUCMSCs were also examed in vivo and vitro. Immunohistochemical and real-time PCR were used to examine the expression of relative pathways. Analysis of variance (ANOVA) and Student-Newman-Keuls post hoc t test were used to analyze the results and determine the statistical significance of the differences. Results showed that titanium particles caused the osteolysis at the mice cranial in vivo and a large number of macrophages that migrated, while local injection of HUCMSCs and exosomes did inhibit the cranial osteolysis and migration. An exosome inhibitor GW4869 significantly increased the osteolysis area in the mice cranium osteolysis model, and increased the number of migrated macrophages. Immunohistochemical results suggested that the expression of CCL2, CCL3 and CD68 in the cranial in Titanium particles mice increased significantly, but was significantly reduced by HUCMSCs or exosomes. HUCMSC and exosomes down-regulate the expression of CCL3 in vitro and in vivo. HUCMSCs and HUCMSC-derived exosomes could suppress the titanium particles-induced osteolysis in mice through inhibiting chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3.

Metabolic effects of CCL5 deficiency in lean and obese mice.

Accumulation and activation of immunocytes in adipose tissues are essential to obesity-induced inflammation and insulin resistance. Chemokines are pivotal for the recruitment of immunocytes in adipose tissue during obesity. Chemokine (C-C motif) ligand 5 (CCL5) plays a vital role in the recruitment of immunocytes to sites of inflammation. CCL5 expression level is increased in obese adipose tissue from humans and mice. However, the role of CCL5 in obesity-induced adipose inflammation remains unclear. Our study found that the CCL5 expression level was increased in the epididymal white adipose tissue (eWAT) of obese mice, particularly in CD8+ T cells. CCL5 knockout (KO) mice exhibited better glucose tolerance than wild-type (WT) mice under lean conditions. In contrast, CCL5 KO mice were more insulin resistant and had severe hepatic steatosis than WT mice under obese conditions. Increased T cells in adipose tissue heaven adipose inflammation in obese CCL5 KO mice. The compensatory increased T cell-associated chemokines may account for increased T cell content in the eWAT of obese CCL5 KO mice. These findings imply that CCL5 deficiency exacerbates adipose inflammation and impairs insulin sensitivity in the metabolic tissues of obese mice.

Glucocorticoids and medroxyprogesterone acetate synergize with inflammatory stimuli to selectively upregulate CCL20 transcription

The pro-inflammatory cytokine, chemokine (C-C motif) ligand 20 (CCL20), is emerging as a therapeutic target for immune-based therapies. Cooperative regulation of CCL20 by glucocorticoids and progestins used in endocrine therapy and pro-inflammatory mediators could modulate immune function and affect disease outcomes. We show that glucocorticoids as well as medroxyprogesterone acetate (MPA), the progestin widely used in injectable contraception in sub-Saharan Africa, cooperate with pro-inflammatory mediators to upregulate CCL20 protein and/or mRNA in human peripheral blood mononuclear cells (PBMCs) and human cervical cell lines. Changes in CCL20 mRNA levels were shown to be synergistic, as assessed by Chou analysis, cell- and gene-specific and to involve transcriptional regulation, with a requirement for a nuclear factor kappa B (NF-κB) site and glucocorticoid receptor (GR) involvement. The novel results suggest a mechanism whereby MPA, like glucocorticoids, may impact inflammation both systemically and in the genital tract in patients using MPA and/or glucocorticoid therapy.

IFN-λ3 and CCL17 as predictors of disease progression in patients with mild to moderate COVID-19: A cohort study in a real-world setting

Coronavirus disease 2019 (COVID-19) has overwhelmed hospitals worldwide. In Japan, serum interferon lambda 3 (IFN-λ3) and C–C motif ligand (CCL) 17 levels have been used as predictive markers for disease progression to severe COVID-19. However, the relationship between these predictive markers and the disease progression of COVID-19 has not been well evaluated. We retrospectively evaluated the patient characteristics, serum IFN-λ3 and CCL17 levels, and comorbidities of 92 patients with mild (n = 20) and moderate (n = 72) COVID-19 who were hospitalized in our institution. The results of the multivariable analysis showed that the positive rates of IFN-λ3, CCL17, and the combination of these markers were significantly elevated in patients with progressed COVID-19. Furthermore, patients who were negative for both markers did not experience disease progression. This study illustrates the importance of measuring these markers to predict disease severity and progression in patients with COVID-19.

Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages.

Basal-like breast cancer (BLBC) has a clinically aggressive nature. It is prevalent in young women and is known to often relapse rapidly. To date, the molecular mechanisms regarding the aggressiveness of BLBC have not been fully understood. In the present study, mechanisms of aggressiveness of BLBC involving EGFR and/or HER2 expression and interactions between tumor and tumor-associated macrophages (TAMs) were explored. The prognosis of breast cancer patients who underwent surgery at Samsung Medical Center was analyzed. It was found that the co-expression of EGFR and HER2 was associated with a worse prognosis. Therefore, we generated EGFR-positive BLBC cells with stable HER2 overexpression and analyzed the profile of secretory cytokines. Chemokine (C-C motif) ligand 2 (CCL2) expression was increased in HER2-overexpressed BLBC cells. Recombinant human CCL2 treatment augmented the motility of TAMs. In addition, the conditioned culture media of HER2-overexpressed BLBC cells increased the motility of TAMs. Furthermore, activation of TAMs by CCL2 or the conditioned culture media of HER2-overexpressed cells resulted in the production of pro-inflammatory cytokines, such as IL-8 and IL-1β. These observations reveal that CCL2 derived from EGFR and HER2 co-expressed BLBC cells can lead to increased TAM recruitment and the induction of IL-8 and IL-1β from recruited TAMs, triggering the tumorigenesis of breast cancer with the expression of both EGFR and HER2. Our findings demonstrate that EGFR+ and HER2+ BLBC aggressiveness is partially mediated through the interaction between BLBC and TAMs recruited by CCL2.

CCL2: An important cytokine in normal and pathological pregnancies: A review.

C-C motif ligand 2 (CCL2), also known as monocytic chemotactic protein 1 (MCP-1), is an integral chemotactic factor which recruits macrophages for the immune response. Together with its receptors (e.g., CCR2, ACKR1, and ACKR2), they exert noticeable influences on various diseases of different systems. At the maternal-fetal interface, CCL2 is detected to be expressed in trophoblasts, decidual tissue, the myometrium, and others. Meanwhile, existing reports have determined a series of physiological regulators of CCL2, which functions in maintaining normal recruitment of immunocytes, tissue remodeling, and angiogenesis. However, abnormal levels of CCL2 have also been reported to be associated with adverse pregnancy outcomes such as spontaneous abortion, preeclampsia and preterm labor. In this review, we concentrate on CCL2 expression at the maternal-fetal interface, as well as its precise regulatory mechanisms and classic signaling pathways, to reveal the multidimensional aspects of CCL2 in pregnancy.

The Effects of 4-Week Aerobic Exercise on the Levels of CCl2, CCl5, and their Respective Receptors in Female BALB/C Mice Suffering from Breast Cancer.

The studies reported that chemokines Chemokine (C-C motif) ligand 2 (CCL2) and Chemokine (C-C motif) ligand 5 (CCL5) have tumor-promoting roles in breast cancer (BC). The aim of the present study was to evaluate the effect of 4 weeks of continuous aerobic exercise (AE) on chemokines CCL2 and CCL5 and their relative receptors in animal model of human BC. BALB/c mice were divided randomly into four groups included cancer control (CC) and three other groups. The total duration of the experiment was 14 weeks, including 2 weeks of familiarization of mice with treadmills and three of 4-week periods of experiment. Tumor inoculation and formation were performed in the second 4-week period. Group 1 received AE in the first 4-week, Group 2 received AE in the second 4-week and Group 3 in the third 4-week. The CCL2 was reduced significantly in Groups 1, 2, and 3 compared to control (F3,12= 4705, P = 0.0001). In terms of CCL5, a significant decrease was seen only between Group 3 and control. Western blot results showed a significant reduction in C-C chemokine receptor Type 2 (CCR2) between Group 1 versus CC and Group 2 versus CC (F3,20= 1.812, P = 0.004). In terms of C-C chemokine receptor Type 5 (CCR5) a significant decrease was observed between Group 2 versus control and Group 3 versus control (F3,20= 273.3, P = 0.042), (P = 0.004). It can be concluded that 4-week AE significantly reduces the chemokines CCL2 and CCL5 and their respective receptors levels CCR5 and CCR2 in different stages, and it may have an inhibitory effect on tumor growth.

Augmented effect of fibroblast growth factor 18 in bone morphogenetic protein 2-induced calvarial bone healing by activation of CCL2/CCR2 axis on M2 macrophage polarization

Fibroblast growth factor (FGF) signaling plays essential roles in various biological events. FGF18 is one of the ligands to be associated with osteogenesis, chondrogenesis and bone healing. The mouse critical-sized calvarial defect healing induced by the bone morphogenetic protein 2 (BMP2)-hydrogel is stabilized when FGF18 is added. Here, we aimed to investigate the role of FGF18 in the calvarial bone healing model. We first found that FGF18 + BMP2 hydrogel application to the calvarial bone defect increased the expression of anti-inflammatory markers, including those related to tissue healing M2 macrophage (M2-Mø) prior to mineralized bone formation. The depletion of macrophages with clodronate liposome hindered the FGF18 effect. We then examined how FGF18 induces M2-Mø polarization by using mouse primary bone marrow (BM) cells composed of macrophage precursors and BM stromal cells (BMSCs). In vitro studies demonstrated that FGF18 indirectly induces M2-Mø polarization by affecting BMSCs. Whole transcriptome analysis and neutralizing antibody treatment of BMSC cultured with FGF18 revealed that chemoattractant chemokine (c-c motif) ligand 2 (CCL2) is the major mediator for M2-Mø polarization. Finally, FGF18-augmented activity toward favorable bone healing with BMP2 was diminished in the calvarial defect in Ccr2-deleted mice. Altogether, we suggest a novel role of FGF18 in M2-Mø modulation via stimulation of CCL2 production in calvarial bone healing.

Shp2 Deficiency in Kupffer Cells and Hepatocytes Aggravates Hepatocarcinogenesis by Recruiting Non-Kupffer Macrophages

Complex communications between hepatocytes and Kupffer cells (KCs) are known to drive or suppress hepatocarcinogenesis, with controversial data in the literature. In previous experiments that aimed to decipher hepatocyte/KC interactions, we unexpectedly unveiled a tumor-suppressing effect of polyinosinic-polycytidylic acid, a widely used inducer of MX dynamin like GTPase 1 (Mx1)-cre expression, which questioned a theory of interleukin 1a/6 cytokine circuit in hepatocyte/KC communication. The goal of this study was to clarify the controversy and decipher unique functions of KCs and non-KC macrophages in liver tumorigenesis. We used the C-type lectin domain family 4 member F (Clec4f)-cre system to delete Src-homology 2 domain-containing tyrosine phosphatase 2 (Shp2)/protein tyrosine phosphatase nonreceptor 11 (Ptpn11) in KCs, and a combination of Clec4f-cre and adeno-associated virus-cre to delete Shp2 in KCs and hepatocytes to investigate the effects on hepatocellular carcinoma development and immune cell compositions/activities. Ablating Shp2 in KCs generated a tumor-promoting niche, which was exacerbated further by concurrent removal of Shp2 in both KCs and hepatocytes. Shp2 deficiency induced KC apoptosis and decreased its numbers, which induced compensatory recruitment of bone marrow-derived monocytes into liver. These newly recruited monocytes differentiated into non-KC macrophages with tumor-associated macrophage function, leading to aggravated tumor progression through down-regulation of CD8 T cells. Tumor-associated macrophage blockade by anti-chemokine (C-C motif) ligand 2 (CCL2) antibody inhibited hepatocellular carcinoma progression, while depletion of all macrophages had a tumor-promoting effect by increasing myeloid-derived suppressor cells(M-MDSCs) and decreasing CD8 T cells. Shp2 loss in KCs or hepatocytes generated a protumorigenic microenvironment, which was exacerbated by its removal in both cell types. These results show the complexity of intercellular signaling events in liver tumorigenesis and raises caution on the use of specific Shp2 inhibitor in liver cancer therapy. Transcript profiling: RNA sequencing data are available at Gene Expression Omnibus (GSE222594).

CCL2 promotes proliferation, migration and angiogenesis through the MAPK/ERK1/2/MMP9, PI3K/AKT, Wnt/β‑catenin signaling pathways in HUVECs.

Severe bone trauma can lead to poor or delayed bone healing and nonunion. Bone regeneration is based on the interaction between osteogenesis and angiogenesis. Angiogenesis serves a unique role in the repair and remodeling of bone defects. Monocyte chemoattractant protein-1, also known as CC motif ligand 2 (CCL2), is a member of the CC motif chemokine family and was the first human chemokine to be revealed to be an effective chemokine of monocytes. However, its underlying mechanism in angiogenesis of bone defect repair remains to be elucidated. Therefore, the present study investigated the detailed mechanism by which CCL2 promoted angiogenesis in bone defects based on cell and animal model experiments. In the present study, CCL2 promoted proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. Western blot analysis revealed that treatment of HUVECs with CCL2 upregulated the protein expression levels of rho-associated coiled-coil-containing protein kinase (Rock)1, Rock2, N-cadherin, c-Myc and VEGFR2. Furthermore, CCL2 promoted the expression of MAPK/ERK1/2/MMP9, PI3K/AKT and Wnt/β-catenin signaling pathway-related proteins, which also demonstrated that CCL2 promoted these functions in HUVECs. Immunohistochemical staining of Sprague Dawley rat femurs following bone defects revealed that VEGF expression was positive in the newly formed bone area in each group, while the expression area of VEGF in the CCL2 addition group was markedly increased. Therefore, CCL2 is a potential therapeutic approach for bone defect repair and reconstruction through the mechanism of angiogenesis-osteogenesis coupling.

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

27: PRE-CARDIOPULMONARY BYPASS BIOMARKERS PREDICT POOR OUTCOMES IN NEONATAL CONGENITAL HEART DISEASE

Introduction: Neonates requiring cardiopulmonary bypass (CPB) for congenital heart disease have higher rates of postoperative mortality and morbidity than older children. However, identified risk factors for poor outcome to date have been limited to non-modifiable demographic or surgical factors. We aimed to identify novel biomarkers measured preoperatively that correlate with poor outcomes. Methods: Plasma samples were collected immediately prior to CPB in 65 consented subjects. Twenty-eight soluble analytes were measured via individual or multiplexed ELISA and tested for association with the validated “ICU-30” composite poor outcome, defined as (1) mortality within 30 days of CPB, (2) ICU stay >30 days, or (3) ICU readmission within 30 days. Differences in biomarker concentrations between subjects with and without ICU-30 were assessed using a Wilcoxon rank sum test. Associations of standardized biomarkers and ICU-30 was tested with logistic regression, adjusting for surgical complexity. Results: Samples for 63 patients were included for analysis (2 subjects did not undergo CPB after blood collection); 18 subjects (28%) had the ICU-30 outcome. Patients with ICU-30 were more likely to have a chromosomal abnormality, with no difference in gestational age or birth weight. There was no difference between age at CPB surgery and CPB time during operation. Seven biomarkers differed between those with and without ICU-30: angiopoietin 2, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, growth differentiation factor-15, C-C motif ligand 2 (CCL2), and neutrophil gelatinase-associated lipocalin (NGAL) were higher in patients with ICU-30, and ADAMTS13 (A disintegrin and metalloproteinase with thrombospondin motifs 13) was lower in those with ICU-30. When controlling for STAT category, TNF-alpha (aOR 5.4, 95% CI 1.6-19.0), IL-6 (aOR 3.0, 95% CI 1.3-6.8), CCL2 (aOR 2.6, 95% CI 1.3-5.2), and NGAL (aOR 2.6, 95% CI 1.3-5.1) were associated with ICU-30. Conclusions: Four novel preoperative plasma biomarkers implicating innate immunity and renal function were associated with worse outcomes in neonates requiring CPB independent of STAT operation. Biomarkers have the potential to identify preoperative risk factors and mechanistic pathways for prognostication and targeted therapeutic interventions.