Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1alpha (CCL3) whose expression was induced by the Th1 cytokines IL-1beta and IFN-gamma. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.     

The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors

Tumor-associated chemokines, including CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2), are thought to play many roles in cancer progression. Here we demonstrate the novel finding that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is a dramatic up-regulation of CCL2 in splenic T cells at both the mRNA and protein levels upon stimulation. Of particular relevance is the finding that tumor-infiltrating T cells also produce high levels of CCL2. While a variety of tumor cell lines have been found to produce CCL2, we found no detectable levels of CCL2 protein in supernatants of the cultured mammary tumor cells. Investigation of the mechanisms involved in CCL2 induction showed that treatment of splenic T cells with the tumor-derived factors GM-CSF and phosphatidyl serine (PS) resulted in increased CCL2 production. This increased production may be involved in the downregulation of IFN-gamma by the T cells of tumor-bearing mice previously reported in this model, as treatment of splenic T lymphocytes with CCL2 resulted in a decreased secretion of IFN-gamma by those cells.     

CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia

Unmethylated CpG oligodeoxynucleotides (CpG-ODNs) interact with Toll-like receptor (TLR) 9 to activate macrophage/microglia in central nervous system (CNS). Here, we investigated the potential involvement of the chemokine CCL9 and its receptor CCR1 in the effects of CpG-ODNs on macrophage/microglial cells. CpG-ODNs enhanced the expression of TLR9 mRNA of RAW264.7 macrophage and BV2 microglia cells time dependently. The expression of CCL9 of macrophages/microglia showed different responsiveness upon stimulation with a variety of CpG-ODN sequences. The CpG-ODNs-mediated induction of CCL9 was TLR9/MyD88 dependent and associated with activation of stress kinases, particularly ERK, p38 MAPK and PI3K. The expression of CCR1 was also significantly increased by CpG-ODNs that increased CCL9 expression. These results reveal the potential involvement of CCL9 and CCR1 in regulation of macrophage and microglial cells by CpG-ODNs and may help improving our understanding about the role of the chemokine/chemokine receptor pairs in macrophage/microglia under physiologic and pathologic conditions.     

Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.     

MicroRNA‐125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age‐associated change is not fully understood. Here, we report the role of microRNA‐125b (miR‐125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T‐cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age‐related increase in CCL4. We then found the level of miR‐125b was inversely correlated with the level of CCL4 in these cells, and the level of miR‐125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock‐down of miR‐125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR‐125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR‐125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR‐125b is a negative regulator of CCL4 and its reduction is partially responsible for the age‐related increase of CCL4.     

Sirt3 is essential for apelin‐induced angiogenesis in post‐myocardial infarction of diabetes

Heart failure following myocardial infarction (MI) is the leading cause of death in diabetic patients. Angiogenesis contributes to cardiac repair and functional recovery in post‐MI. Our previous study shows that apelin (APLN) increases Sirtuin 3 (Sirt3) expression and ameliorates diabetic cardiomyopathy. In this study, we further investigated the direct role of Sirt3 in APLN‐induced angiogenesis in post‐MI model of diabetes. Wild‐type (WT) and Sirt3 knockout (Sirt3KO) mice were induced into diabetes by i.p. streptozotocin (STZ). STZ mice were then subjected to MI followed by immediate intramyocardial injection with adenovirus‐apelin (Ad‐APLN). Our studies showed that Sirt3 expression was significantly reduced in the hearts of STZ mice. Ad‐APLN treatment resulted in up‐regulation of Sirt3, angiopoietins/Tie‐2 and VEGF/VEGFR2 expression together with increased myocardial vascular densities in WT‐STZ+MI mice, but these alterations were not observed in Sirt3KO‐STZ+MI mice. In vitro, overexpression of APLN increased Sirt3 expression and angiogenesis in endothelial progenitor cells (EPC) from WT mice, but not in EPC from Sirt3KO mice. APLN gene therapy increases angiogenesis and improves cardiac functional recovery in diabetic hearts via up‐regulation of Sirt3 pathway.     

The absence of oestrogen receptor beta disturbs collagen I type deposition during Achilles tendon healing by regulating the IRF5‐CCL3 axis

Achilles tendon healing (ATH) remains an unanswered question in the field of sports medicine because it does not produce tissue with homology to the previously uninjured tissue. Oestrogen receptor β (ERβ) is involved in the injury and repair processes of tendons. Our previous study confirmed that ERβ plays a role in the early stage of ATH by affecting adipogenesis, but its role in extracellular matrix (ECM) remodelling is unknown. We established a 4‐week Achilles tendon repair model to investigate the mechanism through which ERβ affects ATH at the very beginning of ECM remodelling phase. In vitro studies were performed using tendon‐derived stem cells (TDSCs) due to their promising role in tendon healing. Behavioural and biomechanical tests revealed that ERβ‐deficient mice exhibit weaker mobility and inferior biomechanical properties, and immunofluorescence staining and qRT‐PCR showed that these mice exhibited an erroneous ECM composition, as mainly characterized by decreased collagen type I (Col I) deposition. The changes in gene expression profiles between ERβ‐knockout and WT mice at 1 week were analysed by RNA sequencing to identify factors affecting Col I deposition. The results highlighted the IRF5‐CCL3 axis, and this finding was verified with CCL3‐treated TDSCs. These findings revealed that ERβ regulates Col I deposition during ATH via the IRF5‐CCL3 axis.     

CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

Metastasis is one of the primary causes for high mortality in patients with squamous cell carcinoma of the head and neck (SCCHN). Our previous study showed that chemokine (C‐C motif) ligand 18 (CCL18), derived from tumour‐associated macrophages (TAMs), regulates SCCHN metastasis by promoting epithelial‐mesenchymal transition (EMT) and preserving stemness. However, the underlying mechanism needs to be further investigation. Interestingly, metadherin (MTDH) expression was induced when SCCHN cells were stimulated with recombinant CCL18 protein in this study. Suppressing MTDH expression reversed CCL18‐induced migration, invasion and EMT in SCCHN cells. Furthermore, the NF‐κB signalling pathway was involved in the MTDH knock‐down cells with CCL18 stimulation. We performed ELISA to evaluate the CCL18 levels in the serums of 132 treatment‐naive SCCHN patients, 25 patients with precancerous lesion and 32 healthy donors. Our results demonstrated that serum CCL18 levels were significantly higher in SCCHN patients than patients with precancerous lesion and healthy individuals. CCL18 levels were found to be significantly correlated with tumour classification, clinical stage, lymph node metastasis and histological grade in SCCHN patients. Thus, our findings suggest that CCL18 may serve as a potential biomarker for diagnosis of SCCHN and promote SCCHN invasion, migration and EMT by MTDH‐NF‐κB signalling pathway.     

Clinical grade production and characterization of a fusion protein comprised of the chemokine CCL2-ligand genetically fused to a mutated and truncated form of the Shiga A1 subunit

First generation chemokine ligand-Shiga A1 (SA1) fusion proteins (leukocyte population modulators, LPMs) were previously only obtained in small quantities due to the ribosomal inactivating protein properties of the SA1 moiety which inhibits protein synthesis in host cells. We therefore employed 4-aminopyrazolo[3,4-d]-pyrimidine, an inhibitor of Shiga A1, to allow the growth of these cells prior to induction and during the expression phase post-induction with IPTG. Scale-up allowed the production of gram quantities of clinical grade material of the lead candidate, OPL–CCL2–LPM. A manufacturing cell bank was established and used to produce OPL–CCL2–LPM in a fed-batch fermentation process. Induction of the expression of OPL–CCL2–LPM led to the production of 22.47 mg/L per OD₆₀₀ unit. The LPM was purified from inclusion bodies using solubilization, renaturation, refolding and chromatography steps. The identity and purity of the OPL–CCL2–LPM was determined using several analytical techniques. The product retained the ability of the SA1 moiety to inhibit protein synthesis as measured in a rabbit reticulocyte lysate cell-free protein synthesis assay and was cytotoxic to target cells. Binding studies established that the protein exerts its effects via CCR2, the cognate receptor for CCL2. Clinical trials in inflammatory nephropathies are planned.     

CCL5 induces a pro-inflammatory profile in microglia in vitro

The chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR2 have been found to be expressed on microglia in many neurodegenerative diseases, such as multiple sclerosis and Alzheimer’s disease. There is emerging evidence that chemokines, besides chemoattraction, might directly modulate reactive profiles of microglia. To address this hypothesis we have investigated the effects of CCL2, CCL3, CCL5, and CXCL1 on cytokine and growth factor production, NO synthesis, and phagocytosis in non-stimulated and lipopolysaccharide-stimulated primary rat microglia. The respective receptors CCR1, CCR5, and CXCR2 were shown to be functionally expressed on microglia. All tested chemokines stimulated chemotaxis whereas only CCL5 increased NO secretion and attenuated IL-10 as well as IGF-1 production in activated microglia. Based on these findings we propose that besides its chemoattractant function CCL5 has a modulatory effect on activated microglia.     

CCR1 and CC chemokine ligand 5 interactions exacerbate innate immune responses during sepsis

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1(-/-)) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1(-/-) mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1(-/-) resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1(-/-) mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-gamma and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.     

CCR5 deficiency aggravates crescentic glomerulonephritis in mice

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.     

CCL5 secreted by senescent theca-interstitial cells inhibits preantral follicular development via granulosa cellular apoptosis

As a fundamental aging mechanism, cellular senescence causes chronic inflammation via the senescence-associated secretory phenotype (SASP). Theca-interstitial cells are an essential but little-studied component of follicle development in the ovarian microenvironment. In the present study, we observed significant cellular senescence in theca-interstitial cells and secretion of chemokine (C-C motif) ligand 5 (CCL5) by these cells during aging. Furthermore, we aimed to investigate whether and how senescence-associated secretory phenotype (SASP)-associated CCL5 may be involved in follicle development. Increased levels of CCL5 in the microenvironment of follicles attenuated preantral follicle growth, survival, and estradiol secretion. Oocyte maturation and the expression of zona pellucida 3 and differentiation factor 9 (GDF9) were also inhibited by CCL5. Granulosa cell apoptosis in follicles was promoted by CCL5, accompanied by the phosphorylation of nuclear factor-κB by CCL5 and inhibition of the PI3K/AKT pathway. These results suggest that SASP-associated CCL5 produced by senescent theca-interstitial cells may impair follicle development and maturation during ovarian aging by promoting granulosa cell apoptosis.     

Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis

Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)alpha1beta2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTalpha1beta2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTbetaR-Fc antagonized development of organized lymphoid structures. LTalpha1beta2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTalpha1beta2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.     

Chemokine receptor CCR5 deficiency exacerbates cerulein-induced acute pancreatitis in mice

Acute pancreatitis (AP) is an inflammatory disease involving the production of different cytokines and chemokines and is characterized by leukocyte infiltration. Because the chemokine receptor CCR5 and its ligands [the CC chemokines CCL3/MIP-1alpha, CCL4/MIP-1beta, and CCL5/regulated upon activation, normal T cell expressed and secreted (RANTES)] regulate leukocyte chemotaxis and activation, we investigated the expression of CCR5 ligands and the role of CCR5 and its ligands in experimental AP in mice. AP was induced by hourly intraperitoneal injections of cerulein in CCR5-deficient (CCR5(-/-)) or wild-type (WT) mice. Induction of AP by cerulein resulted in an early increase of pancreatic CCL2, CCL3, and CCL4 mRNA expression, whereas CCL5 mRNA expression occurred later. CCR5(-/-) mice developed a more severe pancreatic injury than WT mice during cerulein-induced AP, as assessed by a more pronounced increase in serum amylase and lipase levels and by more severe pancreatic edema, inflammatory infiltrates (mainly neutrophils), and necrosis. CCR5(-/-) mice also exhibited increased production of CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-1beta during the course of cerulein-induced AP. In vivo simultaneous neutralization of CC chemokines with monoclonal antibodies in CCR5(-/-) mice reduced the severity of cerulein-induced AP, indicating a role of CC chemokines in exacerbating the course of AP in the absence of CCR5. Moreover, simultaneous neutralization of CCR5 ligands in WT mice also reduced the severity of cerulein-induced AP. In conclusion, lack of the chemokine receptor CCR5 exacerbates experimental cerulein-induced AP and leads to increased levels of CC chemokines and a more pronounced pancreatic inflammatory infiltrate, suggesting that CCR5 expression can modulate severity of AP.     

Osteoclast differentiation gene expression profiling reveals chemokine CCL4 mediates RANKL‐induced osteoclast migration and invasion via PI3K pathway

The migration of osteoclasts (OCs) from circulation and bone marrow into bone surface plays a critical role in the pathogenesis of some bone resorptive diseases, such as rheumatoid arthritis and osteoporosis. To date, how the migration of OCs remains unclear. We investigated gene expression profiling in osteoclastic differentiation of bone marrow–derived macrophages (BMMs) into OCs by microarray analysis. We identified 387 genes overexpressed in osteoclastic differentiation of BMMs. Among them, chemokine CCL4 showed a robust up‐regulation signal. High expression of CCL4 was validated in primary BMMs and OC precursor cell line RAW264.7 during differentiation into OCs. The CCL4 neutralization decreased RANKL‐induced OC precursor cell migration and invasion in Matrigel‐coated transwell membranes assay and in vitro wound healing assay. However, CCL4 inhibition did not affect OCs differentiation and differentiation associated gene expression. The CCL4 inhibition promoted the PI3K phosphorylation at 45 to 60 minutes after RANKL stimulation in RAW264.7. This study indicated that chemokine CCL4 is an important regulator for OCs migration via PI3K pathway, providing a novel therapy target for bone resorptive diseases.     

Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis

Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses. We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic. Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2(+) alveolar macrophage population, and CCR2-deficient mice transfused with CCR2(+) mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin. Our data show that alveolar monocyte recruitment is strictly dependent on CCR2. LPS-induced neutrophil migration to the lungs is CCR2 independent. However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified. We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation.     

Cilia‐localized LKB1 regulates chemokine signaling,macrophage recruitment,and tissue homeostasis in the kidney

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy‐related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell‐autonomous manner and results in peritubular accumulation of CCR2⁺ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.