Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues.

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-l-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

Sex-dependent acrolein sensitivity in mice is associated with differential lung cell, protein, and transcript changes.

Acrolein is a reactive inhalation hazard. Acrolein's initial interaction, which in itself can be function-altering, is followed by time-dependent cascade of complex cellular and pulmonary responses that dictate the severity of the injury. To investigate the pathophysiological progression of sex-dependent acrolein-induced acute lung injury, C57BL/6J mice were exposed for 30 min to sublethal, but toxic, and lethal acrolein. Male mice were more sensitive than female mice. Acrolein of 50 ppm was sublethal to female but lethal to male mice, and 75 ppm was lethal to female mice. Lethal and sublethal acrolein exposure decreased bronchoalveolar lavage (BAL) total cell number at 3 h after exposure. The cell number decrease was followed by progressive total cell and neutrophil number and protein increases. The BAL total cell number in female mice exposed to a sublethal, but not lethal dose, returned to control levels at 16 h. In contrast, BAL protein content and neutrophil number were higher in mice exposed to lethal compared to sublethal acrolein. RNASeq pathway analysis identified greater increased lung neutrophil, glutathione metabolism, oxidative stress responses, and CCL7 (aka MCP-3), CXCL10 (aka IP-10), and IL6 transcripts in males than females, whereas IL10 increased more in female than male mice. Thus, the IL6:IL10 ratio, an indicator of disease severity, was greater in males than females. Further, H3.3 histone B (H3F3B) and pro-platelet basic protein (PPBP aka CXCL7), transcripts increased in acrolein exposed mouse BAL and plasma at 3 h, while H3F3B protein that is associated with neutrophil extracellular traps formation increased at 12 h. These results suggest that H3F3B and PPBP transcripts increase may contribute to extracellular H3F3B and PPBP proteins increase.

Oral Administration of Lipopolysaccharide Prevents Cognitive Impairment in Streptozotocin-induced Diabetic Mice in a Blood Glucose-independent Manner.

BACKGROUND/AIM: Diabetes is a risk factor for dementia. However, no radical preventive method for diabetes-associated dementia has yet been developed. Our previous study revealed that oral administration of lipopolysaccharide (LPS) prevents high-fat diet-induced cognitive impairment. Therefore, we investigated here whether oral administration of LPS (OAL) could also prevent diabetes-associated dementia. MATERIALS AND METHODS: Diabetic mice were produced by intraperitoneal administration of streptozotocin (STZ), and then mice were orally administered LPS. Cognitive ability was evaluated using the Morris water maze, and gene expression was analyzed in isolated microglia. RESULTS: OAL prevented STZ-induced diabetic cognitive impairment, but did not affect blood glucose levels. Moreover, OAL promoted the expression of neuroprotective genes in microglia, such as heat shock protein family 40 (HSP40) and chemokine CCL7. CONCLUSION: OAL prevents diabetes-associated dementia, potentially via promotion of HSP40 and CCL7 expression in microglia.

The increased intrathecal expression of the monocyte-attracting chemokines CCL7 and CXCL12 in tick-borne encephalitis.

Tick-borne encephalitis (TBE) is a relatively severe and clinically variable central nervous system (CNS) disease with a significant contribution of a secondary immunopathology. Monocytes/macrophages play an important role in the CNS inflammation, but their pathogenetic role and migration mechanisms in flavivirus encephalitis in humans are not well known. We have retrospectively analyzed blood and cerebrospinal fluid (CSF) monocyte counts in 240 patients with TBE presenting as meningitis (n = 110), meningoencephalitis (n = 114), or meningoencephalomyelitis (n = 16), searching for associations with other laboratory parameters, clinical presentation, and severity. We have measured concentrations of selected monocytes-attracting chemokines (CCL7, CXCL12, CCL20) in serum and CSF of the prospectively recruited patients with TBE (n = 15), with non-TBE aseptic meningitis (n = 6) and in non-infected controls (n = 8). The data were analyzed with non-parametric tests, p < 0.05 considered significant. Monocyte CSF count correlated with other CSF inflammatory parameters, but not with the peripheral monocytosis, consistent with an active recruitment into CNS. The monocyte count did not correlate with a clinical presentation. The median CSF concentration of CCL7 and CXCL12 was increased in TBE, and that of CCL7 was higher in TBE than in non-TBE meningitis. The comparison of serum and CSF concentrations pointed to the intrathecal synthesis of CCL7 and CXCL12, but with no evident concentration gradients toward CSF. In conclusion, the monocytes are recruited into the intrathecal compartment in concert with other leukocyte populations in TBE. CCL7 and CXCL12 have been found upregulated intrathecally but are not likely to be the main monocyte chemoattractants.

Promoting plaque stability by gene silencing of monocyte chemotactic protein-3 or overexpression of tissue factor pathway inhibitor in ApoE-/- mice.

Chemokines may promote the formation and instability of atherosclerotic plaque, which is the most common cause of acute coronary syndrome. The aim of this study was to clarify the function of monocyte chemotactic protein-3 (MCP-3) in the stability of atherosclerotic plaque, to determine the role of tissue factor pathway inhibitor (TFPI) on the development and stability of atherosclerotic plaques, and to further elucidate the anti-atherosclerotic mechanism of TFPI with the emphasis on chemokine MCP-3. We constructed an adenovirus-mediated shRNA against mouse MCP-3 (Ad-MCP-3-shRNA) and an adenovirus-containing TFPI (Ad-TFPI), and tranferred them in a model of vulnerable plaque in ApoE-/- mice respectively. Here, we reported that MCP-3-shRNA and TFPI could both reduce the plaque area and decrease the content of lipids and macrophages, on the contrary, the fibrous cap thickness and content of collagen and smooth muscle cells were increased. In addition, the expression of MCP-3 and CC chemokine receptor 2 (CCR2) was decreased by TFPI transfer. These data provide the first in vivo evidence that MCP-3 is a major contributor to the unstability of atherosclerotic plaque and TFPI may exert its anti-atherosclerotic effects and promote stabilisation of plaque at least partly through inhibiting MCP-3/CCR2 pathway, which may be a new therapeutic method for atherosclerosis.

Association of High-Mobility Group Box-1 with Inflammationrelated Cytokines in the Aqueous Humor with Acute Primary Angle-Closure Eyes.

AIM: The aim of this study was to measure the levels of High-mobility group box-1 (HMGB1) and inflammation-related cytokines in the aqueous humor of patients with acute primary angle-closure glaucoma (APAG) and age-related cataract eyes (ARC). METHODS: Aqueous humor samples were obtained from 59 eyes of 59 Chinese subjects (APAG, 32 eyes; and ARC, 27eyes). The multiplex bead immunoassay technique was used to measure the levels of HMGB1 and IL-8, IL-6, G-CSF, MCP-3, VEGF, sVEGFR- 1, sVEFGR-2, TNF-α, PDGF, and IL-10 in aqueous. The data of Patients' demographics and preoperative intraocular pressure (IOP) were also collected for detailed analysis. RESULTS: The APAG group showed significantly elevated concentrations of HMGB1, IL- 8, IL-6, G-CSF, VEGF, sVEGFR-1, and TNF-α than those in the ARC group. Aqueous HMGB1 level correlated significantly with IOP, IL-8, IL-6, G-CSF and sVEGFR-1 levels but not with age, TNF-α, or VEGF levels. CONCLUSION: The aqueous level of HMGB1 is elevated in APAG and associated with aqueous level of inflammation-related cytokines, suggesting an association between elevated levels of HMGB1, APAC and certain inflammatory modulators which, of course, should lead to further investigations in order to demonstrate the cause and effect.

CCL7 recruits cDC1 to promote antitumor immunity and facilitate checkpoint immunotherapy to non-small cell lung cancer.

The efficacy of checkpoint immunotherapy to non-small cell lung cancer (NSCLC) largely depends on the tumor microenvironment (TME). Here, we demonstrate that CCL7 facilitates anti-PD-1 therapy for the KrasLSL-G12D/+Tp53fl/fl (KP) and the KrasLSL-G12D/+Lkb1fl/fl (KL) NSCLC mouse models by recruiting conventional DC 1 (cDC1) into the TME to promote T cell expansion. CCL7 exhibits high expression in NSCLC tumor tissues and is positively correlated with the infiltration of cDC1 in the TME and the overall survival of NSCLC patients. CCL7 deficiency impairs the infiltration of cDC1 in the TME and the subsequent expansion of CD8+ and CD4+ T cells in bronchial draining lymph nodes and TME, thereby promoting tumor development in the KP mouse model. Administration of CCL7 into lungs alone or in combination with anti-PD-1 significantly inhibits tumor development and prolongs the survival of KP and KL mice. These findings suggest that CCL7 potentially serves as a biomarker and adjuvant for checkpoint immunotherapy of NSCLC.

The chemokine CCL7 regulates invadopodia maturation and MMP-9 mediated collagen degradation in liver-metastatic carcinoma cells.

Liver metastases remain a major cause of death from gastrointestinal tract cancers and other malignancies, such as breast and lung carcinomas. Understanding the underlying biology is essential for the design of effective therapies. We previously identified the chemokine CCL7 and its receptor CCR3 as critical mediators of invasion and metastasis in lung and colon carcinoma cells. Here we show that the CCL7/CCR3 axis regulates a late stage in invadopodia genesis namely, the targeting of MMP-9 to the invadopodia complex, thereby promoting invadopodia maturation and collagen degradation. We show that this process could be blocked by overexpression of a dominant negative RhoA in highly invasive cells, while a constitutively active RhoA upregulated invadopodia maturation in CCL7-silenced and poorly invasive and metastatic cells and also enhanced their metastatic potential in vivo, collectively, implicating RhoA activation in signaling downstream of CCL7. Blockade of the ERK or PI3K pathways by chemical inhibitors also inhibited invadopodia formation, but affected the initiation stage of invadopodia genesis. Our data implicate CCL7/CCR3 signaling in invadopodia maturation and suggest that chemokine signaling acts in concert with extracellular matrix-initiated signals to promote invasion and liver metastasis.

Intestinal Epithelial Chemokine (C-C Motif) Ligand 7 Overexpression Enhances Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose-induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. The related injury pathogenesis is mainly focused on the liver. Here, the authors report that gut barrier disruption may also be involved in APAP hepatotoxicity. APAP administration led to gut leakiness and colonic epithelial chemokine (C-C motif) ligand 7 (CCL7) up-regulation. Intestinal epithelial cell (IEC)-specific CCL7 transgenic mice (CCL7tgIEC mice) showed markedly increased myosin light chain kinase phosphorylation, and elevated gut permeability and bacterial translocation into the liver compared to wild-type mice. Global transcriptome analysis revealed that the expression of hepatic proinflammatory genes was enhanced in CCL7tgIEC mice compared with wild-type animals. Moreover, CCL7 overexpression in intestinal epithelial cells significantly augmented APAP-induced acute liver injury. These data provide new evidence that dysfunction of CCL7-mediated gut barrier integrity may be an important contributor to APAP-induced hepatotoxicity.

The Microbiota Promotes Arterial Thrombosis in Low-Density Lipoprotein Receptor-Deficient Mice.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient (Ldlr-/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr-/- mice and CONV-R Ldlr-/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr-/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr-/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr-/- mice on HFD were diminished compared to CONV-R Ldlr-/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr-/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr-/- mice relative to CONV-R Ldlr-/- mice. Ex vivo, this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr-/- mice on type III collagen.IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr-/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions.

Hypoxia inducible factor 1α in vascular smooth muscle cells promotes angiotensin II-induced vascular remodeling via activation of CCL7-mediated macrophage recruitment.

The process of vascular remodeling is associated with increased hypoxia. However, the contribution of hypoxia-inducible factor 1α (HIF1α), the key transcription factor mediating cellular hypoxic responses, to vascular remodeling is established, but not completely understood. In the angiotensin II (Ang II)-induced vascular remodeling model, HIF1α was increased and activated in vascular smooth muscle cells (VSMCs). Selective genetic disruption of Hif1a in VSMCs markedly ameliorated Ang II-induced vascular remodeling, as revealed by decreased blood pressure, aortic thickness, collagen deposition, inflammation, and aortic stiffness. VSMC Hif1a deficiency also specifically suppressed Ang II-induced infiltration of CD45+CD11b+F4/80+CD206- M1 macrophages into the vessel. Mechanistically, HIF1α deficiency in VSMCs dramatically suppressed the expression of CCL7, a chemokine critical for macrophage recruitment. Bioinformatic analysis and chromatin immunoprecipitation assays revealed three functional hypoxia-response elements in the Ccl7 promoter, indicating that Ccl7 is a direct HIF1α target gene. Blocking CCL7 with antibody in vivo alleviated Ang II-induced hypertension and vascular remodeling, coincident with decreased macrophage infiltration. This study provides direct evidence that HIF1α activation in VSMCs exacerbates Ang II-induced macrophage infiltration and resultant vascular remodeling via its target gene Ccl7, and thus may serve as a potential therapeutic target for remodeling-related vascular disease.

A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.

FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg2+ and Ca2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.

Effect of Chemokine (C-C Motif) Ligand 7 (CCL7) and Its Receptor (CCR2) Expression on Colorectal Cancer Behaviors.

Colorectal cancer is the source of one of the most common cancer-related deaths worldwide, where the main cause of patient mortality remains metastasis. The aim of this study was to determine the role of CCL7 (chemokine (C-C motif) ligand 7) in tumor progression and finding whether it could predict survival of colorectal cancer patients. Initially, our study focused on the crosstalk between mesenchymal stem cells (MSCs) and CT26 colon carcinoma cells and resulted in identifying CCL7 as a chemokine upregulated in CT26 colon cancer cells cocultured with MSCs, compared with CT26 in monoculture in vitro. Moreover, we showed that MSCs enhance CT26 tumor cell proliferation and migration. We analyzed the effect of CCL7 overexpression on tumor progression in a murine CT26 model, where cells overexpressing CCL7 accelerated the early phase of tumor growth and caused higher lung metastasis rates compared with control mice. Microarray analysis revealed that tumors overexpressing CCL7 had lower expression of immunoglobulins produced by B lymphocytes. Additionally, using Jh mutant mice, we confirmed that in the CT26 model, CCL7 has an immunoglobulin-, and thereby, B-cell-dependent effect on metastasis formation. Finally, higher expression of CCL7 receptor CCR2 (C-C chemokine receptor type 2) was associated with shorter overall survival of colorectal cancer patients. Altogether, we showed that CCL7 is essentially involved in the progression of colorectal cancer in a CT26 mouse model and that the expression of its receptor CCR2 could be related to a different outcome pattern of patients with colorectal carcinoma.

Effects of electroacupuncture combined with stem cell transplantation on anal sphincter injury-induced faecal incontinence in a rat model.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) and acupuncture are known to mitigate tissue damage. This study aimed to investigate the therapeutic effects of combined electroacupuncture (EA) stimulation and BMSC injection in a rat model of anal sphincter injury-induced faecal incontinence (FI). METHODS: 60 Sprague-Dawley rats were randomly divided into five groups: sham-operated control, FI, FI+EA, FI+BMSC, and FI+BMSC+EA. The anorectal tissues were collected on days 1, 3, 7 and 14. Repair of the injured anal sphincter was compared using haematoxylin and eosin (HE) and immunocytochemiscal analyses with sarcomeric α actinin. The expression of stromal cell derived factor-1 (SDF-1) and monocyte chemoattractant protein-3 (MCP-3) was detected by quantitative reverse transcription PCR to evaluate the effects of EA on the homing of BMSCs. RESULTS: The therapeutic effect of combined EA+BMSCs on damaged tissue was the strongest among all the groups as indicated by HE and immunohistochemical staining. The expression of SDF-1 and MCP-3 was significantly increased by combined EA and BMSC treatment when compared with the other groups (P=0.01 to P<0.05), suggesting promotive effects of EA on the homing of BMSCs. CONCLUSION: The combination of EA and BMSC transplantation effectively repaired the impaired anal sphincters. The underlying mechanism might be associated with apparent promotive effects of EA on the homing of BMSCs. Our study provides a theoretical basis for the development of a non-surgical treatment method for FI secondary to muscle impairment.

CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Leishmania major Infection.

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7-/- C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7-/- mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control.

miR-23b Suppresses Leukocyte Migration and Pathogenesis of Experimental Autoimmune Encephalomyelitis by Targeting CCL7.

MicroRNAs (miRNAs) are small, non-coding RNAs involved in immune response regulation. Specific miRNAs have been linked to the development of various autoimmune diseases; however, their contribution to the modulation of CNS-directed cellular infiltration remains unclear. In this study, we found that miR-23b, in addition to its reported functions in the suppression of IL-17-associated autoimmune inflammation, halted the progression of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), by directly inhibiting the migration of pathogenic leukocytes to the CNS. We demonstrated that miR-23b was specifically decreased during the acute phase of EAE and that overexpression of miR-23b resulted in a defect in leukocyte migration and strong resistance to EAE. Furthermore, we found that miR-23b suppressed leukocyte migration of EAE by targeting CCL7, a chemokine that attracts monocytes during inflammation and metastasis. Finally, in the adoptive transfer model, miR-23b reduced the severity of EAE by inhibiting the migration of pathogenic T cells to the CNS rather than diminishing the encephalitogenesis of T cells. Taken together, our results characterize a novel aspect of miR-23b function in leukocyte migration, and they identify miR-23b as a potential therapeutic target in the amelioration of MS and likely other autoimmune diseases.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

Effect of Pregnancy and Delivery on Cytokine Expression in a Mouse Model of Pelvic Organ Prolapse.

OBJECTIVES: The aim of this study was to determine the effect of pregnancy and delivery mode on cytokine expression in the pelvic organs and serum of lysyl oxidase like-1 knockout (LOXL1 KO) mice, which develop pelvic organ prolapse after delivery. METHODS: Bladder, urethra, vagina, rectum, and blood were harvested from female LOXL1 KO mice during pregnancy, after vaginal or cesarean delivery, and from sham cesarean and unmanipulated controls. Pelvic organs and blood were also harvested from pregnant and vaginally delivered wild-type (WT) mice and from unmanipulated female virgin WT controls. Specimens were assessed using quantitative real-time reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS: Both CXCL12 and CCL7 mRNA were significantly up-regulated in the vagina, urethra, bladder, and rectum of pregnant LOXL1 KO mice compared with pregnant WT mice, suggesting systemic dysregulation of both of these cytokines in LOXL1 KO mice as a response to pregnancy.The differences in cytokine expression between LOXL1 KO and WT mice in pregnancy persisted after vaginal delivery. CCL7 gene expression increases faster and to a greater extent in LOXL1 KO mice, translating to longer lasting increases in CCL7 in serum of LOXL1 KO mice after vaginal delivery, compared with pregnant mice. CONCLUSIONS: Lysyl oxidase like-1 KO mice have an increased cytokine response to pregnancy perhaps because they are less able to reform and re-cross-link stretched elastin to accommodate pups, and this resultant tissue stretches during pregnancy. The up-regulation of CCL7 after delivery could provide an indicator of level of childbirth injury, to which the urethra and vagina seem to be particularly vulnerable.

Role of Interleukin-17A on the Chemotactic Responses to CCL7 in a Murine Allergic Rhinitis Model.

BACKGROUND: The proinflammatory cytokine interleukin (IL)-17A is associated with eosinophil infiltration into the nasal mucosa in a mouse model of ovalbumin-induced allergic rhinitis. Chemotaxis of eosinophils is mediated primarily through C-C chemokine receptor type 3 (CCR3). However, the mechanism underlying the IL-17A-mediated enhancement of eosinophil recruitment via chemoattractants/chemokines remains unknown. OBJECTIVES: In this study, we assessed the contribution of IL-17A to eosinophil-related inflammation via the CCL7/CCR3 pathway in experimental allergic rhinitis. METHODS: IL-17A knockout (KO) and wild-type (WT) BALB/c mice were injected intraperitoneally and challenged intranasally with OVA to induce allergic rhinitis. Various parameters of the allergic response were evaluated, and mRNA and protein levels of CCL7 and CCR3 in nasal tissue and serum were compared between the two groups. The chemotactic response to CCL7 with or without IL-17A in bone marrow-derived eosinophils (bmEos) from BALB/c mice was measured. RESULTS: In the allergic rhinitis model, IL-17A deficiency significantly decreased nasal symptoms, serum IgE levels, and eosinophil recruitment to the nasal mucosa. CCL7 and CCR3 mRNA and protein levels were decreased in the nasal mucosa of IL-17A KO mice compared with the WT mice. BmEos showed a significantly increased chemotactic response to -low concentration of CCL7 in the presence of IL-17A compared with its absence. CONCLUSION: The suppression of nasal inflammation due of IL-17A deficiency in allergic rhinitis is partly responsible for the regulation of CCL7 secretion and eosinophil infiltration, which may be regulated via the CCL7/CCR3 pathway.