C-C Motif Chemokine Ligand 2 Research Articles

Abstract 12479: Cardiovascular Stroke Nursing Best Abstract Award: Sex Differences in Relationships Between Inflammation and Symptoms in Persons With Heart Failure

Background: Heart failure (HF) is a complex syndrome involving interplay of pathologic processes, such as inflammation and myocardial remodeling. Sex differences in cardiac remodeling and function are linked to differences in inflammation. Female hearts show a pro-inflammatory shift and increased cytokine expression with aging not seen in male hearts. Yet, sex is not often considered in HF interventions. Previous studies suggest sex may moderate the relationship between inflammation and symptoms in cardiometabolic disease, but the relationship in HF is unknown. Purpose: To examine sex differences in inflammation and symptoms in Black adults living with HF. Methods: Black adults living with HF (N=41) were enrolled in this pilot study. Physical symptoms were measured with PROMIS Dyspnea, Heart Failure Somatic Perception Scale (HFSPS), and Multidimensional Fatigue Inventory (MFI). Mood symptoms were measured with Center for Epidemiological Studies Depression Scale (CESD), State-Trait Anxiety Inventory (STAI), and Perceived Stress Scale (PSS). Inflammatory biomarkers were measured via multiplex immunoassay. Xanthine oxidase (XO) activity, an indicator of oxidative stress, was measured via ELISA. Pearson correlations were stratified by sex. Results: The mean age was 57 ± 11 years, and 66% were female. Dyspnea was positively associated with XO activity in both groups. Among females, dyspnea was positively associated with matrix metalloproteinase (MMP)-12 (r=.519, p=.033), interleukin (IL)-6 (p=.564, p=.018), [C-C motif chemokine ligand(CCL)-3, (r=.509, p=.037), and CCL-11 (r=.520, p=.032). HFSPS was positively associated with XO activity among females only (r=.445, p=.014). CCL-11 was positively associated with MFI (r=.616, p=.008), PSS (p=.0537, r=.032), STAI (r=.714, p=.001), and CESD (r=.635, p=.008) symptoms among females but not males. Conclusions: While physical symptoms were associated with similar inflammatory pathways, fatigue and mood symptoms were associated with inflammation among females only. Women may be more vulnerable to inflammation-induced mood changes and fatigue. Further work is needed to better understand how sex modulates the relationship between inflammation and symptoms in persons with HF.

Intestinal monocarboxylate transporter 1 mediates lactate transport in the gut and regulates metabolic homeostasis of mouse in a sex-dimorphic pattern

Abstract The monocarboxylate transporter 1 (MCT1), encoded by gene Slc16a1, is a proton-coupled transporter for lactate and other monocarboxylates. MCT1-mediated lactate transport was recently found to regulate various biological functions. However, how MCT1 and lactate in the intestine modulate the physiology and pathophysiology of the body is unclear. In this study, we generated a mouse model with specific deletion of Slc16a1 in the intestinal epithelium (Slc16a1IKO mice) and investigated the functions of MCT1 in the gut. When fed a high-fat diet, Slc16a1IKO male mice had improvement in glucose tolerance and insulin sensitivity, while Slc16a1IKO female mice only had increased adiposity. Deficiency of intestinal MCT1 in male mice was associated with downregulation of pro-inflammatory pathways, together with decreased circulating levels of inflammatory cytokines including tumor necrosis factor alpha (TNFα) and C–C motif chemokine ligand 2 (CCL2). Lactate had a stimulatory effect on pro-inflammatory macrophages in vitro. The number of intestinal macrophages was reduced in Slc16a1IKO male mice in vivo. Intestinal deletion of Slc16a1 in male mice reduced interstitial lactate level in the intestine. In addition, treatment of male mice with estrogen lowered interstitial lactate level in the intestine and abolished the difference in glucose homeostasis between Slc16a1IKO and wild-type mice. Deficiency of intestinal MCT1 also blocked the transport of lactate and short-chain fatty acids from the intestine to the portal vein. The effect of Slc16a1 deletion on glucose homeostasis in male mice was partly mediated by alterations in gut microbiota. In conclusion, our work reveals that intestinal MCT1 regulates glucose homeostasis in a sex-dependent manner.

Chronic pain accelerates cognitive impairment by reducing hippocampal neurogenesis may via CCL2/CCR2 signaling in APP/PS1 mice

Patients with chronic pain often have cognitive impairment; this is especially true in elderly patients with neurodegenerative diseases such as Alzheimer's disease (AD), but the mechanism underlying this association remains unclear. This was addressed in the present study by investigating the effect of chronic neuropathic pain on hippocampal neurogenesis and cognitive impairment using amyloid precursor protein/presenilin 1 (APP/PS1) double transgenic mice subjected to spared-nerve injury (SNI). The Von Frey test was performed to determine the mechanical threshold of mouse hind limbs after SNI. The Morris water maze test was used to evaluate spatial learning and memory. Doublecortin-positive (DCX+), 5-bromo-2′-deoxyuridine (BrdU)+, BrdU+/neuronal nuclei (NeuN)+, and C-C motif chemokine ligand 2 (CCL2)+ neurons in the dentate gyrus of the hippocampus were detected by immunohistochemistry and immunofluorescence analysis. CCL2 and C-C chemokine receptor type 2 (CCR2) protein levels in the mouse hippocampus were analyzed by western blotting. The results showed that APP/PS1 mice with chronic neuropathic pain induced by SNI had significant learning and memory impairment. This was accompanied by increased CCL2 and CCR2 expression and decreases in the number of DCX+, BrdU+, and BrdU+/NeuN+ neurons. These results suggest that chronic neuropathic pain is associated with cognitive impairment, which may be caused by CCL2/CCR2 signaling-mediated inhibition of hippocampal neurogenesis. Thus, therapeutic strategies that alleviate neuropathic pain can potentially slow cognitive decline in patients with AD and other neurodegenerative diseases.

Axatilimab Ameliorates Inflammation and Fibrosis By Targeting Macrophages in a Preclinical Model of Chronic GVHD

Introduction: Chronic graft-versus-host disease (cGVHD) is an immune-mediated serious and life-threatening complication after allogeneic hematopoietic stem cell transplantation, occurring in 30%-70% of patients. The pathophysiology of cGVHD may involve inflammation, cell-mediated immunity, humoral immunity, and fibrosis. Axatilimab is a high-affinity humanized IgG4 (kappa light chain) monoclonal antibody targeting colony-stimulating factor 1 receptor (CSF-1R) that is under investigation for the treatment of cGVHD. CSF-1R is a member of the receptor protein tyrosine kinase family of growth factor receptors and is expressed on monocyte precursors, monocytes, and macrophages. Signaling by colony-stimulating factor 1 (CSF-1) or interleukin-34 (IL-34) through CSF-1R has been shown to be the primary regulatory pathway for determining development and differentiation of the mononuclear phagocytic cell lineage. Recent work has established a role for monocyte-derived macrophages as the pathogenic population of cells that establish the fibrotic niche and drive the fibrotic process. Upon entry of monocytes into tissues, the tissue microenvironment regulates their differentiation, resulting in a heterogeneous pool of cells ranging from pro-inflammatory (often referred to as M1 macrophages) to immune suppressive/wound healing (M2 macrophages). An imbalance in the number and activity of these macrophage populations is believed to result in the excess production of fibrotic growth factors, such as transforming growth factor-β and platelet-derived growth factor, leading to uncontrolled fibroblast activation and subsequent fibrosis. Blocking CSF-1R signaling with axatilimab is expected to reduce the circulating levels of pathogenic monocyte-derived macrophage precursors and inhibit their activation in tissues, thereby providing an opportunity to therapeutically intervene in diseases such as cGVHD. The results described herein support this assertion by demonstrating the direct impact of axatilimab exposure to skewing differentiating monocytes away from a pro-inflammatory and pro-fibrotic phenotype. Methods: Primary human monocytes were differentiated in the presence or absence of ascending doses of axatilimab (0.07-7 μM) into distinct phenotypes: activated (M0), pro-inflammatory (M1-like), and suppressive (M2-like) macrophages. Supernatants and cell pellets were then assessed for secreted protein (Luminex Corporation) and gene expression (NanoString Technologies) profiles, respectively. Data were analyzed in R (version 4.1.1). Results: Gene expression profiles of pro-inflammatory cytokines and chemokines (eg, IL-1β, chemokine CC motif ligand 2 [CCL2], CCL7, CCL24, and CCL22) from primary differentiated macrophage subsets were dramatically reduced in the presence of ascending concentrations of axatilimab. Further, macrophages exposed to axatilimab while differentiated under pro-inflammatory conditions showed increased levels of c-MYC expression compared with control cultures. c-MYC is a transcription factor associated with suppressive M2-like macrophages, which suggests that axatilimab interferes with pro-inflammatory macrophage differentiation. Consistent with this, expression of M2-associated cytokines, including IL-10, IL-5, and IL-4, was enhanced in axatilimab-treated macrophage cultures. Conclusions: Blocking CSF-1R signaling in primary, differentiated macrophages dramatically alters the secretomes and gene expression profiles of these cells. Macrophages are key inducers of fibroblast growth factors, which in turn promote fibrotic disease. These data confirm that production of inflammatory and pro-fibrotic factors by pro-inflammatory macrophages is inhibited in the presence of axatilimab, thereby supporting the notion that axatilimab provides a unique opportunity to therapeutically intervene or prevent fibrosis in cGVHD.

Icariin inhibits prostate cancer bone metastasis and destruction via suppressing TAM/CCL5-mediated osteoclastogenesis.

Bone metastasis occurs in nearly 70% of patients with metastatic prostate cancer (PCa), and represents the leading cause of death in patients with PCa. Emerging evidence has demonstrated the potential activities of icariin in modulating bone metabolism and remodelling the tumor microenvironment (TME). However, whether icariin could inhibit PCa bone metastasis and destruction by modulating the TME as well as the underlying mechanisms remains unclear. This study investigated whether icariin could inhibit PCa bone metastasis and destruction by modulating the bone TME as well as the underlying mechanisms. Osteoclasts were induced from mouse bone marrow-derived macrophages (BMMs) or Raw264.7 cells. PCa cells were cultured in the conditional medium (CM) of macrophages in vitro or co-injected with macrophages in vivo to simulate their coexistence in the TME. Multiple molecular biology experiments and the mouse RM1-Luc PCa bone metastasis model were used to explore the inhibitory activity and mechanism of icariin on PCa metastasis and bone destruction. Icariin treatment significantly suppressed PCa growth, bone metastasis and destruction as well as osteoclastogenesis in vivo. Furthermore, icariin remarkably inhibited osteoclast differentiation, even in the presence of the CM of tumor-associated macrophages (TAMs), while exhibiting no obvious effect on osteoblasts. Moreover, icariin suppressed the M2 phenotype polarization of Raw264.7-derived TAMs and transcriptionally attenuated their CC motif chemokine ligand 5 (CCL5) expression and secretion via inhibiting SPI1. Additionally, CCL5 induced the differentiation and chemotaxis of osteoclast precursor cells by binding with its receptor CCR5. The clinicopathological analysis further verified the positive correlation between the TAM/CCL5/CCR5 axis and osteoclastogenesis within the TME of PCa patients. More importantly, icariin remarkably suppressed PCa metastasis-induced bone destruction in vivo by inhibiting osteoclastogenesis via downregulating the TAM/CCL5 pathway. Altogether, these results not only implicate icariin as a promising candidate immunomodulator for PCa bone metastasis and destruction but also shed novel insight into targeting TAM/CCL5-mediated osteoclastogenesis as a potential treatment strategy for osteolytic bone metastasis. This study helps to advance the understanding of the crosstalk between bone TME and bone homeostasis.

Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-β, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

The MYBL2–CCL2 axis promotes tumor progression and resistance to anti-PD-1 therapy in ovarian cancer by inducing immunosuppressive macrophages

BackgroundAn immunosuppressive tumor microenvironment in ovarian cancer facilitates tumor progression and resistance to immunotherapy. The function of MYB Proto-Oncogene Like 2 (MYBL2) in the tumor microenvironment remains largely unexplored.MethodsA syngeneic intraovarian mouse model, flow cytometry analysis, and immunohistochemistry were used to explore the biological function of MYBL2 in tumor progression and immune escape. Molecular and biochemical strategies—namely RNA-sequencing, western blotting, quantitative reverse transcription–polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, multiplex immunofluorescence, chromatic immunoprecipitation assay (CHIP) and luciferase assay—were used to reveal the mechanisms of MYBL2 in the OVC microenvironment.ResultsWe found tumor derived MYBL2 indicated poor prognosis and selectively correlated with tumor associated macrophages (TAMs) in ovarian cancer. Mechanically, C-C motif chemokine ligand 2 (CCL2) transcriptionally activated by MYBL2 induced TAMs recruitment and M2-like polarization in vitro. Using a syngeneic intraovarian mouse model, we identified MYBL2 promoted tumor malignancyand increased tumor-infiltrating immunosuppressive macrophages. Cyclin-dependent kinase 2 (CDK2) was a known upstream kinase to phosphorylate MYBL2 and promote its transcriptional function. The upstream inhibitor of CDK2, CVT-313, reprogrammed the tumor microenvironment and reduced anti-PD-1 resistance.ConclusionsThe MYBL2/CCL2 axis contributing to TAMs recruitment and M2-like polarization is crucial to immune evasion and anti-PD-1 resistance in ovarian cancer, which is a potential target to enhance the efficacy of immunotherapy.

MiR-184-5p represses neuropathic pain by regulating CCL1/CCR8 signaling interplay in the spinal cord in diabetic mice

ABSTRACTBackgroundDiabetic neuropathic pain (DNP) is a serious complication for diabetic patients involving nervous system. MicroRNAs (miRNAs) are small-noncoding RNAs which are dysregulated in neuropathic pain, and might be critical molecules for pain treatment. Our previous study has shown miR-184-5p was significantly downregulated in DNP. Therefore, the mechanism of miR-184-5p in DNP was investigated in this study.MethodsA DNP model was established through streptozotocin (STZ). The pharmacological tools were injected intrathecally, and pain behavior was evaluated by paw withdrawal mechanical thresholds (PWMTs). Bioinformatics analysis, Dual-luciferase reporter assay and fluorescence-in-situ-hybridization (FISH) were used to seek and confirm the potential target genes of miR-184-5p. The expression of relative genes and proteins was analyzed by quantitative reverse transcriptase real-time PCR (qPCR) and western blotting.ResultsMiR-184-5p expression was down-regulated in spinal dorsal on days 7 and 14 after STZ, while intrathecal administration of miR-184-5p agomir attenuates neuropathic pain induced by DNP and intrathecal miR-184-5p antagomir induces pain behaviors in naïve mice. Chemokine CC motif ligand 1 (CCL1) was found to be a potential target of miR-184-5p and the protein expression of CCL1 and the mRNA expression of CCR8 were up-regulated in spinal dorsal on days 7 and 14 after STZ. The luciferase reporter assay and FISH demonstrated that CCL1 is a direct target of miR-184-5p. MiR-184-5p overexpression attenuated the expression of CCL1/CCR8 in DNP; intrathecal miR-184-5p antagomir increased the expression of CCL1/CCR8 in spinal dorsal of naïve mice.ConclusionThis research illustrates that miR-184-5p alleviates DNP through the inhibition of CCL1/CCR8 signaling expression.

The effects of berteroin on inflammatory mediators and antioxidant enzymes expression in human periodontal ligament cells.

Berteroin is a bioactive substance classified as an isothiocyanate found in cruciferous vegetables such as cabbage, arugula, and salad leaves. In this study, we aimed to determine whether berteroin exerts anti-inflammatory effects on human periodontal ligament cells (HPDLCs), a resident cells of periodontal tissue. Berteroin suppressed interleukin (IL)-1β or tumor necrosis factor (TNF)-α-induced chemokines (C-C motif chemokine ligand (CCL)2, CCL20, C-X-C motif chemokine ligand (CXCL)10, IL-8, and IL-6) production and intercellular adhesion molecule (ICAM)-1 expression in HPDLCs. In addition, berteroin inhibited phosphorylation of IκB kinase (IKK)- α/ β, nuclear factor (NF)- κB p65, and IκB- α and degradation of IκB- α in the NF-κB pathway induced by IL-1 β or TNF- α stimulation. Moreover, berteroin could inhibit signal transducer and activator of transcription (STAT)3 phosphorylation in TNF- α -stimulated HPDLC. Furthermore, berteroin increased the expression of the antioxidant enzymes, heme oxygenase (HO)-1 and NAD(P)H quinone dehydrogenase (NQO)1, in IL-1 β or TNF- α -stimulated HPDLCs. These results suggest that berteroin may decrease the production of inflammatory mediators in HPDLCs by suppressing the NF-κB pathway, and may also decrease the local reactive oxygen species (ROS) production in periodontal lesions by increasing the production of antioxidant enzymes.

Peripheral CCL2 induces inflammatory pain via regulation of Ih currents in small diameter DRG neurons.

The C-C motif chemokine ligand 2 (CCL2) has been implicated in chronic pain, but its exact mechanism of peripheral sensitization is unknown. In this study, we aimed to clarify the mechanism of CCL2 regulation of ion channels. Our behavioral experiments revealed that ZD7288, a blocker of Ih current, can inhibit CFA and CCL2-mediated mechanical and thermal nociceptive sensitization. Furthermore, patch clamp studies demonstrated that CFA-induced peripheral sensitization primarily affects the excitability of small-diameter DRG neurons. Further studies revealed that inflammatory pain caused by CFA or incubation of DRG with CCL2 mainly affected Ih currents in small-diameter DRG neurons, which were blocked by co-incubation CCR2 antagonist INCB3344 or adenylate cyclase inhibitor SQ22536. Immunohistochemical staining showed that both intraplantar injection of CFA as well as DRG injection of CCL2 resulted in significant upregulation of CCR2+/HCN2+ expression. In conclusion, we suggest in the inflammatory pain state, CCL2 can act on small-diameter DRG neurons, leading to upregulation of HCN2 expression and consequently Ih, which in turn leads to neuronal hyperexcitability.

Aqueous extract of Epimedium sagittatum mitigates pulmonary fibrosis in mice

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.

Nuclear Factor-Kappa B Regulation of Osteoclastogenesis and Osteoblastogenesis.

Maintenance of skeletal integrity requires the coordinated activity of multinucleated bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts form resorption lacunae on bone surfaces in response to cytokines by fusion of precursor cells. Osteoblasts are derived from mesenchymal precursors and lay down new bone in resorption lacunae during bone remodeling. Nuclear factorkappa B (NF-κB) signaling regulates osteoclast and osteoblast formation and is activated in osteoclast precursors in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which can also control osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors. RANKL and some pro-inflammatory cytokines, including tumor necrosis factor (TNF), activate NF-κB signaling to positively regulate osteoclast formation and functions. However, these cytokines also limit osteoclast and osteoblast formation through NF-κB signaling molecules, including TNF receptor-associated factors (TRAFs). TRAF6 mediates RANKL-induced osteoclast formation through canonical NF-κB signaling. In contrast, TRAF3 limits RANKL- and TNF-induced osteoclast formation, and it restricts transforming growth factor β (TGFβ)-induced inhibition of osteoblast formation in young and adult mice. During aging, neutrophils expressing TGFβ and C-C chemokine receptor type 5 (CCR5) increase in bone marrow of mice in response to increased NF-κB-induced CC motif chemokine ligand 5 (CCL5) expression by mesenchymal progenitor cells and injection of these neutrophils into young mice decreased bone mass. TGFβ causes degradation of TRAF3, resulting in decreased glycogen synthase kinase-3β/β-catenin-mediated osteoblast formation and age-related osteoporosis in mice. The CCR5 inhibitor, maraviroc, prevented accumulation of TGFβ+/CCR5+ neutrophils in bone marrow and increased bone mass by inhibiting bone resorption and increasing bone formation in aged mice. This paper updates current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast and osteoblast formation and activation with a focus on the role of TRAF3 signaling, which can be targeted therapeutically to enhance bone mass.

Dental pulp stem cells accelerate wound healing through CCL2-induced M2 macrophages polarization

The crosstalk between mesenchymal stem cells (MSCs) and the host immune function plays a key role in the efficiency of tissue regeneration and wound healing. However, the difference in immunological modulation and tissue regeneration function between MSCs from different sources remains unclear. Compared to PDLSCs, BMMSCs, and ADSCs, DPSCs exhibited greater tissue regeneration potential and triggered more M2 macrophages invivo. DPSCs elicited the polarization of M2a macrophages by conditioned medium and transwell assay and exhibited higher expression levels of C-C motif chemokine ligand 2 (CCL2). Specific blocking of CCL2 could significantly inhibit the DPSCs-induced polarization of M2 macrophages. DPSCs promoted wound healing of the palatal mucosa and M2 macrophages polarization invivo, which could be significantly impaired by CCL2-neutralized antibody. Our data indicate that DPSCs exert better tissue regeneration potential and immunoregulatory function by secreting CCL2, which can enhance MSCs-mediated tissue regeneration or wound healing.

Predictive value of chemokines (CCL 2) in bronchoalveolar lavage fluid for refractory mycoplasma pneumonia in children

BackgroundThere are relatively few studies investigating C-C motif chemokine ligand 2 (CCL2) level in bronchoalveolar lavage fluid (BALF) in children with Mycoplasma pneumoniae pneumonia (MPP), and the relationship between CCL2 level in BALF and refractory mycoplasma pneumoniae pneumonia (RMPP) is unclear. This study aims to explore the relationship between chemokine CCL2 level in BALF and clinical characteristics and clinical outcome in children with MPP.MethodsA total of 51 children with confirmed acute MPP and requiring bronchoalveolar lavage in Department of Pediatrics, Huanghe Sanmenxia Hospital and The First Clinical College of Xinxiang Medical University from October 2021 to February 2023 were selected as the study group. And 11 children with bronchial foreign body were selected as the control group. The study group was divided into the non-refractory mycoplasma pneumoniae pneumonia (NRMPP) group and the RMPP group based on the response to treatment. BALF and clinical data of the patients were collected. And CCL2 levels were tested in the patients. Differences in CCL2 level in BALF and clinical characteristics were tested and compared.ResultsThe CCL2 level in BALF of the study group was higher than that of the control group, with significant difference (P < 0.05). With ROC curve, the area under the curve (AUC) of CCL2 in BALF predicting RMPP was 0.94, the cut-off value was 0.645 ng/ml, the sensitivity was 85%, and the specificity was 94%, and the diagnostic value was better than that of serum CRP and LDH. Logistic regression analysis was used to build the RMPP prediction model, and CCL2 showed good predictive value.ConclusionThe level of CCL2 in BALF was high in children with MPP and had a high predictive value for RMPP. CCL2 can be used as one of the biomarkers for predicting RMPP.

Effects of Ulipristal Acetate on Reactive Oxygen Species and Proinflammatory Cytokine Release by Epithelial and Stromal Cells from Human Endometrium and Endometriosis.

Endometriosis is a condition characterized by increased oxidative stress and chronic inflammation, which can be treated with progestins and other progesterone receptor ligands. However, some patients are refractory to this treatment and the reason is uncertain. Here we investigated the effects of the selective progesterone receptor modulator ulipristal acetate (UPA) on proliferation, reactive oxygen species (ROS), and proinflammatory cytokine production by endometriotic cells and endometrial cells from women with histologically proven endometriosis (n=22) and endometriosis-free controls (n=6). Epithelial and stromal cells were isolated and treated in triplicate for 24h with 1 μM, 10 μM, or 100 μM UPA. Cells were tested for proliferation and ROS production, while cell supernatants were assayed for interleukin (IL)-6, C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor (TNF)-α concentrations. Proliferation, ROS production, and IL-6 and CCL2 secretion were increased in non-stimulated epithelial and stromal cells from endometriotic lesions compared to endometrial cells from endometriosis patients and controls. UPA induced a dose-dependent increase of cell proliferation only in endometriosis, while enhancing ROS production by all cell types evaluated. UPA reduced CCL2 production in controls but failed to do that in endometriosis, whereas TNF-α was undetectable. We conclude that treatment of endometriotic cells with UPA stimulated in vitro proliferation and ROS production and failed to revert the proinflammatory cytokine excess that characterized these cells, unravelling possible mechanisms of drug resistance in the treatment of endometriosis.

CCL2 is a key regulator and therapeutic target for periodontitis.

Our previous study revealed that the C-C motif chemokine receptor 2 (CCR2) is a promising target for periodontitis prevention and treatment. However, CCR2 is a receptor with multiple C-C motif chemokineligands (CCLs), including CCL2, CCL7, CCL8, CCL13 and CCL16, and which of these ligands plays a key role in periodontitis remains unclear. The aim of the present study was to explore the key functional ligand of CCR2 in periodontitis and to evaluate the potential of the functional ligand as a therapeutic target for periodontitis. The expression levels and clinical relevance of CCR2, CCL2, CCL7, CCL8, CCL13 and CCL16 were studied using human samples. The role of CCL2 in periodontitis was evaluated by using CCL2 knockout mice and overexpressing CCL2 in the periodontium. The effect of local administration of bindarit in periodontitis was evaluated by preventive and therapeutic medication in a mouse periodontitis model. Microcomputed tomography, haematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, bead-based immunoassays and flow cytometry were used for histomorphology, molecular biology and cytology analysis. Among different ligands of CCR2, only CCL2 was significantly up-regulated in periodontitis gingival tissues and was positively correlated with the severity of periodontitis. Mice lacking CCL2 showed milder inflammation and less bone resorption than wild-type mice, which was accompanied by a reduction in monocyte/macrophage recruitment. Adeno-associated virus-2 vectors overexpressing CCL2 in Ccl2-/- mice gingivareversed the attenuation of periodontitis in a CCR2-dependent manner. In ligation-induced experimental periodontitis, preventive or therapeutic administration of bindarit, a CCL2 synthesis inhibitor, significantly inhibited the production of CCL2, decreased the osteoclast number and bone loss and reduced the expression levels of proinflammatory cytokines TNF-α, IL-6 and IL-1β. CCL2 is a pivotal chemokine that binds to CCR2 during the progression of periodontitis, and targeting CCL2 may be a feasible option for controlling periodontitis.

Tumor-associated macrophages confer colorectal cancer 5-fluorouracil resistance by promoting MRP1 membrane translocation via an intercellular CXCL17/CXCL22–CCR4–ATF6–GRP78 axis

Chemotherapy represents a major type of clinical treatment against colorectal cancer (CRC). Aberrant drug efflux mediated by transporters acts as a key approach for tumor cells to acquire chemotherapy resistance. Increasing evidence implies that tumor-associated macrophages (TAMs) play a pivotal role in both tumorigenesis and drug resistance. Nevertheless, the specific mechanism through which TAMs regulate drug efflux remains elusive. Here, we discovered that TAMs endow CRC cells with resistance to 5-fluorouracil (5-FU) treatment via a cell-cell interaction-mediated MRP1-dependent drug efflux process. Mechanistically, TAM-secreted C-C motif chemokine ligand 17 (CCL17) and CCL22, via membrane receptor CCR4, activated the PI3K/AKT pathway in CRC tumor cells. Specifically, phosphorylation of AKT inactivated IP3R and induced calcium aggregation in the ER, resulting in the activation of ATF6 and upregulation of GRP78. Accordingly, excessive GRP78 can interact with MRP1 and promote its translocation to the cell membrane, causing TAM-induced 5-FU efflux. Taken together, our results demonstrated that TAMs promote CRC chemotherapy resistance via elevating the expression of GRP78 to promote the membrane translocation of MRP1 and drug efflux, providing direct proof for TAM-induced drug resistance.

Neutrophils for Revascularization Require Activation of CCR6 and CCL20 by TNFα.

Activation of immune-inflammatory pathways involving TNFα (tumor necrosis factor alpha) signaling is critical for revascularization and peripheral muscle tissue repair after ischemic injury. However, mechanisms of TNFα-driven inflammatory cascades directing recruitment of proangiogenic immune cells to sites of ischemia are unknown. Muscle tissue revascularization after permanent femoral artery ligation was monitored in mutant mice by laser Doppler imaging and light sheet fluorescence microscopy. TNFα-mediated signaling and the role of the CCL20 (C-C motif chemokine ligand 20)-CCR6 (C-C chemokine receptor 6) axis for formation of new vessels was studied in vitro and in vivo using bone marrow transplantation, flow cytometry, as well as biochemical and molecular biological techniques. TNFα-mediated activation of TNFR (tumor necrosis factor receptor) 1 but not TNFR2 was found to be required for postischemic muscle tissue revascularization. Bone marrow-derived CCR6+ neutrophil granulocytes were identified as a previously undescribed TNFα-induced population of proangiogenic neutrophils, characterized by increased expression of VEGFA (vascular endothelial growth factor A). Mechanistically, postischemic activation of TNFR1 induced expression of the CCL20 in vascular cells and promoted translocation of the CCL20 receptor CCR6 to the cell surface of neutrophils, essentially conditioning VEGFA-expressing proangiogenic neutrophils for CCL20-dependent recruitment to sites of ischemia. Moreover, impaired revascularization of ischemic peripheral muscle tissue in diabetic mice was associated with reduced numbers of proangiogenic neutrophils and diminished CCL20 expression. Administration of recombinant CCL20 enhanced recruitment of proangiogenic neutrophils and improved revascularization of diabetic ischemic skeletal muscles, which was sustained by sequential treatment with fluvastatin. We demonstrate that site-specific activation of the CCL20-CCR6 axis via TNFα recruits proangiogenic VEGFA-expressing neutrophils to sites of ischemic injury for initiation of muscle tissue revascularization. The findings provide an attractive option for tissue revascularization, particularly under diabetic conditions.

Quercetin Alleviates Lipopolysaccharide-Induced Cell Oxidative Stress and Inflammatory Responses via Regulation of the TLR4-NF-κB Signaling Pathway in Bovine Rumen Epithelial Cells.

Subacute rumen acidosis (SARA) will cause an increase in endotoxin, which will have a negative effect on the bovine rumen epithelial cells (BREC). Flavonoids are effective in treating inflammation caused by endotoxin. Quercetin is a vital flavonoid widely occurring in fruits and vegetables and has received significant interest as a prospective anti-inflammatory antioxidant. Nonetheless, quercetin's protective machinery against such damage to BREC induced by lipopolysaccharide (LPS) remains unclear. A combined quercetin and LPS-induced BREC inflammation model was utilized to elucidate the effect of quercetin protecting BREC from LPS-induced injury. After treating BREC with different doses of LPS (1, 5, and 10 μg/mL) for 6 h or 24 h, the mRNA expression of inflammatory factors was detected. Our experimental results show the establishment of the BREC inflammation model via mRNA high expression of pro-inflammatory cytokines in BREC following 6 h treatment with 1 µg/mL LPS. The promotive effect of 80 μg/mL quercetin on BREC growth via the cell counting kit-8 (CCK8) assay was observed. The expression of pro-inflammatory cytokines and chemokines, notably tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), IL-6, CC-motif chemokine ligand 2 (CCL2), CCL20, CCL28, and CXC motif chemokine 9 (CXCL9), etc., was significantly reduced by quercetin supplementation. We also analyzed the mRNA detection of related pathways by qRT-PCR. Our validation studies demonstrated that quercetin markedly curbed the mRNA expression of the toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and the nuclear factor-κB (NF-κB) in LPS-treated BREC. In addition, western blot result outcomes confirmed, as expected, that LPS significantly activated phosphorylation of p44/42 extracellular regulated protein kinases (ERK1/2) and NF-κB. Unexpectedly, this effect was reversed by adding quercetin. To complement western blot results, we assessed p-ERK1/2 and p-p65 protein expression using immunofluorescence, which gave consistent results. Therefore, quercetin's capacity to bar the TLR4-mediated NF-κB and MAPK signaling pathways may be the cause of its anti-inflammatory effects on LPS-induced inflammatory reactions in BREC. According to these results, quercetin may be utilized as an anti-inflammatory medication to alleviate inflammation brought on by high-grain feed, and it also lays out a conceptual foundation regarding the development and utilization of quercetin in the later stage.

Meta-Analysis of Single-Cell RNA-Seq Data Reveals the Mechanism of Formation and Heterogeneity of Tertiary Lymphoid Organ in Vascular Disease.

Tertiary lymphoid organs (TLOs) are ectopic lymphoid organs developed in nonlymphoid tissues with chronic inflammation, but little is known about their existence in different types of vascular diseases and the mechanism that mediated their development. To take advantage of single-cell RNA sequencing techniques, we integrated 28 single-cell RNA sequencing data sets containing 5 vascular disease models (atherosclerosis, abdominal aortic aneurysm, intimal hyperplasia, isograft, and allograft) to explore TLOs existence and environment supporting its growth systematically. We also searched Medline, Embase, PubMed, and Web of Science from inception to January 2022 for published histological images of vascular remodeling for histological evidence to support TLO genesis. Accumulation and infiltration of innate and adaptive immune cells have been observed in various remodeling vessels. Interestingly, the proportion of such immune cells incrementally increases from atherosclerosis to intimal hyperplasia, abdominal aortic aneurysm, isograft, and allograft. Importantly, we uncovered that TLO structure cells, such as follicular helper T cells and germinal center B cells, present in all remodeled vessels. Among myeloid cells and lymphocytes, inflammatory macrophages, and T helper 17 cells are the major lymphoid tissue inducer cells which were found to be positively associated with the numbers of TLO structural cells in remodeled vessels. Vascular stromal cells also actively participate in vascular TLO genesis by communicating with myeloid cells and lymphocytes via CCLs (C-C motif chemokine ligands), CXCL (C-X-C motif ligand), lymphotoxin, BMP (bone morphogenetic protein) chemotactic, FGF-2 (fibroblast growth factor-2), and IGF (insulin growth factor) proliferation mechanisms, particularly for lymphoid tissue inducer cell aggregation. Additionally, the interaction between stromal cells and immune cells modulates extracellular matrix remodeling. Among TLO structure cells, follicular helper T, and germinal center B cells have strong interactions via TCR (T-cell receptor), CD40 (cluster of differentiation 40), and CXCL signaling, to promote the development and maturation of the germinal center in TLO. Consistently, by reviewing the histological images from the literature, TLO genesis was found in those vascular remodeling models. Our analysis showed the existence of TLOs across 5 models of vascular diseases. The mechanisms that support TLOs formation in different models are heterogeneous. This study could be a valuable resource for understanding and discovering new therapeutic targets for various forms of vascular disease.