CCL2 Production Research Articles

Expression of CCL2 signaling pathway genes in patients with periodontitis and atherosclerosis.

Periodontitis and atherosclerosis are chronic inflammatory diseases characterized by leukocyte infiltration. We investigated the expression of CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 as well as the possible mechanism involved in the regulation of CCL2 in human periodontitis tissues and atherosclerotic aorta based on previous research on the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathway leading to CCL2 expression in collagen-induced arthritis (CIA) rat. Sixty-five volunteers were recruited and the condition of their gingiva and coronary arteries were assessed. The subjects were divided into four groups: healthy control, chronic periodontitis (CP), coronary artery diseases (CAD), and noncoronary artery diseases (non-CAD). Total RNA was isolated from gingiva in periodontitis patients and control populations and from the aorta in patients with and without CAD. PCR was used to examine CCL4, CCR5, c-Jun, c-Fos, NF-κB, and CCL2 levels. The production of CCL2 in the gingiva and aorta was analyzed by immunostaining. PCR revealed that CCL4, CCR5, and CCL2 mRNA levels were increased in CP patients' gingivae and aortas from coronary artery bypass grafting (CABG) patients. Marked c-Jun, c-Fos, and NF-κB gene productions were detected in CP patients' gingivae but did not show statistical differences between the CAD and non-CAD groups. Stronger immunoreactivity against CCL2 was observed in periodontitis gingiva and aorta from CABG patients. Our findings suggest that the CCL4/CCR5/c-Jun and c-Fos/CCL2 pathways may be involved in CCL2 expression in periodontitis. CCL4, CCR5, and CCL2 might act as possible nodes to link the presence of periodontitis and atherosclerosis.

SRAGE attenuates myocardial ischemia-reperfusion injury by recruiting Tregs through chemokine CCL4

Abstract Background Immune response has recently been shown to play a crucial role in myocardial ischemia-reperfusion (MIR). Our previous studies have confirmed that soluble receptors for advanced glycation end-products (sRAGE) could reduce myocardial ischemia-reperfusion injury (MIRI) by increasing the number of regulatory T cells (Tregs). However, the precise mechanism is still incompletely understood. Purpose The present study aimed to determine which kind of chemokines play a major role in recruiting Tregs during MIRI. The potential mechanism of sRAGE on the chemokine production was further explored. Methods We modeled mice with cardiac-specific overexpression of sRAGE by crossing the floxed sRAGE mouse model with the cardiomyocyte-specific Cre transgenic mouse. Experimental models were established utilizing left anterior descending coronary artery ligation in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Transcriptomics, quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry, and western blotting were used to determine the chemokines and related pathways involved in the recruitment of Tregs during MIRI. Results Using transcriptomics, quantitative real-time polymerase chain reaction, and western blotting approach, we identify the chemokine CCL4 plays a crucial role during regulatory T cell recruitment in the heart after MIRI. In vivo studies show that depletion of CCL4 reduces Tregs infiltration into the ischemic myocardium, subsequently deteriorating cardiac injury post-MIR. Mechanistically, sRAGE promotes cardiomyocytes to express and secrete CCL4 via the JNK/AP-1 pathway in vitro. Selective inhibition of the JNK/AP-1 pathway could decrease CCL4 production in sRAGE overexpression cardiomyocytes. Conclusions sRAGE induces Tregs recruitment and alleviates MIRI via the JNK/AP-1/CCL4 pathway, preserving myocardial function and impeding inflammation and apoptosis, providing new perspectives for immunotherapeutic targets in MIRI treatment.

Pectin-based encapsulation systems for the protection of beneficial bacterial species and impact on intestinal barrier function in vitro

Pectin is a complex polysaccharide important to the pharmaceutical and food industry, and due to its versatility, cheapness, and notable health benefits, it is proposed to be investigated as an encapsulation material to protect sensitive food components such as living bacteria during their passage through the gastrointestinal tract. The main objective of this study was to evaluate both, the ability of pectin-based beads to maintain the viability and functionality of L. plantarum WCFS1 and the protective effect on intestinal epithelial barrier disruption in vitro. Pectin isolated from lemon with two degrees of methylation (DM18 and DM88) was used to form microbeads with either alginate or chitosan, producing four bead types, which were alginate/pectin DM18 (AlgDM18), alginate/pectin DM88 (AlgDM88), chitosan/pectin DM18 (ChitoDM18), and chitosan/pectin DM88 (ChitoDM88).The beads were loaded with L. plantarum, and it was demonstrated that all pectin-based beads could preserve the viability of L. plantarum after encapsulation. While some differences in metabolism was observed, the alginate-pectin beads exhibited higher activity and loading capacity. Then these beads were tested on intestinal epithelial cells to assess their protective impact in the presence of a barrier disruptor A23187. The effect on gut barrier function demonstrates that the composition of the microbeads significantly affected their protective effects. Empty ChitoDM88 beads demonstrated protective effects by delaying barrier disruption and modulating the release of gut epithelial cell-specific cytokines. They increased the production of CCL20 while reducing levels of IL-8 and IL-13, which are markers of inflammation.L. plantarum WCFS1-loaded Chitosan DM88-pectin microbeads provided the strongest protection. These beads not only delayed disruption of the gut epithelial barrier but also increased epithelial barrier function before and after treatment of the monolayer with the A23187 disruptor. Additionally, the loaded ChitoDM88 beads upregulated key genes associated with the tight junction integrity of the intestinal epithelium, including ZO-1, Occludin, and Claudin-1, further supporting their protective role. The immune response was also modulated by the loaded ChitoDM88 beads, as they increased CCL20 production and reduced IL-8 levels, highlighting a bead-type-dependent impact on epithelial immune signaling.This study demonstrates that pectin-based microbeads can preserve the viability and functionality of encapsulated L. plantarum, protecting against gut barrier impairment. The results highlight pectin's potential as a promising polymer for probiotic delivery, with encapsulation effectiveness depending on the pectin's methylation degree and the stabilizing agent used.

Retraction Note: Curcumin inhibits the migration of osteoclast precursors and osteoclastogenesis by repressing CCL3 production

Retraction Note: Curcumin inhibits the migration of osteoclast precursors and osteoclastogenesis by repressing CCL3 production

Osteopontin deficiency promotes cartilaginous endplate degeneration by enhancing the NF-κB signaling to recruit macrophages and activate the NLRP3 inflammasome

Intervertebral disc degeneration (IDD) is a major cause of discogenic pain, and is attributed to the dysfunction of nucleus pulposus, annulus fibrosus, and cartilaginous endplate (CEP). Osteopontin (OPN), a glycoprotein, is highly expressed in the CEP. However, little is known on how OPN regulates CEP homeostasis and degeneration, contributing to the pathogenesis of IDD. Here, we investigate the roles of OPN in CEP degeneration in a mouse IDD model induced by lumbar spine instability and its impact on the degeneration of endplate chondrocytes (EPCs) under pathological conditions. OPN is mainly expressed in the CEP and decreases with degeneration in mice and human patients with severe IDD. Conditional Spp1 knockout in EPCs of adult mice enhances age-related CEP degeneration and accelerates CEP remodeling during IDD. Mechanistically, OPN deficiency increases CCL2 and CCL5 production in EPCs to recruit macrophages and enhances the activation of NLRP3 inflammasome and NF-κB signaling by facilitating assembly of IRAK1-TRAF6 complex, deteriorating CEP degeneration in a spatiotemporal pattern. More importantly, pharmacological inhibition of the NF-κB/NLRP3 axis attenuates CEP degeneration in OPN-deficient IDD mice. Overall, this study highlights the importance of OPN in maintaining CEP and disc homeostasis, and proposes a promising therapeutic strategy for IDD by targeting the NF-κB/NLRP3 axis.

Hdac3 deficiency limits periosteal reaction associated with Western diet feeding in female mice.

Diet-induced obesity is associated with enhanced systemic inflammation that limits bone regeneration. HDAC inhibitors are currently being explored as anti-inflammatory agents. Prior reports show that myeloid progenitor-directed Hdac3 ablation enhances intramembranous bone healing in female mice. In this study, we determined if Hdac3 ablation increased intramembranous bone regeneration in mice fed a high-fat/high-sugar (HFD) diet. Micro-CT analyses demonstrated that HFD-feeding enhanced the formation of periosteal reaction tissue of control littermates, reflective of suboptimal bone healing. We confirmed enhanced bone volume within the defect of Hdac3-ablated females and showed that Hdac3 ablation reduced the amount of periosteal reaction tissue following HFD feeding. Osteoblasts cultured in a conditioned medium derived from Hdac3-ablated cells exhibited a four-fold increase in mineralization and enhanced osteogenic gene expression. We found that Hdac3 ablation elevated the secretion of several chemokines, including CCL2. We then confirmed that Hdac3 deficiency increased the expression of Ccl2. Lastly, we show that the proportion of CCL2-positve cells within bone defects was significantly higher in Hdac3-deficient mice and was further enhanced by HFD. Overall, our studies demonstrate that Hdac3 deletion enhances intramembranous bone healing in a setting of diet-induced obesity, possibly through increased production of CCL2 by macrophages within the defect.

Targeting PADI2 as a potential therapeutic strategy against metastasis in oral cancer via suppressing EMT-mediated migration and invasion and CCL3/5-induced angiogenesis.

Oral squamous cell carcinoma (OSCC) is a prevalent and aggressive malignancy, with metastasis being the leading cause of death in patients. Unfortunately, therapeutic options for metastatic OSCC remain limited. Peptidylarginine deiminases (PADI) are implicated in various tumorigenesis and metastasis processes across multiple cancers. However, the role of PADI2, a type of PADI, in OSCC is not well understood. This study aimed to explore the impact of PADI2 on epithelial-mesenchymal transition (EMT), angiogenesis, and OSCC metastasis. The effect of PADI2 on EMT was evaluated using cell lines by Western blot analysis with shRNA targeting PADI2. In addition, the selective PADI2 inhibitor AFM32a was used to assess the effect of PADI2 on cancer metastasis and angiogenesis in animal models. Our findings indicated that PADI2 expression correlated with EMT changes, and PADI2 knockdown reversed these changes, reducing cell proliferation, cell migration, and invasion. PADI2 inhibition also diminished tube formation in HUVECs and decreased secretion of angiogenesis-related chemokines CCL3, CCL5 and CCL20. In a mouse model, AFM32a markedly reduced lung metastasis and production of CCL3 and CCL5. Our in vitro and in vivo studies suggested inhibiting PADI2 could prevent OSCC metastasis by impeding EMT and angiogenesis via AKT/mTOR signaling pathway. These results highlight PADI2 as a potential therapeutic target for combating OSCC metastasis.

Rabies Virus Regulates Inflammatory Response in BV-2 Cells through Activation of Myd88 and NF-κB Signaling Pathways via TLR7.

Rabies is a fatal neurological infectious disease caused by rabies virus (RABV), which invades the central nervous system (CNS). RABV with varying virulence regulates chemokine expression, and the mechanisms of signaling pathway activation remains to be elucidated. The relationship between Toll-like receptors (TLRs) and immune response induced by RABV has not been fully clarified. Here, we investigated the role of TLR7 in the immune response induced by RABV, and one-way analysis of variance (ANOVA) was employed to evaluate the data. We found that different RABV strains (SC16, HN10, CVS-11) significantly increased CCL2, CXCL10 and IL-6 production. Blocking assays indicated that the TLR7 inhibitor reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). The activation of the Myd88 pathway in BV-2 cells stimulated by RABV was TLR7-dependent, whereas the inhibition of Myd88 activity reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). Meanwhile, the RABV stimulation of BV-2 cells resulted in TRL7-mediated activation of NF-κB and induced the nuclear translocation of NF-κB p65. CCL2, CXCL10 and IL-6 release was attenuated by the specific NF-κB inhibitor used (p < 0.01). The findings above demonstrate that RABV-induced expression of CCL2, CXCL10 and IL-6 involves Myd88 and NF-κB pathways via the TLR7 signal.

Soymilk yogurt fermented using Pediococcus pentosaceus TOKAI 759m improves mice gut microbiota and reduces pro-inflammatory cytokine production.

This study aimed to determine the anti-inflammatory activities and bioactive compounds of soymilk yogurt prepared using Lactiplantibacillus plantarum TOKAI 17 or Pediococcus pentosaceus TOKAI 759m. Mice were divided into five groups: normal diet (ND), soymilk, soymilk yogurt using L. plantarum TOKAI 17 (SY 17) or P. pentosaceus TOKAI 759m (SY 759m), and 0.5×109 cells of each starter strain (BC 17 or BC759m). In the SY 759m group, the serum pro-inflammatory cytokine levels and the cytotoxicity of natural killer cells were attenuated compared to the ND group. In the cecum microbiota, the abundances of butyrate-producing bacteria increased in the SY 759m and BC 17 groups. Furthermore, SY 759m metabolites contained high levels of aglycone isoflavone, adenine and showed a significant decrease in CCL-2 and interleukin-6 production in lipopolysaccharide-induced macrophage. In conclusion, soymilk yogurt produced using P. pentosaceus TOKAI 759m modulates the gut microbiota and can potentially prevent pro-inflammatory cytokine production.

CGAS-activated endothelial cell-T cell cross-talk initiates tertiary lymphoid structure formation.

Aberrant activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway causes autoimmunity in humans and mice; however, the exact mechanism by which the cGAS-STING pathway initiates adaptive immunity and tissue pathology is still not fully understood. Here, we used a cGAS knockin (KI) mouse model that develops systemic autoimmunity. In the lungs of cGAS-KI mice, blood vessels were enclosed by organized lymphoid tissues that resemble tertiary lymphoid structures (TLSs). Cell-intrinsic cGAS induction promoted up-regulation of CCR5 in CD8+ T cells and led to CCL5 production in vascular endothelial cells. Peripheral CD8+ T cells were recruited to the lungs and produced CXCL13 and interferon-γ. The latter triggered endothelial cell death, potentiated CCL5 production, and was essential for TLS establishment. Blocking CCL5 or CCR5, or depleting CD8+ T cells, impaired TLS formation. cGAS-mediated TLS formation also enhanced humoral and antitumor responses. These data demonstrate that cGAS signaling drives a specialized lymphoid structure that underlies autoimmune tissue pathology.

Use of optimized single-cell RNA flow cytometry protocol identifies monocytes as main producers of type I interferon in mouse syngeneic tumors.

The tumor microenvironment (TME) consists of complex interactions between cellular and extracellular components, among which the immune system is known to play an integral role in disease progression and response to therapy. Cytokines and chemokines are cell signaling proteins used by immune cells to communicate with each other as well as with other cell types in the body. These proteins control systemic and local immune responses and levels of cytokines/chemokines in the TME have been associated with tumor outcomes. However, cytokines and chemokines have varied expression across cell types, tumors, and host conditions. Therefore, approaches to effectively study the production of these proteins at the single-cell level in the TME are needed to fully elucidate the mechanisms governing the anti-cancer immune response. Here, we detail a protocol to assess the production of cytokines/chemokines across leukocyte populations in mouse tumors using RNA flow cytometry. Importantly, this method can be adapted with minimal changes to study various mouse and human tumors, other RNA analytes, and non-tumor tissues. With this approach, we characterize single-cell production of Ifnb1, Xcl1 and Ccl5 in mouse tumors and identify monocytes and monocyte-derived macrophages as the main producers of type I interferon transcript Ifnb1 consistent across 4 different syngeneic tumor models.

PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade

Programmed cell death ligand 2 (PD-L2), a ligand for the receptor programmed cell death 1 (PD-1), has an identity of 34% with its twin ligand PD-L1 and exhibits higher binding affinity with PD-1 than PD-L1. However, the role of PD-L2 in non-small cell lung cancer (NSCLC) progression, especially tobacco-induced cancer progression, has not been fully understood. Here, we found that PD-L2 promoted tumor growth in murine models with recruitment of regulatory T cells (Tregs). In patients with NSCLC, PD-L2 expression level in tumor samples was higher than in counterpart normal controls and was positively associated with patients’ response to anti-PD-1 treatment. Mechanismly, PD-L2 bound its receptor Repulsive guidance molecule B (RGMB) on cancer cells and activated extracellular signal-regulated kinase (Erk) and nuclear factor κB (NFκB), leading to increased production of chemokine CCL20, which recruited Tregs and contributed to NSCLC progression. Consistently, knockdown of RGMB or NFκB p65 inhibited PD-L2-induced CCL20 production, and silencing of PD-L2 repressed Treg recruitment by NSCLC cells. Furthermore, cigarette smoke and carcinogen benzo(a)pyrene (BaP) upregulated PD-L2 in lung epithelial cells via aryl hydrocarbon receptor (AhR)-mediated transcription activation, whose deficiency markedly suppressed BaP-induced PD-L2 upregulation. These results suggest that PD-L2 mediates tobacco-induced recruitment of Tregs via the RGMB/NFκB/CCL20 cascade, and targeting this pathway might have therapeutic potentials in NSCLC.

SRGN promotes macrophage recruitment through CCL3 in osteoarthritis

ABSTRACT Background Osteoarthritis (OA) is a degenerative disease that affects synovial joints and leads to significant pain and disability, particularly in older adults. Infiltration of macrophages plays a key role in the progression of OA. However, the mechanisms underlying macrophage recruitment in OA are not fully understood. Methods The Serglycin (SRGN) expression pattern was analyzed, along with its association with macrophage infiltration in OA, using bioinformatic methods. SRGN expression in chondrocytes was altered by small interfering RNA (siRNA) and plasmids. Conditioned media (CM) was obtained from transfected chondrocytes to establish a co-culture model of chondrocytes and THP-1 derived macrophages. The impact of SRGN on macrophage recruitment was evaluated using a transwell assay. Furthermore, the regulatory effect of SRGN on CCL3 was validated through qPCR, WB, and ELISA experiments. Results In OA patients, the upregulation of SRGN positively correlated with K-L grade and macrophage infiltration. It was found that SRGN expression and secretion were up-regulated in OA and that it can promote macrophage migration in vitro. Further investigation showed that SRGN affects macrophage migration by regulating the expression of CCL3. Conclusion SRGN in chondrocytes plays a role in promoting the recruitment of THP-1 derived macrophages in vitro by regulating production of CCL3.

Fusobacterium nucleatum to promote esophageal squamous cell carcinoma progression via CCL20/CCR6-mediated migration of macrophages to the tumour micro environment.

e14668 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive malignant tumor in China. Recent studies have shown that Fusobacterium nucleatum ( F. nucleatum) promotes ESCC progression through various mechanisms. One of them is about changing the tumor micro- environment through cytokine–cytokine receptor interaction. Methods: From January 2022 to June 2023, a total of 34 patients with ESCC from 4th Hospital of Hebei Medical University thoracic surgery were enrolled. All of the patients did not apply any neoadjuvant therapy before and received radical resection. Real-time reverse transcriptase - PCR (RT-PCR) were performed to examine the expressions of F. nucleatum in tumor tissues and para-tumor tissue. According to the CT values of the RT-PCR results, the level of the F. nucleatum infection could be separated in two groups - positive group and negative group. Immunohistochemistry (IHC) staining were used to examine the expressions of chemokine CCL20 in both groups. Immunofluorescence (IF) was characterised homing receptors CCR6 on CD206+ macrophages. In addition, transwell assay was carried on to quantify macrophages migration ability towards chemokines CCL20 in vitro. Results: F. nucleatum DNA positivity was significantly associated with higher expression of chemokine CCL20 and accumulation of CD68+CD206+ macrophages. F. nucleatum DNA positivity was also significantly correlated with worse histopathological grading. Otherwise, there were no significant correlation between F. nucleatum infection and gender, age, tobacco or alcohol use, T or N grade, TNM stage, tumor locations, blood vessel or nerve invasion and tumor size. IHC showed that the expression of CCL20 were higher in F. nucleatum DNA positive tumor tissue. After coculture with F. nucleatum and KYSE30 cell lines in vitro, the CCL20 production by KYSE30 cells were accelerated. In addition, immunofluorescence showed that CCL20 could recruit M2-like macrophages into the tumor environment by bonding CCR6 existed on the cell surface. Furthermore, the migration ability of macrophages was distinctly elevated by CCL20 result from F. nucleatum infection. Conclusions: F. nucleatum promotes esophageal squamous cell carcinoma progression via CCL20/CCR6-mediated migration of M2-like macrophages to the tumor micro- environment and deteriorates the suppression of the local immune micro- environment within the tumor. Such bacteria may be a biomarker to predict the efficacy of immunotherapy in ESCC. [Table: see text]

NAT10-mediated upregulation of GAS5 facilitates immune cell infiltration in non-small cell lung cancer via the MYBBP1A-p53/IRF1/type I interferon signaling axis

Interactions of tumor cells with immune cells in the tumor microenvironment play an important role during malignancy progression. We previously identified that GAS5 inhibited tumor development by suppressing proliferation of tumor cells in non-small cell lung cancer (NSCLC). Herein, we discovered a tumor-suppressing role for tumor cell-derived GAS5 in regulating tumor microenvironment. GAS5 positively coordinated with the infiltration of macrophages and T cells in NSCLC clinically, and overexpression of GAS5 promoted macrophages and T cells recruitment both in vitro and in vivo. Mechanistically, GAS5 stabilized p53 by directly binding to MYBBP1A and facilitating MYBBP1A-p53 interaction, and enhanced p53-mediated transcription of IRF1, which activated type I interferon signaling and increased the production of downstream CXCL10 and CCL5. We also found that activation of type I interferon signaling was associated with better immunotherapy efficacy in NSCLC. Furthermore, the stability of GAS5 was regulated by NAT10, the key enzyme responsible for N4-acetylcytidine (ac4C) modification, which bound to GAS5 and mediated its ac4C modification. Collectively, tumor cell-derived GAS5 could activate type I interferon signaling via the MYBBP1A-p53/IRF1 axis, promoting immune cell infiltration and potentially correlating with immunotherapy efficacy, which suppressed NSCLC progression. Our results suggested GAS5 as a promising predictive marker and potential therapeutic target for combination therapy in NSCLC.A schematic diagram demonstrating the regulatory effect of GAS5 on immune cell infiltration by activating type I interferon signaling via MYBBP1A-p53/IRF1 axis in non-small cell lung cancer. IFN, interferon.

Intermittent fasting alleviates IMQ-induced psoriasis-like dermatitis via reduced γδT17 and monocytes in mice.

Psoriasis is a chronic immune mediated inflammatory skin disease with systemic manifestations. It has been reported that caloric restriction could improve severity of psoriasis patients. However, the mechanism of intermittent fasting effects on psoriasis has not been investigated. Caloric restriction is known to reduce the number of circulating inflammatory monocytes in a CCL2-dependent manner. However, it is still unknown whether caloric restriction can improve psoriasis by regulating monocytes through CCL2. In this study, we used imiquimod (IMQ)-induced psoriasis-like mouse model to explore the effects and the mechanisms of intermittent fasting on psoriasis-like dermatitis. We found that intermittent fasting could significantly improve IMQ-induced psoriasis-like dermatitis, and reduce the number of γδT17 cells and IL-17 production in draining lymph nodes and psoriatic lesion via inhibiting proliferation and increasing death of γδT17 cells. Furthermore, intermittent fasting could significantly decrease monocytes in blood, and this was associated with decreased monocytes, macrophages and DC in psoriasis-like skin inflammation. Reduced monocytes in circulation and increased monocytes in BM of fasting IMQ-induced psoriasis-like mice is through reducing the production of CCL2 from BM to inhibit monocyte egress to the periphery. Our above data shads light on the mechanisms of intermittent fasting on psoriasis.

Exaggerated Kawasaki disease-associated vasculitis in C-C Motif Chemokine Receptor 5 (CCR5) deficient mice is driven by Granulocyte Colony Stimulating Factor (G-CSF) overproduction and neutrophil formation and infiltration

Kawasaki disease (KD) is a rare but serious illness that primarily affects children. It is characterized by inflammation of the blood vessels throughout the body, including the coronary arteries (CA). Polymorphisms in CCR5 influence KD susceptibility and cardiovascular outcome, but the mechanisms are enigmatic yet. Thus, we aimed to explore the mechanisms by which polymorphisms in CCR5 aggravate the vascular injury in KD by using the Candida albicans water soluble (CAWS) fraction model. Male (6-10-weeks-old) CCR5 deficient mice (CCR5−/−) and their counterpart (CCR5+/+) were used. Cardiac single-cell transcriptomics (scRNAseq) analysis of both control and KD mice revealed an inflammatory landscape characterized by an abundance of macrophages, monocytes, and neutrophils. Additionally, our scRNAseq data indicated that CCR5 expression is predominantly observed in macrophages and monocytes in KD mice, whereas its ligands, CCL3 and CCL4, are primarily expressed in neutrophils. Our CellChat analysis uncovered robust intercommunication between neutrophils and macrophages and monocytes. This interaction involves the production of CCL3 and CCL4 by neutrophils, which act on CCR5 receptors in macrophages and monocytes. To validate this scRNAseq data, we analyzed the vasculitis levels in CA and immune cells content in CCR5+/+ and CCR5−/− mice with KD via histology and flow cytometry. CCR5−/− mice with KD exhibited aggravated vasculitis, increased neutrophil accumulation [(% in relation to 10^6 leukocytes) CCR5+/+: 2.5±1.7 n=4 vs. CCR5+/+ KD: 16.1±3.2 n=4], and CCR5−/− KD: 30.2±7.4 n=4), alongside cardiac senescence (3-fold rise in p21 expression) and a 2-fold increase in Caspase-3, indicating increased cell death compared to CCR5+/+ with KD. Mechanistically, our plasma proteome profile cytokine assay revealed that KD increases circulating IL-6 and circulating neutrophil, interestingly CCR5−/− mice with KD showed further circulating IL-6 and neutrophil along with G-CSF, which plays a critical role in stimulating the production and release of granulocytes, particularly neutrophils, from the bone marrow into the bloodstream. Thus, deficiency in CCR5 triggers a worse vasculitis in KD by regulating an over production of neutrophil by bone marrow via G-CSF, which in turn will lead to an exaggerated IL-6 production, further accumulation in CA, and aggravated cardiac damage. FAPESP (2022/06639-2), NHLBI-R00 (R00HL14013903), AHA (CDA857268 and 23DIVSUP1069230), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting the Dendritic Cell-Secreted Immunoregulatory Cytokine CCL22 Alleviates Radioresistance.

Radiation-mediated immune suppression limits efficacy and is a barrier in cancer therapy. Radiation induces negative regulators of tumor immunity including regulatory T cells (Treg). Mechanisms underlying Treg infiltration after radiotherapy (RT) are poorly defined. Given that conventional dendritic cells (cDC) maintain Treg, we sought to identify and target cDC signaling to block Treg infiltration after radiation. Transcriptomics and high dimensional flow cytometry revealed changes in murine tumor cDC that not only mediate Treg infiltration after RT but also associate with worse survival in human cancer datasets. Antibodies perturbing a cDC-CCL22-Treg axis were tested in syngeneic murine tumors. A prototype interferon-anti-epidermal growth factor receptor fusion protein (αEGFR-IFNα) was examined to block Treg infiltration and promote a CD8+ T cell response after RT. Radiation expands a population of mature cDC1 enriched in immunoregulatory markers that mediates Treg infiltration via the Treg-recruiting chemokine CCL22. Blocking CCL22 or Treg depletion both enhanced RT efficacy. αEGFR-IFNα blocked cDC1 CCL22 production while simultaneously inducing an antitumor CD8+ T cell response to enhance RT efficacy in multiple EGFR-expressing murine tumor models, including following systemic administration. We identify a previously unappreciated cDC mechanism mediating Treg tumor infiltration after RT. Our findings suggest blocking the cDC1-CCL22-Treg axis augments RT efficacy. αEGFR-IFNα added to RT provided robust antitumor responses better than systemic free interferon administration and may overcome clinical limitations to interferon therapy. Our findings highlight the complex behavior of cDC after RT and provide novel therapeutic strategies for overcoming RT-driven immunosuppression to improve RT efficacy. See related commentary by Kalinski et al., p. 4260.

Inhibition of SGL2 Prevents Albumin Induced Proximal Tubule Inflammation

Albuminuria is an independent risk factor for cardiovascular disease and renal damage in hypertension. It is associated with a higher risk of cardiovascular morbidity and mortality, and with declining kidney function. The Na+-glucose cotransporter-2 (SGLT2) plays a crucial role in glucose and sodium reabsorption in the proximal tubule. Clinical trials have shown that SGLT2 inhibitors can reduce cardiovascular events, lower blood pressure, and decrease the risk of end-stage kidney disease in individuals with type 2 diabetes mellitus. These trials have also demonstrated the potential of SGLT2 inhibitors to provide additional benefits beyond their glucose control, including decreased albuminuria. In this study, we hypothesized that exposure to elevated albumin causes proximal tubule injury and the release of cytokines from the basolateral membrane. We propose that by reducing transport in the proximal tubule, SGLT2 inhibitors may alleviate kidney damage by reducing cytokine production and subsequently renal inflammation. To test our hypothesis, we conducted experiments using Dahl salt-sensitive (SS) rats and immortalized human proximal tubule cells (RPTEC-TERT1). To determine how early albuminuria and proximal injury occur in the development of salt-sensitive hypertension, 24-hour urine was collected from SS rats fed either a control salt (CS, 0.4% NaCl, n=10) or a high salt (HS, 4.0% NaCl, n=11) diet for 7 days. Significant increases in albuminuria were observed after 1 day of HS (CS: 25±7 vs. HS: 59±13 mg/day p=0.016). This was followed by significant increases in urinary kidney injury molecule-1 (Kim-1) after 2 days of HS (CS: 13±2 vs. HS: 23±3 pg/day p=0.021). Preliminary analysis found substantial increases in urinary CCL2 after 4 days of HS (CS: 58±7 vs. HS: 135±25 pg/mg, n=5). To investigate whether the renoprotective effects of SGLT2 inhibitors are in part due to their ability to reduce inflammation in the proximal tubule, we exposed the apical membrane of RPTEC-TERT1 cells to human serum albumin (HSA, 10mg/ml, n=6) with and without Dapagliflozin (Dapa, 15ng/ml, n=5) for 24-hours and measured the levels of CCL2 in the basolateral media. HSA caused a significant increase in CCL2 levels relative to control cells (Control: 42.5±5.6 vs. HSA: 84.1±8.7 pg/mg, p=0.00). This increase was prevented by co-treatment with Dapa and CCL2 levels were equivalent to those in the control cells (HSA+Dapa: 44.0±4.5 pg/mg, p=0.988). In conclusion, our findings support the hypothesis that albuminuria precedes proximal tubule injury and may contribute to the development of renal inflammation in salt-sensitive hypertension. The ability of Dapa to reduce prevent albumin induced CCL2 production in RPTEC-TERT1 cells suggests that SGLT2 inhibitors may reduce renal inflammation in salt-sensitive hypertension and is an area of ongoing studies. Studies funded by R01HL152166 to LE. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Guanylate-binding protein 1 inhibits inflammatory factors produced by H5N1 virus through Its GTPase activity

The combination of inflammatory factors resulting from an influenza A virus infection is one of the main causes of death in host animals. Studies have shown that guinea pig guanosine monophosphate binding protein 1 (guanylate-binding protein 1, gGBP1) can downregulate cytokine production induced by the influenza virus. Therefore, exploring the innate immune defense mechanism of GBP1 in the process of H5N1 influenza virus infection has important implications for understanding the pathogenic mechanism, disease prevention, and the control of influenza A virus infections. We found that, in addition to inhibiting the early replication of influenza virus, gGBP1 also inhibited the production of CCL2 and CXCL10 cytokines induced by the influenza virus as well as the proliferation of mononuclear macrophages induced by these cytokines. These findings further confirmed that gGBP1 inhibited the production of cytokines through its GTPase activity and cell proliferation through its C-terminal α-helix structure. This study revealed the effect of gGBP1 on the production of cellular inflammatory factors during influenza virus infection and determined the key amino acid residues that assist in the inhibitory processes mediated by gGBP1.