Machine Learning Identifies Key Proteins in Primary Sclerosing Cholangitis Progression and Links High CCL24 to Cirrhosis.

Primary sclerosing cholangitis (PSC) is a rare, progressive disease, characterized by inflammation and fibrosis of the bile ducts, lacking reliable prognostic biomarkers for disease activity. Machine learning applied to broad proteomic profiling of sera allowed for the discovery of markers of disease presence, severity, and cirrhosis and the exploration of the involvement of CCL24, a chemokine with fibro-inflammatory activity. Sera from 30 healthy controls and 45 PSC patients were profiled with proximity extension assay, quantifying the expression of 2870 proteins, and used to train an elastic net model. Proteins that contributed most to the model were tested for correlation to enhanced liver fibrosis (ELF) score and used to perform pathway analysis. Statistical modeling for the presence of cirrhosis was performed with principal component analysis (PCA), and receiver operating characteristics (ROC) curves were used to assess the useability of potential biomarkers. The model successfully predicted the presence of PSC, where the top-ranked proteins were associated with cell adhesion, immune response, and inflammation, and each had an area under receiver operator characteristic (AUROC) curve greater than 0.9 for disease presence and greater than 0.8 for ELF score. Pathway analysis showed enrichment for functions associated with PSC, overlapping with pathways enriched in patients with high levels of CCL24. Patients with cirrhosis showed higher levels of CCL24. This data-driven approach to characterize PSC and its severity highlights potential serum protein biomarkers and the importance of CCL24 in the disease, implying its therapeutic potential in PSC.

CCL24/CCR3 axis plays a central role in angiotensin II-induced heart failure by stimulating M2 macrophage polarization and fibroblast activation.

AIMS: We aimed to investigate the effect and mechanism of pleiotropic chemokine CCL24 in heart failure. METHODS AND RESULTS: Compared with normal donators, the expression of CCL24 and number of cardiac M2 macrophages in heart were higher in heart failure patients, the same as plasma CCL24. Treatment with CCL24 antibody hindered Ang II (1500 ng/kg/min)-induced cardiac adverse remodeling through preventing cardiac hypertrophy and fibrosis. RNA-seq showed that CCL24/CCR3 axis was involved in immune and inflammatory responses. Single-cell analysis of cytometry by time of flight (CyTOF) revealed that CCL24 antibody decreased the M2 macrophage and monocyte polarization during Ang II stimulation. Immunofluorescence co-localization analysis confirmed the expression of CCR3 in macrophage and fibroblasts. Then, in vitro experiments confirmed that CCL24/CCR3 axis was also involved in cardiac primary fibroblast activation through its G protein-coupled receptor function. CONCLUSION: CCL24/CCR3 axis plays a crucial part in cardiac remodeling by stimulating M2 macrophage polarization and cardiac fibroblast activation. Cardiac M2 macrophages, CCL24 and circulation CCL24 increased in heart failure patients. Treatment with CCL24 Ab hindered Ang II induced cardiac structural dysfunction and electrical remodeling. In CCL24 Ab group RNA-seq found that it was related to immune responses and hypertrophic cardiomyopathy, CytoF revealed M2 macrophages and monocytes decreased obviously. In vitro,CCL24 promoted activation and migration of cardiac fibroblast.

Downregulation of Salt-Inducible Kinase 3 Enhances CCL24 Activation in the Placental Environment with Preeclampsia.

Preeclampsia (PE) remains one of the leading causes of maternal and perinatal morbidity and mortality. However, the exact pathophysiology of PE is still unclear. The recent widely accepted notion that successful pregnancy relies on maternal immunological adaptation is of utmost importance. Moreover, salt-inducible kinase 3 (SIK3) is an AMP-activated protein kinase-related kinase, and it has reported a novel regulator of energy and inflammation, and its expression related with some diseases. To explore whether SIK3 expression correlated with PE, we analyzed SIK3 gene expression and its association with PE through GEO datasets. We identified that SIK3 was significantly downregulated in PE across four datasets (p < 0.05), suggesting that SIK3 participated in the pathogenesis of PE. We initially demonstrated the significant downregulation of SIK3 in trophoblast cells of PE. SIK3 downregulation was positively correlated with the increased number of CD204(+) cells in in vivo and in vitro experiments. The increased number of CD204(+) cells could inhibit the migration and invasion of trophoblast cells. We then clarified the potential mechanism of PE with SIK3 downregulation: M2 skewing was triggered by trophoblast cells derived via the CCL24/CCR3 axis, leading to an increase in CD204(+) cells, a decrease in phagocytosis, and the production of IL-10 at the maternal-fetal interface of the placenta with PE. IL-10 further contributed to a reduction in the migration and invasion of trophoblast cells. It also established a feedback loop wherein trophoblast cells increased CCL24 production to maintain M2 dominance in the placental environments of PE.

Hepatic recruitment of eosinophils and their protective function during acute liver injury.

BACKGROUND & AIMS: Beyond the classical description of eosinophil functions in parasite infections and allergic diseases, emerging evidence supports a critical role of eosinophils in resolving inflammation and promoting tissue remodeling. However, the role of eosinophils in liver injury and the underlying mechanism of their recruitment into the liver remain unclear. METHODS: Hepatic eosinophils were detected and quantified using flow cytometry and immunohistochemical staining. Eosinophil-deficient (ΔdblGata1) mice were used to investigate the role of eosinophils in 3 models of acute liver injury. In vivo experiments using Il33-/- mice and macrophage-depleted mice, as well as in vitro cultures of eosinophils and macrophages, were performed to interrogate the mechanism of eotaxin-2 (CCL24) production. RESULTS: Hepatic accumulation of eosinophils was observed in patients with acetaminophen (APAP)-induced liver failure, whereas few eosinophils were detectable in healthy liver tissues. In mice treated with APAP, carbon tetrachloride or concanavalin A, eosinophils were recruited into the liver and played a profound protective role. Mice deficient of macrophages or IL-33 exhibited impaired hepatic eosinophil recruitment during acute liver injury. CCL24, but not CCL11, was increased after treatment of each hepatotoxin in an IL-33 and macrophage-dependent manner. In vitro experiments demonstrated that IL-33, by stimulating IL-4 release from eosinophils, promoted the production of CCL24 by macrophages. CONCLUSIONS: This is the first study to demonstrate that hepatic recruitment of and protection by eosinophils occur commonly in various models of acute liver injury. Our findings support further exploration of eosinophils as a therapeutic target to treat APAP-induced acute liver injury. LAY SUMMARY: The current study unveils that eosinophils are recruited into the liver and play a protective function during acute liver injury caused by acetaminophen overdose. The data demonstrate that IL-33-activated eosinophils trigger macrophages to release high amounts of CCL24, which promotes hepatic eosinophil recruitment. Our findings suggest that eosinophils could be an effective cell-based therapy for the treatment of acetaminophen-induced acute liver injury.

CCL24 Signaling in the Tumor Microenvironment.

Chemokines with their network play an important role in cancer growth, metastasis, and host-tumor interactions. Of many chemokines, C-C motif chemokine ligand 24 (CCL24) has been shown to contribute to tumorigenesis as well as inflammatory diseases like asthma, allergies, and eosinophilic esophagitis. CCL24 is expressed in some tumor cells such as colon cancer, hepatocellular carcinoma, and cutaneous T cell lymphoma. CCL24 can be used as a potential biomarker in several cancers including colon cancer, non-small cell cancer, and nasopharyngeal carcinoma as the plasma level of CCL24 is increased. The various functions of CCL24 contribute to the biology of cancer by M2 macrophage polarization, angiogenesis, invasion and migration, and recruitment of eosinophils.

Increased CCL24 and CXCL7 levels in the cerebrospinal fluid of patients with neurosyphilis.

BACKGROUND: Monocytes are recruited into the cerebrospinal fluid (CSF) of patients with neurosyphilis, suggesting abnormal chemokine expression. We aimed to investigate the aberrant expression of chemokines in the CSF of these patients. METHODS: CSF and serum samples were collected from patients with neurosyphilis between July 2017 and June 2019 in the Dermatology Department, Second Affiliated Hospital of Zhejiang University. Differences in the expression of 38 chemokines between patients with and without neurosyphilis were detected using RayBio® Human Chemokine Antibody Array C1. CCL24 and CXCL7 levels in the patients' CSF and serum were further measured using RayBio® CCL24 and CXCL7 ELISA kits. RESULTS: Ninety-three CSF and serum samples of patients with syphilis were collected. Antibody array analysis showed that the CSF levels of CCL24 (P = .0185), CXCL7 (P < .0001), CXCL13 (P < .0001), CXCL10 (P < .0001), and CXCL8 (P < .0001) were significantly higher in patients with than without neurosyphilis. ELISA confirmed significantly higher CCL24 and CXCL7 levels in the CSF of patients with than without neurosyphilis (CCL24: 6.082 ± 1.137 pg/mL vs 1.773 ± 0.4565 pg/mL, P = .0037; CXCL7: 664.3 ± 73.19 pg/mL vs 431.1 ± 90.54 pg/mL, P = .0118). Increased CCL24 and CXCL7 expression was seen throughout all neurosyphilis stages, had moderate diagnostic efficiency for neurosyphilis, and correlated poorly with CSF cell count and Venereal Disease Research Laboratory titer. CSF CCL24 levels also correlated poorly with CSF protein concentration. CONCLUSION: Abnormally high CSF chemokines levels may play a role in the pathogenesis of neurosyphilis.

Mesenchymal Stromal Cell (MSC)-Derived Combination of CXCL5 and Anti-CCL24 Is Synergistic and Superior to MSC and Cyclosporine for the Treatment of Graft-versus-Host Disease.

The immunosuppressive properties of mesenchymal stromal cells (MSCs) have been clinically proven to be effective in treating graft-versus-host disease (GVHD). However, MSC therapy is limited by the need for laborious and expensive manufacturing processes that are fraught with batch-to-batch variability. Substitution of MSC therapy with key MSC-mediated immunomodulatory factors could be an option for GVHD treatment. Using a simulated in vitro model of the immunosuppressive effects of MSC on allogeneic graft reactions, a synergistic 2-factor combination (2FC) of CXCL5 and anti-CCL24 was identified from a panel of over 100 immunomodulatory factors as being superior to MSCs in the modulation of mixed lymphocyte reactions. This 2FC was superior to cyclosporine in ameliorating both moderate and severe GVHD while being equivalent to MSCs in moderate GVHD and superior to MSCs in severe GVHD. Its immunosuppressive efficacy could be further improved by extended treatment. Mechanistic studies revealed that in vitro the 2FC could only reduce the proliferation of Th 1 and Th 17, whereas in vivo CXCL5 acts in concert with anti-CCL24 antibody to reduce not only transplanted Th 1 and Th 17 but also cytotoxic T lymphocytes and natural killer cells to increase mouse immunosuppressive neutrophils without affecting human hematopoietic stem cell reconstitution. Concurrently, it reduced circulating human proinflammatory cytokines IFN-γ, IL-6, IL-17A, IL-8, macrophage inflammatory protein-1ß, and monocyte chemoattractant protein-1. Both in vitro and in vivo data suggest that CXCL5 and anti-CCL24 antibody act in concert to ameliorate GVHD via suppression of Th 1 and Th 17 responses. We propose that this novel 2FC could substitute for MSC therapy in GVHD treatment.

Melatonin attenuates osteosarcoma cell invasion by suppression of C-C motif chemokine ligand 24 through inhibition of the c-Jun N-terminal kinase pathway.

Osteosarcoma, with its high metastatic potential, is the most prevalent malignant bone tumor in children and adolescents. Melatonin possesses multiple tumor-suppressing properties for a myriad of tumors, but little is known about the effects of melatonin on osteosarcoma metastasis. In this study, we demonstrated that melatonin elicited very low cytotoxicity and significantly inhibited cellular motility, migration, and invasion in human osteosarcoma U2OS and HOS cells. Moreover, using RNA sequencing technology, we revealed that melatonin repressed C-C motif chemokine ligand 24 (CCL24) gene expression in U2OS cells. Manipulation of CCL24 levels influenced the motility of osteosarcoma cells as cell migration and invasion were enhanced by the addition of recombinant human CCL24 and attenuated by the silencing of CCL24. Moreover, melatonin increased and decreased the activation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2, respectively, in a dose-dependent manner in U2OS and HOS cells while exerting no evident influence on the level and activation of p38, Akt, FAK, steroid receptor coactivator, or Raf. In further functional experiments, the use of JNK inhibitors (SP600125 and DN-JNK) confirmed that the pharmaceutic inhibition of JNK augmented the melatonin-mediated CCL24 suppression and migration of U2OS cells. Overall, our results revealed that melatonin attenuated chemokine CCL24 levels through inhibition of the JNK pathway to hinder human osteosarcoma cell invasion, thereby highlighting the therapeutic potential of melatonin for osteosarcoma metastasis.

Tumor progression locus 2 reduces severe allergic airway inflammation by inhibiting Ccl24 production in dendritic cells.

BACKGROUND: The molecular and cellular pathways driving the pathogenesis of severe asthma are poorly defined. Tumor progression locus 2 (TPL-2) (COT, MAP3K8) kinase activates the MEK1/2-extracellular-signal regulated kinase 1/2 MAP kinase signaling pathway following Toll-like receptor, TNFR1, and IL-1R stimulation. OBJECTIVE: TPL-2 has been widely described as a critical regulator of inflammation, and we sought to investigate the role of TPL-2 in house dust mite (HDM)-mediated allergic airway inflammation. METHODS: A comparative analysis of wild-type and Map3k8-/- mice was conducted. Mixed bone marrow chimeras, conditional knockout mice, and adoptive transfer models were also used. Differential cell counts were performed on the bronchoalveolar lavage fluid, followed by histological analysis of lung sections. Flow cytometry and quantitative PCR was used to measure type 2 cytokines. ELISA was used to assess the production of IgE, type 2 cytokines, and Ccl24. RNA sequencing was used to characterize dendritic cell (DC) transcripts. RESULTS: TPL-2 deficiency led to exacerbated HDM-induced airway allergy, with increased airway and tissue eosinophilia, lung inflammation, and IL-4, IL-5, IL-13, and IgE production. Increased airway allergic responses in Map3k8-/- mice were not due to a cell-intrinsic role for TPL-2 in T cells, B cells, or LysM+ cells but due to a regulatory role for TPL-2 in DCs. TPL-2 inhibited Ccl24 expression in lung DCs, and blockade of Ccl24 prevented the exaggerated airway eosinophilia and lung inflammation in mice given HDM-pulsed Map3k8-/- DCs. CONCLUSIONS: TPL-2 regulates DC-derived Ccl24 production to prevent severe type 2 airway allergy in mice.

Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric H,K-ATPase.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by tissue eosinophilia that is associated with poor prognosis. Recent findings that proton pump inhibitors (PPIs) directly modulate the expression of eotaxin-3, an eosinophil chemoattractant, in patients with eosinophilic diseases suggest therapeutic potential for PPIs in those with CRSwNP. OBJECTIVE: We assessed the effect of type 2 mediators, particularly IL-13 and eotaxin-3, on tissue eosinophilia and disease severity in patients with chronic rhinosinusitis (CRS). Further investigation focused on PPI suppression of eotaxin-3 expression in vivo and in vitro, with exploration of underlying mechanisms. METHODS: Type 2 mediator levels in nasal tissues and secretions were measured by using a multiplex immunoassay. Eotaxin-3 and other chemokines expressed in IL-13-stimulated human sinonasal epithelial cells (HNECs) and BEAS-2B cells with or without PPIs were assessed by using ELISA, Western blotting, real-time PCR, and intracellular pH imaging. RESULTS: Nasal tissues and secretions from patients with CRSwNP had increased IL-13, eotaxin-2, and eotaxin-3 levels, and these were positively correlated with tissue eosinophil cationic protein levels and radiographic scores in patients with CRS (P < .05). IL-13 stimulation of HNECs and BEAS-2B cells dominantly induced eotaxin-3 expression, which was significantly inhibited by PPIs (P < .05). Patients with CRS taking PPIs also showed lower in vivo eotaxin-3 levels compared with those without PPIs (P < .05). Using intracellular pH imaging and altering extracellular K+, we found that IL-13 enhanced H+,K+-exchange, which was blocked by PPIs and the mechanistically unrelated H,K-ATPase inhibitor, SCH-28080. Furthermore, knockdown of ATP12A (gene for the nongastric H,K-ATPase) significantly attenuated IL-13-induced eotaxin-3 expression in HNECs. PPIs also had effects on accelerating IL-13-induced eotaxin-3 mRNA decay. CONCLUSION: Our results demonstrated that PPIs reduce IL-13-induced eotaxin-3 expression by airway epithelial cells. Furthermore, mechanistic studies suggest that the nongastric H,K-ATPase is necessary for IL-13-mediated epithelial responses, and its inhibitors, including PPIs, might be of therapeutic value in patients with CRSwNP by reducing epithelial production of eotaxin-3.

Role of chemokine (C-C motif) ligand 24 in spatial arrangement of the inner cell mass of the bovine embryo.

The process of spatial rearrangement of cells of the inner cell mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of GATA6+ cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6+ cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that cell migration is involved in the spatial organization of hypoblast cells in the blastocyst.

Dietary treatment modulates mast cell phenotype, density, and activity in adult eosinophilic oesophagitis.

BACKGROUND: Mast cells (MCs) are abundant in the inflammatory infiltrate in eosinophilic oesophagitis (EoE), but decrease with disease remission. However, their phenotype, role in the pathophysiology of the disease, and modulation after effective dietary therapy are still unclear. OBJECTIVE: To define the phenotype of oesophageal MCs, their modulation through dietary therapy, and their association with clinical manifestations of EoE. METHODS: Oesophageal mucosal samples from 10 adult patients with EoE obtained before and after effective six-food elimination diet (SFED) therapy, as well as from 10 control subjects were analysed. Eosinophil and MC density were quantified. Gene expression of chemoattractants for eosinophils (CCL11, CCL24, and CCL26), MCs (SCF), and their receptors (CCR3 and SCFR, respectively) were assessed by means of qPCR. Gene and protein expression of specific MC proteases (CPA3, CMA, and TPSB2) were evaluated with qPCR and immunofluorescence. Clinical manifestations and atopic background were recorded. RESULTS: MC density was significantly increased in EoE compared with controls, decreasing after dietary treatment (18.6 to 1.44 cells/hpf, respectively; P < 0.001). The MCTC subtype predominated in the oesophageal mucosa (90%) in both patients with EoE and controls. Gene expression of MC-related proteases, eotaxins, and SCF were up-regulated in patients with EoE, but significantly decreased after therapy, regardless of atopic background. Epithelial peaks of MCs and eosinophils were significantly associated (ρ = 0.80) in EoE and correlated with the symptom score (ρ = 0.78). Gene expression of MC proteases and eotaxins also correlated with the symptom score (P < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: MC and its proteases seem to play a relevant role in the pathophysiology and symptoms of EoE, which can be reversed after effective dietary treatment.

IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration.

M2 macrophages have been subdivided into subtypes such as IL-4-induced M2a and IL-10-induced M2c in vitro. Although it was reported that IL-10 stimulation leads to an increase in IL-4Rα, the effect of IL-4 and IL-10 in combination with macrophage subtype differentiation remains unclear. Thus, we sought to clarify whether IL-10 enhanced the M2 phenotype induced by IL-4. In this study, we showed that IL-10 enhanced IL-4Rα expression in M-CSF-induced bone marrow-derived macrophages (BMDMs). Global gene expression analysis of M2 macrophages induced by IL-4, IL-10 or IL-4 + IL-10 showed that IL-10 enhanced gene expression of M2a markers induced by IL-4 in M-CSF-induced BMDMs. Moreover, IL-4 and IL-10 synergistically induced CCL24 (Eotaxin-2) production. Enhanced CCL24 expression was also observed in GM-CSF-induced BMDMs and zymosan-elicited, thioglycolate-elicited and naive peritoneal macrophages. CCL24 is a CCR3 agonist and an eosinophil chemoattractant. In vitro, IL-4 + IL-10-stimulated macrophages produced a large amount of CCL24 and increased eosinophil migration, which was inhibited by anti-CCL24 antibody. We also showed that IL-4 + IL-10-stimulated (but not IL-4 or IL-10 alone) macrophages transferred into the peritoneum of C57BL/6J mice increased eosinophil infiltration into the peritoneal cavity. These results demonstrate that IL-4 + IL-10-simulated macrophages have enhanced M2a macrophage-related gene expression, CCL24 production and eosinophil infiltration-inducing activity, thereby suggesting their contribution to eosinophil-related diseases.

TNF-related apoptosis-inducing ligand (TRAIL) regulates midline-1, thymic stromal lymphopoietin, inflammation, and remodeling in experimental eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. OBJECTIVE: We sought to elucidate the role of TRAIL in EoE. METHODS: We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL(-/-)) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. RESULTS: TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL(-/-) mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL(-/-) mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. CONCLUSION: TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

Chemotaxis of bone marrow derived eosinophils in vivo: a novel method to explore receptor-dependent trafficking in the mouse.

Here, we describe a novel method via which ex vivo cultured mouse bone marrow derived eosinophils (bmEos) can be adoptively transferred into recipient mice in order to study receptor-dependent recruitment to lung tissue in vivo. Intratracheal instillation of recombinant human eotaxin-2 (hCCL24) prior to introduction of bmEos via tail vein injection resulted in an approximately fourfold increase in Siglec F-positive/CD11c-negative eosinophils in the lungs of eosinophil-deficient ΔdblGATA recipient mice compared with controls. As anticipated, bmEos generated from CCR3-gene-deleted mice did not migrate to the lung in response to hCCL24 in this model, indicating specific receptor dependence. BmEos generated from GFP-positive BALB/c mice responded similarly to hCCL24 in vitro and were detected in lung tissue of BALB/c WT as well as BALB/c ΔdblGATA eosinophil-deficient recipient mice, at approximately fourfold (at 5 h post-injection) and approximately threefold (at 24 h postinjection) over baseline, respectively. Comparable results were obtained with GFP-positive C57BL/6 bmEos responding to intratracheal hCCL24 in C57BL/6 ΔdblGATA recipient mice. The use of ex vivo cultured bmEos via one or more of these methods offers the possibility of manipulating bmEos prior to transfer into a WT or gene-deleted recipient host. Thus, this chemotaxis model represents a novel and robust tool for pharmacological studies in vivo.

The Role of CCL24 in Primary Sclerosing Cholangitis: Bridging Patient Serum Proteomics to Preclinical Data.

Primary sclerosing cholangitis (PSC) is an inflammatory and fibrotic biliary disease lacking approved treatment. We studied CCL24, a chemokine shown to be overexpressed in damaged bile ducts, and its involvement in key disease-related mechanisms. Serum proteomics of PSC patients and healthy controls (HC) were analyzed using the Olink® proximity extension assay and compared based on disease presence, fibrosis severity, and CCL24 levels. Disease-related canonical pathways, upstream regulators, and toxicity functions were elevated in PSC patients compared to HC and further elevated in patients with high CCL24 levels. In vitro, a protein signature in CCL24-treated hepatic stellate cells (HSCs) differentiated patients by disease severity. In mice, CCL24 intraperitoneal injection selectively recruited neutrophils and monocytes. Treatment with CM-101, a CCL24-neutralizing antibody, in an α-naphthylisothiocyanate (ANIT)-induced cholestasis mouse model effectively inhibited accumulation of peribiliary neutrophils and macrophages while reducing biliary hyperplasia and fibrosis. Furthermore, in PSC patients, CCL24 levels were correlated with upregulation of monocyte and neutrophil chemotaxis pathways. Collectively, these findings highlight the distinct role of CCL24 in PSC, influencing disease-related mechanisms, affecting immune cells trafficking and HSC activation. Its blockade with CM-101 reduces inflammation and fibrosis and positions CCL24 as a promising therapeutic target in PSC.

Stromal cell and B cell dialogue potentiates IL-33-enriched lymphoid niches to support eosinophil recruitment and function during type 2 immunity.

Eosinophils are involved in host protection against multicellular organisms. However, their recruitment to the mesenteric lymph node (mLN) during type 2 immunity is understudied. Our results demonstrate that eosinophil association with lymphoid stromal niches constructed by fibroblastic reticular cells (FRCs) and lymphatic endothelial cells is diminished in mice selectively lacking interleukin (IL)-4Rα or lymphotoxin-ß (LTß) expression on B cells. Furthermore, eosinophil survival, activation, and enhanced Il1rl1 receptor expression are driven by stromal cell and B cell dialogue. The ligation of lymphotoxin-ß receptor (LTßR) on FRCs improves eosinophil survival and significantly augments IL-33 expression and eosinophil homing to the mLN, thus confirming the significance of lymphotoxin signaling for granulocyte recruitment. Eosinophil-deficient ΔdblGATA-1 mice show diminished mLN expansion, reduced interfollicular region (IFR) alarmin expression, and delayed helminth clearance, elucidating their importance in type 2 immunity. These findings provide insight into dialogue between stromal cells and B cells, which govern mLN eosinophilia, and the relevance of these mechanisms during type 2 immunity.

Dermis resident macrophages orchestrate localized ILC2-eosinophil circuitries to maintain their M2-like properties and promote non-healing cutaneous leishmaniasis.

Tissue-resident macrophages (TRMs) are critical for tissue homeostasis/repair. We previously showed that dermal TRMs produce CCL24 (eotaxin2) which mediates their interaction with IL-4 producing eosinophils, required to maintain their number and M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we unveil another layer of TRM self-maintenance involving their production of TSLP, an alarmin typically characterized as epithelial cell-derived. Both TSLP signaling and IL-5+ innate lymphoid cell 2 (ILC2s) were shown to maintain the number of dermal TRMs and promote infection. Single cell RNA sequencing identified the dermal TRMs as the sole source of TSLP and CCL24. Development of Ccl24-cre mice permitted specific labeling of dermal TRMs, as well as interstitial TRMs from other organs. Genetic ablation of TSLP from dermal TRMs reduced the number of dermal TRMs, and disease was ameliorated. Thus, by orchestrating localized type 2 circuitries with ILC2s and eosinophils, dermal TRMs are self-maintained as a replicative niche for L. major.

Eosinophil trafficking in allergen-mediated pulmonary inflammation relies on IL-13-driven CCL-11 and CCL-24 production by tissue fibroblasts and myeloid cells.

Background: The immunologic mechanisms underlying pulmonary type 2 inflammation, including the dynamics of eosinophil recruitment to the lungs, still need to be elucidated. Objective: We sought to investigate how IL-13-producing TH2 effector cells trigger eosinophil migration in house dust mite (HDM)-driven allergic pulmonary inflammation. Methods: Multiparameter and molecular profiling of murine lungs with HDM-induced allergy was investigated in the absence of IL-13 signaling by using IL-13Rα1-deficient mice and separately through adoptive transfer of CD4+ T cells from IL-5-deficient mice into TCRα-/- mice before allergic inflammation. Results: We demonstrated through single-cell techniques that HDM-driven pulmonary inflammation displays a profile characterized by TH2 effector cell-induced IL-13-dominated eosinophilic inflammation. Using HDM-sensitized IL-13Rα1-/- mice, we found a marked reduction in the influx of eosinophils into the lungs along with a significant downregulation of both CCL-11 and CCL-24. We further found that eosinophil trafficking to the lung relies on production of IL-13-driven CCL-11 and CCL-24 by fibroblasts and Ly6C+ (so-called classical) monocytes. Moreover, this IL-13-mediated eotaxin-dependent eosinophil influx to the lung tissue required IL-5-induced eosinophilia. Finally, we demonstrated that this IL-13-driven eosinophil-dominated pulmonary inflammation was critical for limiting bystander lung transiting Ascaris parasites in a model of allergy and helminth interaction. Conclusion: Our data suggest that IL-5-dependent allergen-specific TH2 effector cell response and subsequent signaling through the IL-13/IL-13Rα1 axis in fibroblasts and myeloid cells regulate the eotaxin-dependent recruitment of eosinophils to the lungs, with multiple downstream consequences, including bystander control of lung transiting parasitic helminths.

Single-Cell RNA-Seq Reveals a Crosstalk between Hyaluronan Receptor LYVE-1-Expressing Macrophages and Vascular Smooth Muscle Cells.

Background: Atherosclerosis is a chronic inflammatory disease where macrophages participate in the progression of the disease. However, the role of resident-like macrophages (res-like) in the atherosclerotic aorta is not completely understood. Methods: A single-cell RNA sequencing analysis of CD45+ leukocytes in the atherosclerotic aorta of apolipoprotein E-deficient (Apoe-/-) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the side-to-specific predisposition to atherosclerosis, was performed. A population of res-like macrophages expressing hyaluronan receptor LYVE-1 was investigated via flow cytometry, co-culture experiments, and immunofluorescence in human atherosclerotic plaques from carotid artery disease patients (CAD). Results: We identified 12 principal leukocyte clusters with distinct atherosclerosis disease-relevant gene expression signatures. LYVE-1+ res-like macrophages, expressing a high level of CC motif chemokine ligand 24 (CCL24, eotaxin-2), expanded under hypercholesteremia in Apoe-/- mice and promoted VSMC phenotypic modulation to osteoblast/chondrocyte-like cells, ex vivo, in a CCL24-dependent manner. Moreover, the abundance of LYVE-1+CCL24+ macrophages and elevated systemic levels of CCL24 were associated with vascular calcification and CAD events. Conclusions: LYVE-1 res-like macrophages, via the secretion of CCL24, promote the transdifferentiation of VSMC to osteogenic-like cells with a possible role in vascular calcification and likely a detrimental role in atherosclerotic plaque destabilization.