CCR5 Expression Research Articles

IL-38 Aggravates Atopic Dermatitis via Facilitating Migration of Langerhans cells.

Atopic dermatitis (AD) is a common inflammation skin disease that involves dysregulated interplay between immune cells and keratinocytes. Interleukin-38 (IL-38), a poorly characterized IL-1 family cytokine, its role and mechanism in the pathogenesis of AD is elusive. Here, we show that IL-38 is mainly secreted by epidermal keratinocytes and highly expressed in the skin and downregulated in AD lesions. We generated IL-38 keratinocyte-specific knockout mice (K14Cre/+-IL-38f/f ) and induced AD models by 2,4-dinitrofluorobenzene (DNFB). Unexpectedly, after treatment with DNFB, K14Cre/+-IL-38f/f mice were less susceptible to cutaneous inflammation of AD. Moreover, keratinocyte-specific deletion of IL-38 suppressed the migration of Langerhans cells (LCs) into lymph nodes which results in disturbed differentiation of CD4+T cells and decreased the infiltration of immune cells into AD lesions. LCs are a type of dendritic cell that reside specifically in the epidermis and regulate immune responses. We developed LC-like cells in vitro from mouse bone marrow (BM) and treated with recombined IL-38. The results show that IL-38 depended on IL-36R, activated the phosphorylated expression of IRAK4 and NF-κB P65 and upregulated the expression of CCR7 to promoting the migration of LCs, nevertheless, the upregulation disappeared with the addition of IL-36 receptor antagonist (IL-36RA), IRAK4 or NF-κB P65 inhibitor. Furthermore, after treatment with IRAK4 inhibitors, the experimental AD phenotypes were alleviated and so IRAK4 is considered a promising target for the treatment of inflammatory diseases. Overall, our findings indicated a potential pathway that IL-38 depends on IL-36R, leading to LCs migration to promote AD by upregulating CCR7 via IRAK4/NF-κB and implied the prevention and treatment of AD, supporting potential clinical utilization of IRAK4 inhibitors in AD treatment.

IL-33-Pretreated Mesenchymal Stem Cells Attenuate Acute Liver Failure by Improving Homing and Polarizing M2 Macrophages.

Mesenchymal stem cells (MSCs) are highly effective in the treatment of acute liver failure (ALF). The efficacy of MSCs is closely related to the inflammatory environment. Therefore, we investigated the functional changes of MSCs in response to interleukin-33 (IL-33) stimulation. The results showed that bone marrow mesenchymal stem cells (BMSCs) pretreated with IL-33 had increased CCR2 expression, targeted CCL2 in the injured liver tissue, and improved the migration ability. Under LPS stimulation, the NF-κB pathway of BMDM was activated, and its phenotype polarized to the M1-type, while BMSCs pretreated with IL-33 inhibited the NF-κB pathway and enhanced M2 macrophage polarization. The M2-type macrophages could further inhibit hepatocytes inflammation, reduce hepatocytes apoptosis, and promote hepatocytes repair. These results suggest that IL-33 can enhance the efficacy of BMSCs in ALF and provide a new strategy for cell therapy of liver diseases.

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

Network pharmacology combines machine learning, molecular simulation dynamics and experimental validation to explore the mechanism of acetylbinankadsurin A in the treatment of liver fibrosis

Ethnopharmacological relevanceThe Kadsura coccinea (Lem.) A. C. Smith is known as “Heilaohu” of the Tujia ethnomedicine in China. It has anti-tumor, anti-oxidation, anti-HIV, anti-inflammatory and liver protective effects, used to treat diseases such as rheumatoid arthritis, cancer, gastritis and hepatitis. In this research, we investigated the anti-fibrotic effect and possible mechanisms of acetylbinankadsurin A (ACBA) in vitro and in vivo, which is a natural compound derived from roots of K. coccinea. Aim of the studyWe try to evaluate the efficacy of ACBA in the treatment of liver fibrosis and to explore the underlying mechanisms of ACBA by network pharmacology, machine learning, molecular docking, molecular dynamics simulations, and experimental assessment. Materials and methodsACBA was isolated from the CH2Cl2 layer of the roots of K. coccinea through column chromatographic techniques. The structure of ACBA was determined by using 1D and 2D NMR. CCl4-induced C57BL/6 mouse liver fibrosis models were established to evaluate the anti-fibrosis effects of ACBA in vivo. The molecular targets of ACBA and liver fibrosis were obtained from various databases, then constructed a protein-protein interaction (PPI) networks through the STRING database. Gene ontology (GO) enrichment and kyoto encyclopedia of genes and genomes (KEGG) analysis were applied using the “clusterProfiler” R package. Furthermore, the key genes for ACBA treatment of liver fibrosis were identified by the least absolute shrinkage and selection operator (LASSO). Molecular docking and molecular dynamics simulations were also carried out. Finally, the target and pathway of ACBA were verified by immunofluorescence staining, RT-PCR and Western blot. ResultFirst, ACBA attenuated CCl4-induced liver injury and fibrosis in vivo. These findings were accompanied by decreased expression of α-SMA and collagen I. Second, ACBA significantly decreased serum levels of ALT, AST, TNF-α and IL-6. Then, we identified 133 potential targets of ACBA and 7987 targets of liver fibrosis. KEGG analysis showed that ACBA could regulate the drug metabolism - cytochrome P450, fructose and mannose metabolism, IL-17 and NF-κB signaling pathways. Next, six core targets was screened by LASSO analysis including AKR1B1, PFKFB3, EPHA3, CDK1, CCR1 and CYP3A4. Molecular docking showed that ACBA has a good binding affinity for CCR1. Furthermore, compared with CCR1 inhibitor BX-471, The results of molecular simulation dynamics showed that ACBA was stable in binding with CCR1. Consistently, ACBA remarkably downregulated the expression of CCR1, p-NF-κBp65, p-IκBα, p-STAT1 and TNF-α proteins, which were upregulated in CCl4-induced hepatic fibrosis and LPS-THP-1 cells. ConclusionOur results suggest that ACBA significantly attenuated CCl4-induced liver fibrosis in histopathological and in serum level. This effect may be mediated by CCR1, NF-κB and STAT1 signalling.

CCL2 Potentiates Inflammation Pain and Related Anxiety-Like Behavior Through NMDA Signaling in Anterior Cingulate Cortex.

Previous studies have shown that the C-C motif chemokine ligand 2 (CCL2) is widely expressed in the nervous system and involved in regulating the development of chronic pain and related anxiety-like behaviors, but its precise mechanism is still unclear. This paper provides an in-depth examination of the involvement of CCL2-CCR2 signaling in the anterior cingulate cortex (ACC) in intraplantar injection of complete Freund's adjuvant (CFA) leading to inflammatory pain and its concomitant anxiety-like behaviors by modulation of glutamatergic N-methyl-D-aspartate receptor (NMDAR). Our findings suggest that local bilateral injection of CCR2 antagonist in the ACC inhibits CFA-induced inflammatory pain and anxiety-like behavior. Meanwhile, the expression of CCR2 and CCL2 was significantly increased in ACC after 14 days of intraplantar injection of CFA, and CCR2 was mainly expressed in excitatory neurons. Whole-cell patch-clamp recordings showed that the CCR2 inhibitor RS504393 reduced the frequency of miniature excitatory postsynaptic currents (mEPSC) in ACC, and CCL2 was involved in the regulation of NMDAR-induced current in ACC neurons in the pathological state. In addition, local injection of the NR2B inhibitor of NMDAR subunits, Ro 25-6981, attenuated the effects of CCL2-induced hyperalgesia and anxiety-like behavior in the ACC. In summary, CCL2 acts on CCR2 in ACC excitatory neurons and participates in the regulation of CFA-induced pain and related anxiety-like behaviors through upregulation of NR2B. CCR2 in the ACC neuron may be a potential target for the treatment of chronic inflammatory pain and pain-related anxiety.

Metformin-induced reduction of CCR8 enhances the anti-tumor immune response of PD-1 immunotherapy in glioblastoma

Immunotherapy strategies targeting the programmed cell death 1 (PD-1) in clinical treatments have shown limited success in controlling glioblastoma malignancies. Metformin exserts antitumor function, yet the underlying mechanisms remain unclear. Here, we investigated whether metformin could enhance the effectiveness of anti-PD-1 therapy by activating the immune system. Whether combination of an anti-PD-1 antibody or not, metformin significantly increased tumor-infiltrating CD4+ T cells while decreased regulatory T (Treg) cells in a mouse GBM model. Additionally, metformin reduced CC motif chemokine receptor CCR8 and elevated Interleukin 17 A (IL-17 A) expressions. Mechanistically, metformin reduces histone acetylation at the CCR8 promotor and inhibits CCR8 expression by upregulating AMP-activated protein kinase (AMPK)-activated sirtuin 2 (SIRT2). Metformin enhances the effectiveness of anti-PD-1 immunotherapy by reducing CCR8 expression on tumor-infiltrating Treg cells, suggesting that metformin has an antitumor effect by alleviating immunosuppression and promoting T cell-mediated immune response.

The effects of supplemental dietary chitosan on broiler performance and myopathic features of white striping

White striping (WS) is a common myopathy seen in fast-growing broilers. Studies have demonstrated that chitosan is effective as an antioxidant and has antiobesity and fat-absorption reduction properties. We hypothesized that the dietary supplementation of chitosan would have similar effects when fed to fast-growing broilers and would thus lower WS incidence and improve meat quality. One hundred twenty-six broilers were fed corn-soy diets. The grower and finisher diets contained either 0, 0.2, or 0.4% chitosan. After a 6 wk growth period, birds were euthanized, and then WS and gross pathology scores were assessed. Pectoralis major tissues were collected to evaluate cook loss, drip loss, histopathology scores, and the gene expression of CCR7, LECT2, CD36, PPARG, and PTGS2. There were no significant differences between the broiler weights, thus chitosan did not appear to compromise the overall growth of the broilers. Female broilers fed 0.4% chitosan had the lowest WS incidence, while male broiler fed 0.4% chitosan had the least cook loss. However, gene expression analyses did not offer insight into any grossly or histologically visualized differences in the muscles. Thus, while we can postulate that chitosan could have some positive effect in reducing WS incidence and improving meat quality, further studies are required to better scrutinize the mechanisms by which chitosan affects WS and other such myopathies in fast-growing broilers.

Targeting the CCL2-CCR2 signaling pathway: potential implications of statins beyond cardiovascular diseases.

The chemokine ligand CCL2 and its cognate receptor CCR2 have been implicated in the pathogenesis of a wide variety of diseases. Hence, the inhibition of the CCL2/CCR2 signaling pathway has been of great attention in recent studies. Among suggested medications, statins known as HMG-COA reductase inhibitors with their pleiotropic effects are widely under investigation. A comprehensive literature search on Scopus and PubMed databases was conducted using the keywords 'CCL2', 'CCR2', 'monocyte chemoattractant protein-1', 'HMG-COA reductase inhibitor', and 'statin'. Both experimental and clinical studies measuring CCL2/CCR2 expressions following statin therapy were identified excluding the ones focused on cardiovascular diseases. Herein, we summarized the effects of statins on CCL2 and CCR2 expression in various pathologic conditions including immune-mediated diseases, nephropathies, diabetes, rheumatic diseases, neuroinflammation, inflammatory bowel diseases, gynecologic diseases, and cancers. For the most part, statins play an inhibitory role on the CCL2-CCR2 axis which implies their potential to be further developed as therapeutic options in non-cardiovascular diseases either alone or in combination with other conventional treatments. However, the existing literature mostly focused on experimental models and is therefore inadequate to reach a conclusion.

Patients with Chronic Spinal Cord Injury Display a Progressive Alteration over the Years of the Activation Stages of the T Lymphocyte Compartment.

Spinal cord injury (SCI) is a serious medical condition associated with severe morbidities and disability. Chronic SCI patients present an enhanced susceptibility to infections and comorbidities with inflammatory pathogenesis. Chronic SCI appears to be associated with a systemic dysfunction of the immune system. We investigated the alteration of the pivotal CD4+ and CD8+ T lymphocytes in patients with chronic SCI at different years of evolution. A clinically homogenous population of 105 patients with chronic SCI (31 with time of evolution less than 5 years (SCI SP); 32 early chronic (SCI ECP) with time of evolution between 5 and 15 years; and 42 late chronic (SCI LCP) with time of evolution more than 15 years) and 38 healthy controls were enrolled. SCI ECP and SCI LCP patients showed significant CD4+ and CD8+ T lymphopenia, ascribed to a reduction in naïve and CM subsets. Furthermore, SCI ECP and SCI LCP patients showed a significant reduction in the expression of CD28 on CD8+ T lymphocytes. The expression of CCR6 by CD4+ T lymphocytes was decreased during the evolution of chronic SCI, but on CD8+ T lymphocytes, it was observed during the first 15 years of evolution. In conclusion, the chronic SCI course with severe damage to T lymphocytes mainly worsens over the years of disease evolution.

P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory Microglia Activation.

Microglia are activated following cerebral ischemic insult. P-glycoprotein (P-gp) is an efflux transporter on microvascular endothelial cells and upregulated after cerebral ischemia. This study evaluated the effects and possible mechanisms of P-gp on microglial polarization/activation in mice after ischemic stroke. P-gp-specific siRNA and adeno-associated virus (p-AAV) were used to silence and overexpress P-gp, respectively. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were performed in mice and cerebral microvascular endothelial cells (bEnd.3) in vitro, respectively. OGD/R-injured bEnd.3 cells were cocultured with mouse microglial cells (BV2) in Transwell. Influences on acute ischemic stroke outcome, the expression of inflammatory cytokines, and chemokines and chemokines receptors, microglial polarization, glucocorticoid receptor (GR) nuclear translocation, and GR-mediated mRNA decay (GMD) activation were evaluated via reverse transcription real-time polymerase chain reaction, western blot, or immunofluorescence. Silencing P-gp markedly alleviated experimental ischemia injury as indicated by reduced cerebral infarct size, improved neurological deficits, and reduced the expression of interleukin-6 (IL-6) and IL-12 expression. Silencing P-gp also mitigated proinflammatory microglial polarization and the expression of C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 expression, whereas promoted anti-inflammatory microglia polarization. Additionally, P-gp silencing promoted GR nuclear translocation and the expression of GMD relative proteins in endothelial cells. Conversely, overexpressing P-gp via p-AAV transfection offset all these effects. Furthermore, silencing endothelial GR counteracted all effects mediated by silencing or overexpressing P-gp. Elevated P-gp expression aggravated inflammatory response and brain damage after ischemic stroke by augmenting proinflammatory microglial polarization in association with increased endothelial CCL2 release due to GMD inhibition by P-gp.

C5a enhances inflammation and chemotaxis of γδ T cells in malignant pleural effusion

BackgroundThe inhibitory effect of γδT17 cells on the formation of murine malignant pleural effusions (MPE) has been established. However, there is limited understanding regarding the phenotypic characterization of γδ T cells in MPE patients and their recruitment to the pleural cavity. MethodsWe quantified γδ T cell prevalence in pleural effusions and corresponding peripheral blood from malignant and benign patients using immunohistochemistry and flow cytometry. The expression of effector memory phenotype, stimulatory/inhibitory/chemokine receptors and cytokines on γδ T cells in MPE was analyzed using multicolor flow cytometry. The infiltration of γδ T cells in MPE was assessed through immunofluorescence, ELISA, flow cytometry and transwell migration assay. ResultsWe observed a significant infiltration of γδ T cells in MPE, surpassing the levels found in blood and benign pleural effusion. γδ T cells in MPE exhibited heightened expression of CD56 and an effector memory phenotype, while displaying lower levels of PD-1. Furthermore, γδ T cells in MPE showed higher levels of cytokines (IFN-γ, IL-17A and IL-22) and chemokine receptors (CCR2, CCR5 and CCR6). CCR2 expression was notably higher in the Vδ2 subtype compared to Vδ1 cells. Moreover, the complement C5a enhanced cytokine release by γδ T cells, upregulated CCR2 expression in Vδ2 subsets, and stimulated the production of chemokines (CCL2, CCL7 and CCL20) in MPE. In vitro utilizing CCR2 neutralising and C5aR antagonist significantly reduced the recruitment of γδ T cells. Conclusionsγδ T cells infiltrate MPE by overexpressing CCR2 and exhibit hightened inflammation, which is further augmented by C5a.

Abstract B046: Racial differences in CCR9 signaling and its association with disparity in chemotherapeutic response in triple negative breast cancer

Abstract African American (AA) women are more frequently diagnosed with Triple Negative Breast Cancer (TNBC) and have a poorer prognosis as compared to European American (EA) women. The standard treatment choice fails to achieve a similar response in AA compared to EA. We hypothesize that CCR9 signaling dictates response to therapeutics, and race-specific differences in CCR9 expression and G protein coupling may be contributing to the disparity in clinical and therapeutic outcomes among the two races. Hence, the focus of this study was to determine the race-specific biological differences in CCR9-CCL25 signaling and its association with the observed disparity in chemotherapeutic response. Our data show differences in G-protein decoupling from CCR9 after activation with its natural ligand, CCL25, in cell lines derived from AA lines (MDA-MB-468 and HCC 1806) compared to EA (HCC 1143 and BT 549). Race-specific differences in G-protein decoupling activate the signaling in promoting stemness; overcoming chemotherapy-induced cell death and high cell, survival collectively plays a crucial role in the chemotherapeutic response. Based on our in vitro results, we tested if blocking CCR9-CCL25 addresses the observed racial difference in chemotherapeutic response. To address this, we developed a xenograft model using TNBC cell lines derived from AA (HCC1806) and EA (MDA-MB-231). Tumor-bearing mice were treated with Carboplatin with or without the CCL25 neutralization antibody. CCL25 neutralization showed a reduction in tumor size in both AA and EA xenografts. This reduction in tumor size was more prominent and statistically significant in EA xenografts (p= 0.01), while the reduction wasn’t significant in AA xenografts (p=0.09). Carboplatin as a single agent effectively reduced tumor size in both AA and EA xenografts, but the response was better in EA (p=0.07) compared to AA. The therapeutic response of Carboplatin was significantly higher when given with an anti-CCL25 neutralization antibody (p=0.001) compared to controls. Carboplatin response was better in AA xenograft after CCL25 neutralization compared to vehicle control, CCL25 neutralization, or Carboplatin alone. In conclusion, our results show that in addition to higher expression, signaling downstream to CCR9 in AA differed from EA primarily due to differential G-protein decoupling. Thus, the difference in CCR9-mediated molecular signaling between AA and EA contributes to the observed racial gap in chemotherapeutic response. Therefore, blocking the CCR9-CCL25 axis is critical to addressing the disparity in chemotherapeutic response and clinical outcome in TNBC. Citation Format: Hina Mir, Rajeev Samant, Shailesh Singh. Racial differences in CCR9 signaling and its association with disparity in chemotherapeutic response in triple negative breast cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B046.

Immune checkpoint status and exhaustion-related phenotypes of CD8+ T cells from the tumor-draining regional lymph nodes in breast cancer.

Functional status of T cells determines the responsiveness of cancer patients to immunotherapeutic interventions. Even though T cell-mediated immunity is inaugurated in the tumor-adjacent lymph nodes, peripheral blood has been routinely sampled for testing the immunological assays. The purpose of this study is to determine the immune checkpoint molecule expression and the exhaustion-related phenotype of cytotoxic T cells in the regional lymph nodes from breast cancer patients. Multicolor immunophenotyping was used to determine the expression of PD-1, TIM-3, LAG3, CTLA-4, CCR7, CD45RO, CD127, CD25, CXCR5, and ICOS molecules on CD3+ CD4- CD56- CD8+ cytotoxic T cells freshly obtained from the lymph nodes and the peripheral blood samples of the breast cancer patients. The results were assessed together with the clinical data. A population of cytotoxic T cells was noted with high PD-1 and CXCR5 expression in the lymph nodes of the breast cancer patients. Co-expression of PD-1, CXCR5, TIM-3, and ICOS indicated a follicular helper T cell (Tfh)-like, exhaustion-related immunophenotype in these cytotoxic T cells. Only a minor population with CTLA-4 and LAG3 expression was noted. The PD-1+ CXCR5+ cytotoxic T cells largely displayed CD45RO+ CCR7+ central memory markers. The amount of CXCR5-expressing PD-1- cytotoxic T cells was elevated in the lymph nodes of the patients. The regional lymph nodes of breast cancer patients harbor Tfh-like exhausted cytotoxic T lymphocytes with high PD-1 and TIM-3 checkpoint molecule expression. The immunological conditions in the regional lymph nodes should be implicated for immune checkpoint immunotherapy (ICI) of cancer.

Identification of leukocyte surface biomarkers associated with late‐onset Alzheimer’s disease

AbstractBackgroundAltered innate immunity has been long associated with late‐onset Alzheimer’s disease (AD) and its associated blood‐based biomarkers are important for AD diagnosis and prognosis.MethodWe collected 38 participants from the AIBL study, including 22 cognitive normal (CN) individuals with negative amyloid burden (Centiloid [CL] < 15), 5 CN individuals with positive amyloid burden (CL > 15), and 11 mild cognitive impairment (MCI) and AD cases. A total of 17 leukocyte surface antigens were examined by flow cytometry immunophenotyping and compared between healthy controls (CN Ab ‐ve), pre‐clinical patients (CN Ab +ve), and clinical cases (MCI & AD), including CD36, MerTK, Clec7a, RAGE, Tyro3, CR1 (CD35), CX3CR1, CCR2, Axl, LILRB2, LILRB3, LILRB4, PILRA, and P2×7.ResultWe identified leukocyte surface markers differentially expressed between HC, pre‐clinical patients, and clinical cases. Mean fluorescence intensities of CD85d and CD85k were significantly reduced in pre‐clinical patients and cases compared with HC, implicating CD85d and CD85k downregulation in AD pathogenesis. Significant upregulation of RAGE, Tyro3, CCR2, CD85a, and PILRA was also noted in cases compared with HC. Leukocyte surface expressions of RAGE, CCR2, PILRA, and CD85k were significantly associated with the Preclinical Alzheimer’s Cognitive Composite (PACC), which is one of the most accurate estimates of cognition in AD diagnosis.ConclusionOur preliminary investigations into these leukocyte markers demonstrated their dysregulations in pre‐clinical stage of AD, implicating early deficits in innate immunity, as supported by genomic studies of AD. This project will continue collecting more AIBL participants up to 200 to evaluate leukocyte surface markers more comprehensively, which would benefit AD screening and diagnosis.

Increased gene expression of CCR6 and RORγt in peripheral blood cells of rheumatoid arthritis patients and their correlation with anti-cyclic citrullinated peptide and disease activity.

The significance of T helper 17 (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated in many studies. Retinoic acid receptor-related orphan receptor γt (RORγt) is a transcription factor that is specifically involved in the generation of Th17 cells. Besides, the chemokine receptor CCR6, the receptor for CCL20, is characteristically expressed by these cells. Considering the pivotal roles of Th17 cells in RA pathogenesis, in this study, we assessed the gene expression of CCR6 and RORγt in the peripheral blood leukocytes of new case RA patients. Also, we evaluated their association with anticyclic citrullinated peptide (anti-CCP) antibodies and disease activity. Forty-five new case RA patients and 45 healthy persons have been recruited in this investigation. The gene expression of CCR6 and RORγt was evaluated by quantitative real-time PCR (qRT-PCR), and anti-CCP antibodies plasma levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Disease activity was measured according to the disease activity score-28 (DAS-28) formula. The gene expression of CCR6 and RORγt increased remarkably in new case RA patients compared to healthy controls (p < .05 and p < .01, respectively). Moreover, there was a positive correlation between RORγt gene expression and parameters, including gene expression of CCR6 (p = .001, r = .461), plasma levels of CCL20 (p = .0009, r = .477), ESR (p = .004, r = .419), DAS-28 (p = .006, r = .402), anti-CCP (p = .019, r = .346), and RF (p = .001, r = .451). Also, CCR6 gene expression was positively associated with the DAS-28 (p = .037, r = .310), plasma levels of anti-CCP (p = .037, r = .312), and ESR (p = .029, r = .327). Increased gene expression of CCR6 and RORγt in peripheral blood leukocytes of new case RA patients may contribute to the exacerbation and pathogenesis of RA.

North American Adult T-Cell Leukemia/Lymphoma Has Frequent Mutations in CCR4 and Responds in Vitro to a Small Molecule CCR4 Antagonist, CCR4-351

Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive disease of malignant CD4+ T cells that develops in human T-lymphotropic virus-1 (HTLV-1) carriers. Patients diagnosed with ATLL have the worst survival among all peripheral T-cell lymphomas. There is an urgent need for in-depth understanding of its pathogenesis and development of new treatment strategies. We and others have shown that ATLLs diagnosed in the Japanese (J-ATLL) and North American (NA-ATLL) patients have differences in clinical characteristics and immune-genetic landscape. Among the most frequently mutated genes in J-ATLL cohorts is CCR4, a chemokine receptor that is targeted by C-terminal truncation mutations in one third of the cases. These mutations result in a more stabilized surface CCR4 and have been reported to cause elevated Akt activation following chemokine stimulation. Since our previous targeted exon sequencing study did not include CCR4, this work was initiated to assess CCR4 mutation frequency in NA-ATLL cases and to evaluate the preclinical activity of a small molecule CCR4 antagonist. This is because, although an anti-CCR4 mAb, mogamulizumab, has been approved in Japan to treat ATLL, this therapy failed in a Phase 2 trial among patients outside of Japan. Thus, mogamulizumab is not available as a routine treatment for NA-ATLL patients. We compiled CCR4 gene status data from bulk RNA-seq (n=10) and whole genome sequencing (WGS) analysis (n = 10). Out of a total of 17 NA-ATLL cases, 8 (47.1%) carried CCR4 gene alterations that included in-frame and frameshift indels, as well as nonsense mutations targeted to the C-terminus cytoplasmic tail and also the trans-membrane domain. In subtype analysis, 6 out 14 acute cases (42.9%) and 2 out of 3 (66.7%) of chronic cases carried at least one copy of mutated CCR4 gene, indicating that the mutation frequency in NA-ATLL is possibly even higher than that reported for the Japanese cohorts ( Figure 1). We then evaluated cell surface CCR4 expression using flow cytometry. The primary ATLL cells (CD4 +CD7 -CD8 -) from all 7 diagnostic samples uniformly express markedly increased levels of CCR4 relative to healthy control CD4 T cells. Interestingly, CCR4 expression among 8 ATLL cell lines is highly variable, implying that in vivo, tumor microenvironment factors may also contribute to sustained CCR4 upregulation in addition to the influence from the HTLV-1 oncoprotein, HBZ. FLX475 is an orally active small molecule CCR4 antagonist that is currently in a Phase 1/2 study (NCT03674567) in several types of advanced cancer including EBV+ NK/T cell lymphoma. So far, favorable clinical antitumor activity has been observed that includes complete responses with FLX475 monotherapy and encouraging combination activity (Lin C.-C. et al ESMO-IO 2022 Dec; #187P). Using several CCR4-positive cell lines, we examined in vitro anti-ATLL activity of CCR4-351, a CCR4 antagonist that is a highly related analog of FLX475. CCR4 is the exclusive homing receptor for two chemokine molecules, CCL17 and CCL22. In a transwell assay, both chemokines can elicit strong chemotaxis activity in a J-ATLL cell line ED40515(+). However, only CCL22 but not CCL17 can induce significant chemotaxis in two NA-ATLL cell lines, ATL#13 and ATL#29. The exact cause of this difference between these cell lines awaits future studies. In these transwell-based assays, CCR4-351 exhibited potent chemotaxis inhibitory activity with IC 50 values ranging from 7-60 nM ( Figure 2). In addition, when the cell lines were cultured under growth factor-deprivation conditions, CCR4-351 treatment caused a mild reduction in total cell viability, suggesting an additional anti-ATLL mechanism of action. In summary, in this first study of the CCR4 pathway status in NA-ATLL, we found that nearly half of the cases in our ATLL cohort carried a mutated CCR4 gene. Contrary to primary ATLL cells, ATLL cell lines exhibit a wide variation in CCR4 expression. CCL22 but not CCL17 induced strong chemotaxis behavior in NA-ATLL cell lines, which was potently inhibited by a small molecule CCR4 antagonist, CCR4-351. Since extramedullary presentation is frequently seen in NA-ATLL and central nervous system involvement is an adverse prognostic feature, inhibiting chemotaxis with a CCR4 antagonist such as FLX475 may be an effective therapeutic approach.

HDAC3 Is Required for Pathognomonic Features of Langerhans Cell Histiocytes

Langerhans cell histiocytosis (LCH) is a pediatric inflammatory myeloid neoplasm that develops due to dysregulated myeloid cell development. BRAFV600E is the most common disease-causing mutation and constitutively activates the mitogen-activated protein kinase (MAPK) pathway in myeloid lineage precursors leading to the key pathognomonic features of LCH cells. Enhanced myelopoiesis and reduced CCR7 expression promote accumulation of LCH cells in tissues by simultaneously increasing the production of pathological DCs and preventing tissue egress. Furthermore, LCH cells acquire an oncogene induced senescence-associated secretory phenotype (SASP) that depends on mammalian target of rapamycin (mTOR), which is hallmarked by increased expression of anti-apoptotic proteins, inflammatory cytokines, and matrix metalloproteinases enhancing survival of LCH cells and promoting recruitment of inflammatory immune cells forming characteristic granulomatous lesions. These pathognomonic features result in the accumulation of LCH cells in any organ causing a wide range of clinical symptoms. Frontline therapy for LCH involves combination chemotherapy and steroidal anti-inflammatories, or MAPK inhibitors, which have significant toxicity and fail to eliminate disease causing precursors. New therapeutic approaches are urgently needed. Here, using multiple genetic mouse models, we show that normal epidermal Langerhans cells (LCs) depend on HDAC3 for their development, differentiation, and survival. Integrative RNA and chromatin-immunoprecipitation-sequencing show loss of HDAC3 abrogates the expression of master regulators of myeloid development and function including Csf1r, Spi1, Id2 and Runx3. LCH cells are also known to rely on Csf1r and Pu.1 for their development and homeostasis, thus we hypothesized that LCH cells similarly rely on HDAC3 for their development and survival. CD11c Cre LSL-BRAFV600E ( BRAFV600E CD11c) mice develop severe multifocal LCH with pronounced lesion development in their livers and lungs, hepatosplenomegaly, and a reduced lifespan due to the accumulation of pathological dendritic cells (DCs). We generated BRAFV600E CD11c HDAC3 fl/fl ( BRAFV600E HD3KO) mice, which produce pathological DCs that simultaneously express BRAFV600E and harbor a conditional deletion in the deacetylase domain of HDAC3. Compared to BRAFV600E CD11c mice, BRAFV600E HD3KO mice exhibited significantly less hepatosplenomegaly, reduced lesional burden (Panel A), attenuated disease progression, and improved survival indicating reduced LCH disease burden. Compared to BRAFV600E CD11c flow cytometry showed BRAFV600E HD3KO had reduced numbers of LCH cells in lungs and livers linking improved disease outcomes to abrogation of pathological DCs. Flow cytometric analysis of circulating myeloid cells further found reduced frequency of circulating DCs and DC progenitors, indicating that a lack of HDAC3 activity prevents the development of pathological DCs (Panel B). LCH-like cells can be generated in vitro by culturing BRAFV600E CD11c bone marrow with granulocyte-monocyte colony-stimulating factor (GM-CSF), providing a valuable drug screening tool. Treating LCH-like cells with RGFP966, an HDAC3-specific inhibitor, increased apoptosis indicated by annexin-V and DAPI staining, reduced expression of Bcl-2, increased CCR7 expression, and decreased S6 phosphorylation (an indication of decreased mTOR activity), showing that pharmacological inhibition of HDAC3 may prove therapeutically efficacious by abrogating pathognomonic features of LCH cells. Together, our findings identify HDAC3 as a critical epigenetic regulator for both healthy and pathological LCs. We further show, HDAC3 is required for multiple pathognomonic features of LCH cells and could be a promising drug target. Furthermore, if HDAC3 is required for the development of pathological DC and DC progenitors, HDAC3 blockade would address a great need in treatment of patients with LCH.

CCR1-Targeting CAR T Cells for Acute Myeloid Leukemia

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is highly effective in patients with B cell malignancies and multiple myeloma, but initial clinical trials with CARs targeting acute myeloid leukemia (AML) have shown less successful results. Challenges inherent to using CAR T cells for AML include lack of a leukemia-specific target antigen due to heterogeneous antigen expression and potential on-target/off-tumor toxicity due to co-expression of target antigens on normal myeloid cells. C-C chemokine receptor 1 (CCR1, also known as CD191), a G protein-coupled receptor that binds to members of the C-C chemokine family, is a promising AML-associated antigen that is reported to be expressed on over 75% of AML samples and minimally expressed on hematopoietic stem/progenitor cells and T cells. The present study aimed to explore the potential of CCR1 as a novel CAR target antigen using a syngeneic mouse model that allows investigation of both efficacy and toxicity of the CAR T cells. METHODS AND RESULTS We developed a CAR based on the single chain variable fragment (scFv) KM5907 targeting both mouse and human CCR1. We constructed a second-generation CCR1-CAR containing a CD28 hinge, a CD28 transmembrane domain, a CD3 zeta signaling domain, and a CD28 co-stimulatory domain. We mutated the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 zeta chain to retain only a single active membrane-proximal (1XX) ITAM, based on a prior study showing that the 1XX ITAM format enhanced CAR T cell activity by reducing T cell exhaustion. Primary murine T cells were retrovirally transduced and CCR1-CAR expression was assessed by flow cytometry. CCR1-CAR T cells promoted effective elimination of BM185 leukemia cells overexpressing murine CCR1 in vitro. Similarly, primary human T cells transduced with the CCR1-CAR exhibited potent cytotoxicity against C1498 leukemia cells overexpressing human CCR1 in vitro. To evaluate anti-leukemia activity of the CCR1-CAR T cells in vivo, BALB/c mice were sublethally irradiated and engrafted with 1x10 5 BM185 leukemia cells modified to overexpress murine CCR1. Two days later, mice were treated with 2x10 6 CCR1-CAR T cells and monitored for weight changes and survival. Growth of BM185 leukemia cells (expressing firefly luciferase) and expansion of CAR T cells (expressing gaussia luciferase) were assessed via two-color bioluminescence imaging (BLI). On day 6, mice were bled retro-orbitally and serum was analyzed for cytokine levels. The CCR1-CAR T cells demonstrated potent anti-leukemia activity and T cell expansion in vivo, promoting improved overall survival compared with control mice that received only tumor cells (Figure 1). CCR1-CAR T cell activity was accompanied by mild, self-limited toxicity, characterized by transient weight loss (Figure 2), but not significant elevation of serum levels of cytokines associated with cytokine release syndrome (CRS) such as IL-6 and TNF-α. BLI of the CCR1-CAR T cells revealed enhanced trafficking into the bone marrow and spleen followed by proliferation in irradiated mice even in the absence of tumor, suggesting that CCR1-CAR T cells are stimulated by CCR1 antigen expressed on non-leukemic host cells in addition to leukemia cells. CONCLUSIONS In summary, CCR1-CAR T cells represent a promising therapeutic strategy for AML that to our knowledge has not yet been evaluated in preclinical or clinical studies. We generated CCR1-CAR T cells that demonstrated potent anti-leukemia activity in vitro and in vivo in a syngeneic mouse model. Clinical signs of self-limited toxicity were not associated with elevated levels of inflammatory cytokines and might be caused by an on-target/off-tumor effect, which warrants further investigations to better characterize the safety profile of CCR1-CAR T cells. The CCR1-CAR T cells also displayed potent cytotoxicity in vitro via human CCR1, which will facilitate clinical translation of the findings. A CCR1-CAR may also offer benefit in other hematologic malignancies with reported CCR1 expression such as multiple myeloma or Hodgkin's lymphoma.

Expression of constitutively active TβRI leads to attenuation of ovalbumin-induced allergic airway inflammation associated with augmented M2 polarization of alveolar macrophage

BackgroundTransforming growth factor-β (Tgf-β) plays an important role in the pathogenesis of asthma through the regulation of T cells and airway epithelium. Its functions in alveolar macrophage (AM) during allergic airway inflammation remain unknown. MethodsA murine asthma model was induced with ovalbumin (ova) in TβRICA/Fsp1-Cre transgenic mice expressing constitutively active Tgf-β receptor type I (TβRICA) under the control of Fsp1-Cre transgene. Cells in the bronchoalveolar lavage (BAL) were collected to study immune cell infiltration in the lungs. Cytokine levels in BAL fluid were measured by enzyme-linked immunoassay (ELISA). Lungs were sectioned and stained with hematoxylin and eosin, periodic acid-Schiff, and trichrome for histopathologic evaluation. AMs were assessed by flow cytometry and were sorted for quantitative polymerase chain reaction analysis. ResultsOur data indicated that TβRICA transcripts were induced in AMs of TβRICA/Fsp1-Cre mice. Following the ova challenges, TβRICA/Fsp1-Cre mice exhibited reduced cellular infiltration of the airway, reduced pulmonary fibrosis, and reduced bronchial mucus secretion as compared to ova-challenged wild-type mice. An alternatively activated macrophage (M2) polarization was significantly elevated in the lungs of ova-challenged TβRICA/Fsp1-Cre mice as reflected by increased numbers of AMs expressing M2 subtype marker, CD163, in the lungs and enhanced expression of CCR2 and CD206 in AMs. Moreover, TβRICA/Fsp1-Cre AMs showed augmented expression of transcription factors, Foxo1, and IRF4, which are known to be positive regulators for M2 polarization. ConclusionsExpression of TβRICA in AMs promoted M2 polarization and ameliorated allergic airway inflammation in an ova-induced asthma mouse model.

High-dimensional phenotyping of the peripheral immune response in community-acquired pneumonia.

Community-acquired pneumonia (CAP) represents a major health burden worldwide. Dysregulation of the immune response plays an important role in adverse outcomes in patients with CAP. We analyzed peripheral blood mononuclear cells by 36-color spectral flow cytometry in adult patients hospitalized for CAP (n=40), matched control subjects (n=31), and patients hospitalized for COVID-19 (n=35). We identified 86 immune cell metaclusters, 19 of which (22.1%) were differentially abundant in patients with CAP versus matched controls. The most notable differences involved classical monocyte metaclusters, which were more abundant in CAP and displayed phenotypic alterations reminiscent of immunosuppression, increased susceptibility to apoptosis, and enhanced expression of chemokine receptors. Expression profiles on classical monocytes, driven by CCR7 and CXCR5, divided patients with CAP into two clusters with a distinct inflammatory response and disease course. The peripheral immune response in patients with CAP was highly similar to that in patients with COVID-19, but increased CCR7 expression on classical monocytes was only present in CAP. CAP is associated with profound cellular changes in blood that mainly relate to classical monocytes and largely overlap with the immune response detected in COVID-19.