Chemokine Receptor Research Articles

Multi-omic characteristics of longitudinal immune profiling after breakthrough infections caused by Omicron BA.5 sublineages

Omicron sub-variants breakthrough infections (BTIs) have led to millions of coronavirus disease 2019 (COVID-19) cases worldwide. The acute-phase immune status is critical for prognosis, however, the dynamic immune profiling of COVID-19 during the first month after BTIs remains unclear. In this study, we monitored the immune dynamics at various timepoints in a longitudinal cohort during the first month post-BTIs through clinical evaluation, single-cell RNA sequencing (scRNA-seq), T cell receptor (TCR)/B cell receptor (BCR) sequencing, and antibody mass spectrometry. Serological analysis revealed limited impairment to functions of major organs, active cellular and humoral immunity at 2 weeks post-BTI, with significant increases in cytokines (CKs) and neutralizing antibody levels. However, 1 month post-BTI, organ function parameters and CK levels reverted to pre-infection levels, whereas neutralizing antibody levels remained high. Notably, scRNA-seq showed that lymphocytes maintained strong antiviral activity and cell depletion at 2 weeks and 1 month post-BTI, with genes CD81, ABHD17A, CXCR4, DUSP1, etc. upregulated, and genes PFDN5, DYNLRB1, CD52, etc. downregulated, indicating that lymphocytes status take longer to recover to normal levels than that routine blood tests revealed. Additionally, T cell-exhaustion associated genes, including LAG3, TIGIT, PDCD1, CTLA4, HAVCR2, and TOX, were upregulated after BTI. TCRs and BCRs exhibited higher clonotypes, mainly in CD8Tem or plasmablast cells, at 2 weeks post-BTI comparing 1 month. More IgG and IgA-type BCRs were found in the groups of 1 month post-BTI, with higher somatic hypermutation, indicating greater maturity. Verification of monoclonal antibodies corresponding to amplified BCRs highlighted the antigen-specific and broad-spectrum characteristics. Our study elucidated the dynamic immune profiling of individuals after Omicron BA.5 sublineages BTI. Strong immune activation, antiviral response, antibody maturation and class transition at 2 weeks and 1 month after BTI may provide essential insights into pathogenicity, sequential immune status, recovery mechanisms of Omicron sublineage BTI. This study was supported by the National Key R&D Program of China, the China Postdoctoral Science Foundation, Guangdong Basic and Applied Basic Research Foundation, the National Natural Science Foundation of China, CAS Project for Young Scientists in Basic Research, and the Air Force Special Medical Center Science and Technology Booster Program.

C-C motif chemokine receptor-2 blockade ameliorates pulmonary hypertension in rats and synergizes with a pulmonary vasodilator

Abstract Aims We investigated whether the disruption of C-C motif chemokine receptor (CCR) 2 may attenuate the development of pulmonary arterial hypertension (PAH) in any rat models with the reversal of the associated pro-inflammatory state and vascular dysfunction, and synergize with a conventional pulmonary vasodilator. Methods and Results Using Ccr2(-/-) rats generated by CRISPR/Cas9, we investigated pulmonary hypertension (PH) in Ccr2(+/+) or Ccr2(-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx) and chronic hypoxia (CH). Ccr2(-/-) decreased the right ventricular systolic pressure, an index of right ventricular hypertrophy and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (interleukin-6, tumor necrosis factor-α, C-C motif chemokine receptor (CCL)-2, interleukin-1β, transforming growth factor-β) in rats 3weeks after MCT injection, but not in SuHx or CH models. Consistently, Ccr2(-/-) decreased indices of pulmonary vascular diseases (PVD) and perivascular macrophage infiltration, as well as reversed impaired bone morphogenetic protein receptor type 2 signaling, increased endothelial apoptosis and impaired nitric oxide signaling and decreased phosphodiesterase-5 (PDE5) expression in lungs in MCT-treated rats. Gene expression of receptors for prostaglandin I2 and endothelin was not changed by Ccr2(-/-) in MCT-treated rats. In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2(-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. The whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2(-/-) PASMCs, which are related to regulation of cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2(-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2(-/-) rats but not in Ccr2(+/+) rats. Tadalafil further improved survival in MCT-treated Ccr2(-/-) rats. Conclusion The present findings demonstrated that CCR2 disruption ameliorated PAH in MCT-treated rats, which was associated with the reversal of dysregulated inflammatory pathways and vascular dysfunction and synergized with tadalafil. These findings suggest that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

Abstract C040: Spatial transcriptomics unveils disparities in tumor microenvironment dynamics between breast cancer responders and non-responders

Abstract Spatial transcriptomics is revolutionizing our understanding of cellular dynamics and interactions in the tumor microenvironment (TME). By mapping distinct cellular populations and their interactions, we can gain valuable insights into tumor behavior. To investigate these dynamics in breast cancer, we conducted Xenium In-situ spatial transcriptomics on twenty formalin-fixed paraffin-embedded (FFPE) samples taken from eight patients undergoing neoadjuvant chemotherapy. These samples have been taken pre-treatment, mid-treatment and post-treatment from four cases with pathological complete response (pCR), and four cases with residual disease (RD) at time of surgery. Our analyses have found distinct cellular composition changes between pCR and RD cases. Patients with RD demonstrated an increase in endothelial, perivascular-like (PVL) and epithelial cells from pre- to post-treatment. This was accompanied by a decrease in myeloid and malignant epithelial cells at mid-treatment. In contrast, patients with pCR demonstrated increases in cancer-associated fibroblasts (CAFs) and myeloid cells, with reductions in plasmablasts and cancer epithelial cells at mid-treatment. This suggests that an increase in CAF and myeloid cells observed between pre-treatment and mid-treatment samples may be predictive of response. We performed ligand-receptor (LR) analyses, which demonstrated diverse myeloid interactions in RD cases and diverse CAF interactions in pCR cases in pre-treatment samples. Mid-treatment samples exhibited a decrease in interactions for CAFs, myeloid and T cells in pCR and RD cases. The most frequently identified CAF LR interactions, as senders, were between TGFB1/TGFB3 and TGFBR1/TGFBR2, while the most frequently identified myeloid LR interactions were between PDCD1LG2 - PDCD1 (encoding PDL2 & PD1), CCL8 - ACKR1 (encoding Atypical Chemokine Receptor 1) and CCL2 - ACKR1. Our findings indicate that the dynamic changes in cellular composition and interactions within the TME differs between responders and non-responders. To further explore these differences, we plan to perform spatially constrained analyses to identify unique cellular niches that may serve as potential biomarkers for informing novel therapeutic strategies. Citation Format: Jeremy Mo, Hanyun Zhang, Kate Harvey, Alexander Swarbrick. Spatial transcriptomics unveils disparities in tumor microenvironment dynamics between breast cancer responders and non-responders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C040.

99mTc]-PentixaTec SPECT/CT for Imaging of Chemokine Receptor 4 Expression After Myocardial Infarction.

Accumulation of CXCR4 (C-X-C motif chemokine receptor 4)-positive immune cells after acute myocardial infarction (AMI) can be visualized by positron emission tomography. For a broader clinical application, there is a need for CXCR4-directed radiotracers labeled with isotopes that can be used with single-photon emission computed tomography (SPECT). We report on the detection of CXCR4 expression after AMI in humans using the novel tracer [99mTc]-PentixaTec. In this retrospective analysis, 9 patients with AMI after mechanical revascularization underwent myocardial inflammation imaging with [99mTc]-PentixaTec SPECT/computed tomography and rest perfusion SPECT imaging. Tracer uptake in the infarcted area, spleen, bone marrow, and blood pool were used for semiquantitative analysis and calculation of signal-to-background ratios. The extent and intensity of SPECT-derived inflammatory changes were compared with serological markers and perfusion defects. CXCR4-directed SPECT was positive in all patients. Increased CXCR4 expression was only detected in areas with diminished perfusion corresponding to the affected vessel in coronary angiography, with a signal-to-background ratio (infarcted area-to-blood pool) of 2.36±0.74. Uptake in bone marrow and spleen showed a significant correlation with CXCR4 expression in the infarcted areas (r=0.73 and P=0.03 for spleen and r=0.81 and P=0.008 for bone marrow, respectively). The extent and intensity of SPECT-derived inflammatory changes showed no significant association with serum troponin, CK (creatine kinase), leukocyte, or CRP (C-reactive protein) levels. This is the first report of in vivo CXCR4 imaging after AMI using a 99mTc-labeled tracer. Increased CXCR4 expression was observed locally in the infarcted region and was related to a systemic inflammatory response in the reticuloendothelial system. This proof-of-concept investigation demonstrates the general feasibility of evaluating the inflammation-related CXCR4 expression in the myocardium after AMI using conventional scintigraphy or SPECT and might, thus, broaden its worldwide application in clinical practice.

CXCR3-expressing myeloid cells recruited to the hypothalamus protect against diet-induced body mass gain and metabolic dysfunction.

Microgliosis plays a critical role in diet-induced hypothalamic inflammation. A few hours after a high-fat diet (HFD), hypothalamic microglia shift to an inflammatory phenotype, and prolonged fat consumption leads to the recruitment of bone marrow-derived cells to the hypothalamus. However, the transcriptional signatures and functions of these cells remain unclear. Using dual-reporter mice, this study reveals that CX3CR1-positive microglia exhibit minimal changes in response to a HFD, while significant transcriptional differences emerge between microglia and CCR2-positive recruited myeloid cells, particularly affecting chemotaxis. These recruited cells also show sex-specific transcriptional differences impacting neurodegeneration and thermogenesis. The chemokine receptor CXCR3 is emphasized for its role in chemotaxis, displaying notable differences between recruited cells and resident microglia, requiring further investigation. Central immunoneutralization of CXCL10, a ligand for CXCR3, resulted in increased body mass and decreased energy expenditure, especially in females. Systemic chemical inhibition of CXCR3 led to significant metabolic changes, including increased body mass, reduced energy expenditure, elevated blood leptin, glucose intolerance, and decreased insulin levels. This study elucidates the transcriptional differences between hypothalamic microglia and CCR2-positive recruited myeloid cells in diet-induced inflammation and identifies CXCR3-expressing recruited immune cells as protective in metabolic outcomes linked to HFD consumption, establishing a new concept in obesity-related hypothalamic inflammation.

CXCR3-expressing myeloid cells recruited to the hypothalamus protect against diet-induced body mass gain and metabolic dysfunction

Microgliosis plays a critical role in diet-induced hypothalamic inflammation. A few hours after a high-fat diet (HFD), hypothalamic microglia shift to an inflammatory phenotype, and prolonged fat consumption leads to the recruitment of bone marrow-derived cells to the hypothalamus. However, the transcriptional signatures and functions of these cells remain unclear. Using dual-reporter mice, this study reveals that CX3CR1-positive microglia exhibit minimal changes in response to a HFD, while significant transcriptional differences emerge between microglia and CCR2-positive recruited myeloid cells, particularly affecting chemotaxis. These recruited cells also show sex-specific transcriptional differences impacting neurodegeneration and thermogenesis. The chemokine receptor CXCR3 is emphasized for its role in chemotaxis, displaying notable differences between recruited cells and resident microglia, requiring further investigation. Central immunoneutralization of CXCL10, a ligand for CXCR3, resulted in increased body mass and decreased energy expenditure, especially in females. Systemic chemical inhibition of CXCR3 led to significant metabolic changes, including increased body mass, reduced energy expenditure, elevated blood leptin, glucose intolerance, and decreased insulin levels. This study elucidates the transcriptional differences between hypothalamic microglia and CCR2-positive recruited myeloid cells in diet-induced inflammation and identifies CXCR3-expressing recruited immune cells as protective in metabolic outcomes linked to HFD consumption, establishing a new concept in obesity-related hypothalamic inflammation.

Relation of CXCL10 and CXCR3 Gene Polymorphisms with COVID-19 Severity and its Serum Biochemical Markers

CXCL10 (rs201830102) is a chemokine involved in immune cell recruitment, while its receptor, CXCR3 (rs779120264), mediates immune responses through the activation of T cells. These genes are critical in the immune response to viral infections, including COVID-19. The study aimed to explore the relationship between polymorphisms in the CXCL10 gene and CXCR3 receptor with disease severity in COVID-19 patients. In this cross-sectional analytical study, 100 COVID-19 patients were enrolled after ethical approval, and written informed consent was obtained from each participant. Polymorphisms in CXCL10 and CXCR3 were analyzed by sequencing, while biochemical and hematological parameters were assessed using appropriate methods. Descriptive and inferential statistics were employed to analyze continuous and categorical data. Significant associations were observed between severe COVID-19 cases and elevated levels of serum D-dimer, ferritin, random blood sugar (RBS), neutrophils, and erythrocyte sedimentation rate (ESR), along with reduced hemoglobin levels. Lymphocyte and platelet counts were significantly lower with increased disease severity. The wild genotype of CXCL10 was notably associated with elevated ferritin levels, suggesting that certain gene variants may offer protective effects. However, no significant correlation was found between CXCR3 and CXCL10 polymorphisms and other serum biomarkers. Our study confirmed a significant rise in serum D-dimer, ferritin, RBS, neutrophils, and ESR, along with a reduction in hemoglobin, lymphocyte, and platelet counts in severe COVID-19 cases compared to mild ones. Notably, mutations in the CXCL10 gene were linked to less severe COVID-19 outcomes.

Abstract 4137693: CC-Chemokine Receptor 2 Inhibition Prevents Monocyte/Macrophages Recruitment in Abdominal Aortic Aneurysms

Intro: Abdominal aortic aneurysm (AAA) is a prevalent condition in the elderly, with a significant risk of mortality if it ruptures. Despite this, there is currently no effective medical therapy to prevent AAA growth and rupture. The monocyte chemoattractant protein (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis appears to play a role in AAA development. We demonstrated the potential of CCR2 as a theranostic marker for predicting AAA rupture, along with the effectiveness of a CCR2 inhibitor (RS-504393) in mitigating rupture. We further hypothesized that CCR2 inhibition can impact monocytes/macrophages trafficking in aortic tissue. Mehtods: Sprague-Dawley rats (N=54) underwent intraluminal aortic exposure to porcine pancreatic elastase (PPE) to induce aneurysmal degeneration. To stimulate AAA rupture, rats also recieved β-aminopropionitrile (BAPN). After 3 days of PPE exposure, a group of rats with AAA were administered a CCR2 inhibitor (CCR2i) either daily until the end of the study (CCR2i Full), or discontinued at day 5 (CCR2i Short), while the remaining rats proceeded without intervention (Fig1A). Ultrasound (12MHz) was performed at 7&14 days to assess diameter, while another cohort of animals were euthanized at 6 days, prior to rupture, for single-cell RNA sequencing analysis. The relative content of inflammatory cells within the aortic tissue was analyzed. Results: Both CCR2i Short and CCR2i Full treated, exhibited a significant reduction in AAA diameter at day 7 and 14 compared to controls (p<0.05; Fig1B&C). Notably, rats treated with CCR2i Full did not experience any AAA ruptures, while controls had a 56% risk of rupture. In contrast, CCR2i Short delayed the occurrence of ruptures without reducing the rupture rate (p<0.01; Fig1D). Single-cell RNA seq analysis of aortic tissue revealed that CCR2 inhibition led to a decrease in the number of recruited monocytes/macrophages. Cell recruitment was also relative to the duration of CCR2i treatment (Fig1E-G). Conclusions: Our study findings highlight the impact of CCR2-targeting in reducing AAA growth and the risk of rupture. Interestingly, length of CCR2i was a crucial variable for AAA rupture prevention. Furthermore, treatment length impacted the relative recruitment of monocytes/macrophages to AAA tissue, highlighting the role these cells in AAA disease pathology. Future analysis of CCR2+ inflammatory cell subgroups may help elucidate how CCR2 mechanistically modulates AAA progression and rupture risk.

Abstract 4115947: Empagliflozin Inhibits the Mobilization of CCR2+ Monocyte-Derived Macrophages by Reducing CCL2 Expression in Fibroblasts and Slows the Progression of Heart Failure After Pressure Overload.

Background: The positive effects of sodium glucose co-transporter-2 (SGLT2) inhibitors on reducing cardiovascular mortality and hospitalizations due to heart failure have been well established. Although numerous theories propose explanations for the cardioprotective actions of SGLT2 inhibitors, these hypotheses primarily rely on initial findings and require further validation. Studies have shown that immune cells, particularly macrophages marked by C-C chemokine receptor type 2 (CCR2), contribute to the progression of heart failure by infiltrating the myocardium and inducing detrimental structural changes following myocardial infarction or pressure overload. Purpose: This study aims to investigate whether SGLT2 inhibitors can mitigate heart damage by inhibiting the migration of CCR2+ monocyte-derived macrophages. Methods: In this experiment, male B6 mice were subjected to transverse aortic constriction (TAC) to induce heart failure. Two weeks post-TAC, with heart function confirmed as equal across all groups by echocardiography, one group began receiving daily oral doses of empagliflozin, while another served as the control. At four weeks post-TAC, cardiac function was reassessed. The animals were then euthanized, and their hearts were analyzed using flow cytometry, staining, and qPCR to evaluate heart failure and the involvement of immune cells. Additionally, isolated cardiac fibroblasts were subjected to cyclic stretching to mimic post-TAC mechanical stress. Results: Treatment with empagliflozin resulted in improved cardiac function and reduced congestion in the heart and lungs, indicating alleviation of heart failure. The treatment notably decreased myocardial fibrosis, evident from Picrosirius-red staining and reduced expression of fibrosis-related mRNA. Importantly, empagliflozin specifically reduced the number of CCR2-positive macrophages in the heart, without affecting other types of immune cells. In cardiac fibroblasts that underwent cyclic stretching, empagliflozin pre-treatment diminished the expression of C-C motif chemokine ligand 2 (CCL2), the ligand for CCR2. Conclusion: Empagliflozin’s ability to suppress CCL2 expression in fibroblasts and curtail the recruitment of CCR2+ macrophages may represent a potential strategy to decelerate heart failure progression. These findings advance our understanding of the cardioprotective mechanisms of SGLT2 inhibitors and propose novel therapeutic avenues for heart failure management.

Abstract 4132968: CCR5 modulation enhances M-MDSCs functionality in early atherosclerosis

Background: The role of immune suppressive microenvironment, promulgated by the presence of Myeloid Derived Suppressor Cells (MDSC) have been the major focus of research delineating the underlying mechanisms of cardiovascular diseases (CVD). CCR5, a chemokine receptor, has been associated with both immunosuppressive and inflammatory phenotypes, however, the possible role of CCR5 pertaining to MDSCs in the development of atherosclerosis has not been elucidated yet. Therefore, we investigated the therapeutic potential of CCR5 modulation in early atherosclerosis progression. Methods: CCR5 receptor-ligand expression was studied with flow cytometry, real time PCR and immunocytochemistry in 65 individuals (25 Healthy controls, 20 young hypercholesterolemic individuals, and 20 stable [Coronary Artery Disease] CAD patients). To induce atherosclerosis, mice were fed high fat diet (HFD) for 6 weeks following partial ligation of left carotid artery in C57BL6 mice. MDSCs phenotype and functionality were assessed using CCR5 inhibitor in vitro (10 -8 M) and in vivo (i.p 3ng/day for 15 days) . M-MDSCs phenotype switch, atherosclerotic lesion development and plaque phenotype was studied in vivo . Results: We observed CCR5 elevation on Monocytic-MDSCs in the early phase of atherosclerosis. M-MDSCs displayed an intermediate phenotype with expression of both pro and anti-inflammatory mediators in early atherosclerosis, a similar trend was observed in inflammatory cytokines stimulated M-MDSCs ( in vitro ). Notably, DAPTA treatment shifted phenotype of M-MDSCs to anti-inflammatory profile with elevated expression of IL-10 and reduced expression of IFNγ, IL4, and IL22. Further, CCR5 modulation expanded IL10+M-MDSC in vivo . Additionally, DAPTA reduced the migratory potential, reduced cholesterol uptake and improved the functionality (suppression of T cell) of M-MDSCs. Blocking CCR5 with DAPTA led to the formation of stable plaque in HFD-fed mice. Conclusion: Increased expression of CCR5 coupled with inflammatory cytokine secretion by regulatory cells lead to dysfunctional M-MDSCs in CVD risk factor population. Possibly this dysfunctionality contributes to the development and progression of CVD including atherosclerosis. Our findings show targeting of CCR5 with DAPTA reduces atherogenesis, increases plaque stabilization and corrects phenotypic alterations in immune suppressive M-MDSCs. The functionality and phenotype of M-MDSCs in atherosclerosis, may be ameliorated by targeting CCR5.

Abstract 4132249: Preoperative identification of respiratory viral genomes in the trachea but not in the nasopharynx is a risk factor for postoperative pulmonary hypertension in patients with congenital cardiac communications

Introduction: Pulmonary vascular remodeling is a key determinant of outcomes in patients undergoing surgery for congenital cardiac communications (CCCs). Respiratory epithelial cells infected by viruses express cytokines and growth factors with potential impact on airway and vascular remodeling. Hypothesis: Preoperative exposure to respiratory viruses renders CCC patients susceptible to postoperative pulmonary hypertension. Aims: To investigate if asymptomatic patients carrying respiratory viral genomes (RVGs) in the airways are at risk for pulmonary hypertension following cardiac surgery. To analyze the role of postoperative inflammatory reaction. Methods: The study comprised patients with unrestrictive CCCs undergoing surgery under cardiopulmonary bypass (CPB). In the absence of any signs of recent infection, RVGs were searched for in nasopharyngeal aspirates 2-3 days before surgery, and in tracheal aspirates collected in the operating room (RT-PCR, panel for 19 viruses). Serum levels of 36 inflammatory proteins were measured by chemiluminescence preoperatively and 4 hours post CPB. Pulmonary/systemic mean arterial pressure ratio (PAP/SAP) computed during the first 12 hours postoperatively (invasive assessment) was defined as the study outcome. A cutoff level was set at 0.40. Results: Sixty patients were enrolled (age 11 [7-16] months, median with interquartile range). Preoperative PAP/SAP was 0.78 (0.63-0.88). RVGs were present in nasopharyngeal and tracheal aspirates in 64% and 38% of patients, respectively. The presence of RVGs in tracheal aspirates was associated with an upward shift of postoperative PAP/SAP curve (p=0.006). The same was not observed for positive tests in nasopharyngeal aspirates (p=0.343). Of all factors analyzed as possible predictors of postoperative PAP/SAP >0.40, only preoperative peripheral oxygen saturation and the presence of RVGs in the trachea were selected at multivariate analysis (respective OR with 95% CI, 0.40 [0.23-0.72] and 4.42 [1.17-16.78]. Heightened post-CPB levels of interleukin-1 receptor antagonist and chemokines MIF and MCP-1 were found to increase the risk of having a PAP/SAP >0.40 in RVG carriers (respective OR with 95% CI, 2.04 [1.09-3.81], 2.34 [1.15-4.74] and 2.18 [1.07-4.43]. Conclusion: The observed relation of postoperative hemodynamics to respiratory viruses and post-CPB inflammatory reaction may have pathophysiological and therapeutic implications in pulmonary hypertension associated with congenital heart disease.

Abstract 4122957: C-C motif chemokine receptor-2 disruption Synergizes with Phosphodiesterase Type 5 Inhibition in Ameliorating Pulmonary Hypertension in Monocrotaline-treated Rats: Proof-of-Concept Study for a Combination therapy and Potential Mechanisms

Background: Whether and how any anti-inflammatory agents and their combination with conventional pulmonary vasodilators protect against pulmonary arterial hypertension (PAH) is unknown. Hypothesis: The disruption of C-C motif chemokine receptor (CCR) -2, a receptor for CCL-2, ameliorates PAH in any models with the reversal of the associated pro-inflammatory state and vascular cell phenotypes and synergizes with any conventional pulmonary vasodilators. Methods and Results: By using Ccr2 (-/-) rats generated by CRISPR/ Cas9 , we investigated PAH in Ccr2 (+/+) or Ccr2 (-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx), and chronic hypoxia (CH). Ccr2 (-/-) decreased the right ventricular systolic pressure, right ventricular hypertrophy, and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (IL-6, TNF-α, CCL2, IL-1β, TGF-β) in MCT-treated rats, but not in SuHx or CH models. Consistently, Ccr2 (-/-) decreased indices of pulmonary vascular diseases (PVDs) and perivascular macrophage infiltration, as well as reversed impaired BMPR2 signaling, increased endothelial apoptosis, impaired NO signaling (eNOS, peNOS/eNOS) and decreased phosphodiesterase (PDE)-5 expression in lungs in MCT-treated rats. Gene expression of the receptor for prostaglandin I 2 and endothelin was not altered in MCT-treated rats by Ccr2 (-/-). In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2 (-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. Consistently, the whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2 (-/-) PASMCs, which are related to cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2 (-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2 (-/-) rats but not in Ccr2 (+/+) rats, and further improved survival in Ccr2 (-/-) rats. Conclusions: PDE5 inhibition synergized with Ccr2 disruption in protecting against PAH in MCT-treated rats, which was mechanistically related to the restoration of proinflammatory states and associated vascular cell phenotypes. These findings confer a fundamental basis supporting that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

Abstract 4137240: MHCII hi LYVE1 lo CCR2 hi Interstitial Macrophages Promote Medial Fibrosis in Pulmonary Arterioles and Contribute to Pulmonary Hypertension

Background: Pulmonary hypertension (PH) is a lethal disease characterized in part by progressive pulmonary arteriole (PA) remodeling. Excessive PA fibrosis and macrophage infiltration are often present in PH, but potential association is obscure. We invesitgated the link between interstitial macrophage (iMΦ) infiltration and PA fibrosis in PH and idiopathic pulmonary arterial hypertension (IPAH). Methods: Lung tissue samples from patients with IPAH (n=11) and experimental PH (sugen-5416 and hypoxia, hypoxia, or monocrotaline, n=5) animals (adult male and female SD rats) were obtained to analyze the extent of fibrosis in PA adventitia and media and their correlation to disease severity. Single-cell RNA sequencing, immunohistological analyses, iMΦs and PA smooth muscle cells (PASMCs) coculture, and transgenic animal experiments were used to investigate the cell heterogeneity and molecular mechanisms by which iMΦs promote PA medial fibrosis. Data in this study were analyzed with unpaired two-tailed t-tests with Welch’s correction, Kruskal-Wallis one-way ANOVA followed by Dunn’s test, RM ANOVA with Geisser-Greenhouse correction, or Pearson correlation test, and p < 0.05 was considered statistically significant. Results: We found that increased collagen deposition and fibrosis in the PA media were correlated with PH severity, and iMΦ medial infiltration may be involved in these pathological processes. Single-cell transcriptomics suggested that ligand-receptor associations between MHCII hi LYVE1 lo CCR2 hi iMΦ and fibroblast-like vascular SMC subclusters contributed to PA medial fibrosis. Mechanistically, MHCII hi LYVE1 lo CCR2 hi iMΦs regulated PASMC phenotypic transition and collagen production through the wingless member 11 (WNT11)/planar cell polarity (PCP) pathway. Deletion of iMΦ-enriched Wnt11 in myeloid cells of PH rats ( Itgam CreERT2 ; Wnt11 fl/fl ) normalized the fibrotic PASMC phenotype and decreased PA medial fibrosis, thereby protecting against PH. Moreover, myeloid-specific C-C motif chemokine receptor 2 ( Ccr2 ) deficiency ( Itgam CreERT2 ; Ccr2 fl/fl ) in PH-PAs inhibited MHCII hi LYVE1 lo CCR2 hi iMΦ infiltration. Conclusions: PA medial fibrosis is a causative factor of PH, and the inhibition of MHCII hi LYVE1 lo CCR2 hi iMΦ pathogenicity or infiltration reverses PA medial fibrosis and thus alleviates PH.

Unraveling the Multifaceted Roles of Atypical Chemokine Receptors in Breast Cancer.

Breast cancer (BC) remains one of the most prevalent and deadly malignancies among women globally. A deeper understanding of the molecular mechanisms driving BC progression and metastasis is essential for the development of effective therapeutic strategies. While traditional chemokine receptors are well known for their roles in immune cell migration and positioning, atypical chemokine receptors (ACKRs) have recently gained attention as key modulators in cancer-related processes. Unlike conventional receptors, ACKRs-comprising ACKR1, ACKR2, ACKR3, and ACKR4-primarily function by scavenging chemokines, regulating their availability, and modulating receptor signaling in a ligand-independent manner. This review aims to elucidate the roles of ACKRs in BC, focusing on their influence on the tumor microenvironment (TME), cancer cell proliferation, survival, metastasis, and angiogenesis. Additionally, we will explore the potential of ACKRs as diagnostic and prognostic markers and assess their viability as therapeutic targets. By synthesizing recent research findings and highlighting future research directions, this review seeks to provide a comprehensive understanding of the significance of ACKRs in BC and underscore the need for continued investigation into their therapeutic potential.

TMIC-81. AN IMMUNOSUPPRESSIVE MACROPHAGE NICHE MAY DRIVE T CELL DYSFUNCTION AND IMMUNOTHERAPY RESISTANCE IN MELANOMA BRAIN METASTASES

Abstract Nearly half of metastatic melanoma patients develop brain metastases (MBM). Local therapies like surgery and radiotherapy provide limited survival benefit. While targeted therapies and immune checkpoint inhibitors (ICI) improved outcomes in patients with asymptomatic MBM, the prognosis is still grim for the majority of patients. To decipher resistance mechanism to immunotherapy in MBM, we integrated datasets from both single-cell RNAseq (scRNAseq) and spatial transcriptomics (Visium). Our scRNAseq analysis of 25 MBM patients (7 treated pre-surgery with ICI) showed that lymphoid cells increased by 8.1-fold post-ICI (p=0.006). Notably, 34% of these were CXCL13+CXCR6+ putative tumor-reactive T cells (TR-T), known for their critical role as tumor-killing effectors. This led us to investigate why, despite their prevalence, TR-T don’t translate to effective immunotherapy in MBM. Our scRNAseq interactome analysis revealed that ICI intensified IFN-II interactions between TR-T and interferon-stimulated macrophage/microglia (Mφ.ISG). However, it also boosted immunosuppressive interactions, like the TIGIT-NECTIN2 interaction, which increased 20 fold, potentially diminishing TR-T’s effectiveness. To overcome the lack of spatial context in scRNAseq, we applied spatial transcriptomics on 27 MBM sections, including 12 post-ICI treatments. Remarkably, TR-T were primarily found (63%) in proximity with Mφ.ISG cells—not with tumor cells. These TR-T-Mφ.ISG niches were 1.7 times more prevalent in ICI-treated samples; these niches are rich in chemokine receptors like CCR1 and immunosuppressive molecules like NECTIN2. This suggests that Mφ.ISG cells are chemotactically attracted to TR-T, forming a barrier that impedes TR-T’s tumor interaction and impairs their functionality. Our current efforts focus on pinpointing key pathways enriched within TR-T-Mφ.ISG niches, particularly in ICI-treated MBM. We are also assessing the clinical impact of these niches in relation to patient survival. Our goal is to elucidate how specific immunosuppressive subsets and molecules within the MBM microenvironment restrict TR-T effectiveness, with the ultimate objective of enhancing ICI efficacy for these patients.

The potential of ESCO2 as a prognostic and immunotherapeutic marker of pan-cancer and its role in anti-PD1 treatment of bladder cancer.

Background Establishment of sister chromatid cohesion N-acetyltransferase 2 (ESCO2), a member of the EFO2 family, is implicated in the pathogenesis and progression of various cancers. However, there has been limited comprehensive pan-cancer analysis conducted on ESCO2 thus far. Methods Publicly available databases, such as the UCSC Xena database, were utilized to examine differential expression patterns across various cancer types. In addition, variations in expression levels were investigated across distinct clinical stages. Univariate Cox regression and Kaplan-Meier survival analyses were conducted to evaluate the impact on overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) at a pan-cancer level. The correlation between ESCO2 expression and immune cell infiltration was examined to gain insight into the tumor microenvironment in different cancers. The results of the bioinformatic analysis were validated using immunotherapy clinical trials and pathological specimens. CCK-8 and transwell assay experiments were performed to investigate the biological function of ESCO2. Results ESCO2 expression was found to be up-regulated in most cancers, with a correlation to TNM stages. Prognostic analysis indicated that overexpression of ESCO2 was associated with poor prognosis in various cancers. Furthermore, the correlation between ESCO2 expression and immune cell infiltration suggested its potential as a predictor for immunotherapy efficacy. Notably, ESCO2 expression showed positive associations with immunoinhibitor, immnostimulator, MHC molecule, chemokine receptor, tumor mutation burden (TMB) and microsatellite instability (MSI) levels in bladder cancer (BLCA). The validation cohort for immunotherapy corroborated these findings and substantiated that ESCO2 could function as an autonomous prognostic biomarker and a promising target for cancer treatment via immunotherapy. In addition, in vitro experiments confirmed the role of ESCO2 in influcing the proliferation, invasion and migration of BLCA cells. Conclusion ESCO2 participates in regulating the immune infiltration and affecting the prognosis of patients in many cancers, especially in BLCA. ESCO2 may serve as a prognostic and immunotherapy biomarker in future treatment of human cancer.

CXCR4 antagonism ameliorates leukocyte abnormalities in a preclinical model of WHIM syndrome

BackgroundWHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is an ultra-rare, combined primary immunodeficiency and chronic neutropenic disorder characterized by a range of clinical presentations, including peripheral neutropenia, lymphopenia, and recurrent infections. WHIM syndrome is most often caused by gain-of-function mutations in the gene encoding C-X-C chemokine receptor 4 (CXCR4). As such, inhibition of CXCR4 with XOLREMDI® (mavorixafor), an orally bioavailable CXCR4 antagonist, demonstrated clinically meaningful increases in absolute neutrophil and lymphocyte counts and concomitant reduction in infections in patients with WHIM syndrome, resulting in its recent U.S. Food and Drug Administration approval. The impact of CXCR4 antagonism on other aspects of the pathobiology in WHIM syndrome, such as lymphopoiesis and leukocyte trafficking between primary and secondary lymphoid organs, is less understood.MethodsIn the current study, the effects of CXCR4 antagonism on leukocyte trafficking and distribution in primary and secondary lymphoid organs were investigated in a mouse model of WHIM syndrome carrying the heterozygous Cxcr41013 mutation. Cxcr4+/1013 and Cxcr4 wild-type mice received the orally bioavailable CXCR4 antagonist X4-185. Blood, spleen and bone marrow samples were collected for numeration, flow cytometry, and functional studies.ResultsCxcr4+/1013 mice exhibited profound peripheral blood leukopenia as seen in patients with WHIM syndrome. CXCR4 antagonism corrected circulating leukopenia and mobilized functional neutrophils without disrupting granulopoiesis in the bone marrow of Cxcr4+/1013 mice. Furthermore, Cxcr4+/1013 displayed aberrant splenic T and B-cell counts and frequency. Treatment with X4-185 normalized splenic T-cell abnormalities, correcting the reduced CD8+ T-cell numbers, restoring the CD4/CD8 T-cell ratio, and ameliorating peripheral blood T-cell lymphopenia. In addition, CXCR4 antagonism was able to correct the abnormal frequencies and numbers of splenic marginal zone and follicular B cells in Cxcr4+/1013 mice, and ultimately normalize B-cell lymphopenia in the peripheral circulation.ConclusionsOur study provides comprehensive evidence that oral dosing with a CXCR4 antagonist can effectively correct WHIM-associated neutrophil and lymphocyte abnormalities in a mouse model of WHIM syndrome. These findings extend our understanding of how targeting the dysregulated CXCR4 signaling pathway can ameliorate the pathogenesis of WHIM syndrome.

R-Spondin 1 Suppresses Inflammatory Cytokine Production in Human Cortical Astrocytes

Background/Objectives: Wnt signaling pathways are essential in various biological processes, including embryonic development and tissue homeostasis, and are implicated in many diseases. The R-Spondin (RSpo) family, particularly RSpo1, plays a significant role in modulating Wnt signaling. This study aims to explore how RSpo1 binding to astrocytic LGR6 receptors influences central nervous system (CNS) homeostasis, particularly in the context of inflammation. Methods: Human-induced pluripotent stem cell-derived astrocytes were treated with RSpo1 to assess its impact on inflammatory cytokine release. A proteomic analysis was conducted using a Human Cytokine Array Kit to measure differential protein expression. Pathway enrichment analysis was performed to identify affected signaling pathways. Results: RSpo1 treatment led to a suppression of inflammatory cytokines such as IL-10, IFN-γ, and IL-23 in astrocytes, while TNF-α and CXCL12 levels were increased. Pathway analysis revealed significant alterations in key signaling pathways, including cytokine–cytokine receptor interaction, chemokine signaling, and TNF signaling pathways, suggesting RSpo1’s role in modulating immune responses within the CNS. Conclusions: RSpo1 significantly influences inflammatory responses in astrocytes by modulating cytokine release and altering key signaling pathways. These findings enhance our understanding of the interaction between cell-specific Wnt signaling and CNS inflammation, suggesting potential therapeutic applications of RSpo1 in neuroinflammatory and neurodegenerative diseases.

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization.

Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.

CXCR6 expression predicts prognosis and immunotherapeutic benefit in muscle-invasive bladder cancer

BackgroundIncreasing evidence suggests that the CXC chemokine receptor 6 (CXCR6) is involved in tumor progression and the regulation of tumor immunity. However, its role in muscle-invasive bladder cancer (MIBC) remains largely unexplored.MethodsData from 391 MIBC patients in the TCGA, 212 patients from GEO, 131 patients from our center, 195 patients in the IMvigor210 cohort, and single-cell RNA sequencing (scRNA-seq) data from 9 bladder cancer patients (GSE222315) were analyzed. Additionally, data from the GEPIA 2, TISCH2, TIMER2.0, and UALCAN platforms were utilized to investigate the prognostic and immunotherapeutic significance of CXCR6 in MIBC.ResultsWe observed that CXCR6 expression was significantly reduced in bladder cancer tumors and correlated with tumor stage and grade. Low CXCR6 expression was associated with poor recurrence-free survival (RFS) and overall survival (OS) in the TCGA cohort, a finding validated in both the meta-GEO dataset and our center’s cohort. Multivariate analysis confirmed that low CXCR6 expression was an independent predictor of poor OS and RFS. A nomogram incorporating CXCR6 expression and other independent prognostic factors was developed to accurately predict 3- and 5-year OS. Gene set enrichment analysis indicated that immune activation-related pathways were significantly enriched in tumors with high CXCR6 expression. CIBERSORT analysis revealed that CXCR6 expression was positively correlated with CD8+ T cells, CD4+ T cells, activated NK cells, M1 macrophages, and activated dendritic cells in TCGA, findings further validated by TIMER2.0. scRNA-seq data showed that CXCR6 was predominantly expressed in T and NK cells and facilitated T/NK-myeloid interaction via the CXCR6-CXCL16 axis. Importantly, CXCL16+ macrophages and dendritic cells recruited CXCR6+ T and NK cells, which exhibited enhanced cytotoxicity, thereby amplifying anti-tumor immunity. Clinically, in the IMvigor210 immunotherapy cohort, higher CXCR6 expression was associated with improved anti-PD-L1 therapeutic outcomes.ConclusionOur findings highlight CXCR6 as a critical biomarker for predicting prognosis and immunotherapeutic response in MIBC.