Signaling In Macrophages Research Articles

Hydroxygenkwanin suppresses peritoneal metastasis in colorectal cancer by modulating tumor-associated macrophages polarization

Peritoneal metastasis is an important cause of high mortality and poor prognosis in colorectal cancer (CRC) patients. Therefore, the development of compounds with unique anti-CRC Peritoneal metastasis activities is urgently needed to improve the survival of CRC patients. Hydroxygenkwanin (HGK),a natural flavonoid compound, have been shown to display anti-inflammatory, antioxidant, antitumor, and immunoregulatory effects. Here, we employed CRC peritoneal metastasis mouse model with MC38 cells to examine the antitumor activity of HGK. The result showed that HGK not only inhibited peritoneal metastasis, but also significantly increased the proportion of M1-like macrophages while decreasing the proportion of M2-like macrophages within the tumor microenvironment (TME). Furthermore, we demonstrated that the inhibitory effect of HGK on peritoneal metastasis of CRC depended on macrophages in vitro and in vivo. Moreover, we revealed that HGK promoted the polarization of TAMs into M1-like macrophages and inhibited their polarization into M2-like macrophages in a LPS- or IL-4-induced bone marrow-derived macrophages (BMDMs) model and co-culture system. Finally, we also investigated the regulatory mechanism of HGK on TAMs polarization that HGK may active p-STAT5, p–NF–κB signaling in M1-like macrophages and inhibit p-STAT6, JMJD3, PPARγ expression in M2-like macrophages. Taken together, our findings suggest that HGK is a natural candidate for effective prevention of peritoneal metastasis in colorectal cancer, which provides a potential strategy for clinical treatment of colorectal cancer.

IRF7 Exacerbates Candida albicans Infection by Compromising CD209-Mediated Phagocytosis and Autophagy-Mediated Killing in Macrophages.

IFN regulatory factor 7 (IRF7) exerts anti-infective effects by promoting the production of IFNs in various bacterial and viral infections, but its role in highly morbid and fatal Candida albicans infections is unknown. We unexpectedly found that Irf7 gene expression levels were significantly upregulated in tissues or cells after C. albicans infection in humans and mice and that IRF7 actually exacerbates C. albicans infection in mice independent of its classical function in inducing IFNs production. Compared to controls, Irf7-/- mice showed stronger phagocytosis of fungus, upregulation of C-type lectin receptor CD209 expression, and enhanced P53-AMPK-mTOR-mediated autophagic signaling in macrophages after C. albicans infection. The administration of the CD209-neutralizing Ab significantly hindered the phagocytosis of Irf7-/- mouse macrophages, whereas the inhibition of p53 or autophagy impaired the killing function of these macrophages. Thus, IRF7 exacerbates C. albicans infection by compromising the phagocytosis and killing capacity of macrophages via regulating CD209 expression and p53-AMPK-mTOR-mediated autophagy, respectively. This finding reveals a novel function of IRF7 independent of its canonical IFNs production and its unexpected role in enhancing fungal infections, thus providing more specific and effective targets for antifungal therapy.

Abstract PO3-03-01: Impact of Environmental Temperature on Clinical Outcomes and Tumor Microenvironment of Early-Stage Breast Cancer

Abstract Background Preclinical evidence using mouse model suggests that thermal/cold stress increases tumor growth by modulating the tumor microenvironment (TME); however, the clinical relevance of temperature on breast cancer (BC) outcomes is unknown. Studies show that residing in cold regions is associated with higher incidence of BC. We aim to study the impact of environmental temperature on the pathological complete response (pCR) and survival of early-stage BC patients (pts). Methodology A multi-institutional study was conducted within the Oncology Research Information Exchange Network (ORIEN). We analyzed the clinical and genomic data for early-stage BC pts from 12 centers in different environment zones (5 warm and 7 cold) (based on average annual regional temperature obtained from National Centers for Environmental Information). Cox regression was used to measure the association of climate and overall/relapse-free survival (OS/RFS) after adjusting for age, race, body mass index, grade, stage, treatments, comorbidities, and subtype. Raw feature counts were normalized, and differential expression analysis was carried out using DESeq2, while adjusting for all of the covariates. Differential expression rank order was used for subsequent gene set enrichment analysis (GSEA), performed using the cluster profile package in R. Gene sets queried included the Hallmark, Canonical pathways, and GO Biological Processes Ontology collections available through the Molecular Signatures Database (MSigDB). Results Out of the 1,304 early-stage BC pts, 271 pts received neoadjuvant chemotherapy (NAC) (186 warm, 85 cold). Higher clinical T- and N-stages were observed in pts from warm compared to cold regions (p< 0.001). Pts residing in cold regions had more comorbidities (57.6% vs 4.8%, p< 0.001). Pts in warm regions had higher pCR, though not statistically significant (8% vs 2.5%, p= 0.1). In the overall population, the OS (univariate (UV) HR= 0.48, 95% CI 0.27-0.64, p < 0.001; adjusted HR (aHR)= 0.56, 95% CI 0.32 - 0.96, p= 0.03) and RFS (UV HR= 0.51, 95% CI 0.38 - 0.68, p< 0.001; aHR= 0.52, 95% CI 0.36 - 0.75, p= 0.0005) were higher in pts from warm compared to cold regions (Table 1). Among the patient subgroup treated with NAC, the OS (aHR= 0.35, p= 0.02) and RFS (aHR= 0.49, p= 0.02) of pts from warmer regions was also higher than cold regions. RNA sequencing was performed on 826 tumors (443 warm, 383 cold) using pre-treatment samples. Naïve B-cells (mean: 0.09 vs 0.08, p=0.004), CD4 naïve T cells (0.03 vs 0.02, p= 0.0008), CD4+ memory T cells (0.03 vs 0.02, p= 0.04), gamma-delta T-cells (0.007 vs 0.004, p= 0.006) were higher in pts residing in cold compared to warm regions while CD8 T cells, T regulatory cells, macrophages, dendritic cells, natural killer cells were similar in both regions. Macrophage signaling, cholesterol metabolism and the pathway for negative regulation of cell migration in angiogenesis were upregulated in pts living in warm compared to cold regions. Conclusion Early-stage BC pts living in cold have worse OS and RFS compared to warm regions. Our study is the first to report significant differences in the TME among these pts. However, it is important to acknowledge that the limited availability of comprehensive data regarding patients' relocation to different environmental temperature zones during treatments, and individual temperature preferences may be considered a potential limitation of our study. To ensure the validity and robustness of these intriguing findings, larger multi-center studies should be conducted, encompassing detailed information on the duration of exposure to both warm and cold weather conditions. Table 1: OS and RFS of patients living in warm vs cold environments Citation Format: Arya Mariam Roy, Spencer Rosario, Anthony George, Qiang Hu, Brian Czerniecki, John Carpten, Virginia Borges, Bryan Schneider, Jill Kolesar, Christopher Moskaluk, Craig Shriver, Cindy Matsen, Shridar Ganesan, Sneha Phadke, Wajeeha Razaq, Kristopher Attwood, Shipra Gandhi. Impact of Environmental Temperature on Clinical Outcomes and Tumor Microenvironment of Early-Stage Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-03-01.

Abstract PO3-14-06: Immunologic features and association with prognosis in hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC) treated with chemotherapy (CT) or CDK4/6-inhibitors (CDK4/6i) + endocrine therapy (ET)

Abstract Purpose: Little is known regarding the prognostic and predictive role of tumor immunological features in HR+/HER2- MBC treated with CT or CDK4/6i+ET. Methods: The Italian KENDO phase 2 trial randomized CT-naïve patients with HR+/HER2-neg. MBC with aggressive characteristics to receive CDK4/6i+ET (arm A) or CT+/-ET (arm B). Primary endpoint was progression-free survival (PFS). A tumor sample from the primary or metastatic tumor (archived or newly-obtained) was mandatory for inclusion. In this correlative biomarker analysis, tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLS) were detected on baseline tumor samples by hematoxylin&eosin staining, while immune genomic signatures were assessed with the nCounter BC360 panel. Immune pattern at immunohistochemistry was defined as inflamed (IF), excluded (IE) or desert (ID). TILs and signatures were dichotomized (high vs. low) with the maximally-selected rank statistics (MSRS) method. Survival analyses were conducted with the Kaplan-Meier (KM) method and differences were tested with log-rank test. Hazard ratios (HR) with 95% confidence intervals (CI) were estimated with Cox regression. Significance was set at p≤0.05. Since the trial was stopped earlier due to slow accrual all analyses were exploratory. Results: Forty-nine patients were randomized, 17 (34.7%) in arm A and 32 (65.3%) in arm B. No significant clinicopathological differences were observed at baseline between the two arms, except for tumors in arm B showing more TLS (50.0% vs. 12.5%, p=0.013). Median PFS (mPFS) with CT+/-ET was numerically shorter than mPFS with CDK4/6i+ET (11.2 vs. 19.9 months, HR: 1.41, 95%CI: 0.75-2.64, p=0.289). The median OS (mOS) for arm A was not estimable (NE) vs. 30.6 months in arm B (p=0.283). In arm A, median TILs levels were 3% (interquartile range [IQR]: 1-5%) and TLS, with/without germinal centers, were present in 2 cases (12.5%). These were also the only IF tumors in the arm, whereas the rest (87.5%) were ID. In arm B, 7 (25.0%) tumors were IF, 19 (87.5%) ID and 2 (7.1%) IE. Median TILs were 4.5% (IQR: 2-12.8%) and TLS were present in 50% cases. In arm A, ID tumors showed lower mPFS than IF (15.8 vs. 27.5 months), with concordant trend at the OS KM curve. In arm B there was no clear difference in PFS, but IF tumors did not reach mOS, differently from ID (28.7 months) and IE (28.1 months). High vs. low TILs levels were significantly associated with better PFS (p=0.003) and OS (p=0.005) in arm A. High levels of a TGFβ gene expression signature were significantly associated with better PFS (p=0.020) and OS (p=0.005) in arm A and PFS (p=0.03) in arm B; higher levels of a cytokine/chemokine signature were associatedwith better PFS (p=0.02) in arm A, and higher levels of a mastcells signature was associated with worse OS in both arms (p=0.02 and p=0.03). In arm B, higher levels of a macrophage (p < 0.001) and antigen presentation signatures (p=0.04) were associated with worse and better OS, respectively, whereas higher levels of an immune infiltration and a cytotoxic cells signature were associated with better PFS (p=0.03 both). TLS presence was numerically associated to longer mPFS (23.5 vs. 15.8 months) in arm A, with consistent OS KM curve’s trend, and longer mOS (44.5 vs. 28.1 months) in arm B. Conclusions: The KENDO trial further supports CDK4/6i+ET use in aggressive CT-naïve HR+/HER2- MBC. Biomarkers of immune activation such as IF tumors, higher TILs and presence of TLS pointed towards better survival outcomes. Genomic immunological features also showed prognostic effect, with differences according to treatment arm and/or the immune process or cell line tracked. Further research on larger cohorts is needed to confirm these preliminary findings. Citation Format: Francesco Schettini, Michela Palleschi, Francesca Mannozzi, Fara Brasó-Maristany, Lorenzo Cecconetto, Patricia Galván, Marita Mariotti, Alessia Ferrari, Emanuela Scarpi, Anna Miserocchi, Oriana Nanni, Esther Sanfeliu, Aleix Prat, Andrea Rocca, Ugo De Giorgi. Immunologic features and association with prognosis in hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC) treated with chemotherapy (CT) or CDK4/6-inhibitors (CDK4/6i) + endocrine therapy (ET) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-06.

Mapping the cellular landscape of the maternal-fetal interface in women with preterm birth and chronic chorioamnionitis

Abstract Preterm birth (PTB) affects over 15 million infants yearly. In up to 40% of late PTBs, placental examination reveals chronic chorioamnionitis (CCA), maternal mononuclear cell infiltration in the chorioamniotic membranes; yet, the cellular landscape remains fully unknown. Herein, we first used imaging mass cytometry (IMC) to map the phenotype and spatial distribution of immune cells at the maternal-fetal interface of women with PTB and CCA and non-CCA controls (n = 4 each). Cases exhibited a 5-fold increase in T cells, including effector memory CD4+ (p=0.02) and proliferative CD8+ T cells (p=0.04), and a 7-fold rise in CD163+CD206+ macrophages (p=0.05) compared to controls. NK cells were also present in both groups. Neighborhood analysis revealed that CD8+ T cells are in proximity to macrophages and CD4+Tbet+ T cells, and distanced from NK cells. Next, we performed spatial transcriptomics (STx) in each study group (n = 8 PTB with CCA, n = 7 PTB no CCA). STx highlighted diversity in several clusters of T cells, macrophages, and other immune cells found to be transcriptionally active in CCA. Gene ontology analysis indicated enrichment for CD8+ T cell activation, as well as IL-12 and IFNγ signaling in macrophages in CCA. Ongoing efforts are focused on validation and functional testing of such immune cells in order to unveil the complex interactions at the maternal-fetal interface in CCA, a placenta pathology associated with adverse outcomes in the mother and offspring.

Lung homeostasis is maintained by the interactions between CX3CR1-expressing interstitial macrophage and lung commensal bacteria.

Abstract As an anti-inflammatory cytokine, IL10 plays an important role in modulating immune responses. However, it remains relatively unknown about the importance of IL10-IL10 receptor (IL10R) signaling in maintaining lung homeostasis. Here we investigated that IL10R signaling of lung interstitial macrophages (IMs) contributes to the maintenance of tissue homeostasis by preventing fibrosing lung inflammation. We observed that IL10-deficient mice spontaneously induced lung inflammation in adulthood. In parallel, IL10R-deficiency of IMs identically induced the inflammatory phenotypes observed in IL10-deficient mice. Antibiotics treatments, 16S rRNA-Sequencing analysis, and germ-free (GF) animals allowed us to determine that the lung inflammation occurring in the absence of IL10 or IL10R was triggered by lung commensal dysbiosis. Furthermore, we identified that two distinct commensal microbes (D. Acidovorans and R. Erythropolis) dominantly colonized in the lung of mice lacking IL10R in IMs, where these commensals initiate lung inflammation. Collectively, our data highlight that IL10R signaling of IMs contributes to lung homeostasis by restricting commensal dysbiosis and prevents progressive lung fibrosis.

Abstract 1049: Long Noncoding RNA Regulation Of Inflammation In Atherosclerosis

Atherosclerosis is a chronic inflammatory disease driven by cholesterol deposition in the artery wall. Resolution of atherosclerotic inflammation and plaque regression are important clinical goals. As long noncoding RNAs (lncRNAs) are key regulators of gene expression, there is an intense interest in understanding their roles in atherosclerosis and their potential as therapeutic targets. To identify lncRNAs regulated during plaque progression and regression, we performed single cell RNA-sequencing of CD45 + immune cells isolated from aortic arch plaques of Ldlr –/– mice under conditions of plaque progression (16 wks Western diet; WD) or regression (16 wks WD + 4 wks apoB ASO). We screened for lncRNAs acting in cis to modify the expression of proximal protein-coding genes and identified Caslr ( C tsc a nti s ense l nc R NA) , a novel lncRNA transcribed from the opposite strand of Cathepsin C gene ( Ctsc ) encoding a pro-atherosclerotic lysosomal protease. Caslr–Ctsc pair is upregulated in macrophages of progressing plaques and in response to pro-inflammatory stimuli ( i.e . LPS, IL-6) in vitro , and conversely downregulated during atherosclerosis regression and in response to anti-inflammatory stimulus ( i.e. IL-4). LncRNA knockdown approaches combined with transcriptomic analyses and functional assays showed that Caslr regulates Ctsc expression and associated inflammatory responses, including NF-κB signaling and inflammasome-dependent IL-1β release. RNA purification strategies combined with mass-spectrometry revealed that Caslr interacts with spliceosome-associated proteins to coordinate splicing of Ctsc mRNA. Notably, Caslr shares functional homology with a human lncRNA, CASLR, proximal to the CTSC gene, which regulates CTSC expression and protease activity. Human CASLR and CTSC are highly expressed in the blood of hyperlipidemic patients and their expression is coordinately decreased upon aggressive cholesterol lowering by anti-PCSK9 treatment. Collectively, our data identify Caslr as an evolutionary conserved lncRNA acting in cis to drive the expression of cathepsin C with downstream consequences on inflammatory signaling in plaque macrophages, and suggest that targeting this lncRNA may be beneficial in treating atherosclerosis.

Verapamil and tangeretin enhances the M1 macrophages to M2 type in lipopolysaccharide-treated mice and inhibits the P-glycoprotein expression by downregulating STAT1/STAT3 and upregulating SOCS3

LPS induced sepsis is a complex process involving various immune cells and signaling molecules. Dysregulation of macrophage polarization and ROS production contributed to the pathogenesis of sepsis. PGP is a transmembrane transporter responsible for the efflux of a number of drugs and also expressed in murine macrophages. Natural products have been shown to decrease inflammation and expression of efflux transporters. However, no treatment is currently available to treat LPS induced sepsis. Verapamil and Tangeretin also reported to attenuate lipopolysaccharide-induced inflammation. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis and its detailed anti-inflammatory mechanism have not been reported. Here, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages populations and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in macrophages. An hour before LPS (10 mg/kg) was administered; mice were given intraperitoneal injections of either verapamil (5 mg/kg) or tangeretin (5 mg/kg). The peritoneal macrophages from different experimental groups of mice were isolated. Hepatic, pulmonary and splenic morphometric analyses revealed that verapamil and tangeretin decreased the infiltration of neutrophils into the tissues. Verapamil and tangeritin also enhanced the activity of SOD, CAT, GRX and GSH level in all the tissues tested. verapamil or tangeretin pre-treated mice shifted M1 macrophages to M2 type possibly through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression. Hence, both these drugs have shown protective effects in sepsis via suppressing iNOS, COX-2, oxidative stress and NF-κB signaling in macrophages. Therefore, in our study we can summarize that mice were treated with either Vera or Tan before LPS administration cause an elevated IL-10 by the macrophages which enhances the SOCS3 expression, and thereby able to limits STAT1/STAT3 inter-conversion in the macrophages. As a result, NF-κB activity is also getting down regulated and ultimately mitigating the adverse effect of inflammation caused by LPS in resident macrophages. Whether verapamil or tangeretin offers such protection possibly through the inhibition of P-glycoprotein expression in macrophages needs clarification with the bio availability of these drugs under PGP inhibited conditions is a limitation of this study.

PPARγ attenuates cellular senescence of alveolar macrophages in asthma-COPD overlap

BackgroundAsthma-chronic obstructive pulmonary disease (COPD) overlap (ACO) represents a complex condition characterized by shared clinical and pathophysiological features of asthma and COPD in older individuals. However, the pathophysiology of ACO remains unexplored. We aimed to identify the major inflammatory cells in ACO, examine senescence within these cells, and elucidate the genes responsible for regulating senescence.MethodsBioinformatic analyses were performed to investigate major cell types and cellular senescence signatures in a public single-cell RNA sequencing (scRNA-Seq) dataset derived from the lung tissues of patients with ACO. Similar analyses were carried out in an independent cohort study Immune Mechanisms Severe Asthma (IMSA), which included bulk RNA-Seq and CyTOF data from bronchoalveolar lavage fluid (BALF) samples.ResultsThe analysis of the scRNA-Seq data revealed that monocytes/ macrophages were the predominant cell type in the lung tissues of ACO patients, constituting more than 50% of the cells analyzed. Lung monocytes/macrophages from patients with ACO exhibited a lower prevalence of senescence as defined by lower enrichment scores of SenMayo and expression levels of cellular senescence markers. Intriguingly, analysis of the IMSA dataset showed similar results in patients with severe asthma. They also exhibited a lower prevalence of senescence, particularly in airway CD206 + macrophages, along with increased cytokine expression (e.g., IL-4, IL-13, and IL-22). Further exploration identified alveolar macrophages as a major subtype of monocytes/macrophages driving cellular senescence in ACO. Differentially expressed genes related to oxidation-reduction, cytokines, and growth factors were implicated in regulating senescence in alveolar macrophages. PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) emerged as one of the predominant regulators modulating the senescent signature of alveolar macrophages in ACO.ConclusionThe findings suggest that senescence in macrophages, particularly alveolar macrophages, plays a crucial role in the pathophysiology of ACO. Furthermore, PPARγ may represent a potential therapeutic target for interventions aimed at modulating senescence-associated processes in ACO.Key words ACO, Asthma, COPD, Macrophages, Senescence, PPARγ.

Cross-Domain Text Mining of Pathophysiological Processes Associated with Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide. This study's goal was to identify the signaling drivers and pathways that modulate glomerular endothelial dysfunction in DKD via artificial intelligence-enabled literature-based discovery. Cross-domain text mining of 33+ million PubMed articles was performed with SemNet 2.0 to identify and rank multi-scalar and multi-factorial pathophysiological concepts related to DKD. A set of identified relevant genes and proteins that regulate different pathological events associated with DKD were analyzed and ranked using normalized mean HeteSim scores. High-ranking genes and proteins intersected three domains-DKD, the immune response, and glomerular endothelial cells. The top 10% of ranked concepts were mapped to the following biological functions: angiogenesis, apoptotic processes, cell adhesion, chemotaxis, growth factor signaling, vascular permeability, the nitric oxide response, oxidative stress, the cytokine response, macrophage signaling, NFκB factor activity, the TLR pathway, glucose metabolism, the inflammatory response, the ERK/MAPK signaling response, the JAK/STAT pathway, the T-cell-mediated response, the WNT/β-catenin pathway, the renin-angiotensin system, and NADPH oxidase activity. High-ranking genes and proteins were used to generate a protein-protein interaction network. The study results prioritized interactions or molecules involved in dysregulated signaling in DKD, which can be further assessed through biochemical network models or experiments.

Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages

Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor Tcell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered Tcell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

Corosolic acid attenuates platelet-derived growth factor signaling in macrophages and smooth muscle cells of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease that is characterized by vascular remodeling of the pulmonary artery. Pulmonary vascular remodeling is primarily caused by the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are facilitated by perivascular inflammatory cells including macrophages. Corosolic acid (CRA) is a natural pentacyclic triterpenoid that exerts anti-inflammatory effects. In the present study, the effects of CRA on the viability of macrophages were examined using monocrotaline (MCT)-induced PAH rats and human monocyte-derived macrophages. Although we previously reported that CRA inhibited signal transducer and activator of transcription 3 (STAT3) signaling and ameliorated pulmonary vascular remodeling in PAH, the inhibitory mechanism remains unclear. Therefore, the underlying mechanisms were investigated using PASMCs from idiopathic PAH (IPAH) patients. In MCT-PAH rats, CRA inhibited the accumulation of macrophages around remodeled pulmonary arteries. CRA reduced the viability of human monocyte-derived macrophages. In IPAH-PASMCs, CRA attenuated cell proliferation and migration facilitated by platelet-derived growth factor (PDGF)-BB released from macrophages and PASMCs. CRA also downregulated the expression of PDGF receptor β and its signaling pathways, STAT3 and nuclear factor-κB (NF-κB). In addition, CRA attenuated the phosphorylation of PDGF receptor β and STAT3 following the PDGF-BB simulation. The expression and phosphorylation levels of PDGF receptor β after the PDGF-BB stimulation were reduced by the small interfering RNA knockdown of NF-κB, but not STAT3, in IPAH-PASMCs. In conclusion, CRA attenuated the PDGF-PDGF receptor β-STAT3 and PDGF-PDGF receptor β-NF-κB signaling axis in macrophages and PASMCs, and thus, ameliorated pulmonary vascular remodeling in PAH.

A Cell Specific Effect of MBOAT7 MAFLD-risk Variant on Immune Cells.

Disease risk variants are likely to affect gene expression in a context- and cell-type specific manner. The membrane bound O-acyltransferase domain containing 7 (MBOAT7) rs8736 metabolic-dysfunction-associated fatty liver disease (MAFLD)-risk variant was recently reported to be a negative regulator of toll-like receptors (TLRs) signalling in macrophages. Whether this effect is generic or cell-type specific in immune cells is unknown. We investigated the impact of modulating TLR signaling on MBOAT7 expression in peripheral blood mononuclear cells (PBMCs). We also examined whether the rs8736 polymorphism in MBOAT7 regulates this effect. Furthermore, we measured the allele-specific expression of MBOAT7 in various immune cell populations under both unstimulated and stimulated conditions. We show that MBOAT7 is down-regulated by TLRs in PBMCs. This effect is modulated by the MBOAT7 rs8736 polymorphism. Additionally, we provide evidence that MBOAT7 acts primarily as a modulator of TLR signalling in mononuclear phagocytes. Our results highlight the importance of studying Genome-Wide Association Studies (GWAS) signals in the specific cell types in which alterations of gene expression are found.

Single-cell RNA sequencing reveals aberrant sphingolipid metabolism in non-small cell lung cancer impacts tumor-associated macrophages and stimulates angiogenesis via macrophage inhibitory factor signaling.

Sphingolipids not only serve as structural components for maintaining cell membrane fluidity but also function as bioactive molecules involved in cell signaling and the regulation of various biological processes. Their pivotal role in cancer cell development, encompassing cancer cell proliferation, migration, angiogenesis, and metastasis, has been a focal point for decades. However, the contribution of sphingolipids to the complexity of tumor microenvironment promoting cancer progression has been rarely investigated. Through the integration of publicly available bulk RNA-seq and single-cell RNA-seq data, we conducted a comprehensive analysis to compare the transcriptomic features between tumors and adjacent normal tissues, thus elucidating the intricacies of the tumor microenvironment (TME). Disparities in sphingolipid metabolism (SLM)-associated genes were observed between normal and cancerous tissues, with the TME characterized by the enrichment of sphingolipid signaling in macrophages. Cellular interaction analysis revealed robust communication between macrophages and cancer cells exhibiting low SLM, identifying the crucial ligand-receptor pair, macrophage inhibitory factor (MIF)-CD74. Pseudo-time analysis unveiled the involvement of SLM in modulating macrophage polarization towards either M1 or M2 phenotypes. Categorizing macrophages into six subclusters based on gene expression patterns and function, the SPP1+ cluster, RGS1+ cluster, and CXCL10+ cluster were likely implicated in sphingolipid-induced M2 macrophage polarization. Additionally, the CXCL10+, AGER+, and FABP4+ clusters were likely to be involved in angiogenesis through their interaction with endothelial cells. Based on multiple scRNA-seq datasets, we propose that a MIF-targeted strategy could potentially impede the polarization from M1 to M2 and impair tumor angiogenesis in low-SLM non-small cell lung cancer (NSCLC), demonstrating its potent antitumor efficacy.

Abstract CT053: Exploration of potential biomarkers correlated with efficacy of ociperlimab (anti-TIGIT) plus tislelizumab (anti-PD-1) in 1L PD-L1+ non-small cell lung cancer (NSCLC)

Abstract Background: PD-L1 expression was associated with anti-immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) + anti-PD-(L)1 treatment, in which a high PD-L1 subgroup showed improved efficacy. We investigated if anti-TIGIT mechanism of action (MOA)-related markers were associated with the efficacy of ociperlimab + tislelizumab in Cohort 3 (1L PD-L1+ NSCLC) of the phase 1/1b AdvanTIG-105 trial (NCT04047862) and evaluated a potential patient-enrichment strategy based on tumor tissue gene expression profile (GEP). Methods: Tumor tissue GEP was tested using TruSeq RNA Access technology. Ventana SP263 PD-L1 immunohistochemistry (IHC) assay was used to evaluate PD-L1 expression. Median progression-free survival (mPFS) by investigator was calculated descriptively by Kaplan-Meier methodology. 95% confidence intervals for mPFS were generated using the Brookmeyer method. The primary inferential PFS comparison used unstratified log-rank test with 2-sided descriptive P-values. Results: At data cutoff (Feb 2, 2023), 24 of 45 patients had GEP results. Anti-TIGIT MOA-related genes and signatures correlated with ociperlimab + tislelizumab treatment response. Patients with high (H) vs low (L) expression of TIGIT, CD226, CCR8, or a tumor-associated macrophage (TAM) signature had significantly longer mPFS (Table). Dual biomarkers combining both anti-PD-L1 (PD-L1 IHC) and one of the anti-TIGIT MOA-related factors (TIGIT, CCR8, TAM signature GEP) identified subgroups of PD-L1 H + TIGIT MOA-related factor H patients with improved PFS vs other subgroups (Table). A highly overlapped PD-L1 H + TIGIT H + CCR8 H + TAM signature H patient population was observed in dual biomarker analyses. Conclusions: Anti-TIGIT MOA-related genes and signatures correlated with efficacy in ociperlimab + tislelizumab-treated 1L PD-L1+ NSCLC. Combining anti-TIGIT MOA-related factors with PD-L1 expression identified a subgroup of patients with improved efficacy. TABLE 1. NAND Table. Efficacy Analyses in Patient Subgroups TIGITa CD226b CCR8b TAMb Subgroup H L H L H L H L n 8 16 12 12 12 12 12 12 mPFS, months (95% CI) NR (2.6, NR) 5.26 (2.07, 11.86) NR (4.21, NR) 4.68 (1.41, 15.05) 15.21 (2.6, NR) 4.7 (1.71, 7.16) 15.21 (4.21, NR) 4.17 (1.71, 5.45) PFS, P-value 0.0326 0.0327 0.0131 0.0153 PD-L1c + TIGITb PD-L1c + CCR8b PD-L1c + TAMb Subgroup TIGITHPD-L1H TIGITHPD-L1L TIGITLPD-L1H TIGITL PD-L1L CCR8HPD-L1H CCR8HPD-L1L CCR8LPD-L1H CCR8L PD-L1L TAMHPD-L1H TAMHPD-L1L TAMLPD-L1H TAML PD-L1L n 8 4 8 4 11 1 5 7 9 3 7 5 mPFS, months (95% CI) NR (1.41,NR) 4.76 (2.6,NR) 8.62 (2.07,NR) 2.94 (1.25,NR) NR (1.41,NR) 2.6 (NR,NR) 5.19 (2.07,NR) 4.21 (1.25,7.16) 23.89 (1.41,NR) 5.32 (4.21,NR) 5.29 (2.07,NR) 2.6 (1.25,NR) Cutoff: aTop 1/3; bMedian; cTC≥25% Citation Format: Se Hyun Kim, Jiayuan Zhang, Wei Tan, Han Yan, Tian Tian, Hao Zheng, Ziqi Zhou, Ruihua Wang, Yun Zhang, Zhirong Shen, Hye Ryun Kim, Diansheng Zhong, Shun Lu. Exploration of potential biomarkers correlated with efficacy of ociperlimab (anti-TIGIT) plus tislelizumab (anti-PD-1) in 1L PD-L1+ non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT053.

Artificial periosteum promotes bone regeneration through synergistic immune regulation of aligned fibers and BMSC-recruiting phages

Given the crucial role of periosteum in bone repair, the use of artificial periosteum to induce spontaneous bone healing instead of using bone substitutes has become a potential strategy. Also, the proper transition from pro-inflammatory signals to anti-inflammatory signals is pivotal for achieving optimal repair outcomes. Hence, we designed an artificial periosteum loaded with a filamentous bacteriophage clone named P11, featuring an aligned fiber morphology. P11 endowed the artificial periosteum with the capacity to recruit bone marrow mesenchymal stem cells (BMSCs). The artificial periosteum also regulated the immune microenvironment at the bone injury site through the synergistic effects of biochemical factors and topography. Specifically, the inclusion of P11 preserved inflammatory signaling in macrophages and additionally facilitated the migration of BMSCs. Subsequently, aligned fibers stimulated macrophages, inducing alterations in cytoskeletal and metabolic activities, resulting in the polarization into the M2 phenotype. This progression encouraged the osteogenic differentiation of BMSCs and promoted vascularization. In vivo experiments showed that the new bone generated in the AP group exhibited the most efficient healing pattern. Overall, the integration of biochemical factors with topographical considerations for sequential immunomodulation during bone repair indicates a promising approach for artificial periosteum development. Statement of significanceThe appropriate transition of macrophages from a pro-inflammatory to an anti-inflammatory phenotype is pivotal for achieving optimal bone repair outcomes. Hence, we designed an artificial periosteum featuring an aligned fiber morphology and loaded with specific phage clones. The artificial periosteum not only fostered the recruitment of BMSCs but also achieved sequential regulation of the immune microenvironment through the synergistic effects of biochemical factors and topography, and improved the effect of bone repair. This study indicates that the integration of biochemical factors with topographical considerations for sequential immunomodulation during bone repair is a promising approach for artificial periosteum development.

Acidovorax temperans skews neutrophil maturation and polarizes Th17 cells to promote lung adenocarcinoma development

Change within the intratumoral microbiome is a common feature in lung and other cancers and may influence inflammation and immunity in the tumor microenvironment, affecting growth and metastases. We previously characterized the lung cancer microbiome in patients and identified Acidovorax temperans as enriched in tumors. Here, we instilled A. temperans in an animal model driven by mutant K-ras and Tp53. This revealed A. temperans accelerates tumor development and burden through infiltration of proinflammatory cells. Neutrophils exposed to A. temperans displayed a mature, pro-tumorigenic phenotype with increased cytokine signaling, with a global shift away from IL-1β signaling. Neutrophil to monocyte and macrophage signaling upregulated MHC II to activate CD4+ T cells, polarizing them to an IL-17A+ phenotype detectable in CD4+ and γδ populations (T17). These T17 cells shared a common gene expression program predictive of poor survival in human LUAD. These data indicate bacterial exposure promotes tumor growth by modulating inflammation.

STING-Pathway Inhibiting Nanoparticles (SPINs) as a Platform for Treatment of Inflammatory Diseases.

Aberrant activation of the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway has been implicated in the development and progression of a myriad of inflammatory diseases including colitis, nonalcoholic steatohepatitis, amyotrophic lateral sclerosis (ALS), and age-related macular degeneration. Thus, STING pathway inhibitors could have therapeutic application in many of these inflammatory conditions. The cGAS inhibitor RU.521 and the STING inhibitor H-151 have shown promise as therapeutics in mouse models of colitis, ALS, and more. However, these agents require frequent high-dose intraperitoneal injections, which may limit translatability. Furthermore, long-term use of systemically administered cGAS/STING inhibitors may leave patients vulnerable to viral infections and cancer. Thus, localized or targeted inhibition of the cGAS/STING pathway may be an attractive, broadly applicable treatment for a variety of STING pathway-driven ailments. Here we describe STING-Pathway Inhibiting Nanoparticles (SPINS)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with RU.521 and H-151-as a platform for enhanced and sustained inhibition of cGAS/STING signaling. We demonstrate that SPINs are equally or more effective at inhibiting type-I interferon responses induced by cytosolic DNA than free H-151 or RU.521. Additionally, we describe a SPIN formulation in which PLGA is coemulsified with poly(benzoyloxypropyl methacrylamide) (P(HPMA-Bz)), which significantly improves drug loading and allows for tunable release of H-151 over a period of days to over a week by varying P(HPMA-Bz) content. Finally, we find that all SPIN formulations were as potent or more potent in inhibiting cGAS/STING signaling in primary murine macrophages, resulting in decreased expression of inflammatory M1-like macrophage markers. Therefore, our study provides an in vitro proof-of-concept for nanoparticle delivery of STING pathway inhibitors and positions SPINs as a potential platform for slowing or reversing the onset or progression of cGAS/STING-driven inflammatory conditions.

NFĸB signaling drives myocardial injury via CCR2+ macrophages in a preclinical model of arrhythmogenic cardiomyopathy.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.

Abstract 1602: Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy marked by a highly immunosuppressive tumor microenvironment (TME). Macrophages are known to promote immunosuppression and exhibit a profibrotic transcriptional profile with potential involvement in extracellular matrix (ECM) remodeling. Previously, we identified hyperactivation of cancer cell intrinsic cyclic AMP response element binding protein 1 (CREB) regulates leukemia inhibitory factor (LIF) to mediate macrophage infiltration and polarization within the TME of PDAC. Subsequently, we observed that disrupting LIF-LIF receptor (LIFR) signaling therapeutically not only diminishes the infiltration of tumor-associated macrophages (TAMs) but also results in a decrease in ECM deposition. This study delves into the potential regulatory pathway through which LIF induces remodeling of the ECM within the PDAC TME. Methods: We targeted LIF-induced macrophage-stromal crosstalk using the LIFR antagonist (EC359) in an orthotopically tumor implanted LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice model of PDAC. Sirius red staining in tumor sections was performed to assess ECM (collagen) deposition. Moreover, we performed RNA transcriptomics-based analysis of recombinant (rLIF) induced bone marrow derived macrophages harvested from syngeneic C57BL/6 mice. The Cancer Genome Atlas (TCGA) pancreatic cancer (PAAD) data set along with single-cell RNA sequencing (scRNA seq) were employed to assess the expression of major downstream targets of LIF. To validate the RNA-seq results, we conducted immunohistochemical analysis (IHC) on mouse PDAC tissue sections and quantitative polymerase (qPCR) analysis on RAW 264 macrophage cell line treated with rLIF or KPC CREBWT PDAC tumor cells conditioned media. Results: We observed a drastic decrease in both TAM infiltration and, of note, ECM deposition in orthotopically implanted KPC pancreas tumor mice tissue treated with EC359 as compared to vehicle. Mechanistically, our RNA-seq analysis unveiled a significant upregulation of Factor XIIIA (FXIIIa), a major downstream target of LIF. Notably, TCGA data revealed that FXIIIa mRNA expression is upregulated across PDAC tumors marked by poor survival outcomes. Moreover, scRNA seq and IHC established that TAMs highly express FXIIIa in the PDAC TME. In exploring the effect of LIF-LIFR signaling in the ECM, mouse RAW 264.7 macrophage lines incubated in high LIF conditions confirmed transcriptional upregulation of ECM-associated proteins such as FXIIIa as well as fibronectin. Conclusion: Our investigation provides valuable insights into the possible mechanism by which LIF triggers ECM remodeling, thereby contributing to the desmoplastic stroma. Significantly, our findings propose that LIF-regulated FXIIIa signaling in macrophages serves as a plausible mediator of the LIF-induced ECM remodeling in PDAC. Citation Format: Nishah Jaferi, Siddharth Mehra, Varun Krishnamoorthy, Sudhakar Jinka, Anna Bianchi, Vanessa T. Garrido, Luis A. Nivelo, Ban Yuguang, Nagaraj S. Nagathihalli. Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1602.