Macrophage Depletion Research Articles

Abstract 2010: Atherosclerotic Plaque Macrophages Drive Abdominal Aortic Aneurysm Formation

Introduction: Cigarette smoking (CS) contributes to the most deaths in abdominal aortic aneurysms (AAA) and tobacco use associates with early damage to the abdominal aorta. Associated risk factors, such as hyperlipidemia, link clinical AAA to the atherosclerotic process, suggesting common pathogenetic features with plaque development. We hypothesized that CS exposure exacerbates atherosclerotic disease in the abdominal aorta by increasing the recruitment of inflammatory macrophages that mediate arterial degradation and AAA formation. Methods: Adult male Apoe deficient mice commenced a high cholesterol diet and were concomitantly exposed to CS or room air (RA) for 4, 8, 12, and 16 weeks. As plaque macrophages are highly prevalent in advanced AAA, additional CS exposed mice received a selective CSF1R inhibitor, PLX3397, for up to 16 weeks to deplete macrophages. AAA incidence, atherosclerotic plaque burden and lesion composition were assessed in the aorta by immunofluorescence, Movat, EVG, and Oil Red O staining. Results: CS induced AAA at all timepoints with the highest incidence of 37% at 16 weeks of exposure (n=151 CS, n=75 RA p< 0.05). Aneurysms always coincided with atherosclerosis (n=7 CS, n=7 CS/AAA p<0.05), and severe elastin fragmentation was consistently overlaid by plaque (n=20 CS p<0.05, Fig 1A and B). In some cases, lesions also associated with aortic rupture, causing death in ~11% of animals (n = 35 RA, n = 46 CS p<0.04). CD68+ macrophages associated highly with severe elastin damage but less at intact regions (2.6-fold p<0.0001, Fig 1C). PLX3397-mediated macrophage depletion attenuated plaque development and prevented AAA (n=20 treated, n=27 control p<0.05, Fig 1D). Conclusion: CS exacerbates atherosclerosis and increases the accumulation of macrophages at sites of arterial injury. Depletion of macrophages results in a lack of aneurysm formation, highlighting their capacity to directly injure the aortic wall and necessity to mediate AAA.

Neuro-inflammatory interactions as a mediator of the Exaggerated Exercise Pressor Reflex (EPR) in a mouse model of Peripheral Artery Disease

Introduction and rationale: The Exercise pressor reflex (EPR) is a physiological mechanism in which the contraction of skeletal muscle results in a rise in heart rate (HR) and mean arterial pressure (MAP). In patients with Peripheral Artery Disease (PAD), this reflex is exaggerated, leading to abnormally high elevations in blood pressure during exercise. These cardiovascular responses in patients with PAD have been linked to increased morbidity and mortality, thus limiting the prescription of exercise for these patients. Using a novel murine model of the EPR, our laboratory has demonstrated that mice with femoral artery ligation (a well-established model of PAD), exhibit augmented increases in MAP which is mediated by PIEZO2 positive, mechanosensitive afferent neurons. We have demonstrated that inhibition of PIEZO2 expressing channels normalizes the MAP response to exercise. Patients with PAD also display increased macrophage infiltration in their ischemia-injured limbs, indicating a pro-inflammatory environment. Sensory neurons have been reported to detect various signals produced by immune cells including IL-1β and TNFα. The immune-derived signals can sensitize the peripheral terminals of the sensory neurons, thereby reducing their firing threshold and increasing their responsiveness. Previous studies have also reported the enhancement of Piezo2-mediated mechanosensitive currents in the presence of inflammatory signals. Hypothesis: We, therefore, hypothesize that in mice with femoral artery ligation, muscle inflammation increases the expression and activation of resident ion channels (e.g., Piezo2) in mechanosensitive afferent neurons to mediate the exaggerated EPR in PAD.Study objective: To mechanistically investigate the interaction between muscle inflammation and Piezo2 in mechanosensitive afferent neurons.Methods: We performed sham surgery or ligation of the femoral artery ipsilaterally in C57BL/6 mice. After 72 hours, we harvested the gastrocnemius muscle for histological and molecular analyses.Data and summary and results: Our preliminary findings reveal that there is significantly increased expression of pro-inflammatory genes and infiltrating immune cells in the ischemic muscle when compared to non-ischemic muscle.Specifically, H&E staining revealed the presence and accumulation of inflammatory cells, including macrophages, in the ischemic muscle compared to control, along with observed necrosis. Transcript analysis of the ischemic gastrocnemius muscle showed significantly increased IL-1β and TNFα, two pan-proinflammatory genes that encode cytokines released by macrophages. TNFα is known to induce the synthesis of adhesion molecules (E-selectin and ICAM-1) on endothelial cells, allowing leukocytes to adhere and cross the endothelial wall into the injured tissue. Indeed, we observed significantly increased ICAM-1 and E-selectin transcripts in the ischemic muscle compared to controls. MCP1, a well-established chemokine, was also significantly increased, further emphasizing the recruitment of leukocytes to the ischemic muscle. In addition, inducible NOS ( iNOS), which is known to be expressed in IL-1β- and TNFα-rich proinflammatory environment was also significantly increased in our FAL model compared to Sham. These data establish that the ischemic muscle of a femoral-artery ligated mouse has a high proinflammatory phenotype. We are currently exploring the impact of proinflammatory mediators on Piezo2 channel expression and activity, as well as the effect of macrophage depletion on Piezo2 in afferent neurons. The data from these studies will provide novel insights regarding the influence of neuroimmune interactions on cardiovascular responses to exercise in PAD. These data hold the potential to reveal innovative therapies enabling safe prescription of exercise, thus alleviating the burden of PAD, and improving the quality of life for those affected by this condition. Internal sources. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal macrophages induce hypertension and kidney fibrosis in Angiotensin II salt mice model

The immune system is involved in hypertension development with different immune cells reported to have either pro or anti–hypertensive effects. In hypertension, immune cells have been thought to infiltrate blood pressure-regulating organs, resulting in either elevation or reduction of blood pressure. There is controversy over whether macrophages play a detrimental or beneficial role in the development of hypertension, and the few existing studies have yielded conflicting results. This study aimed to determine the effects of angiotensin II (Ang II) salt-induced hypertension on renal immune cells and to determine whether renal macrophages are involved in the induction of hypertension. Hypertension was induced by administration of Ang II and saline for two weeks. The effects of hypertension on kidney immune cells were assessed using flow cytometry. Macrophage infiltration in the kidney was assessed by immunohistochemistry and kidney fibrosis was assessed using trichrome stain and kidney real time-qPCR. Liposome encapsulated clodronate was used to deplete macrophages in C57BL/6J mice and investigate the direct role of macrophages in hypertension induction. Ang II saline mice group developed hypertension, had increased renal macrophages, and had increased expression of Acta2 and Col1a1 and kidney fibrotic areas. Macrophage depletion blunted hypertension development and reduced the expression of Acta2 and Col1a1 in the kidney and kidney fibrotic areas in Ang II saline group. The results of this study demonstrate that macrophages infiltrate the kidneys and increase kidney fibrosis in Ang II salt-induced hypertension, and depletion of macrophages suppresses the development of hypertension and decreases kidney fibrosis. This indicates that macrophages play a direct role in hypertension development. Hence macrophages have a potential to be considered as therapeutic target in hypertension management.

Lipopolysaccharide modification enhances the inhibitory effect of clodronate liposomes on hepatic fibrosis by depletion of macrophages and hepatic stellate cells

Hepatic fibrosis is a complex chronic liver disease in which both macrophages and hepatic stellate cells (HSCs) play important roles. Many studies have shown that clodronate liposomes (CLD-lipos) effectively deplete macrophages. However, no liposomes have been developed that target both HSCs and macrophages. This study aimed to evaluate the therapeutic efficacy of lipopolysaccharide-coupled clodronate liposomes (LPS-CLD-lipos) and the effects of liposomes size on hepatic fibrosis. Three rat models of hepatic fibrosis were established in vivo; diethylnitrosamine (DEN), bile duct ligation (BDL), and carbon tetrachloride (CCl4). Hematoxylin and eosin staining and serological liver function indices were used to analyze pathological liver damage. Masson's trichrome and Sirius red staining were used to evaluate the effect of liposomes on liver collagen fibers. The hydroxyproline content in liver tissues was determined. In vitro cell counting kit-8 (CCK-8) and immunofluorescence assays were used to further explore the effects of LPS modification and liposomes size on the killing of macrophages and HSCs. Both in vitro and in vivo experiments showed that 200 nm LPS-CLD-lipos significantly inhibited hepatic fibrosis and the abnormal deposition of collagen fibers in the liver and improved the related indicators of liver function. Further results showed that 200 nm LPS-CLD-lipos increased the clearance of macrophages and induced apoptosis of hepatic stellate cells, significantly. The present study demonstrated that 200 nm LPS-CLD-lipos could significantly inhibit hepatic fibrosis and improve liver function-related indices and this study may provide novel ideas and directions for hepatic fibrosis treatment.

Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and chemotherapy still serves as the cornerstone treatment functioning by inducing cytotoxic cell death. Notably, emerging evidence suggests that dying cell-released signals may induce cancer progression and metastasis by modulating the surrounding microenvironment. However, the underlying molecular mechanisms and targeting strategies are yet to be explored.MethodsApoptotic TNBC cells induced by paclitaxel or adriamycin treatment were sorted and their released extracellular vesicles (EV-dead) were isolated from the cell supernatants. Chemokine array analysis was conducted to identify the crucial molecules in EV-dead. Zebrafish and mouse xenograft models were used to investigate the effect of EV-dead on TNBC progression in vivo.ResultsIt was demonstrated that EV-dead were phagocytized by macrophages and induced TNBC metastasis by promoting the infiltration of immunosuppressive PD-L1+ TAMs. Chemokine array identified CXCL1 as a crucial component in EV-dead to activate TAM/PD-L1 signaling. CXCL1 knockdown in EV-dead or macrophage depletion significantly inhibited EV-dead-induced TNBC growth and metastasis. Mechanistic investigations revealed that CXCL1EV-dead enhanced TAM/PD-L1 signaling by transcriptionally activating EED-mediated PD-L1 promoter activity. More importantly, TPCA-1 (2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide) was screened as a promising inhibitor targeting CXCL1 signals in EVs to enhance paclitaxel chemosensitivity and limit TNBC metastasis without noticeable toxicities.ConclusionsOur results highlight CXCL1EV-dead as a novel dying cell-released signal and provide TPCA-1 as a targeting candidate to improve TNBC prognosis.Graphical abstract

NLRP3 inflammasome activation triggers severe inflammatory liver injury in N, N-dimethylformamide-exposed mice

N,N-dimethylformamide (DMF) is a widely utilized chemical solvent with various industrial applications. Previous studies have indicated that the liver is the most susceptible target to DMF exposure, whereas the underlying mechanisms remain to be elucidated. This study aimed to investigate the role of NLRP3 inflammasome in DMF-induced liver injury in mice by using two NLRP3 inflammasome inhibitors, Nlrp3−/− mice, Nfe2l2−/− mice, and a macrophage-depleting agent. RNA sequencing revealed that endoplasmic reticulum (ER) stress and NLRP3 inflammasome-associated pathways were activated in the mouse liver after acute DMF exposure, which was validated by Western blotting. Interestingly, DMF-induced liver injury was effectively suppressed by two inflammasome inhibitors, MCC950 and Dapansutrile. In addition, knockout of Nlrp3 markedly attenuated DMF-induced liver injury without affecting the metabolism of DMF. Furthermore, silencing Nfe2l2 aggravated the liver injury and the NLRP3 inflammasome activation in mouse liver. Finally, the depletion of hepatic macrophages by clodronate liposomes significantly reduced the liver damage caused by DMF. These results suggest that NLRP3 inflammasome activation is the upstream molecular event in the development of acute liver injury induced by DMF.

Modulation of alveolar macrophage and mitochondrial fitness by medicinal plant-derived nanovesicles to mitigate acute lung injury and viral pneumonia

Acute lung injury (ALI) is generally caused by severe respiratory infection and characterized by overexuberant inflammatory responses and inefficient pathogens-containing, the two major processes wherein alveolar macrophages (AMs) play a central role. Dysfunctional mitochondria have been linked with distorted macrophages and hence lung disorders, but few treatments are currently available to correct these defects. Plant-derive nanovesicles have gained significant attention because of their therapeutic potential, but the targeting cells and the underlying mechanism remain elusive. We herein prepared the nanovesicles from Artemisia annua, a well-known medicinal plant with multiple attributes involving anti-inflammatory, anti-infection, and metabolism-regulating properties. By applying three mice models of acute lung injury caused by bacterial endotoxin, influenza A virus (IAV) and SARS-CoV-2 pseudovirus respectively, we showed that Artemisia-derived nanovesicles (ADNVs) substantially alleviated lung immunopathology and raised the survival rate of challenged mice. Macrophage depletion and adoptive transfer studies confirmed the requirement of AMs for ADNVs effects. We identified that gamma-aminobutyric acid (GABA) enclosed in the vesicles is a major molecular effector mediating the regulatory roles of ADNVs. Specifically, GABA acts on macrophages through GABA receptors, promoting mitochondrial gene programming and bioenergy generation, reducing oxidative stress and inflammatory signals, thereby enhancing the adaptability of AMs to inflammation resolution. Collectively, this study identifies a promising nanotherapeutics for alleviating lung pathology, and elucidates a mechanism whereby the canonical neurotransmitter modifies AMs and mitochondria to resume tissue homeostasis, which may have broader implications for treating critical pulmonary diseases such as COVID-19.

Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2

Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

The crosstalk between macrophages and cancer cells potentiates pancreatic cancer cachexia

With limited treatment options, cachexia remains a major challenge for patients with cancer. Characterizing the interplay between tumor cells and the immune microenvironment may help identify potential therapeutic targets for cancer cachexia. Herein, we investigate the critical role of macrophages in potentiating pancreatic cancer induced muscle wasting via promoting TWEAK (TNF-like weak inducer of apoptosis) secretion from the tumor. Specifically, depletion of macrophages reverses muscle degradation induced by tumor cells. Macrophages induce non-autonomous secretion of TWEAK through CCL5/TRAF6/NF-κB pathway. TWEAK promotes muscle atrophy by activating MuRF1 initiated muscle remodeling. Notably, tumor cells recruit and reprogram macrophages via the CCL2/CCR2 axis and disrupting the interplay between macrophages and tumor cells attenuates muscle wasting. Collectively, this study identifies a feedforward loop between pancreatic cancer cells and macrophages, underlying the non-autonomous activation of TWEAK secretion from tumor cells thereby providing promising therapeutic targets for pancreatic cancer cachexia.

Endoplasmic Reticulum Stress Activates Hepatic Macrophages through PERK-hnRNPA1 Signaling.

Endoplasmic reticulum (ER) stress plays a crucial role in liver diseases, affecting various types of hepatic cells. While studies have focused on the link between ER stress and hepatocytes as well as hepatic stellate cells (HSCs), the precise involvement of hepatic macrophages in ER stress-induced liver injury remains poorly understood. Here, we examined the effects of ER stress on hepatic macrophages and their role in liver injury. Acute ER stress led to the accumulation and activation of hepatic macrophages, which preceded hepatocyte apoptosis. Notably, macrophage depletion mitigated liver injury induced by ER stress, underscoring their detrimental role. Mechanistic studies revealed that ER stress stimulates macrophages predominantly via the PERK signaling pathway, regardless of its canonical substrate ATF4. hnRNPA1 has been identified as a crucial mediator of PERK-driven macrophage activation, as the overexpression of hnRNPA1 effectively reduced ER stress and suppressed pro-inflammatory activation. We observed that hnRNPA1 interacts with mRNAs that encode UPR-related proteins, indicating its role in the regulation of ER stress response in macrophages. These findings illuminate the cell type-specific responses to ER stress and the significance of hepatic macrophages in ER stress-induced liver injury. Collectively, the PERK-hnRNPA1 axis has been discovered as a molecular mechanism for macrophage activation, presenting prospective therapeutic targets for inflammatory hepatic diseases such as acute liver injury.

Abstract LB077: CSF-1R inhibition with Pimicotinib (ABSK021) enhanced anti-tumor efficacy of KRASG12C inhibitors in preclinical non-small cell lung cancer mouse models

Abstract Background: KRAS mutations are highly prevalent oncogenic drivers in human cancers and KRASG12C is the most frequent mutations found in KRAS-mutant non-small lung cancer (NSCLC). It is reported that tumor associated macrophages (TAMs) are enriched in KRAS-driven transgenic lung cancer models. Depletion of macrophage results in a significant decrease in tumor burden and increased survival. Sotorasib (AMG510), the first KRASG12C inhibitor approved by FDA, has been found to modulate tumor immunity, impacting the recruitment of macrophages. CSF-1R signaling is critical for macrophage proliferation and survival. CSF-1R inhibition depletes TAMs and reprograms tumor microenvironment (TME), resulting in enhancement of anti-tumor immunity. Therefore, the combination of KRASG12C and CSF-1R inhibitors may synergize to enhance anti-tumor efficacy. Pimicotinib (ABSK021) is a potential best-in-class small molecule inhibitor of CSF-1R in clinical development of multiple indications. Here, we conducted in vivo experiments to explore the combined effects of KRASG12C and CSF-1R inhibitors. Methods: In vivo efficacy studies were performed to evaluate the combined treatment of KRASG12C inhibitors (AMG510 and ABSK071, a next-generation inhibitor targeting KRASG12C with better inhibitory activity) and CSF-1R inhibitor (ABSK021) in NCI-H2122 PBMC humanized mice and LLC syngeneic mice. RNA sequencing was employed to investigate the mechanism of action underlying the synergistic anti-tumor effect. Immunohistochemistry (IHC) was utilized to examine the changes of immune cell populations in TME. Results: The combinatory regimen of ABSK021 and KRASG12C inhibitors showed improved anti-tumor effects than each single agent alone in both mouse NSCLC models. Bioinformatics and follow-up analysis revealed a reduction in immune suppressive TAMs in tumors treated with ABSK021 alone or in combination with KRASG12C inhibitors. Furthermore, modulation of TME related to anti-tumor immunity, such as activated CD4 T, CD8 T and NK cells, was increased, and pro-tumor immunity, such as MDSC, regulatory T cells, was decreased in combination group. IHC results confirmed that ABSK021 addition led to macrophage depletion and more CD8 T cell infiltration in TME. Conclusions: For the first time, we demonstrate that the combined inhibition of KRASG12C and CSF-1R leads to superior therapeutic efficacy in preclinical NSCLC mouse models. This results suggests a potential novel therapeutic regimen that could yield improved clinical benefit to patients. Citation Format: Nannan Zhang, Bin Shen, Shenyan Liu, Cheng Dai, Haiyan Ying, Fushen Guo, Zhixuan Zhu, Wan Shi, Min Tu, Jie Wang, Jie Zhang, Manqi Liu, Qi Zhang, Zhui Chen. CSF-1R inhibition with Pimicotinib (ABSK021) enhanced anti-tumor efficacy of KRASG12C inhibitors in preclinical non-small cell lung cancer mouse models [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 LB077.

Destruction of vascular endothelial glycocalyx during formation of pre-metastatic niches

A special microenvironment called the “pre-metastatic niche” is thought to help primary tumor cells migrate to new tissues and invade them, in part because the normal barrier function of the vascular endothelium is compromised. While the primary tumor itself can promote the creation of such niches by secreting pro-metastatic factors, the underlying molecular mechanisms are still poorly understood. Here, we show that the injection of primary tumor-secreted pro-metastatic factors from B16F10 melanoma or 4T1 breast cancer cells into healthy mice can induce the destruction of the vascular endothelial glycocalyx, which is a polysaccharide coating on the vascular endothelial lumen that normally inhibits tumor cell passage into and out of the circulation. However, when human umbilical vein endothelial cultures were treated in vitro with these secreted pro-metastatic factors, no significant destruction of the glycocalyx was observed, implying that this destruction requires a complex in vivo microenvironment. The tissue section analysis revealed that secreted pro-metastatic factors could clearly upregulate macrophage-related molecules such as CD11b and tumor necrosis factor-α (TNF-α) in the heart, liver, spleen, lung, and kidney, which is associated with the upregulation and activation of heparanase. In addition, macrophage depletion significantly attenuated the degradation of the vascular endothelial glycocalyx induced by secreted pro-metastatic factors. This indicates that the secreted pro-metastatic factors that destroy the vascular endothelial glycocalyx rely primarily on macrophages. Our findings suggest that the formation of pre-metastatic niches involves degradation of the vascular endothelial glycocalyx, which may hence be a useful target for developing therapies to inhibit cancer metastasis.

Immunomodulation in allergic rhinitis: Insights from Th2 cells and NLRP3/IL-18pathway.

Allergic rhinitis (AR) is characterized by nasal symptoms such as rubbing and sneezing, often triggered by allergen exposure. The purpose of this study is to dissect the roles of NLRP3-mediated immune modulation and macrophage pyroptosis in modulating T cell differentiation within the context of ovalbumin (OVA)-induced AR in mice. OVA-induced AR was established in mice, evaluating nasal symptoms, macrophage infiltration, cytokine levels, and T cell differentiation. Manipulations using NLRP3-/-, ASC-/- mice, clodronate liposome treatment, and NLRP3 inhibitor MCC950 were performed to assess their impact on AR symptoms and immune responses. Following OVA stimulation, increased nasal symptoms were observed in the OVA group along with augmented GATA3 expression and elevated IL-4 and IL-1b levels, indicative of Th2 polarization and cellular pyroptosis involvement. NLRP3-/- and ASC-/- mice exhibited reduced CD3+ T cells post OVA induction, implicating cellular pyroptosis in AR. Macrophage depletion led to decreased IgE levels, highlighting their involvement in allergic responses. Further investigations revealed enhanced macrophage pyroptosis, influencing Th1/Th2 differentiation in AR models. IL-18 released through NLRP3-mediated pyroptosis induced Th2 differentiation, distinct from IL-1b. Additionally, MCC950 effectively mitigated AR symptoms by modulating Th2 responses and reducing macrophage infiltration. This comprehensive study unravels the pivotal role of NLRP3-mediated immune modulation and macrophage pyroptosis in Th1/Th2 balance regulation in OVA-induced AR. Targeting NLRP3 pathways with MCC950 emerged as a promising strategy to alleviate AR symptoms, providing insights for potential therapeutic interventions in AR management.

IL6/adiponectin/HMGB1 feedback loop mediates adipocyte and macrophage crosstalk and M2 polarization after myocardial infarction.

Differences in border zone contribute to different outcomes post-infarction, such as left ventricular aneurysm (LVA) and myocardial infarction (MI). LVA usually forms within 24h of the onset of MI and may cause heart rupture; however, LVA surgery is best performed 3 months after MI. Few studies have investigated the LVA model, the differences in border zones between LVA and MI, and the mechanism in the border zone. The LVA, MI, and SHAM mouse models were used. Echocardiography, Masson's trichrome staining, and immunofluorescence staining were performed, and RNA sequencing of the border zone was conducted. The adipocyte-conditioned medium-treated hypoxic macrophage cell line and LVA and MI mouse models were employed to determine the effects of the hub gene, adiponectin (ADPN), on macrophages. Quantitative polymerase chain reaction (qPCR), Western blot analysis, transmission electron microscopy, and chromatin immunoprecipitation (ChIP) assays were conducted to elucidate the mechanism in the border zone. Human subepicardial adipose tissue and blood samples were collected to validate the effects of ADPN. A novel, simple, consistent, and low-cost LVA mouse model was constructed. LVA caused a greater reduction in contractile functions than MI owing to reduced wall thickness and edema in the border zone. ADPN impeded cardiac edema and promoted lymphangiogenesis by increasing macrophage infiltration post-infarction. Adipocyte-derived ADPN promoted M2 polarization and sustained mitochondrial quality via the ADPN/AdipoR2/HMGB1 axis. Mechanistically, ADPN impeded macrophage HMGB1 inflammation and decreased interleukin-6 (IL6) and HMGB1 secretion. The secretion of IL6 and HMGB1 increased ADPN expression via STAT3 and the co-transcription factor, YAP, in adipocytes. Based on ChIP and Dual-Glo luciferase experiments, STAT3 promoted ADPN transcription by binding to its promoter in adipocytes. In vivo, ADPN promoted lymphangiogenesis and decreased myocardial injury after MI. These phenotypes were rescued by macrophage depletion or HMGB1 knockdown in macrophages. Supplying adipocytes overexpressing STAT3 decreased collagen disposition, increased lymphangiogenesis, and impaired myocardial injury. However, these effects were rescued after HMGB1 knockdown in macrophages. Overall, the IL6/ADPN/HMGB1 axis was validated using human subepicardial tissue and blood samples. This axis could serve as an independent factor in overweight MI patients who need coronary artery bypass grafting (CABG) treatment. The IL6/ADPN/HMGB1 loop between adipocytes and macrophages in the border zone contributes to different clinical outcomes post-infarction. Thus, targeting the IL6/ADPN/HMGB1 loop may be a novel therapeutic approach for cardiac lymphatic regulation and reduction of cell senescence post-infarction.

Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit.

Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-p-STAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-p-STAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

Abstract 4909: An RNA-based model for tertiary lymphoid structure (TLS) prediction and classification in pancreatic adenocarcinoma (PDAC)

Abstract Although TLS status possesses prognostic significance in PDAC and can potentially affect chemotherapy outcomes, there is currently a notable lack of RNA sequencing (RNA-seq) models that specialize in TLS identification and classification in PDAC. Here, we developed a model for predicting TLS status (high or low) based on RNA-seq data. Design: Hematoxylin and eosin (H&E) whole slide images of PDAC samples from The Cancer Genome Atlas (TCGA, n = 118) and Clinical Proteomic Tumor Analysis Consortium (CPTAC, n = 129) were used to detect intratumoral and borderline TLSs followed by TLS density measurements (units/mm2) by an experienced pathologist. The samples were then stratified into TLS-high and TLS-low groups based on median density values. Next, we used deconvolution by Kassandra algorithm to identify cell subtypes enriched in each TLS group based on gene expression (RNA-seq) data. Calculation of ssGSEA scores for gene signatures corresponding to cell subtypes and TLS structures was performed, along with survival analysis. Differential expression analysis between TLS-high and TLS-low samples, followed by functional enrichment (|logFC| > 2; padj < 0.01), was conducted. The LightGBM gradient boosting classifier was then trained on ranked expression data with sequential feature selection to predict TLS-high and TLS-low groups. We trained the model with H&E staining annotations and ranked RNA expression data from TCGA or CPTAC samples (total n = 167). The remaining 80 were designated as hold-out samples. The weighted F1 score was computed as a performance metric. Findings: Median density of detected TLSs in the TCGA and CPTAC samples was 0.012 units/mm2. Kassandra deconvolution revealed B-cell enrichment, but fibroblast and M2 macrophage depletion in the TLS-high group. Our calculated ssGSEA scores of previously described TLS gene signatures, along with those of different B-cell subtypes and follicular dendritic cells, showed significant association with the TLS-high group. Genes associated with B-cell proliferation, differentiation, and signaling (CD19, CD22, CD79A, CD79B, and CR2) were also upregulated in this group. Comparing the performance of our RNA-based model on the validation dataset with manual TLS classification by a pathologist, we obtained an F1 weighted score of 0.72 and ROC-AUC score of 0.77. Thus, the TLS predictions by our model concurred with the TLS classification based on H&E annotations and pathological evaluation. Moreover, patients in the predicted TLS-low group had worse overall survival (OS) compared to the TLS-high group (Log(HR) = 0.76; 95% CI [0.04; 1.48]; p<0.05). We present an RNA-based model that stratifies PDAC samples as TLS-high or TLS-low, with predictions that conform to pathological findings. We also found TLS-low samples to associate with worse OS, thus offering an objective means to predict prognoses of PDAC patients based on TLS status. Citation Format: Alexandra Livanova, Andrey Tyshevich, Andrey Kravets, Stanislav Kurpe, Nadezhda Lukashevich, Dmitry Ivchenkov, Daniil Dymov, Anna Belozerova, Kirill Kryukov, Aleksandr Sarachakov, Viktor Svekolkin, Vladimir Kushnarev. An RNA-based model for tertiary lymphoid structure (TLS) prediction and classification in pancreatic adenocarcinoma (PDAC) [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 4909.

ICOS costimulation in combination with CTLA-4 blockade remodels tumor-associated macrophages toward an antitumor phenotype.

We have previously demonstrated synergy between ICOS costimulation (IVAX; ICOSL-transduced B16-F10 cellular vaccine) and CTLA-4 blockade in antitumor therapy. In this study, we employed CyTOF and single-cell RNA sequencing and observed significant remodeling of the lymphoid and myeloid compartments in combination therapy. Compared with anti-CTLA-4 monotherapy, the combination therapy enriched Th1 CD4 T cells, effector CD8 T cells, and M1-like antitumor proinflammatory macrophages. These macrophages were critical to the therapeutic efficacy of anti-CTLA-4 combined with IVAX or anti-PD-1. Macrophage depletion with clodronate reduced the tumor-infiltrating effector CD4 and CD8 T cells, impairing their antitumor functions. Furthermore, the recruitment and polarization of M1-like macrophages required IFN-γ. Therefore, in this study, we show that there is a positive feedback loop between intratumoral effector T cells and tumor-associated macrophages (TAMs), in which the IFN-γ produced by the T cells polarizes the TAMs into M1-like phenotype, and the TAMs, in turn, reshape the tumor microenvironment to facilitate T cell infiltration, immune function, and tumor rejection.

Abstract 3959: Unraveling the anti-tumor partnership of CD169+ macrophages and CD8+ T cells in a preclinical breast cancer model

Abstract In this study, we describe both the individual and the cooperative mechanisms by which CD169+ macrophages (MP) and CD8+ T cells support an anti-tumor immune response against breast cancer tumors. Breast cancer is the second leading cause of death in women of all ages each year, thus a deeper understanding of the molecular determinants that promote tumor development and suppress the anti-tumor immune response is still greatly needed. Here we use high dimensional flow cytometry, functional assays, and new mouse models to investigate the MP - T cell anti-tumor partnership. With our collaborators, we developed a preclinical mouse model of breast cancer by genetically engineering mammary stem cells to harbor three cancer driver mutations that are frequently associated in human patients: functional loss of the tumor suppressor genes p53 and mixed-lineage leukemia protein 3, and constitutive activation of the PI3 kinase. To characterize the immune cells in this tumor model, we developed a powerful 30-color high dimensional spectral flow cytometry panel to track and phenotype most known immune cell subsets. We found that CD169+ MP rapidly infiltrated and outnumbered all other populations during tumor growth, suggesting that they play an important role in shaping the tumor microenvironment. Surprisingly, although current literature describes breast cancer MP to be highly immunosuppressive, our phenotypic analysis showed only low expression of the pro-tumor MP markers TREM2 and CD206, but extremely high expression of the anti-tumor markers FOLR2 and CD64. To further investigate if these MP promoted or prevented tumor growth, we selectively depleted CD169+ MP in tumor-bearing mice and observed significantly accelerated tumor growth compared to the non-depleted control group, suggesting that these MP promoted anti-tumor responses. MP depletion also affected tumor-infiltrating CD8+ T cells, which produced lower effector functions (e.g., secretion of IFNγ, CCL3, TNF), indicating that MP may also boost anti-tumor CD8+ T cell responses. To further test if CD8+ T cells control tumor growth, we depleted either CD8+ or CD4+ T cells in tumor-bearing mice and observed that only CD8+ but not CD4+ T cell depletion accelerated tumor growth compared to the control group. MP have been documented to provide type I IFN signals to CD8+ T cells via the triggering of the STING pathway. To explore this mechanism in our model, we created a CD169creSTINGflox/KO mouse in which MP lack functional STING. Tumors in these mice grew much faster compared to those in the control group, consistent with the hypothesis that STING sensing occurs in tumor MP and promotes anti-tumor effector CD8+ T cell responses. Overall, our data provide evidence for an unexpected anti-tumor role for CD169+ MP in breast tumors, presumably through enhancing anti-tumor effector CD8+ T cell responses. Citation Format: Nicole Couturier, Marie Boutet, Kenta Nishitani, Wenjun Guo, Gregoire Lauvau. Unraveling the anti-tumor partnership of CD169+ macrophages and CD8+ T cells in a preclinical breast cancer model [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 3959.