Mouse Monocytes Research Articles

Colostrum-derived extracellular vesicles: potential multifunctional nanomedicine for alleviating mastitis

Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation.Graphical

The aryl hydrocarbon receptor shapes monocyte transcriptional responses to interleukin-4 by prolonging STAT6 binding to promoters.

Cytokines induce functional and metabolic adaptations in immune cells, typically through transcriptional responses that can be influenced by other extracellular signals and by intracellular factors. The binding of the cytokine interleukin-4 (IL-4) to its receptor induces the phosphorylation and activation of the transcription factor STAT6. The aryl hydrocarbon receptor (AhR), a transcription factor activated by various endogenous and microbe-derived metabolites, modulates the responses of immune cells to danger signals or inflammatory mediators such as cytokines. Here, we investigated cross-talk between the AhR and signaling stimulated by IL-4 in human and mouse monocytes. AhR activation was required for a subset of IL-4-induced transcriptional responses and inhibited the IL-4-induced metabolic switch to fatty acid β-oxidation. The promoters of the genes that were induced by IL-4 in an AhR-dependent manner lacked canonical AhR binding sites, implying a nongenomic mechanism of AhR action. Mechanistically, AhR activation reduced the activity of SHP-1, a phosphatase that targets and inhibits STAT6, and prolonged STAT6 phosphorylation and binding to specific target loci, thus extending the duration of STAT6 activity. Our results identify AhR as a key player in the molecular control of responses to IL-4 in monocytes and suggest a nongenomic mechanism through which AhR ligands may influence the functional responses of cells to IL-4.

Monocyte-driven inflamm-aging reduces intestinal barrier function in females.

The intestinal barrier encompasses physical and immunological components that act to compartmentalize luminal contents, such as bacteria and endotoxins, from the host. It has been proposed that an age-related decline of intestinal barrier function may allow for the passage of luminal contents into the bloodstream, triggering a low-grade systemic inflammation termed inflamm-aging. Although there is mounting evidence to support this hypothesis in model species, it is unclear if this phenomenon occurs in humans. In addition, despite being well-established that biological sex impacts aging physiology, its influence on intestinal barrier function and inflamm-aging has not been explored. In this study, we observed sex differences in markers of intestinal barrier integrity, where females had increased epithelial permeability throughout life as compared to males. With age, females had an age-associated increase in circulating bacterial products and metabolites such as LPS and kynurenine, suggesting reduced barrier function. Females also had age-associated increases in established markers of inflamm-aging, including peripheral blood monocytes as well as TNF and CRP. To determine if impaired barrier function was driving inflamm-aging, we performed a mediation analysis. The results show that the loss of intestinal barrier integrity was not the mediator of inflamm-aging in humans. Instead, persistent, low-grade inflammation with age preceded the increase in circulating bacterial products, which we confirmed using animal models. We found, as in humans, that sex modified age-associated increases in circulating monocytes in mice, and that inflammation mediates the loss of intestinal barrier function. Taken together, our results suggest that higher basal intestinal permeability in combination with age-associated inflammation, increases circulating LPS in females. Thus, targeting barrier permeability in females may slow the progression of inflamm-aging, but is unlikely to prevent it.

GRN Activates TNFR2 to Promote Macrophage M2 Polarization Aggravating Mycobacterium Tuberculosis Infection.

The polarization of macrophages plays a critical role in the immune response to infectious diseases, with M2 polarization shown to be particularly important in various pathological processes. However, the specific mechanisms of M2 macrophage polarization in Mycobacterium tuberculosis (Mtb) infection remain unclear. In particular, the roles of Granulin (GRN) and tumor necrosis factor receptor 2 (TNFR2) in the M2 polarization process have not been thoroughly studied. To investigate the effect of macrophage M2 polarization on Mtb infection and the mechanism of GRN and TNFR2 in M2 polarization. Forty patients with pulmonary tuberculosis (PTB) and 40 healthy volunteers were enrolled in this study, and peripheral blood samples were taken to detect the levels of TNFR2 and GRN mRNA by Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR); monocytes were isolated and then assessed by Flow Cytometry (FC) for M1 and M2 macrophage levels. To further validate the function of TNFR2 in macrophage polarization, we used interleukin 4 (IL-4) to induce mouse monocyte macrophages RAW264.7 to M2 polarized state. The expression of TNFR2 was detected by Western Blot and RT-qPCR. Next, we constructed a GRN knockdown plasmid and transfected it into IL-4-induced mouse monocyte macrophage RAW264.7, and detected the expression of TNFR2, M1 macrophage-associated factors tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL-6), and the M2 macrophage-associated factors CD206, IL-10, and Arginase 1 (Arg1); Immunofluorescence staining was used to monitor the expression of CD86+ and CD206+, and FC was used to analyze the macrophage phenotype. Subsequently, immunoprecipitation was used to detect the binding role of GRN and TNFR2. Finally, the effects of GRN and TNFR2 in macrophage polarization were further explored by knocking down GRN and simultaneously overexpressing TNFR2 and observing the macrophage polarization status. The results of the study showed elevated expression of TNFR2 and GRN and predominance of M2 type in macrophages in PTB patients compared to healthy volunteers (p < 0.05). Moreover, TNFR2 was highly expressed in M2 macrophages (p < 0.05). Additionally, GRN knockdown was followed by elevated expression of M1 polarization markers TNF-α, iNOS and IL-6 (p < 0.05), decreased levels of M2 polarization-associated factors CD206, IL-10 and Arg1 (p < 0.05), and macrophage polarization towards M1. Subsequently, we found that GRN binds to TNFR2 and that GRN upregulates TNFR2 expression (p < 0.05). In addition, knockdown of GRN elevated M1 polarization marker expression, decreased M2 polarization marker expression, and increased M1 macrophages and decreased M2 macrophages, whereas concurrent overexpression of TNFR2 decreased M1 polarization marker expression, elevated M2 polarization marker expression, and decreased M1 macrophages and increased M2 macrophages. TNFR2 and GRN are highly expressed in PTB patients and GRN promotes macrophage M2 polarization by upregulating TNFR2 expression.

Oncolytic Tanapoxvirus Variants Expressing mIL-2 and mCCL-2 Regress Human Pancreatic Cancer Xenografts in Nude Mice.

Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death and presents the lowest 5-year survival rate of any form of cancer in the US. Only 20% of PDAC patients are suitable for surgical resection and adjuvant chemotherapy, which remains the only curative treatment. Chemotherapeutic and gene therapy treatments are associated with adverse effects and lack specificity/efficacy. In this study, we assess the oncolytic potential of immuno-oncolytic tanapoxvirus (TPV) recombinants expressing mouse monocyte chemoattractant protein (mMCP-1 or mCCL2) and mouse interleukin (mIL)-2 in human pancreatic BxPc-3 cells using immunocompromised and CD-3+ T-cell-reconstituted mice. Intratumoral treatment with TPV/∆66R/mCCL2 and TPV/∆66R/mIL-2 resulted in a regression in BxPc-3 xenograft volume compared to control in immunocompromised mice; mCCL-2 expressing TPV OV resulted in a significant difference from control at p < 0.05. Histological analysis of immunocompromised mice treated with TPV/∆66R/mCCL2 or TPV/∆66R/mIL-2 demonstrated multiple biomarkers indicative of increased severity of chronic, active inflammation compared to controls. In conclusion, TPV recombinants expressing mCCL2 and mIL-2 demonstrated a therapeutic effect via regression in BxPc-3 tumor xenografts. Considering the enhanced oncolytic potency of TPV recombinants demonstrated against PDAC in this study, further investigation as an alternative or combination treatment option for human PDAC may be warranted.

Discovery of quaternized pyridine-thiazole-ruthenium complexes as potent anti-Staphylococcus aureus agents

Quaternization of ruthenium complexes may be a promising strategy for the development of new antibiotics. In response to the increasing bacterial resistance, we integrated the quaternary amine structure into the design of ruthenium complexes and evaluated their antibacterial activity. All the ruthenium complexes showed good antibacterial activity against the tested Staphylococcus aureus (S. aureus). Ru-8 was the most effective antibacterial agent that displayed excellent antibacterial activity against S. aureus (MIC = 0.78–1.56 μg/mL). In vitro experiments showed that all nine ruthenium complexes had low hemolytic toxicity to rabbit erythrocytes. Notably, Ru-8 was found to disrupt bacterial cell membranes, alter their permeability, and induce ROS production in bacteria, all the above leading to the death of bacteria without inducing drug resistance. To further explore the antibacterial activity of Ru-8in vivo, we established a mouse skin wound infection model and a G. mellonella larvae infection model. Ru-8 exhibited significant antibacterial efficacy against S. aureus in vivo and low toxicity to mouse tissues. The Ru-8 showed low toxicity to Raw264.7 cells (mouse monocyte macrophage leukemia cells). This study indicates that the ruthenium complex ruthenium quaternary was a promising strategy for the development of new antibacterial agents.

Uncovering the connection between Ly6ChiCD103+ myeloid cells and radiation-induced cardiac fibrosis by CyTOF

Several immune cell populations participate in the development of radiation-induced cardiac fibrosis. However, the underlying mechanism remains to be elucidated. Male C57BL/6 mice (8–12 weeks old) were anesthetized and exposed to a single cardiac radiation dose of 20 Gy using the small-animal radiation research platform. We conducted echocardiography and histological analysis to identify cardiac fibrosis from a functional and pathological perspective. Time-of-flight mass cytometry (CyTOF) was used to describe the compositional changes of immune cells 1, 4, and 8 weeks after cardiac irradiation in mouse peripheral blood and cardiac tissue samples. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure CD14 and CD105 mRNA levels from peripheral blood cells in eight patients who received thoracic radiotherapy 6 months ago. We observed reduced cardiac systolic function and increased collagen deposition in cardiac tissue after irradiation. Transforming growth factor-β, tumor necrosis factor-α, and interleukin-6 expression significantly elevated in the plasma. We identified a significant increase in CD45+ immune cell levels four weeks after cardiac irradiation using CyTOF, and neutrophil, monocyte, and macrophage levels elevated in blood samples after cardiac irradiation (four weeks). In cardiac tissue samples, monocyte and macrophage levels increased after cardiac irradiation (four weeks) compared with control group. Macrophages and monocytes were further analyzed, and the proportion of Ly6ChiCD103+ myeloid cells increased after cardiac irradiation in tissue samples (four weeks). RT-qPCR revealed that CD14 expression was higher in patients with cardiac injured group than in the control group (Previous studies reported that Ly6Chi monocytes in mice are similar to CD14 + CD16− monocytes in humans). We found that Ly6ChiCD103+ myeloid cells are critical subsets in radiation-induced cardiac fibrosis and might play protective roles in the process. Ly6ChiCD103+ myeloid cells may be considered an early biomarker for detecting radiation-induced cardiac fibrosis.

The regulatory effect of TiO2 nanotubes loaded with graphene oxide on macrophage polarization in an inflammatory environment

BackgroundExcessive inflammation is a major cause of implant failure. The surface morphology, hydrophilicity, and loading of biomaterials are major properties modulating anti-inflammatory macrophage activation. This paper investigates the regulatory effects of modifying the surface of Titanium dioxide nanotubes (TNTs) with graphene oxide (GO) on the polarization of mouse monocyte macrophages (RAW264.7).MethodsTNT was produced by the anodic oxidation of titanium. GO was subsequently electrodeposited on the TNT to obtain a TNT–GO composite. The samples were characterised through scanning electron microscopy (SEM), Raman spectroscopy, and X-ray diffraction. RAW264.7 cells were separately seeded onto the surface of three groups of samples: pure Ti, TNT, and TNT–GO. Under the condition of lipopolysaccharide stimulation, the influence of the sample surfaces on the gene expression profiles was investigated through RNA sequence analysis. In addition, cell spreading was observed through SEM, cell adhesion and proliferation were analysed using the CCK8 assay, and the expression of inflammation-related factors was investigated by ELISA and cellular immunofluorescence staining. The production of reactive oxygen species (ROS) in the RAW264.7 cells on the surface of the three groups was detected via immunofluorescence staining.ResultsThe CCK8 results indicated that the adhesion and proliferation of the RAW264.7 cells were reduced on the TNT and TNT–GO surfaces. ELISA results revealed significant differences in the pro-inflammatory factors tumour necrosis factor-α and interleukin-6 secretion among the three groups at 24 h (p < 0.05). The secretion of pro-inflammatory factors significantly reduced and the expression of anti-inflammatory factor IL-10 increased on the TNT and TNT–GO surfaces. The RNA sequencing, ELISA, and cell immunofluorescence staining test results suggested that the inflammatory response of M1 polarization was reduced and the M2 polarization of macrophages was induced on the TNT–GO surface, which may be attributed to the reduction in ROS production.ConclusionsUnder lipopolysaccharide stimulation, the inflammatory response of the RAW264.7 cells was reduced and the M2 polarization of macrophages was promoted on the TNT–GO surface, which may be caused by the reduced ROS production. Consequently, the designed TNT–GO material is promising for implants owing to its excellent inflammation regulation ability.

Changes in hematological parameters when using silver preparations in laboratory ICR mice infected with the Newcastle disease virus

The results of blood tests are important indicators for assessing and monitoring the health status, adaptation processes and metabolic states of the body. Currently, there is insufficient data on the effect of the Newcastle disease virus on peripheral blood parameters in animals, and there is also no information on changes in these parameters during Newcastle disease and the simultaneous use of silver preparations in animals. Although the mechanisms of the antiviral action of silver nanoparticles are not yet fully studied, researches show their effectiveness. The purpose of research was to study changes in hematological parameters with using silver preparations in laboratory ICR mice infected with Newcastle disease virus. The object of the study was the blood of laboratory ICR mice, infected with the Newcastle disease virus, and healthy ones as a control, and also silver preparations: Argosil and ArgovitMaxi. As a result of hematological studies, a significant effect of the Newcastle disease virus on hematological parameters was established. Deviations of the main blood parameters in infected animals were demonstrated: there was an increase in the content of granulocytes, the number of erythrocytes, the hemoglobin content and hematocrit; the number of lymphocytes and the average erythrocytes volume were reduced. It was also found that the use of the preparation Argosil significantly reduces the content of monocytes in ICR mice against the background of Newcastle disease viral infection. The use of the preparation Argovit significantly affects the decrease in monocytes and erythrocyte blood parameters: it increases the number of erythrocytes, hemoglobin, hematocrit, and reduces the average hemoglobin content in an erythrocyte, the average hemoglobin concentration in an erythrocyte.

The role of inflammation in takotsubo syndrome: A new therapeutic target?

Takotsubo syndrome (TTS) is a particular form of acute heart failure that can be challenging to distinguish from acute coronary syndrome at presentation. TTS was previously considered a benign self-limiting condition, but it is now known to be associated with substantial short- and long-term morbidity and mortality. Because of the poor understanding of its underlying pathophysiology, there are few evidence-based interventions to treat TTS. The hypotheses formulated so far can be grouped into endogenous adrenergic surge, psychological stress or preexisting psychiatric illness, coronary vasospasm with microvascular dysfunction, metabolic and energetic alterations, and inflammatory mechanisms. Current evidence demonstrates that the infiltration of immune cells such as macrophages and neutrophils play a pivotal role in TTS. At baseline, resident macrophages were the dominant subset in cardiac macrophages, however, it underwent a shift from resident macrophages to monocyte-derived infiltrating macrophages in TTS. Depletion of macrophages and monocytes in mice strongly protected them from isoprenaline-induced cardiac dysfunction. It is probable that immune cells, especially macrophages, may be new targets for the treatment of TTS.

Low to moderate dose 137Cs (γ) radiation promotes M2 type macrophage skewing and reduces atherosclerotic plaque CD68+ cell content in ApoE(−/−) mice

The effects of low doses of ionizing radiation on atherosclerosis remain uncertain, particularly as regards the generation of pro- or anti-inflammatory responses, and the time scale at which such effects can occur following irradiation. To explore these phenomena, we exposed atheroprone ApoE(−/−) mice to a single dose of 0, 0.05, 0.5 or 1 Gy of 137Cs (γ) administered at a 10.35 mGy min−1 dose rate and evaluated short-term (1–10 days) and long-term consequences (100 days). Bone marrow-derived macrophages were derived from mice 1 day after exposure. Irradiation was associated with a significant skewing of M0 and M2 polarized macrophages towards the M2 phenotype, as demonstrated by an increased mRNA expression of Retnla, Arg1, and Chil3 in cells from mice exposed to 0.5 or 1 Gy compared with non-irradiated animals. Minimal effects were noted in M1 cells or M1 marker mRNA. Concurrently, we observed a reduced secretion of IL-1β but enhanced IL-10 release from M0 and M2 macrophages. Effects of irradiation on circulating monocytes were most marked at day 10 post-exposure, when the 1 Gy dose was associated with enhanced numbers of both Ly6CHigh and Ly6Low cells. By day 100, levels of circulating monocytes in irradiated and non-irradiated mice were equivalent, but anti-inflammatory Ly6CLow monocytes were significantly increased in the spleen of mice exposed to 0.05 or 1 Gy. Long term exposures did not affect atherosclerotic plaque size or lipid content, as determined by Oil red O staining, whatever the dose applied. Similarly, irradiation did not affect atherosclerotic plaque collagen or smooth muscle cell content. However, we found that lesion CD68+ cell content tended to decrease with rising doses of radioactivity exposure, culminating in a significant reduction of plaque macrophage content at 1 Gy. Taken together, our results show that short- and long-term exposures to low to moderate doses of ionizing radiation drive an anti-inflammatory response, skewing bone marrow-derived macrophages towards an IL-10-secreting M2 phenotype and decreasing plaque macrophage content. These results suggest a low-grade athero-protective effect of low and moderate doses of ionizing radiation.

Intermittent fasting alleviates IMQ-induced psoriasis-like dermatitis via reduced γδT17 and monocytes in mice.

Psoriasis is a chronic immune mediated inflammatory skin disease with systemic manifestations. It has been reported that caloric restriction could improve severity of psoriasis patients. However, the mechanism of intermittent fasting effects on psoriasis has not been investigated. Caloric restriction is known to reduce the number of circulating inflammatory monocytes in a CCL2-dependent manner. However, it is still unknown whether caloric restriction can improve psoriasis by regulating monocytes through CCL2. In this study, we used imiquimod (IMQ)-induced psoriasis-like mouse model to explore the effects and the mechanisms of intermittent fasting on psoriasis-like dermatitis. We found that intermittent fasting could significantly improve IMQ-induced psoriasis-like dermatitis, and reduce the number of γδT17 cells and IL-17 production in draining lymph nodes and psoriatic lesion via inhibiting proliferation and increasing death of γδT17 cells. Furthermore, intermittent fasting could significantly decrease monocytes in blood, and this was associated with decreased monocytes, macrophages and DC in psoriasis-like skin inflammation. Reduced monocytes in circulation and increased monocytes in BM of fasting IMQ-induced psoriasis-like mice is through reducing the production of CCL2 from BM to inhibit monocyte egress to the periphery. Our above data shads light on the mechanisms of intermittent fasting on psoriasis.

Discrepant Phenotyping of Monocytes Based on CX3CR1 and CCR2 Using Fluorescent Reporters and Antibodies.

Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.

Exaggerated Kawasaki disease-associated vasculitis in C-C Motif Chemokine Receptor 5 (CCR5) deficient mice is driven by Granulocyte Colony Stimulating Factor (G-CSF) overproduction and neutrophil formation and infiltration

Kawasaki disease (KD) is a rare but serious illness that primarily affects children. It is characterized by inflammation of the blood vessels throughout the body, including the coronary arteries (CA). Polymorphisms in CCR5 influence KD susceptibility and cardiovascular outcome, but the mechanisms are enigmatic yet. Thus, we aimed to explore the mechanisms by which polymorphisms in CCR5 aggravate the vascular injury in KD by using the Candida albicans water soluble (CAWS) fraction model. Male (6-10-weeks-old) CCR5 deficient mice (CCR5−/−) and their counterpart (CCR5+/+) were used. Cardiac single-cell transcriptomics (scRNAseq) analysis of both control and KD mice revealed an inflammatory landscape characterized by an abundance of macrophages, monocytes, and neutrophils. Additionally, our scRNAseq data indicated that CCR5 expression is predominantly observed in macrophages and monocytes in KD mice, whereas its ligands, CCL3 and CCL4, are primarily expressed in neutrophils. Our CellChat analysis uncovered robust intercommunication between neutrophils and macrophages and monocytes. This interaction involves the production of CCL3 and CCL4 by neutrophils, which act on CCR5 receptors in macrophages and monocytes. To validate this scRNAseq data, we analyzed the vasculitis levels in CA and immune cells content in CCR5+/+ and CCR5−/− mice with KD via histology and flow cytometry. CCR5−/− mice with KD exhibited aggravated vasculitis, increased neutrophil accumulation [(% in relation to 10^6 leukocytes) CCR5+/+: 2.5±1.7 n=4 vs. CCR5+/+ KD: 16.1±3.2 n=4], and CCR5−/− KD: 30.2±7.4 n=4), alongside cardiac senescence (3-fold rise in p21 expression) and a 2-fold increase in Caspase-3, indicating increased cell death compared to CCR5+/+ with KD. Mechanistically, our plasma proteome profile cytokine assay revealed that KD increases circulating IL-6 and circulating neutrophil, interestingly CCR5−/− mice with KD showed further circulating IL-6 and neutrophil along with G-CSF, which plays a critical role in stimulating the production and release of granulocytes, particularly neutrophils, from the bone marrow into the bloodstream. Thus, deficiency in CCR5 triggers a worse vasculitis in KD by regulating an over production of neutrophil by bone marrow via G-CSF, which in turn will lead to an exaggerated IL-6 production, further accumulation in CA, and aggravated cardiac damage. FAPESP (2022/06639-2), NHLBI-R00 (R00HL14013903), AHA (CDA857268 and 23DIVSUP1069230), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. 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.

Abstract 5536: The role of p38 MAPK in the tumor-induced immune suppressive microenvironment in metastatic breast cancer

Abstract The tumor microenvironment (TME) in Metastatic Breast Cancer (MBC) is a major factor contributing to therapy resistance and suppression of antitumor immune response. Tumors promote expansion and recruitment of immune suppressive cells such as myeloid-derived suppressor cells (MDSCs) contributing to tumor invasion and suppressing anti-tumor T cells. Our prior work suggested a critical role of p38 MAPK in tumor-induced expansion and mobilization of myeloid cell populations thereby facilitating metastasis. The current study examined the role of p38 MAPK in tumor-induced changes in immune landscape and explored the mechanisms by which p38 mediates tumor-immune interactions. First, the contribution of T cells to anti-metastatic activity of p38 inhibitor (p38i) was addressed by depletion of CD8 T cells. Depletion of CD8+ T cells negated the effects of p38i on tumor growth and metastasis in the syngeneic 4T1 model. Next, we examined whether p38i exhibits a direct effect on T cells in vitro or in vivo. The in vitro assays showed that p38 blockade increased levels of CD44+ CD62L+ CD8+ T cells indicating enhancement of T cell differentiation. To determine the effects of p38 blockade on the immune landscape in vivo, single-cell RNA-seq and flow cytometry studies were performed in the 4T1 model with p38i or p38-deficient cells in which p38α/Mapk14 was inactivated by CRISPR/Cas9 (p38KO). The scRNA-seq data showed that p38 blockade increased the expression of T cell differentiation markers in the TME. Furthermore, p38 blockade significant decreased IL-6 signaling in myeloid cells, an important mediator of MDSC function. Assessment of myeloid cell populations showed a decreased expression of the immune suppressive signature. This observation was further validated in MDSCs isolated from the spleens of 4T1 tumor-bearing mice treated with p38i and in p38KO cohorts. Our prior work showed that p38i does not affect the generation of MDSCs in vitro. Therefore, we examined whether blockade of p38 affects the chemotactic capacity of conditioned media from MBC cells using mouse monocytes. The chemotaxis assays showed that p38i and p38KO largely reduced the ability of conditioned media to stimulate transwell migration of mouse monocytes. Notably, p38i and p38KO largely reduced expression of chemotactic cytokines in MBC cell models. Furthermore, p38i and p38KO reduced production and chemotactic ability of exosomes isolated from tumor cells. Together, these observations suggest that tumor p38 MAPK signaling promotes immune-suppressive TME and metastasis by facilitating expansion and mobilization of myeloid cell populations through the mechanisms involving production of exosomes and pro-myeloid chemokines/cytokines. This work highlights that p38 blockade can be utilized in combination with immune therapy or chemotherapy to enhance clinical benefits of immune therapy and reduce promyeloid effects of chemotherapy. Citation Format: Priyanka Rajan, Robert Zollo, Mackenzie Lieberman, Yanqi Guo, Mohammed Alruwaili, Mohammed Alqarni, Brian Morreale, Scott Olejniczak, Joseph Barbi, Scott Abrams, Andrei Bakin. The role of p38 MAPK in the tumor-induced immune suppressive microenvironment in metastatic breast 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 5536.

Synthesis and biological evaluation of novel benzothiazole derivatives as potential anticancer and antiinflammatory agents.

Introduction: Cancer, a significant global health concern, necessitates innovative treatments. The pivotal role of chronic inflammation in cancer development underscores the urgency for novel therapeutic strategies. Benzothiazole derivatives exhibit promise due to their distinctive structures and broad spectrum of biological effects. This study aims to explore new anti-tumor small molecule drugs that simultaneously anti-inflammatory and anticancer based on the advantages of benzothiazole frameworks. Methods: The compounds were characterized by nuclear magnetic resonance (NMR), liquid chromatograph-mass spectrometer (LC-MS) and high performance liquid chromatography (HPLC) for structure as well as purity and other related physicochemical properties. The effects of the compounds on the proliferation of human epidermoid carcinoma cell line (A431) and human non-small cell lung cancer cell lines (A549, H1299) were evaluated by MTT method. The effect of compounds on the expression levels of inflammatory factors IL-6 and TNF-α in mouse monocyte macrophages (RAW264.7) was assessed using enzyme-linked immunosorbent assay (ELISA). The effect of compounds on apoptosis and cell cycle of A431 and A549 cells was evaluated by flow cytometry. The effect of compounds on A431 and A549 cell migration was evaluated by scratch wound healing assay. The effect of compounds on protein expression levels in A431 and A549 cells was assessed by Western Blot assay. The physicochemical parameters, pharmacokinetic properties, toxicity and drug similarity of the active compound were predicted using Swiss ADME and admetSAR web servers. Results: Twenty-five novel benzothiazole compounds were designed and synthesized, with their structures confirmed through spectrogram verification. The active compound 6-chloro-N-(4-nitrobenzyl) benzo[d] thiazol-2-amine (compound B7) was screened through a series of bioactivity assessments, which significantly inhibited the proliferation of A431, A549 and H1299 cancer cells, decreased the activity of IL-6 and TNF-α, and hindered cell migration. In addition, at concentrations of 1, 2, and 4μM, B7 exhibited apoptosis-promoting and cell cycle-arresting effects similar to those of the lead compound 7-chloro-N-(2, 6-dichlorophenyl) benzo[d] thiazole-2-amine (compound 4i). Western blot analysis confirmed that B7 inhibited both AKT and ERK signaling pathways in A431 and A549 cells. The prediction results of ADMET indicated that B7 had good drug properties. Discussion: This study has innovatively developed a series of benzothiazole derivatives, with a focus on compound B7 due to its notable dual anticancer and anti-inflammatory activities. B7 stands out for its ability to significantly reduce cancer cell proliferation in A431, A549, and H1299 cell lines and lower the levels of inflammatory cytokines IL-6 and TNF-α. These results position B7B7 as a promising candidate for dual-action cancer therapy. The study's mechanistic exploration, highlighting B7's simultaneous inhibition of the AKT and ERK pathways, offers a novel strategy for addressing both the survival mechanisms of tumor cells and the inflammatory milieu facilitating cancer progression.

Biomimetic nanoparticles for effective Celastrol delivery to targeted treatment of rheumatoid arthritis through the ROS-NF-κB inflammasome axis

Previous study has indicated that Celastrol (Cel) has various physiological and pharmacological effects, including antibacterial, antioxidant, pro-apoptotic, anticancer and anti-rheumatoid arthritis (RA) effects. However, low water solubility, low oral bioavailability, narrow treatment window, and high incidence of systemic adverse reactions still limit the further clinical application of Cel. Here, aiming at effectively overcome those shortcomings of Cel to boost its beneficial effects for treating RA, we developed the leukosome (LEUKO) coated biomimetic nanoparticles (NPs) for the targeted delivery of Cel to arthritis injury area in RA. LEUKO were synthesized using membrane proteins purified from activated J774 macrophage. LEUKO and Cel-loaded LEUKO (Cel@LEUKO) were characterized using dynamic light scattering and transmission electron microscopy. Our results demonstrated that Cel@LEUKO can inhibit the inflammatory response of lipopolysaccharide (LPS) induced mouse monocyte macrophage leukemia cells (RAW264.7 cells) and human rheumatoid arthritis synovial fibroblasts (MH7A) cells through the inhibition of reactive oxygen species (ROS)-NF-κB pathway. In addition, research has shown that LEUKO effectively targets and transports Cel to the inflammatory site of RA, increased drug concentration in affected areas, reduced systemic toxicity of Cel, and reduced clinical symptoms, inflammatory infiltration, bone erosion, and serum inflammatory factors in collagen-induced arthritis (CIA) rats.

Type III secretion system effector YfiD inhibits the activation of host poly(ADP-ribose) polymerase-1 to promote bacterial infection

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida’s proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1’s activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection.

Myeloid and lymphoid expression of C9orf72 regulates IL-17A signaling in mice.

A mutation in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Patients with ALS or FTD often develop autoimmunity and inflammation that precedes or coincides with the onset of neurological symptoms, but the underlying mechanisms are poorly understood. Here, we knocked out murine C9orf72 in seven hematopoietic progenitor compartments by conditional mutagenesis and found that myeloid lineage C9orf72 prevents splenomegaly, loss of tolerance, and premature mortality. Furthermore, we demonstrated that C9orf72 plays a role in lymphoid cells to prevent interleukin-17A (IL-17A) production and neutrophilia. Mass cytometry identified early and sustained elevation of the costimulatory molecule CD80 expressed on C9orf72-deficient mouse macrophages, monocytes, and microglia. Enrichment of CD80 was similarly observed in human spinal cord microglia from patients with C9ORF72-mediated ALS compared with non-ALS controls. Single-cell RNA sequencing of murine spinal cord, brain cortex, and spleen demonstrated coordinated induction of gene modules related to antigen processing and presentation and antiviral immunity in C9orf72-deficient endothelial cells, microglia, and macrophages. Mechanistically, C9ORF72 repressed the trafficking of CD80 to the cell surface in response to Toll-like receptor agonists, interferon-γ, and IL-17A. Deletion of Il17a in C9orf72-deficient mice prevented CD80 enrichment in the spinal cord, reduced neutrophilia, and reduced gut T helper type 17 cells. Last, systemic delivery of an IL-17A neutralizing antibody augmented motor performance and suppressed neuroinflammation in C9orf72-deficient mice. Altogether, we show that C9orf72 orchestrates myeloid costimulatory potency and provide support for IL-17A as a therapeutic target for neuroinflammation associated with ALS or FTD.

Target Cell Activation of a Structurally Novel NOD-Like Receptor Pyrin Domain-Containing Protein 3 Inhibitor NT-0796 Enhances Potency.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1β and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.