Murine Monocytes Research Articles

Regulation of Bacillus Calmette-Guérin-induced macrophage autophagy and apoptosis by the AMPK–mTOR–ULK1 pathway

Tuberculosis (TB) is a chronic wasting infectious disease caused by Mycobacterium tuberculosis (MTB) or Mycobacterium bovis that can be transmitted among people and domestic animals. During the development ofTB, macrophages of the innate immune system can act against MTB via autophagy and apoptosis to prevent the spread of the disease. Among the many autophagy regulatory pathways, the adenosine monophosphate (AMP)–activated protein kinase (AMPK)–mammalian rapamycin target protein(mTOR)–Unc-51-like kinase 1(ULK1) pathway has received considerable attention. This study investigates the regulatory role of the AMPK–mTOR–ULK1 pathway in attenuating M. bovis Bacillus Calmette–Guérin (BCG)-induced autophagy and apoptosis in murine monocyte macrophages (RAW264.7). Changes in macrophage autophagy and apoptosis were analyzed using the AMPK activator AICAR and inhibitor Compound C to interfere with the AMPK–mTOR–ULK1 pathway and siRNA to silence the pathway. Consequently, BCG stimulation of macrophages significantly activated the AMPK–mTOR–ULK1 pathway while BCG-induced macrophage AMPK activation promoted macrophage autophagy and apoptosis. Activation of the AMPK–mTOR–ULK1 pathway by AICAR significantly improved autophagy occurrence in BCG-induced macrophages and increased apoptosis while Compound C with siRNA produced opposing effects by attenuating autophagy and apoptosis in BCG-induced macrophages. Thus, the AMPK–mTOR–ULK1 pathway has a dual regulatory role in BCG-induced macrophage autophagy and apoptosis and may have synergistic effects. This study analyzes the mechanism of resistance of host cells to MTB and provides a theoretical basis for new therapeutic strategies and related drug development.

Determining the Feasibility of a Cadmium Exposure Model to Activate the Inflammatory Arm of PANoptosis in Murine Monocytes.

A prevalence of cigarette smoking can cause the accumulation of cadmium (Cd2+) in the lungs, kidneys, and blood. The effects of exposure can cause multiple chronic disease types to emerge in the affected organ systems. The only moderately effective therapeutic option is chelation therapy; the health risks associated with this therapy have caused much criticism. The disease types associated with Cd2+ toxicity have inflammatory components and greatly impact innate immunity. These factors are affected at the cellular level and cause pathways like apoptosis, pyroptosis, and necroptosis. A development in understanding these pathways stipulates that these three pathways act as one complex of pathways, known together as PANoptosis. The inflammatory mechanisms of PANoptosis are particularly interesting in Cd2+ toxicity due to its inflammatory effects. Proteins in the gasdermin family act to release inflammatory cytokines, like interleukin-1β, into the extracellular environment. Cytokines cause inflammatory disease pathologies like fibrosis and cancer. RAW 264.7 monocytes are key in the murine immune system and provide an excellent model to investigate Cd2+ toxicity. Exposure of 0-15 µM CdCl2 was sufficient to increase expression of cleaved gasdermin D (GSDMD) and gasdermin E (GSDME) in this cell type. Cd2+ also exhibits a dose-dependent cytotoxicity in this cell type.

Optimizing Biocompatibility and Gene Delivery with DMAEA and DMAEAm: A Niacin-Derived Copolymer Approach.

Gene therapy is pivotal in nanomedicine, offering a versatile approach to disease treatment. This study aims to achieve an optimal balance between biocompatibility and efficacy, which is a common challenge in the field. A copolymer library is synthesized, incorporating niacin-derived monomers 2-acrylamidoethyl nicotinate (AAEN) or 2-(acryloyloxy)ethyl nicotinate (AEN) with N,N-(dimethylamino)ethyl acrylamide (DMAEAm) or hydrolysis-labile N,N-(dimethylamino)ethyl acrylate (DMAEA). Evaluation of the polymers' cytotoxicity profiles reveals that an increase in AAEN or DMAEA molar ratios correlates with improved biocompatibility. Remarkably, an increase in AAEN in both DMAEA and DMAEAm copolymers demonstrated enhanced transfection efficiencies of plasmid DNA in HEK293T cells. Additionally, the top-performing polymers demonstrate promising gene expression in challenging-to-transfect cells (THP-1 and Jurkat cells) and show no significant effect on modulating immune response induction in ex vivo treated murine monocytes. Overall, the best performing candidates exhibit an optimal balance between biocompatibility and efficacy, showcasing potential advancements in gene therapy.

In vitro and in vivo studies on exogenous polyamines and α-difluoromethylornithine to enhance bone formation and suppress osteoclast differentiation

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.

CCR1 and CCR2 Coexpression on Monocytes Is Nonredundant and Delineates a Distinct Monocyte Subpopulation.

The interactions between chemokines and their receptors, particularly in the context of inflammation, are complex, with individual receptors binding multiple ligands and individual ligands interacting with multiple receptors. In addition, there are numerous reports of simultaneous coexpression of multiple inflammatory chemokine receptors on individual inflammatory leukocyte subtypes. Overall, this has previously been interpreted as redundancy and proposed as a protective mechanism to ensure that the inflammatory response is robust. By contrast, we have hypothesized that the system is not redundant but exquisitely subtle. Our interests relate to the receptors CCR1, CCR2, CCR3, and CCR5, which, together, regulate nonneutrophilic myeloid cell recruitment to inflammatory sites. In this study, we demonstrate that although most murine monocytes exclusively express CCR2, there is a small subpopulation that is expanded during inflammation and coexpresses CCR1 and CCR2. Combinations of transcript and functional analysis demonstrate that this is not redundant expression and that coexpression of CCR1 and CCR2 marks a phenotypically distinct population of monocytes characterized by expression of genes otherwise typically associated with neutrophils. Single-cell RNA sequencing confirms this as a monodisperse population of atypical monocytes. This monocytic population has previously been described as having immunosuppressive activity. Overall, our data confirm combinatorial chemokine receptor expression by a subpopulation of monocytes but demonstrate that this is not redundant expression and marks a discrete monocytic population.

Ly6Chi Monocytes Are Metabolically Reprogrammed in the Blood during Inflammatory Stimulation and Require Intact OxPhos for Chemotaxis and Monocyte to Macrophage Differentiation.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b+Ly6G-Ly6Chi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

Abstract 1036: Mechanisms Of Foamy Monocyte Formation In Atherosclerosis

Diseases linked to atherosclerosis are the leading cause of death in the United States. Transmigration of monocytes across the endothelial layer and their differentiation into macrophages play an important role in the initiation and progression of atherosclerosis. These monocyte-derived macrophages internalize modified LDL in the arterial wall and drive the pathogenesis of atherosclerosis. Recent studies have shown that pharmacological depletion of foamy monocytes inhibits atherosclerosis development in mice, identifying monocyte uptake of plasma LDL as a therapeutic target in atherosclerosis. However, the precise mechanisms by which monocytes internalize plasma LDL remain unknown. The goal of our study was to investigate the role, and relative contribution, of macropinocytosis and scavenger receptors ( Cd36 and Sr-a ) to foamy monocyte formation. The presence of foamy monocytes was confirmed in hypercholesterolemic mice and humans via flow cytometry analysis of Nile Red fluorescence. High resolution scanning electron microscopy and flow cytometry analysis of dextran internalization confirmed macropinocytosis stimulation in THP-1 and primary murine monocytes. Stimulation of macropinocytosis in monocytes induced uptake of both native LDL and oxLDL, leading to foamy monocyte formation in vitro . Pharmacological (EIPA) and genetic inhibition of macropinocytosis ( Lysm - CreER T2+/- Nhe1 -/- ) inhibited foamy monocyte formation (58.9%) in hypercholesterolemic mice in vivo . RT-PCR confirmed expression of Cd36 and Sr-a in monocytes and deletion of Cd36 and Sr-a inhibited foamy monocyte formation in hypercholesterolemic mice (10.3%) compared to wild type controls. FACS and bulk RNA sequencing characterized monocyte subsets including classical, intermediate, and non-classical monocytes in hypercholesterolemic Cd36 -/- / Sr-a -/- and Lysm - CreER T2+/- Nhe1 -/- mice and their respective wild type controls. Mechanistic studies identified NADPH oxidase 2 (Nox2) and downstream redox regulation of actin-cytoskeleton as a major stimulator of monocyte macropinocytosis. These results provide novel insights into the mechanisms of foamy monocyte formation and potentially identify new therapeutic targets in the treatment of atherosclerosis.

A Robust In Vitro Culture Model and Generation of Memory Monocytes.

Innate monocytes can be trained or reprogrammed to adopt distinct memory states, such as low-grade inflammation and immune exhaustion, bearing fundamental relevance to the pathogenesis of both acute diseases such as sepsis as well as chronic diseases such as atherosclerosis. Therefore, it is critically important to develop a regimen for generating memory monocytes in vitro in order to better define key monocyte memory states with diverse potentials for proliferation, differentiation, and activation, as well as underlying mechanisms. Here, we describe an efficient in vitro system to propagate a large number of highly purified murine memory monocytes through sustaining bone marrow-derived monocytes with macrophage colony-stimulating factor (M-CSF, 10ng/mL)-containing medium, together with other polarization agents such as lipopolysaccharide (LPS) for a 5-day period. This method can yield high-purity monocytes, capable of exhibiting dynamic memory behaviors upon training with various polarizing agents.

Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

Exploring biological activity and In-vitro anticancer effects of a new biomaterial derived from Schiff base isolated from Homarus americanus (Lobster) shell waste

In this study, chitin, a valuable biopolymer material, was extracted from lobster (Homarus americanus) shell waste using a chemical method. Afterward, the biopolymer was added to a mixture of the precursor (β-diketone and acidhydrazide) and monochloroacetic acid to prepare the biomaterial/hydrazone-based O-carboxymethyl chitosan Schiff base compounds. The prepared biomaterials were studied for potential biological applications, such as antimicrobial, antioxidant, wound healing, cytotoxicity, phosphate inhibition, and anticancer studies. The inhibition of alkaline phosphatase (ALP) enzyme was recorded using UV–Vis spectrophotometry. Wound healing studies of biopolymer materials and their derivatives were studied to RAW 264.7 (murine monocyte / macrophage) cells by scratch wound healing analysis. Additionally, using MTT analysis the in-vitro, anticancer efficacies (cell viability/cell proliferation/cytotoxicity) were conducted for SK-MEL-28 (human melanoma cells), MDA-MB-231 (human breast cancer cells), and the U87 (human glioma brain cells). The half maximal inhibitory concentration (IC50) values for SK-MEL-28 cells had 0.71 μg/mL at 24 h, and was a potent activity as compared to U-87 (35.98 μg/mL) and MDA-MB-231 (18.31 μg/mL) cells. The water-soluble / swelling nature of prepared biomaterials is utilized in the bio-ink formulation by the incorporation of the biopolymers such as cellulose, sodium alginate, gelatin, hyaluronic acid, and laponite RDS for potential biomedical applications.

Phosphoprotein phosphatase activity positively regulates oligomeric pyrin to trigger inflammasome assembly in phagocytes.

Pyrin, a unique cytosolic receptor, initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors like Yersinia's YopE and YopT. Understanding pyrin regulation is crucial due to its association with dysregulated inflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. FMF mutations historically acted as a defense mechanism against plague. Negative regulation of pyrin through PKN phosphorylation is well established, with Yersinia using the YopM effector to promote pyrin phosphorylation and counteract its activity. This study highlights the importance of phosphoprotein phosphatase activity in positively regulating pyrin inflammasome assembly in phagocytic cells of humans and mice. Oligomeric murine pyrin has S205 phosphorylated before inflammasome assembly, and this study implicates the dephosphorylation of murine pyrin S205 by two catalytic subunits of PP2A in macrophages. These findings offer insights for investigating the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity in pyrin-associated infectious and autoinflammatory diseases.

Aikeqing, a kidney- and spleen-tonifying compound Chinese medicine granule, prevented ovariectomy-induced bone loss in rats via the suppression of osteoclastogenesis

Postmenopausal women are prone to osteoporosis due to increased osteoclast activation and bone resorption caused by oestrogen deficiency. In Traditional Chinese Medicine theory, medicines with spleen- and kidney-nourishing effects are commonly used in postmenopausal osteoporosis (PMOP) treatment. Aikeqing (AKQ) is a compound Chinese medicinal granule with spleen- and kidney-nourishing effects. Herein, we investigate the in vitro and in vivo anti-osteoporotic effects of AKQ, its underlying mechanisms and pharmacodynamic basis. In vitro antiosteoporotic effects of AKQ were assessed by its ability to promote osteoblastogenesis in MC3T3-E1 and/or inhibit RANKL-induced osteoclastogenesis in murine bone marrow monocytes (BMMs). The protective effect of AKQ on bone loss induced by oestrogen deficiency was evaluated in ovariectomized rats. The underlying mechanisms were studied in BMMs by detecting the effects of AKQ on the RANKL-induced expression of genes and proteins involved in the regulation of osteoclastogenesis. The main chemical constituents of AKQ in the granule were analyzed by UPLC-QTOF-MS. Our findings show that AKQ did not affect osteoblastogenesis, but it inhibited RANKL-induced osteoclastogenesis. In the ovariectomized rats, oral administration of AKQ (4 g/kg/d) for 90 d effectively prevented oestrogen deficiency-induced bone loss. Mechanistic studies in BMMs revealed that AKQ inhibited RNAKL-induced activation of NF-κB (p65) and MAPKs (p38 and JNK) via blocking the RANK-TRAF6 interaction, subsequently suppressing the translocation and expression of NFATc1 and c-Fos. UPLC-QTOF-MS analysis quantified the 123 main components of AKQ. Taken together, AKQ was demonstrated for the first time as a novel alternative therapy for osteoclast-associated bone diseases.

Comparative analyses of monocyte memory dynamics from mice to humans.

Innate monocytes can adopt dynamic "memory" states ranging from low-grade inflammation to pathogenic exhaustion, dependent upon signal strength and history of challenges. Low-grade inflammatory monocytes facilitate the pathogenesis of chronic inflammatory diseases, while exhausted monocytes drive the pathogenesis of severe sepsis. Although clinical and basic studies suggest the conservation of key features of exhausted monocytes from human and murine sepsis, systems analyses of monocyte exhaustion among human and murine monocytes are lacking. We performed cross examination of septic monocytes scRNAseq data recently collected from human sepsis patients as well as experimental septic mice, in reference to monocytes experimentally exhausted in vitro. Furthermore, we performed pseudo-time analyses of in vitro programmed monocytes following prolonged challenges causing either low-grade inflammation or exhaustion. Additional comparative analyses of low-grade inflammatory monocytes were performed with scRNAseq data from selected human patients with chronic low-grade inflammatory diseases. Our systems analyses reveal key features of monocyte exhaustion including reduced differentiation, pathogenic inflammation and immune suppression that are highly conserved in human and murine septic monocytes, and captured by in vitro experimental exhaustion. Pseudo-time analyses reveal that monocytes initially transition into a less-differentiated state with proliferative potential. The expansion of proliferative monocytes can be observed not only in experimentally challenged monocytes, but also in tissues of murine sepsis and human septic blood. We observed that monocytes similarly transition into the less-differentiated state when challenged with a subclinical dose endotoxin under chronic inflammatory conditions. Instead of being exhausted, monocytes with prolonged challenges with super-low dose endotoxin bifurcate into the low-grade inflammatory immune-enhancing or the chemotactic/adhesive state, often see in atherosclerosis or auto-immune diseases. Key features of monocyte memory dynamics are identified and conserved in human and murine monocytes, which can be captured by prolonged challenges of innate signals with varying signal strength.

BCG component mycolic acid protects against monocyte exhaustion in vitro

Abstract Monocyte exhaustion reflected in pathogenic inflammation and immune suppression underlies the pathogenesis of sepsis. The objective of this study is to define novel approaches in alleviating monocyte exhaustion, based on intriguing clinical reports that BCG components may be beneficial in reducing polymicrobial infection-triggered sepsis severity. We recapitulated in vitrothe development of exhausted murine monocytes through prolonged culture with high dose bacterial lipopolysaccharide (LPS), measured by the expansion of immature Ly6C hipopulation with elevated expression of pathogenic mediator CD38 and immune suppressor PD-L1, and reduced expression of CD86. Intriguingly, we observed that co-culture of murine bone marrow monocytes with Mycobacterium tuberculosis(BCG) component mycolic acid (MA) can significantly alleviate the development of exhausted monocytes. We found that monocytes co-stimulated with MA and LPS exhibited significantly reduced induction of CD38 and PD-L1 as compared to monocytes challenged with LPS alone. On the other hand, monocytes co-treated with MA and LPS exhibited enhanced expression of CD86 as compared to cells treated with LPS. In the monocyte-T cell coculture system, we found that T cells co-cultured with MA and LPS-treated monocytes had a significantly higher proliferation potential as compared to the T cells co-cultured with LPS-treated monocytes. Mechanically, we demonstrated that MA exerts its protective effect by selectively activating P-AKT and inhibiting STAT1/P-STAT1. In conclusion, we demonstrate that the BCG component mycolic acid can potently alleviate monocyte exhaustion and may serve as a future promising agent in treating sepsis. Supported by grants from NIH (AI136386)

Pharmacological Targeting of the RAGE-NFκB Signalling Axis Impedes Monocyte Activation under Diabetic Conditions through the Repression of SHP-2 Tyrosine Phosphatase Function.

Monocytes play a vital role in the development of cardiovascular diseases. Type 2 diabetes mellitus (T2DM) is a major CVD risk factor, and T2DM-induced aberrant activation and enhanced migration of monocytes is a vital pathomechanism that leads to atherogenesis. We recently reported the upregulation of SHP-2 phosphatase expression in mediating the VEGF resistance of T2DM patient-derived monocytes or methylglyoxal- (MG, a glucose metabolite and advanced glycation end product (AGE) precursor) treated monocytes. However, the exact mechanisms leading to SHP-2 upregulation in hyperglycemic monocytes are unknown. Since inflammation and accumulation of AGEs is a hallmark of T2DM, we hypothesise that inflammation and AGE-RAGE (Receptor-for-AGEs) signalling drive SHP-2 expression in monocytes and blockade of these pathways will repress SHP-2 function. Indeed, monocytes from T2DM patients revealed an elevated SHP-2 expression. Under normoglycemic conditions, the serum from T2DM patients strongly induced SHP-2 expression, indicating that the T2DM serum contains critical factors that directly regulate SHP-2 expression. Activation of pro-inflammatory TNFα signalling cascade drove SHP-2 expression in monocytes. In line with this, linear regression analysis revealed a significant positive correlation between TNFα expression and SHP-2 transcript levels in T2DM monocytes. Monocytes exposed to MG or AGE mimetic AGE-BSA, revealed an elevated SHP-2 expression and co-treatment with an NFκB inhibitor or genetic inhibition of p65 reversed it. The pharmacological inhibition of RAGE was sufficient to block MG- or AGE-BSA-induced SHP-2 expression and activity. Confirming the importance of RAGE-NFκB signalling in regulating SHP-2 expression, the elevated binding of NFκB to the SHP-2 promoter-induced by MG or AGE-BSA-was reversed by RAGE and NFκB inhibition. Besides, we detected elevated RAGE levels in human and murine T2DM monocytes and monocytes exposed to MG or AGE-BSA. Importantly, MG and AGE-BSA treatment of non-T2DM monocytes phenocopied the aberrant pro-migratory phenotype of T2DM monocytes, which was reversed entirely by either SHP-2- or RAGE inhibition. In conclusion, these findings suggest a new therapeutic approach to prevent accelerated atherosclerosis in T2DM patients since inhibiting the RAGE-NFκB-SHP-2 axis impeded the T2DM-driven, SHP-2-dependent monocyte activation.

Inflammatory Chemokine Receptors Support Inflammatory Macrophage and Dendritic Cell Maturation.

Dendritic cells form clusters in vivo, but the mechanism behind this has not been determined. In this article, we demonstrate that monocytes from mice deficient in the chemokine receptors CCR1, CCR2, CCR3, and CCR5 display reduced clustering in vitro, which is associated with impaired dendritic cell and macrophage differentiation. We further show that the differentiating cells themselves produce ligands for these receptors that function, in a redundant manner, to regulate cell clustering. Deletion of, or pharmacological blockade of, more than one of these receptors is required to impair clustering and differentiation. Our data show that chemokines and their receptors support clustering by increasing expression of, and activating, cell-surface integrins, which are associated with cell-cell interactions and, in the context of monocyte differentiation, with reduced expression of Foxp1, a known transcriptional suppressor of monocyte differentiation. Our data therefore provide a mechanism whereby chemokines and their receptors typically found in inflammatory environments can interact to promote murine monocyte differentiation to macrophages and dendritic cells.

Interleukin-4 receptor alpha signaling regulates monocyte homeostasis.

Interleukin‐4 (IL‐4) and its receptors (IL‐4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL‐4R also influences monocyte homeostasis and if steady state IL‐4 levels are sufficient to affect monocytes. Employing full IL‐4 receptor alpha knockout mice (IL‐4Rα−/−) and mice with a myeloid‐specific deletion of IL‐4Rα (IL‐4Rαf/f LysMcre), we show that IL‐4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL‐4Rα, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte‐derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti‐apoptotic factors including BIRC6 in IL‐4Rα−/− knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL‐4Rα−/− mice, whereas subcutaneously applied IL‐4 prolonged it by 75%. Treatment of human monocytes with IL‐4 reduced the amount of dying monocytes in vitro. Furthermore, IL‐4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NFκBp65 protein. In a cohort of human patients, serum IL‐4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL‐4Rαf/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL‐4Rα in regulating the lifespan of monocytes in vivo.

Calcium channel antagonists interfere with the mechanism of action of elastin-derived peptide VGVAPG in mouse cortical astrocytes in vitro

Elastin-derived peptides (EDPs) contain replications of the Val-Gly-Val-Ala-Pro-Gly (VGVAPG) hexapeptide. It has been described that the VGVAPG peptide induces reactive oxygen species (ROS) production in murine monocytes and astrocytes, human fibroblasts, and the human neuroblastoma (SH-SY5Y) cell line. To date, there is growing evidence that calcium channel blockers (CCBs) reduce oxidative stress and development of inflammation in the nervous system. Therefore, the aim of the present study was to evaluate the impact of such CCBs as Nifedipine, Verapamil, and MK-801 on the expression of peroxisome proliferator-activated receptor (Pparγ), i.e. ROS-related and inflammation-related proteins, in mouse astrocytes exposed in vitro to the VGVAPG peptide. The experiments showed that Nifedipine or MK-801 used in co-treatment with the VGVAPG peptide potentiated the effect of this peptide on the Pparγ level after the 24-h and 48-h treatment. Moreover, all studied compounds decreased the VGVAPG-induced caspase-1 activity in both time intervals. The data also showed that the VGVAPG peptide decreased the interleukin 1 beta (IL-1β) level in both studied time intervals. Upon a short-time exposure, the use of CCBs intensified the decrease in IL-1β stimulated by the VGVAPG peptide, opposite to the longer treatment. Moreover, the VGVAPG peptide decreased the IL-1βR1 level in both studied time intervals. After 24 h, Nifedipine and Verapamil potentiated the effect of the VGVAPG peptide. The VGVAPG peptide decreased the catalase (Cat) protein expression only after 24 h, whereas CCBs did not affect the expression of Cat induced by the VGVAPG peptide. The VGVAPG peptide increased the expression of the superoxide dismutase 1 (Sod1) protein. After 24 h of exposure, Nifedipine and Verapamil potentiated the increase in the Sod1 protein expression. Finally, our data showed that VGVAPG did not change the level of estradiol (E2) in the astrocytes. Interestingly, Nifedipine and Verapamil in co-treatment with VGVAPG increased the E2 level. Summarizing, it can be assumed that increased amounts of the VGVAPG during lifetime can play a certain role in calcium channel functioning in neurodegenerative diseases.

MRSA Infection in the Thigh Muscle Leads to Systemic Disease, Strong Inflammation, and Loss of Human Monocytes in Humanized Mice.

MRSA (Methicillin-resistant Staphylococcus aureus) is the second-leading cause of deaths by antibiotic-resistant bacteria globally, with more than 100,000 attributable deaths annually. Despite the high urgency to develop a vaccine to control this pathogen, all clinical trials with pre-clinically effective candidates failed so far. The recent development of “humanized” mice might help to edge the pre-clinical evaluation closer to the clinical situation and thus close this gap. We infected humanized NSG mice (huNSG: (NOD)-scid IL2Rγ null mice engrafted with human CD34+ hematopoietic stem cells) locally with S. aureus USA300 LAC* lux into the thigh muscle in order to investigate the human immune response to acute and chronic infection. These mice proved not only to be more susceptible to MRSA infection than wild-type or “murinized” mice, but displayed furthermore inferior survival and signs of systemic infection in an otherwise localized infection model. The rate of humanization correlated directly with the severity of disease and survival of the mice. Human and murine cytokine levels in blood and at the primary site of infection were strongly elevated in huNSG mice compared to all control groups. And importantly, differences in human and murine immune cell lineages surfaced during the infection, with human monocyte and B cell numbers in blood and bone marrow being significantly reduced at the later time point of infection. Murine monocytes in contrast behaved conversely by increasing cell numbers. This study demonstrates significant differences in the in vivo behavior of human and murine cells towards S. aureus infection, which might help to sharpen the translational potential of pre-clinical models for future therapeutic approaches.

Abstract 1316: Tamm-Horsfall protein as a novel antitumor therapy for triple negative breast cancer

Abstract Tamm-Horsfall Protein (THP) is expressed exclusively in the kidney and is the most abundant protein in urine. THP has a role in antibacterial host defense but also in inflammatory disorders of the urogenital tract. THP has been shown to regulate the innate and adaptive immunity in the urinary tract by potently activating macrophages and dendritic cells. Given these immunostimulatory effects, we hypothesize that systemic THP administration can promote an antitumor and antimetastatic response. THP was isolated from human urine of healthy individuals at a purity of >99% with less than < 1 UE/mg de THP of endotoxin. We treated murine J744 macrophages and human monocytes with 10 and 100 µg/ml THP for 18h and TNFα levels were determined by ELISA. In J744 macrophages TNFα levels reached 0.9 and 1.4 ng/ml, respectively and in human monocytes 4.5 and 6 ng/ml, respectively, confirming the macrophage-stimulating properties of THP. We then evaluated proliferation and migration by cell count and wound healing, respectively, in the 4T1 murine triple negative breast cancer (TNBC) model and the MDA-MB-231 human TNBC model. We cultured 4T1 and MDA-MB-231 cells for 4 days with 100 µg/ml THP and observed an inhibition in proliferation of 62% and 40%, respectively. Cell migration was evaluated under treatment with 100 µg/ml THP for 18h. Migration was inhibited by 70% in 4T1 cells and by 30% in MDA-MB-231 cells. These data highlight the antitumoral and antimetastatic effect of THP in TNBC. The in vivo experiments were performed by administering THP 3 times a week and/or docetaxel (Dx) at 15 mg/kg twice a week in BALB/c mice bearing 4T1 tumors (50-75 mm2), and tumor growth, lung metastasis and survival were determined. A dose-response curve (0.3-3 mg/kg) was performed using THP administered either s.c. or i.p. THP s.c. at 1.5 µg/kg and THP i.p. at 3 µg/kg induced 40% and 38% tumor growth inhibition, respectively. THP also reduced metastasis measured as the number of lung nodules, and the addition of Dx deepened the effect even more. In the case of survival studies, the tumors were surgically removed after reaching 50 mm3 and two days later the animals were treated with vehicle, Dx, or Dx+THP. Log rank analysis of Kaplan-Meier plots showed a significant improvement of overall survival in mice treated with Dx+THP (P= 0.011) and only a trend with Dx treatment (P=0.067). Here we show that THP decreases TNBC proliferation, reduces metastasis dissemination and improves Dx's effect. These evidences are especially important in TNBC whose standard of care is only chemotherapy. In conclusion, THP can be a valuable agent to enhance the use of Dx in the chemotherapy treatment of TNBC tumors. However, beneficial effects over other tumors should be confirmed. Citation Format: María Florencia Mercogliano, Sofia Bruni, Mara De Martino, Elena Cavanagh, Liliana Balanian, Emilio Sojo, Felipe Inserra, Jose Groisman, Roxana Schillaci. Tamm-Horsfall protein as a novel antitumor therapy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1316.