Role of macrophages and their exosomes in orthopedic diseases.

Exosomes are vesicles with a lipid bilayer structure that carry various active substances, such as proteins, DNA, non-coding RNA, and nucleic acids; these participate in the immune response, tissue formation, and cell communication. Owing to their low immunogenicity, exosomes play a key role in regulating the skeletal immune environment. Macrophages are important immune cells that swallow various cellular and tissue fragments. M1-like and M2-like macrophages differentiate to play pro-inflammatory, anti-inflammatory, and repair roles following stimulation. In recent years, the increase in the population base and the aging of the population have led to a gradual rise in orthopedic diseases, placing a heavy burden on the social medical system and making it urgent to find effective solutions. Macrophages and their exosomes have been demonstrated to be closely associated with the pathogenesis and prognosis of orthopedic diseases. An in-depth understanding of their mechanisms of action and the interaction between them will be helpful for the future clinical treatment of orthopedic diseases. This review focuses on the mechanisms of action, diagnosis, and treatment of orthopedic diseases involving macrophages and their exosomes, including fracture healing, diabetic bone damage, osteosarcoma, and rheumatoid arthritis. In addition, we discuss the prospects and major challenges faced by macrophages and their exosomes in clinical practice.

Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1ß, IL-6, IL-12p40, IFN-ß) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Upregulation of GPR133 expression impaired the phagocytosis of macrophages in recurrent spontaneous miscarriage.

Decidual macrophages are the second-largest immune cell group at the maternal-foetal interface. They participate in apoptotic cell removal, and protect the foetus from microorganisms or pathogens. Dysfunction of decidual macrophages gives rise to pregnancy complications such as preeclampsia and recurrent spontaneous miscarriage (RSM). However, the mechanisms by which decidual macrophages are involved in the occurrence of adverse pregnancy outcomes have not been elucidated. Here we integrated DNA methylation and gene expression data from decidua macrophages to identify potential risk factors related to RSM. GPR133 was significantly hypomethylated and upregulated in decidual macrophages from RSM patients. Further demethylation analysis demonstrated that GPR133 expression in decidual macrophages was significantly increased by 5-Aza-dC treatment. In addition, the influence of GPR133 on the phagocytic ability of macrophages was explored. Phagocytosis was impaired in the decidual macrophages of RSM patients with increased GPR133 expression. Increased GPR133 expression induced by demethylation treatment in the decidual macrophages of healthy control patients led to a significant decrease in phagocytic function. Importantly, knockdown of GPR133 resulted in a significant improvement in the phagocytic function of THP-1 macrophages. In conclusion, the existing studies have shown the influence of GPR133 on the phagocytic function of decidual macrophages and pregnancy outcomes, providing new data and ideas for future research on the role of decidual macrophages in RSM.

Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Modelling of macrophage responses to biomaterials in vitro: state-of-the-art and the need for the improvement.

The increasing use of medical implants in various areas of medicine, particularly in orthopedic surgery, oncology, cardiology and dentistry, displayed the limitations in long-term integration of available biomaterials. The effective functioning and successful integration of implants requires not only technical excellence of materials but also consideration of the dynamics of biomaterial interaction with the immune system throughout the entire duration of implant use. The acute as well as long-term decisions about the efficiency of implant integration are done by local resident tissue macrophages and monocyte-derived macrophages that start to be recruited during tissue damage, when implant is installed, and are continuously recruited during the healing phase. Our review summarized the knowledge about the currently used macrophages-based in vitro cells system that include murine and human cells lines and primary ex vivo differentiated macrophages. We provided the information about most frequently examined biomarkers for acute inflammation, chronic inflammation, foreign body response and fibrosis, indicating the benefits and limitations of the model systems. Particular attention is given to the scavenging function of macrophages that controls dynamic composition of peri-implant microenvironment and ensures timely clearance of microorganisms, cytokines, metabolites, extracellular matrix components, dying cells as well as implant debris. We outline the perspective for the application of 3D systems for modelling implant interaction with the immune system in human tissue-specific microenvironment avoiding animal experimentation.

Current research on the interaction between Helicobacter pylori and macrophages.

Helicobacter pylori (H. pylori) is a gram-negative bacteria with a worldwide infection rate of 50%, known to induce gastritis, ulcers and gastric cancer. The interplay between H. pylori and immune cells within the gastric mucosa is pivotal in the pathogenesis of H. pylori-related disease. Following H. pylori infection, there is an observed increase in gastric mucosal macrophages, which are associated with the progression of gastritis. H. pylori elicits macrophage polarization, releases cytokines, reactive oxygen species (ROS) and nitric oxide (NO) to promote inflammatory response and eliminate H. pylori. Meanwhile, H. pylori has developed mechanisms to evade the host immune response in order to maintain the persistent infection, including interference with macrophage phagocytosis and antigen presentation, as well as induction of macrophage apoptosis. Consequently, the interaction between H. pylori and macrophages can significantly impact the progression, pathogenesis, and resolution of H. pylori infection. Moreover, macrophages are emerging as potential therapeutic targets for H. pylori-associated gastritis. Therefore, elucidating the involvement of macrophages in H. pylori infection may provide novel insights into the pathogenesis, progression, and management of H. pylori-related disease.

CD74 as a prognostic and M1 macrophage infiltration marker in a comprehensive pan-cancer analysis.

CD74 is a type-II transmembrane glycoprotein that has been linked to tumorigenesis. However, this association was based only on phenotypic studies, and, to date, no in-depth mechanistic studies have been conducted. In this study, combined with a multi-omics study, CD74 levels were significantly upregulated in most cancers relative to normal tissues and were found to be predictive of prognosis. Elevated CD74 expression was associated with reduced levels of mismatch-repair genes and homologous repair gene signatures in over 10 tumor types. Multiple fluorescence staining and bulk, spatial, single-cell transcriptional analyses indicated its potential as a marker for M1 macrophage infiltration in pan-cancer. In addition, CD74 expression was higher in BRCA patients responsive to conventional chemotherapy and was able to predict the prognosis of these patients. Potential CD74-activating drugs (HNHA and BRD-K55186349) were identified through molecular docking to CD74. The findings indicate activation of CD74 may have potential in tumor immunotherapy.

[Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells].

Objective: To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6). Methods: Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 µg/L transforming growth factor ß1 (TGF-ß1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 µmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-ß1 stimulation group, the TGF-ß1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-ß1 stimulation group, and the M2-type + TGF-ß1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results: Intracellular ROS increased significantly following TGF-ß1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F = 686.1, P = 0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased (P < 0.05). After the addition of GKT137831, ROS, and NOX4, α-SMA mRNA and protein expression were comparatively decreased in the TGF-ß1 stimulation group (P < 0.05), while mRNA and protein expressions of Nrf2 and HO-1 were increased (P < 0.05). The expression of ROS and NOX4, as well as α-SMA mRNA and protein, produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-ß1 stimulation (P < 0.05). After the addition of GKT137831, ROS, NOX4, α-SMA mRNA, and protein expression were further reduced in the co-culture group compared with the single culture group (P < 0.05), while the mRNA and protein expression of Nrf2 and HO-1 were further increased (P < 0.05). Conclusion: NOX4 inhibitor GKT137831 can reduce RO, NOX4, and α-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells. After M2-type macrophage co-culture, GKT137831 assists in lowering ROS, NOX4, and α-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells, which regulates the balance between oxidative stress and anti-oxidative stress systems, thereby antagonizing the fibrosis process.

Dopamine promotes Klebsiella quasivariicola proliferation and inflammatory response in the presence of macrophages.

Background: Dopamine, a frequently used therapeutic agent for critically ill patients, has been shown to be implicated in clinical infections recently, however, the precise mechanisms underlying this association remain elusive. Klebsiella quasivariicola, a novel strain belonging to the Klebsiella species, exhibits potential pathogenic attributes. The impact of dopamine on K. quasivariicola infection has aroused our interest. Objective: Considering the contribution of host immune factors during infection, this study aimed to investigate the intricate interactions between K. quasivariicola, dopamine, and macrophages were explored. Methods: RAW264.7 cells and C57/BL6 mice were infected with K. quasivariicola, and the bacterial growth within macrophage, the production of inflammatory cytokines and the pathological changes in mice lungs were detected, in the absence or presence of dopamine. Results: Dopamine inhibited the growth of K. quasivariicola in the medium, but promoted bacterial growth when co-cultured with macrophages. The expression of proinflammatory cytokines increased in RAW 264.7 cells infected with K. quasivariicola, and a significant rise was observed upon the addition of dopamine. The infection of K. quasivariicola in mice induced an inflammatory response and lung injury, which were exacerbated by the administration of dopamine. Conclusions: Our findings suggest that dopamine may be one of the potential risk factors associated with K. quasivariicola infection. This empirical insight provides solid references for clinical precision medicine. Furthermore, an in vitro model of microbes-drugs-host immune cells for inhibitor screening was proposed to more accurately replicate the complex in vivo environment. This fundamental work had contributed to the present understanding of the crosstalk between pathogen, dopamine and host immune cells.

Extracellular vesicles as carriers for noncoding RNA-based regulation of macrophage/microglia polarization: an emerging candidate regulator for lung and traumatic brain injuries.

Macrophage/microglia function as immune defense and homeostatic cells that originate from bone marrow progenitor cells. Macrophage/microglia activation is historically divided into proinflammatory M1 or anti-inflammatory M2 states based on intracellular dynamics and protein production. The polarization of macrophages/microglia involves a pivotal impact in modulating the development of inflammatory disorders, namely lung and traumatic brain injuries. Recent evidence indicates shared signaling pathways in lung and traumatic brain injuries, regulated through non-coding RNAs (ncRNAs) loaded into extracellular vesicles (EVs). This packaging protects ncRNAs from degradation. These vesicles are subcellular components released through a paracellular mechanism, constituting a group of nanoparticles that involve exosomes, microvesicles, and apoptotic bodies. EVs are characterized by a double-layered membrane and are abound with proteins, nucleic acids, and other bioactive compounds. ncRNAs are RNA molecules with functional roles, despite their absence of coding capacity. They actively participate in the regulation of mRNA expression and function through various mechanisms. Recent studies pointed out that selective packaging of ncRNAs into EVs plays a role in modulating distinct facets of macrophage/microglia polarization, under conditions of lung and traumatic brain injuries. This study will explore the latest findings regarding the role of EVs in the progression of lung and traumatic brain injuries, with a specific focus on the involvement of ncRNAs within these vesicles. The conclusion of this review will emphasize the clinical opportunities presented by EV-ncRNAs, underscoring their potential functions as both biomarkers and targets for therapeutic interventions.

M1 macrophages polarized by crude polysaccharides isolated from Auricularia polytricha exhibit anti-tumor effect on human breast cancer cells.

Breast cancer has been reported to correlate with the infiltration of tumor-associated macrophages (TAMs) or M2-like macrophages in tumor microenvironment (TME) that could promote breast cancer progression. In contrast, M1-like macrophages displayed anti-tumor activity toward cancer. This study was focused on Auricularia polytricha (AP), a cloud ear mushroom, which has been reported for anti-tumor activity and immunomodulation. AP extracts were screened on differentiated THP-1 macrophages (M0). Results demonstrated that water extract (APW) and crude polysaccharides (APW-CP) could upregulate M1-related genes and cytokines production (IL-6, IL-1 ß and TNF-α) significantly. Moreover, APW and APW-CP showed a high expression of CD86 (M1 marker) compared to M0. The NF-κB signaling pathway is crucial for pro-inflammatory gene regulation. The APW and APW-CP treatment showed the induction of the NF-κB pathway in a dose-dependent manner, which related to the ß-glucan content in the extracts. Furthermore, APW-CP polarized macrophages were investigated for anti-tumor activity on human breast cancer cells (MCF-7 and MDA-MB-231). Results showed that APW-CP could inhibit the invasion of breast cancer cells and induce apoptosis. Therefore, M1 macrophages polarized by APW-CP showed anti-tumor activity against the breast cancer cells and ß-glucan may be the potential M1-phenotype inducer.

Induction of therapeutic immunity and cancer eradication through biofunctionalized liposome-like nanovesicles derived from irradiated-cancer cells.

Immunotherapy has revolutionized the treatment of cancer. However, its efficacy remains to be optimized. There are at least two major challenges in effectively eradicating cancer cells by immunotherapy. Firstly, cancer cells evade immune cell killing by down-regulating cell surface immune sensors. Secondly, immune cell dysfunction impairs their ability to execute anti-cancer functions. Radiotherapy, one of the cornerstones of cancer treatment, has the potential to enhance the immunogenicity of cancer cells and trigger an anti-tumor immune response. Inspired by this, we fabricate biofunctionalized liposome-like nanovesicles (BLNs) by exposing irradiated-cancer cells to ethanol, of which ethanol serves as a surfactant, inducing cancer cells pyroptosis-like cell death and facilitating nanovesicles shedding from cancer cell membrane. These BLNs are meticulously designed to disrupt both of the aforementioned mechanisms. On one hand, BLNs up-regulate the expression of calreticulin, an "eat me" signal on the surface of cancer cells, thus promoting macrophage phagocytosis of cancer cells. Additionally, BLNs are able to reprogram M2-like macrophages into an anti-cancer M1-like phenotype. Using a mouse model of malignant pleural effusion (MPE), an advanced-stage and immunotherapy-resistant cancer model, we demonstrate that BLNs significantly increase T cell infiltration and exhibit an ablative effect against MPE. When combined with PD-1 inhibitor (α-PD-1), we achieve a remarkable 63.6% cure rate (7 out of 11) among mice with MPE, while also inducing immunological memory effects. This work therefore introduces a unique strategy for overcoming immunotherapy resistance.

[Tumor-associated Macrophage: 
Emerging Targets for Modulating the Tumor Microenvironment].

Tumor-associated macrophage (TAM) play a crucial role in the immune microenvironment of lung cancer. Through changes in their phenotype and phagocytic functions, TAM contribute to the initiation and progression of lung cancer. By promoting the formation of an immune-suppressive microenvironment and accelerating the growth of abnormal tumor vasculature, TAM facilitate the invasion and metastasis of lung cancer. Macrophages can polarize into different subtypes with distinct functions and characteristics in response to various stimuli, categorized as anti-tumor M1 and pro-tumor M2 types. In tumor tissues, TAM typically polarize into the alternatively activated M2 phenotype, exhibiting inhibitory effects on tumor immunity. This article reviews the role of anti-angiogenic drugs in modulating TAM phenotypes, highlighting their potential to reprogram M2-type TAM into an anti-tumor M1 phenotype. Additionally, the functional alterations of TAM play a significant role in anti-angiogenic therapy and immunotherapy strategies. In summary, the regulation of TAM polarization and function opens up new avenues for lung cancer treatment and may serve as a novel target for modulating the immune microenvironment of tumors.
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Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures.

BACKGROUND: Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear. METHODS: CCR2 RFP+ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 µg), or gram-positive peptidoglycan (PGN, 100 µg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey'smultiple comparison) and nonparametric statistical (Kruskal-Wallis, Dunn's multiple comparison) tests were used following Shapiro-Wilk testing for normality. RESULTS: Intratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2+ CD11cintCD11bhi monocytes/macrophages and both CCR2+ and CCR2- CD11c-CD11bhi monocytes at 48 h. There were also increases in CCR2+ CD4+ and CD8+ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cintCD11bhi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cintCD11bhi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome. CONCLUSION: Highly concentrated environmental/occupational exposures induced the recruitment of CCR2+ and CCR2- transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.

C-type lectin 4 of Toxocara canis activates NF-ĸB and MAPK pathways by modulating NOD1/2 and RIP2 in murine macrophages in vitro.

Toxocara canis is a parasitic zoonose that is distributed worldwide and is one of the two pathogens causing toxocariasis. After infection, it causes serious public health and safety problems, which pose significant veterinary and medical challenges. To better understand the regulatory effects of T. canis infection on the host immune cells, murine macrophages (RAW264.7) were incubated with recombinant T. canis C-type lectin 4 (rTc-CTL-4) protein in vitro. The quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to analyze the nucleotide-binding oligomerization domain-containing protein 1/2 (NOD1/2), receptor-interacting protein 2 (RIP2), nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) on mRNA level and protein expression level in macrophages. Our results indicated that 10 µg/mL rTc-CTL-4 protein could modulate the expression of NOD1, NOD2, and RIP2 at both the transcriptional and translational levels. The protein translation levels of NF-κB, P-p65, p38, and P-p38 in macrophages were also modulated by rTc-CTL-4 protein. Macrophages were co-incubated with rTc-CTL-4 protein after siRNA silencing of NOD1, NOD2, and RIP2. The expression levels of NF-κB, P-p65, p38, and P-p38 were significantly changed compared with the negative control groups (Neg. Ctrl.). Taken together, rTc-CTL-4 protein seemed to act on NOD1/2-RIP2-NF-κB and MAPK signaling pathways in macrophages and might activate MAPK and NF-κB signaling pathways by regulating NOD1, NOD2, and RIP2. The insights from the above studies could contribute to our understanding of immune recognition and regulatory mechanisms of T. canis infection in the host animals.

Jiedu Xiaozheng Yin Inhibits the Progression of Colitis Associated Colorectal Cancer by Stimulating Macrophage Polarization Towards an M1 Phenotype via the TLR4 Pathway.

To investigate the effect of Jiedu Xiaozheng Yin (JXY) on the polarization of macrophages in colitis-associated colon cancer (CAC). An orthotopic model of CAC was established to monitor changes in the pathological state of mice. Colon length, number of colon tumors were recorded, and indices for liver, spleen, and thymus were calculated. Hematoxylin and eosin (H&E) staining was employed to observe intestinal mucosal injury and tumor formation. Immunohistochemistry (IHC) staining was utilized to investigate the effect of JXY on M1 and M2 polarization of macrophages in the colonic mucosa of CAC mice. For in vitro experiments, RT-qPCR (Reverse Transcription-quantitative PCR) and flow cytometry were used to observe the effect of JXY on various M1-related molecules such as IL-1ß, TNF-α, iNOS, CD80, CD86, and its phagocytic function as well as M2-related molecules including Arg-1, CD206, and IL-10. Subsequently, after antagonizing the TLR4 pathway with antagonists (TAK242, PDTC, KG501, SR11302, LY294002), the expression of IL-6, TNF-α, iNOS, and IL-1ß mRNA were detected by RT-qPCR. In vivo experiments, the results showed that JXY improved the pathological condition of mice in general. And JXY treatment decreased the shortening of colon length and number of tumors as compared to non-treated CAC mice. Additionally, JXY treatment improved the lesions in the colonic tissue and induced a polarization of intestinal mucosal macrophages towards the M1 phenotype, while inhibiting polarization towards the M2 phenotype. In vitro experiments further confirmed that JXY treatment promoted the activation of macrophages towards the M1 phenotype, leading to increased expression of IL-1ß, TNF-α, iNOS, CD80, CD86, as well as enhanced phagocytic function. JXY treatment concomitantly inhibited the expression of M2-phenotype related molecules Arginase-1 (Arg-1), CD206, and IL-10. Furthermore, JXY inhibited M1-related molecules such as IL-6, TNF-α, iNOS, and IL-1ß after antagonizing the TLR4 pathway. Obviously, JXY could exhibit inhibitory effects on the development of colon tumors in mice with CAC by promoting M1 polarization through TLR4-mediated signaling and impeding M2 polarization of macrophages.

A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis.

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Empowering macrophages: the cancer fighters within the tumour microenvironment in mantle cell lymphoma.

In Mantle Cell Lymphoma (MCL), the role of macrophages within the tumour microenvironment (TME) has recently gained attention due to their impact on prognosis and response to therapy. Despite their low absolute number in MCL tumour tissue, recent findings reveal an association between the levels of macrophages and prognosis, consistent with trends observed in other lymphoma subtypes. M2-like macrophages, identified by markers such as CD163, contribute to angiogenesis and suppression of the immune response. Clinical trials with MCL patients treated with chemoimmunotherapy and targeted treatments underscore the adverse impact of high levels of M2-like macrophages. Immunomodulatory drugs like lenalidomide reduce the levels of MCL-associated CD163+ macrophages and enhance macrophage phagocytic activity. Similarly, clinical approaches targeting the CD47 "don't eat me" signalling, in combination with the anti-CD20-antibody rituximab, demonstrate increased macrophage activity and phagocytosis of MCL tumour cells. Cell-based therapies such as chimeric antigen receptor (CAR) T-cell have shown promise but various challenges persist, leading to a potential interest in CAR-macrophages (CAR-M). When macrophages are recruited to the TME, they offer advantages including phagocytic function and responsiveness to microenvironment alterations, suggesting their potential as a manipulable and inducible alternative when CAR T-cell therapies fails in the complex landscape of MCL treatment.

Uterine macrophages and NK cells exhibit population and gene-level changes after implantation but maintain pro-invasive properties.

Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.