Spatial distribution pattern of immune cells is associated with patient prognosis in colorectal cancer.

BACKGROUND: The spatial context of tumor-infiltrating immune cells (TIICs) is important in predicting colorectal cancer (CRC) patients' clinical outcomes. However, the prognostic value of the TIIC spatial distribution is unknown. Thus, we aimed to investigate the association between TIICs in situ and patient prognosis in a large CRC sample. METHODS: We implemented multiplex immunohistochemistry staining technology in 190 CRC samples to quantify 14 TIIC subgroups in situ. To delineate the spatial relationship of TIICs to tumor cells, tissue slides were segmented into tumor cell and microenvironment compartments based on image recognition technology, and the distance between immune and tumor cells was calculated by implementing the computational pipeline phenoptr. RESULTS: MPO+ neutrophils and CD68+IDO1+ tumor-associated macrophages (TAMs) were enriched in the epithelial compartment, and myeloid lineage cells were located nearest to tumor cells. Except for CD68+CD163+ TAMs, other cells were all positively associated with favorable prognosis. The prognostic predictive power of TIICs was highly related to their distance to tumor cells. Unsupervised clustering analysis divided colorectal cancer into three subtypes with distinct prognostic outcomes, and correlation analysis revealed the synergy among B cells, CD68+IDO1+TAMs, and T lineage cells in producing an effective immune response. CONCLUSIONS: Our study suggests that the integration of spatial localization with TIIC abundance is important for comprehensive prognostic assessment.

Graphene oxide quantum dots-loaded sinomenine hydrochloride nanocomplexes for effective treatment of rheumatoid arthritis via inducing macrophage repolarization and arresting abnormal proliferation of fibroblast-like synoviocytes.

The characteristic features of the rheumatoid arthritis (RA) microenvironment are synovial inflammation and hyperplasia. Therefore, there is a growing interest in developing a suitable therapeutic strategy for RA that targets the synovial macrophages and fibroblast-like synoviocytes (FLSs). In this study, we used graphene oxide quantum dots (GOQDs) for loading anti-arthritic sinomenine hydrochloride (SIN). By combining with hyaluronic acid (HA)-inserted hybrid membrane (RFM), we successfully constructed a new nanodrug system named HA@RFM@GP@SIN NPs for target therapy of inflammatory articular lesions. Mechanistic studies showed that this nanomedicine system was effective against RA by facilitating the transition of M1 to M2 macrophages and inhibiting the abnormal proliferation of FLSs in vitro. In vivo therapeutic potential investigation demonstrated its effects on macrophage polarization and synovial hyperplasia, ultimately preventing cartilage destruction and bone erosion in the preclinical models of adjuvant-induced arthritis and collagen-induced arthritis in rats. Metabolomics indicated that the anti-arthritic effects of HA@RFM@GP@SIN NPs were mainly associated with the regulation of steroid hormone biosynthesis, ovarian steroidogenesis, tryptophan metabolism, and tyrosine metabolism. More notably, transcriptomic analyses revealed that HA@RFM@GP@SIN NPs suppressed the cell cycle pathway while inducing the cell apoptosis pathway. Furthermore, protein validation revealed that HA@RFM@GP@SIN NPs disrupted the excessive growth of RAFLS by interfering with the PI3K/Akt/SGK/FoxO signaling cascade, resulting in a decline in cyclin B1 expression and the arrest of the G2 phase. Additionally, considering the favorable biocompatibility and biosafety, these multifunctional nanoparticles offer a promising therapeutic approach for patients with RA.

ER-to-lysosome Ca2+ refilling followed by K+ efflux-coupled store-operated Ca2+ entry in inflammasome activation and metabolic inflammation.

We studied lysosomal Ca2+ in inflammasome. Lipopolysaccharide (LPS) + palmitic acid (PA) decreased lysosomal Ca2+ ([Ca2+]Lys) and increased [Ca2+]i through mitochondrial ROS, which was suppressed in Trpm2-KO macrophages. Inflammasome activation and metabolic inflammation in adipose tissue of high-fat diet (HFD)-fed mice were ameliorated by Trpm2 KO. ER→lysosome Ca2+ refilling occurred after lysosomal Ca2+ release whose blockade attenuated LPS + PA-induced inflammasome. Subsequently, store-operated Ca2+entry (SOCE) was activated whose inhibition suppressed inflammasome. SOCE was coupled with K+ efflux whose inhibition reduced ER Ca2+ content ([Ca2+]ER) and impaired [Ca2+]Lys recovery. LPS + PA activated KCa3.1 channel, a Ca2+-activated K+ channel. Inhibitors of KCa3.1 channel or Kcnn4 KO reduced [Ca2+]ER, attenuated increase of [Ca2+]i or inflammasome activation by LPS + PA, and ameliorated HFD-induced inflammasome or metabolic inflammation. Lysosomal Ca2+ release induced delayed JNK and ASC phosphorylation through CAMKII-ASK1. These results suggest a novel role of lysosomal Ca2+ release sustained by ER→lysosome Ca2+ refilling and K+ efflux through KCa3.1 channel in inflammasome activation and metabolic inflammation.

CCR7 and CD48 as Predicted Targets in Acute Rejection Related to M1 Macrophage after Pediatric Kidney Transplantation.

Background: Kidney transplantation (KT) is the best treatment for end-stage renal disease. Although long and short-term survival rates for the graft have improved significantly with the development of immunosuppressants, acute rejection (AR) remains a major risk factor attacking the graft and patients. The innate immune response plays an important role in rejection. Therefore, our objective is to determine the biomarkers of congenital immunity associated with AR after KT and provide support for future research. Materials and Methods: A differential expression genes (DEGs) analysis was performed based on the dataset GSE174020 from the NCBI gene Expression Synthesis Database (GEO) and then combined with the GSE5099 M1 macrophage-related gene identified in the Molecular Signatures Database. We then identified genes in DEGs associated with M1 macrophages defined as DEM1Gs and performed gene ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) enrichment analysis. Cibersort was used to analyze the immune cell infiltration during AR. At the same time, we used the protein-protein interaction (PPI) network and Cytoscape software to determine the key genes. Dataset, GSE14328 derived from pediatric patients, GSE138043 and GSE9493 derived from adult patients, were used to verify Hub genes. Additional verification was the rat KT model, which was used to perform HE staining, immunohistochemical staining, and Western Blot. Hub genes were searched in the HPA database to confirm their expression. Finally, we construct the interaction network of transcription factor (TF)-Hub genes and miRNA-Hub genes. Results: Compared to the normal group, 366 genes were upregulated, and 423 genes were downregulated in the AR group. Then, 106 genes related to M1 macrophages were found among these genes. GO and KEGG enrichment analysis showed that these genes are mainly involved in cytokine binding, antigen binding, NK cell-mediated cytotoxicity, activation of immune receptors and immune response, and activation of the inflammatory NF-κB signaling pathway. Two Hub genes, namely CCR7 and CD48, were identified by PPI and Cytoscape analysis. They have been verified in external validation sets, originated from both pediatric patients and adult patients, and animal experiments. In the HPA database, CCR7 and CD48 are mainly expressed in T cells, B cells, macrophages, and tissues where these immune cells are distributed. In addition to immunoinfiltration, CD4+T, CD8+T, NK cells, NKT cells, and monocytes increased significantly in the AR group, which was highly consistent with the results of Hub gene screening. Finally, we predicted that 19 TFs and 32 miRNAs might interact with the Hub gene. Conclusions: Through a comprehensive bioinformatic analysis, our findings may provide predictive and therapeutic targets for AR after KT.

Exosome-shuttled miR-150-5p from LPS-preconditioned mesenchymal stem cells down-regulate PI3K/Akt/mTOR pathway via Irs1 to enhance M2 macrophage polarization and confer protection against sepsis.

Rationale: Sepsis is a life-threatening organ dysfunction and lack of effective measures in the current. Exosomes from mesenchymal stem cells (MSCs) reported to alleviate inflammation during sepsis, and the preconditioning of MSCs could enhance their paracrine potential. Therefore, this study investigated whether exosomes secreted by lipopolysaccharide (LPS)-pretreated MSCs exert superior antiseptic effects, and explored the underlying molecular mechanisms. Methods: Exosomes were isolated and characterized from the supernatants of MSCs. The therapeutic efficacy of normal exosomes (Exo) and LPS-pretreated exosomes (LPS-Exo) were evaluated in terms of survival rates, inflammatory response, and organ damage in an LPS-induced sepsis model. Macrophages were stimulated with LPS and treated with Exo or LPS-Exo to confirm the results of the in vivo studies, and to explain the potential mechanisms. Results: LPS-Exo were shown to inhibit aberrant pro-inflammatory cytokines, prevent organ damages, and improve survival rates of the septic mice to a greater extent than Exo. In vitro, LPS-Exo significantly promoted the M2 polarization of macrophages exposed to inflammation. miRNA sequencing and qRT-PCR analysis identified the remarkable expression of miR-150-5p in LPS-Exo compared to that in Exo, and exosomal miR-150-5p was transferred into recipient macrophages and mediated macrophage polarization. Further investigation demonstrated that miR-150-5p targets Irs1 in recipient macrophages and subsequently modulates macrophage plasticity by down-regulating the PI3K/Akt/mTOR pathway. Conclusion: The current findings highly suggest that exosomes derived from LPS pre-conditioned MSCs represent a promising cell-free therapeutic method and highlight miR-150-5p as a novel molecular target for regulating immune hyperactivation during sepsis.

Immune cells and checkpoints in pancreatic adenocarcinoma: Association with clinical and pathological characteristics.

INTRODUCTION: Pancreatic adenocarcinoma is an extremely aggressive neoplasm, with many challenges to be overcome in order to achieve a truly effective treatment. It is characterized by a mostly immunosuppressed environment, with dysfunctional immune cells and active immunoinhibitory pathways that favor tumor evasion and progression. Thus, the study and understanding of the tumor microenvironment and the various cells subtypes and their functional capacities are essential to achieve more effective treatments, especially with the use of new immunotherapeutics. METHODS: Seventy cases of pancreatic adenocarcinoma divided into two groups 43 with resectable disease and 27 with unresectable disease were analyzed using immunohistochemical methods regarding the expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), and human leukocyte antigen G (HLA-G) molecules as well as the populations of CD4+ and CD8+ T lymphocytes, regulatory T cells (Tregs), and M2 macrophages (MM2). Several statistical tests, including multivariate analyses, were performed to examine how those immune cells and immunoinhibitory molecules impact the evolution and prognosis of pancreatic adenocarcinoma. RESULTS: CD8+ T lymphocytes and M2 macrophages predominated in the group operated on, and PD-L2 expression predominated in the unresectable group. PD-L2 was associated with T stage, lymph node metastasis, and clinical staging, while in survival analysis, PD-L2 and HLA-G were associated with a shorter survival. In the inoperable cases, Tregs cells, MM2, PD-L1, PD-L2, and HLA-G were positively correlated. CONCLUSIONS: PD-L2 and HLA-G expression correlated with worse survival in the cases studied. Tumor microenvironment was characterized by a tolerant and immunosuppressed pattern, mainly in unresectable lesions, where a broad positive influence was observed between immunoinhibitory cells and immune checkpoint proteins expressed by tumor cells.

Antimicrobial peptide 2K4L inhibits the inflammatory response in macrophages and Caenorhabditis elegans and protects against LPS-induced septic shock in mice.

2K4L is a rationally designed analog of the short α-helical peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L displayed improved and broad-spectrum antibacterial activity than temporin-1CEc in vitro. Here, the antibacterial and anti-inflammatory activities of 2K4L in macrophages, C. elegans and mice were investigated. The results demonstrated that 2K4L could enter THP-1 cells to kill a multidrug-resistant Acinetobacter baumannii strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22933), as well as reduce proinflammatory responses induced by MRAB 0227 by inhibiting NF-κB signaling pathway. Similarly, 2K4L exhibited strong bactericidal activity against A. baumannii uptake into C. elegans, extending the lifespan and healthspan of the nematodes. Meanwhile, 2K4L alleviated the oxidative stress response by inhibiting the expression of core genes in the p38 MAPK/PMK-1 signaling pathway and downregulating the phosphorylation level of p38, thereby protecting the nematodes from damage by A. baumannii. Finally, in an LPS-induced septic model, 2K4L enhanced the survival of septic mice and decreased the production of proinflammatory cytokines by inhibiting the signaling protein expression of the MAPK and NF-κB signaling pathways and protecting LPS-induced septic mice from a lethal inflammatory response. In conclusion, 2K4L ameliorated LPS-induced inflammation both in vitro and in vivo.

SerpinB2 deficiency is associated with delayed mammary tumor development and decreased pro-tumorigenic macrophage polarization.

The in vivo functions of SerpinB2 in tumor cells and tumor-associated macrophages (TAMs) during breast cancer development and metastasis remain elusive. SerpinB2-deficient MMTV-PyMT mice (PyMTSB2-/-) were previously produced to explore the biological roles of SerpinB2 in breast cancer. Compared with MMTV-PyMT wild-type (PyMTWT) mice, PyMTSB2-/- mice showed delayed tumor progression and reduced CK8 + tumor cell dissemination to lymph nodes. RNA-Seq data revealed significantly enriched genes associated with inflammatory responses, especially upregulated M1 and downregulated M2 macrophage marker genes in PyMTSB2-/- tumors. Decreased CD206+M2 and increased NOS2+M1 markers were detected in the primary tumors and metastatic lymph nodes of PyMTSB2-/- mice. In an in vitro study, SerpinB2 knockdown decreased the sphere formation and migration of MDA-MB-231 cells and suppressed protumorigenic M2 polarization of RAW264.7 cells. The combination of low SerpinB2, high NOS2, and low CD206 expression was favorable for survival in patients with breast cancer, as assessed in the BreastMark dataset. Our study demonstrates that SerpinB2 deficiency delays mammary tumor development and metastasis in PyMTWT mice, along with reduced sphere formation and migration abilities of tumor cells and decreased macrophage protumorigenic polarization.

The bioinfomatics analysis of the M1 macrophage-related gene CXCL9 signature in cervical cancer.

BACKGROUND: The expression and function of coexpression genes of M1 macrophage in cervical cancer have not been identified. And the CXCL9-expressing tumour-associated macrophage has been poorly reported in cervical cancer. METHODS: To clarify the regulatory gene network of M1 macrophage in cervical cancer, we downloaded gene expression profiles of cervical cancer patients in TCGA database to identify M1 macrophage coexpression genes. Then we constructed the protein-protein interaction networks by STRING database and performed functional enrichment analysis to investigate the biological effects of the coexpression genes. Next, we used multiple bioinformatics databases and experiments to overall investigate coexpression gene CXCL9, including western blot assay and immunohistochemistry assay, GeneMANIA, Kaplan-Meier Plotter, Xenashiny, TISCH2, ACLBI, HPA, TISIDB, GSCA and cBioPortal databases. RESULTS: There were 77 positive coexpression genes and 5 negative coexpression genes in M1 macrophage. The coexpression genes in M1 macrophage participated in the production and function of chemokines and chemokine receptors. Especially, CXCL9 was positively correlated with M1 macrophage infiltration levels in cervical cancer. CXCL9 expression would significantly decrease and high CXCL9 levels were linked to good prognosis in the cervical cancer tumour patients, it manifestly expressed in blood immune cells, and was positively related to immune checkpoints. CXCL9 amplification was the most common type of mutation. The CXCL9 gene interaction network could regulate immune-related signalling pathways, and CXCL9 amplification was the most common mutation type in cervical cancer. Meanwhile, CXCL9 may had clinical significance for the drug response in cervical cancer, possibly mediating resistance to chemotherapy and targeted drug therapy. CONCLUSION: Our findings may provide new insight into the M1 macrophage coexpression gene network and molecular mechanisms in cervical cancer, and indicated that M1 macrophage association gene CXCL9 may serve as a good prognostic gene and a potential therapeutic target for cervical cancer therapies.

Cervical cancer is a common gynaecological malignancy, investigating the precise gene expression regulation of M1 macrophage is crucial for understanding the changes in the immune microenvironment of cervical cancer. In our study, a total of 82 coexpression genes with M1 macrophages were identified, and these genes were involved in the production and biological processes of chemokines and chemokine receptors. Especially, the chemokine CXCL9 was positively correlated with M1 macrophage infiltration levels in cervical cancer. CXCL9 as a protective factor, it manifestly expressed in blood immune cells, and was positively related to immune checkpoints. CXCL9 amplification was the most common type of mutation. And CXCL9 expression could have an effect on the sensitivity of some chemicals or targeted drugs against cervical cancer. These findings may provide new insight into the M1 macrophage coexpression gene network and molecular mechanisms, and shed light on the role of CXCL9 in cervical cancer.

IFNγ-IL12 axis regulates intercellular crosstalk in metabolic dysfunction-associated steatotic liver disease.

Obesity is a major cause of metabolic dysfunction-associated steatohepatitis (MASH) and is characterized by inflammation and insulin resistance. Interferon-γ (IFNγ) is a pro-inflammatory cytokine elevated in obesity and modulating macrophage functions. Here, we show that male mice with loss of IFNγ signaling in myeloid cells (Lyz-IFNγR2-/-) are protected from diet-induced insulin resistance despite fatty liver. Obesity-mediated liver inflammation is also attenuated with reduced interleukin (IL)-12, a cytokine primarily released by macrophages, and IL-12 treatment in vivo causes insulin resistance by impairing hepatic insulin signaling. Following MASH diets, Lyz-IFNγR2-/- mice are rescued from developing liver fibrosis, which is associated with reduced fibroblast growth factor (FGF) 21 levels. These results indicate critical roles for IFNγ signaling in macrophages and their release of IL-12 in modulating obesity-mediated insulin resistance and fatty liver progression to MASH. In this work, we identify the IFNγ-IL12 axis in regulating intercellular crosstalk in the liver and as potential therapeutic targets to treat MASH.

DKK1 loss promotes endometrial fibrosis via autophagy and exosome-mediated macrophage-to-myofibroblast transition.

INTRODUCTION: Intrauterine adhesions (IUA) manifest as endometrial fibrosis, often causing infertility or recurrent miscarriage; however, their pathogenesis remains unclear. OBJECTIVES: This study assessed the role of Dickkopf WNT signaling pathway inhibitor 1 (DKK1) and autophagy in endometrial fibrosis, using clinical samples as well as in vitro and in vivo experiments. METHODS: Immunohistochemistry, immunofluorescence and western blot were used to determine the localization and expression of DKK1 in endometrium; DKK1 silencing and DKK1 overexpression were used to detect the biological effects of DKK1 silencing or expression in endometrial cells; DKK1 gene knockout mice were used to observe the phenotypes caused by DKK1 gene knockout. RESULTS: In patients with IUA, DKK1 and autophagy markers were down-regulated; also, α-SMA and macrophage localization were increased in the endometrium. DKK1 conditional knockout (CKO) mice showed a fibrotic phenotype with decreased autophagy and increased localization of α-SMA and macrophages in the endometrium. In vitro studies showed that DKK1 knockout (KO) suppressed the autophagic flux of endometrial stromal cells. In contrast, ectopic expression of DKK1 showed the opposite phenotype. Mechanistically, we discovered that DKK1 regulates autophagic flux through Wnt/ß-catenin and PI3K/AKT/mTOR pathways. Further studies showed that DKK1 KO promoted the secretion of interleukin (IL)-8 in exosomes, thereby promoting macrophage proliferation and metastasis. Also, in DKK1 CKO mice, treatment with autophagy activator rapamycin partially restored the endometrial fibrosis phenotype. CONCLUSION: Our findings indicated that DKK1 was a potential diagnostic marker or therapeutic target for IUA.

Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Interleukin-5 alleviates cardiac remodelling via the STAT3 pathway in angiotensin II-infused mice.

Interleukin-5 (IL-5) has been reported to be involved in cardiovascular diseases, such as atherosclerosis and cardiac injury. This study aimed to investigate the effects of IL-5 on cardiac remodelling. Mice were infused with angiotensin II (Ang II), and the expression and source of cardiac IL-5 were analysed. The results showed that cardiac IL-5 expression was time- and dose-dependently decreased after Ang II infusion, and was mainly derived from cardiac macrophages. Additionally, IL-5-knockout (IL-5-/-) mice were used to observe the effects of IL-5 knockout on Ang II-induced cardiac remodelling. We found knockout of IL-5 significantly increased the expression of cardiac hypertrophy markers, elevated myocardial cell cross-sectional areas and worsened cardiac dysfunction in Ang II-infused mice. IL-5 deletion also promoted M2 macrophage differentiation and exacerbated cardiac fibrosis. Furthermore, the effects of IL-5 deletion on cardiac remodelling was detected after the STAT3 pathway was inhibited by S31-201. The effects of IL-5 on cardiac remodelling and M2 macrophage differentiation were reversed by S31-201. Finally, the effects of IL-5 on macrophage differentiation and macrophage-related cardiac hypertrophy and fibrosis were analysed in vitro. IL-5 knockout significantly increased the Ang II-induced mRNA expression of cardiac hypertrophy markers in myocardial cells that were co-cultured with macrophages, and this effect was reversed by S31-201. Similar trends in the mRNA levels of fibrosis markers were observed when cardiac fibroblasts and macrophages were co-cultured. In conclusions, IL-5 deficiency promote the differentiation of M2 macrophages by activating the STAT3 pathway, thereby exacerbating cardiac remodelling in Ang II-infused mice. IL-5 may be a potential target for the clinical prevention of cardiac remodelling.

Differential signalling requirements for RIPK1-dependent pyroptosis in neutrophils and macrophages.

TLR4 and TNFR1 signalling promotes potent proinflammatory signal transduction events, thus, are often hijacked by pathogenic microorganisms. We recently reported that myeloid cells retaliate Yersinia blockade of TAK1/IKK signalling by triggering RIPK1-dependent caspase-8 activation that promotes downstream GSDMD and GSDME-mediated pyroptosis in macrophages and neutrophils respectively. However, the upstream signalling events for RIPK1 activation in these cells are not well defined. Here, we demonstrate that unlike in macrophages, RIPK1-driven pyroptosis and cytokine priming in neutrophils are driven through TNFR1 signalling, while TLR4-TRIF signalling is dispensable. Furthermore, we demonstrate that activation of RIPK1-dependent pyroptosis in neutrophils during Yersinia infection requires IFN-γ priming, which serves to induce surface TNFR1 expression and amplify soluble TNF secretion. In contrast, macrophages utilise both TNFR1 and TLR4-TRIF signalling to trigger cell death, but only require TRIF but not autocrine TNFR1 for cytokine production. Together, these data highlight the emerging theme of cell type-specific regulation in cell death and immune signalling in myeloid cells.

Baseline data collections of lipopolysaccharide content in 414 herbal extracts and its role in innate immune activation.

Some herbal extracts contain relatively high amounts of lipopolysaccharide (LPS). Because orally administered LPS activates innate immunity without inducing inflammation, it plays a role as an active ingredient in herbal extracts. However, the LPS content in herbal extracts remains extensively unevaluated. This study aimed to create a database of LPS content in herbal extracts; therefore, the LPS content of 414 herbal extracts was measured and the macrophage activation potential was evaluated. The LPS content of these hot water extracts was determined using the kinetic-turbidimetric method. The LPS concentration ranged from a few ng/g to hundreds of µg/g (Standard Escherichia coli LPS equivalent). Twelve samples had a high-LPS-content of > 100 µg/g, including seven samples from roots and three samples from leaves of the herbal extracts. These samples showed high phagocytosis and NO production capacity, and further investigation using polymyxin B, an LPS inhibitor, significantly inhibited macrophage activation. This study suggests that some herbal extracts contain sufficient LPS concentration to activate innate immunity. Therefore, a new approach to evaluate the efficacy of herbal extracts based on their LPS content was proposed. A database listing the LPS content of different herbal extracts is essential for this approach.

Glutamate oxaloacetate transaminase 1 is dispensable in macrophage differentiation and anti-pathogen response.

Macrophages play a pivotal role in orchestrating the immune response against pathogens. While the intricate interplay between macrophage activation and metabolism remains a subject of intense investigation, the role of glutamate oxaloacetate transaminase 1 (Got1) in this context has not been extensively assessed. Here, we investigate the impact of Got1 on macrophage polarization and function, shedding light on its role in reactive oxygen species (ROS) production, pathogen defense, and immune paralysis. Using genetically modified mouse models, including both myeloid specific knockout and overexpression, we comprehensively demonstrate that Got1 depletion leads to reduced ROS production in macrophages. Intriguingly, this impairment in ROS generation does not affect the resistance of Got1 KO mice to pathogenic challenges. Furthermore, Got1 is dispensable for M2 macrophage differentiation and does not influence the onset of LPS-induced immune paralysis. Our findings underscore the intricate facets of macrophage responses, suggesting that Got1 is dispensable in discrete immunological processes.

An injectable magnesium-loaded hydrogel releases hydrogen to promote osteoporotic bone repair via ROS scavenging and immunomodulation.

Background: The repair of osteoporotic bone defects remains challenging due to excessive reactive oxygen species (ROS), persistent inflammation, and an imbalance between osteogenesis and osteoclastogenesis. Methods: Here, an injectable H2-releasing hydrogel (magnesium@polyethylene glycol-poly(lactic-co-glycolic acid), Mg@PEG-PLGA) was developed to remodel the challenging bone environment and accelerate the repair of osteoporotic bone defects. Results: This Mg@PEG-PLGA gel shows excellent injectability, shape adaptability, and phase-transition ability, can fill irregular bone defect areas via minimally invasive injection, and can transform into a porous scaffold in situ to provide mechanical support. With the appropriate release of H2 and magnesium ions, the 2Mg@PEG-PLGA gel (loaded with 2 mg of Mg) displayed significant immunomodulatory effects through reducing intracellular ROS, guiding macrophage polarization toward the M2 phenotype, and inhibiting the IκB/NF-κB signaling pathway. Moreover, in vitro experiments showed that the 2Mg@PEG-PLGA gel inhibited osteoclastogenesis while promoting osteogenesis. Most notably, in animal experiments, the 2Mg@PEG-PLGA gel significantly promoted the repair of osteoporotic bone defects in vivo by scavenging ROS and inhibiting inflammation and osteoclastogenesis. Conclusions: Overall, our study provides critical insight into the design and development of H2-releasing magnesium-based hydrogels as potential implants for repairing osteoporotic bone defects.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

PCLAF induces bone marrow adipocyte senescence and contributes to skeletal aging.

Bone marrow adipocytes (BMAds) affect bone homeostasis, but the mechanism remains unclear. Here, we showed that exercise inhibited PCNA clamp-associated factor (PCLAF) secretion from the bone marrow macrophages to inhibit BMAds senescence and thus alleviated skeletal aging. The genetic deletion of PCLAF in macrophages inhibited BMAds senescence and delayed skeletal aging. In contrast, the transplantation of PCLAF-mediated senescent BMAds into the bone marrow of healthy mice suppressed bone turnover. Mechanistically, PCLAF bound to the ADGRL2 receptor to inhibit AKT/mTOR signaling that triggered BMAds senescence and subsequently spread senescence among osteogenic and osteoclastic cells. Of note, we developed a PCLAF-neutralizing antibody and showed its therapeutic effects on skeletal health in old mice. Together, these findings identify PCLAF as an inducer of BMAds senescence and provide a promising way to treat age-related osteoporosis.

The impact of cholesteryl ester transfer protein on the progression of cutaneous leishmaniasis.

Introduction: Pathogenesis of cutaneous leishmaniases involves parasite growth, persistent inflammation, and likely participation of lipoproteins (LP). The cholesteryl ester transfer protein (CETP), involved in LP remodeling, has been shown to participate in the inflammatory response and the evolution of infectious conditions. Methods: We evaluated the impact of the presence of CETP on infection by Leishmania (L.) amazonensis in an experimental model of cutaneous leishmaniasis using C57BL6/J mice transgenic for human CETP (CETP), having as control their littermates that do not express the protein, wild-type (WT) mice. The progression of the lesion after infection in the footpad was monitored for 12 weeks. Two groups of animals were formed to collect the plantar pad in the 4th and 12th week post-infection. Results: The lesion increased from the 3rd week onwards, in both groups, with a gradual decrease from the 10th week onwards in the CETP group compared to the WT group, showing a reduction in parasitism and an improvement in the healing process, a reduction in CD68+ cells, and an increase in CD163+ and CD206, characterizing a population of M2 macrophages. A reduction in ARG1+ cells and an increase in INOS+ cells were observed. During infection, the LP profile showed an increase in triglycerides in the VLDL fraction in the CETP group at 12 weeks. Gene expression revealed a decrease in the CD36 receptor in the CETP group at 12 weeks, correlating with healing and parasite reduction. In vitro, macrophages derived from bone marrow cells from CETP mice showed lower parasite load at 48 h and, a reduction in arginase activity at 4 h accompanied by increased NO production at 4 and 24 h compared to WT macrophages, corroborating the in vivo findings. Discussion: The data indicate that the presence of CETP plays an important role in resolving Leishmania (L.) amazonensis infection, reducing parasitism, and modulating the inflammatory response in controlling infection and tissue repair.