Macrophages as a Source and Target of GDF-15.

Growth differentiation factor 15 (GDF-15) is a multifunctional cytokine that belongs to the transforming growth factor-beta (TGF-ß) superfamily. GDF-15 is involved in immune tolerance and is elevated in several acute and chronic stress conditions, often correlating with disease severity and patient prognosis in cancer172 and metabolic and cardiovascular disorders. Despite these clinical associations, the molecular mechanisms orchestrating its effects remain to be elucidated. The effects of GDF-15 are pleiotropic but cell-specific and dependent on the microenvironment. While GDF-15 expression can be stimulated by inflammatory mediators, its predominant effects were reported as anti-inflammatory and pro-fibrotic. The role of GDF-15 in the macrophage system has been increasingly investigated in recent years. Macrophages produce high levels of GDF-15 during oxidative and lysosomal stress, which can lead to fibrogenesis and angiogenesis at the tissue level. At the same time, macrophages can respond to GDF-15 by switching their phenotype to a tolerogenic one. Several GDF-15-based therapies are under development, including GDF-15 analogs/mimetics and GDF-15-targeting monoclonal antibodies. In this review, we summarize the major physiological and pathological contexts in which GDF-15 interacts with macrophages. We also discuss the major challenges and future perspectives in the therapeutic translation of GDF-15.

Dual-Specificity Phosphatases in Regulation of Tumor-Associated Macrophage Activity.

The regulation of protein kinases by dephosphorylation is a key mechanism that defines the activity of immune cells. A balanced process of the phosphorylation/dephosphorylation of key protein kinases by dual-specificity phosphatases is required for the realization of the antitumor immune response. The family of dual-specificity phosphatases is represented by several isoforms found in both resting and activated macrophages. The main substrate of dual-specificity phosphatases are three components of mitogen-activated kinase signaling cascades: the extracellular signal-regulated kinase ERK1/2, p38, and Janus kinase family. The results of the study of model tumor-associated macrophages supported the assumption of the crucial role of dual-specificity phosphatases in the formation and determination of the outcome of the immune response against tumor cells through the selective suppression of mitogen-activated kinase signaling cascades. Since mitogen-activated kinases mostly activate the production of pro-inflammatory mediators and the antitumor function of macrophages, the excess activity of dual-specificity phosphatases suppresses the ability of tumor-associated macrophages to activate the antitumor immune response. Nowadays, the fundamental research in tumor immunology is focused on the search for novel molecular targets to activate the antitumor immune response. However, to date, dual-specificity phosphatases received limited discussion as key targets of the immune system to activate the antitumor immune response. This review discusses the importance of dual-specificity phosphatases as key regulators of the tumor-associated macrophage function.

Macrophage diversity in human cancers: New insight provided by single-cell resolution and spatial context.

M1/M2 paradigm of macrophage plasticity has existed for decades. Now it becomes clear that this dichotomy doesn't adequately reflect the diversity of macrophage phenotypes in tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are a major population of innate immune cells in the TME that promotes tumor cell proliferation, angiogenesis and lymphangiogenesis, invasion and metastatic niche formation, as well as response to anti-tumor therapy. However, the fundamental restriction in therapeutic TAM targeting is the limited knowledge about the specific TAM states in distinct human cancer types. Here we summarized the results of the most recent studies that use advanced technologies (e.g. single-cell RNA sequencing and spatial transcriptomics) allowing to decipher novel functional subsets of TAMs in numerous human cancers. The transcriptomic profiles of these TAM subsets and their clinical significance were described. We emphasized the characteristics of specific TAM subpopulations - TREM2+, SPP1+, MARCO+, FOLR2+, SIGLEC1+, APOC1+, C1QC+, and others, which have been most extensively characterized in several cancers, and are associated with cancer prognosis. Spatial transcriptomics technologies defined specific spatial interactions between TAMs and other cell types, especially fibroblasts, in tumors. Spatial transcriptomics methods were also applied to identify markers of immunotherapy response, which are expressed by macrophages or in the macrophage-abundant regions. We highlighted the perspectives for novel techniques that utilize spatial and single cell resolution in investigating new ligand-receptor interactions for effective immunotherapy based on TAM-targeting.

ROS signaling in innate immunity via oxidative protein modifications.

The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in various aspects of innate immune function, which involves respiratory bursts and inflammasome activation. These reactive species widely distributed within the cellular environment are short-lived intermediates that play a vital role in cellular signaling and proliferation and are likely to depend on their subcellular site of formation. NADPH oxidase complex of phagocytes is known to generate superoxide anion radical (O2 •-) that functions as a precursor for antimicrobial hydrogen peroxide (H2O2) production, and H2O2 is utilized by myeloperoxidase (MPO) to generate hypochlorous acid (HOCl) that mediates pathogen killing. H2O2 modulates the expression of redox-responsive transcriptional factors, namely NF-kB, NRF2, and HIF-1, thereby mediating redox-based epigenetic modification. Survival and function of immune cells are under redox control and depend on intracellular and extracellular levels of ROS/RNS. The current review focuses on redox factors involved in the activation of immune response and the role of ROS in oxidative modification of proteins in macrophage polarization and neutrophil function.

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.

Hyperglycemia amplifies TLR-mediated inflammatory response of M(IL4) macrophages to dyslipidemic ligands.

Hyperglycemia is critical for initiation of diabetic vascular complications. We systemically addressed the role of hyperglycemia in the regulation of TLRs in primary human macrophages. Expression of TLRs (1-9) was examined in monocyte-derived M(NC), M(IFNγ), and M(IL4) differentiated in normoglycemic and hyperglycemic conditions. Hyperglycemia increased expression of TLR1 and TLR8 in M(NC), TLR2 and TLR6 in M(IFNγ), and TLR4 and TLR5 in M(IL4). The strongest effect of hyperglycemia in M(IL4) was the upregulation of the TLR4 gene and protein expression. Hyperglycemia amplified TLR4-mediated response of M(IL4) to lipopolysaccharide by significantly enhancing IL1ß and modestly suppressing IL10 production. In M(IL4), hyperglycemia in combination with synthetic triacylated lipopeptide (TLR1/TLR2 ligand) amplified expression of TLR4 and production of IL1ß. In summary, hyperglycemia enhanced the inflammatory potential of homeostatic, inflammatory, and healing macrophages by increasing specific profiles of TLRs. In combination with dyslipidemic ligands, hyperglycemia can stimulate a low-grade inflammatory program in healing macrophages supporting vascular diabetic complications.

Titanium induces pro-inflammatory and tissue-destructive responses in primary human macrophages.

Implants and medical devices are efficient and practical therapeutic solutions for a multitude of pathologies. Titanium and titanium alloys are used in orthopedics, dentistry, and cardiology. Despite very good mechanical properties, and corrosion resistance titanium implants can fail due to inflammatory or tissue-degradation related complications. Macrophages are major immune cells that control acceptance of failure of the implant. In this study, for the first time, we have performed a systematic analysis of the response of differentially activated human macrophages (M(Control), M(IFNγ) and M(IL-4)) to the polished and porous titanium surfaces in order to identify the detrimental effect of titanium leading to the tissue destruction and chronic inflammation. Transcriptome analysis revealed that the highest number of differences between titanium and control settings are found in M(IL-4) that model healing type of macrophages. RT-qPCR analysis confirmed that both polished and porous titanium affected expression of cytokines, chitinases/chitinase-like proteins and matrix metalloproteinases. Titanium-induced release and activation of MMP7 by macrophages was enhanced by fibroblasts in both juxtacrine and paracrine cell interaction models. Production of titanium-induced MMPs and cytokines associated with chronic inflammation were independent of the presence of Staphylococcus aureus. MMP7, one of the most pronounced tissue-destroying factor and chitinase-like protein YKL-40 were expressed in CD68+ macrophages in peri-implant tissues of patients with orthopedic implants. In summary, we demonstrated that titanium induces pro-inflammatory and tissue-destructing responses mainly in healing macrophages, and the detrimental effects of titanium surfaces on implant-adjacent macrophages are independent on the bacterial contamination.

KAT8-mediated H4K16ac is essential for sustaining trophoblast self-renewal and proliferation via regulating CDX2.

Abnormal trophoblast self-renewal and differentiation during early gestation is the major cause of miscarriage, yet the underlying regulatory mechanisms remain elusive. Here, we show that trophoblast specific deletion of Kat8, a MYST family histone acetyltransferase, leads to extraembryonic ectoderm abnormalities and embryonic lethality. Employing RNA-seq and CUT&Tag analyses on trophoblast stem cells (TSCs), we further discover that KAT8 regulates the transcriptional activation of the trophoblast stemness marker, CDX2, via acetylating H4K16. Remarkably, CDX2 overexpression partially rescues the defects arising from Kat8 knockout. Moreover, increasing H4K16ac via using deacetylase SIRT1 inhibitor, EX527, restores CDX2 levels and promoted placental development. Clinical analysis shows reduced KAT8, CDX2 and H4K16ac expression are associated with recurrent pregnancy loss (RPL). Trophoblast organoids derived from these patients exhibit impaired TSC self-renewal and growth, which are significantly ameliorated with EX527 treatment. These findings suggest the therapeutic potential of targeting the KAT8-H4K16ac-CDX2 axis for mitigating RPL, shedding light on early gestational abnormalities.

Microarray Analysis for Transcriptomic Profiling of Myocardium in Patients with Fatal Myocardial Infarction.

Transcriptomic evidence from human myocardium in myocardial infarction (MI) is still not sufficient. Thus, there is a need for studies on human cardiac samples in relation to the clinical data of patients. The purpose of our pilot study was to investigate the transcriptomic profile of myocardium in the infarct zone, in comparison to the remote myocardium, in patients with fatal MI, via microarray analysis. This study included four patients with fatal MI type 1. We selected histologically verified samples from within the infarct area (n = 4) and remote myocardium (n = 4). The whole transcriptome was evaluated using microarray analysis. Differentially expressed genes (DEGs) clustered in the infarct area and in the remote myocardium allowed their differentiation. We identified a total of 1785 DEGs (8.32%) in the infarct area, including 1692 up-regulated (94.79%) and 93 down-regulated (5.21%) genes. The top 10 up-regulated genes were TRAIL, SUCLA2, NAE1, PDCL3, OSBPL5, FCGR2C, SELE, CEP63, ST3GAL3 and C4orf3. In the infarct area, we found up-regulation of seventeen apoptosis-related genes, eleven necroptosis-related, and six necrosis-related genes. Transcriptome profiling of the myocardium in patients with MI remains a relevant area of research for the formation of new scientific hypotheses and a potential way to increase the translational significance of studies into myocardial infarction.

Specific changes in amino acid profiles in monocytes of patients with breast, lung, colorectal and ovarian cancers.

Introduction: Immunometabolism is essential factor of tumor progression, and tumor-associated macrophages are characterized by substantial changes in their metabolic status. In this study for the first time, we applied targeted amino acid LC-MS/MS analysis to compare amino acid metabolism of circulating monocytes isolated from patients with breast, ovarian, lung, and colorectal cancer. Methods: Monocyte metabolomics was analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/ MS) analysis of amino acid extracts. The targeted analysis of 26 amino acids was conducted by LCMS/MS on an Agilent 6460 triple quadrupole mass spectrometer equipped with an electrospray ionization source and an Agilent 1260 II liquid chromatograph. Results: Comparison of monocytes of cancer patients with monocytes of healthy control individuals demonstrated that in breast cancer most pronounced changes were identified for tryptophan (AUC = 0.76); for ovarian cancer, aminobutyric acid was significantly elevated (AUC= 1.00); for lung cancer significant changes we indented for citrulline (AUC = 0.70). In order to identify key amino acids that are characteristic for monocytes in specific cancer types, we compared each individual cancer with other 3 types of cancer. We found, that aspartic acid and citrulline are specific for monocytes of patients with colorectal cancer (p<0.001, FC = 1.40 and p=0.003, FC = 1.42 respectively). Citrulline, sarcosine and glutamic acid are ovarian cancer-specific amino acids (p = 0.003, FC = 0.78, p = 0.003, FC = 0.62, p = 0.02, FC = 0.78 respectively). Glutamine, methionine and phenylalanine (p = 0.048, FC = 1.39. p = 0.03, FC = 1.27 and p = 0.02, FC = 1.41) are lung cancer-specific amino acids. Ornithine in monocytes demonstrated strong positive correlation (r = 0.63) with lymph node metastasis incidence in breast cancer patients. Methyl histidine and cysteine in monocytes had strong negative correlation with lymph node metastasis in ovarian cancer patients (r = -0.95 and r = -0.95 respectively). Arginine, citrulline and ornithine have strong negative correlation with tumor size (r = -0.78, citrulline) and lymph node metastasis (r = -0.63 for arginine and r = -0.66 for ornithine). Discussion: These alterations in monocyte amino acid metabolism can reflect the reaction of systemic innate immunity on the growing tumor. Our data indicate that this metabolic programming is cancer specific and can be inhibiting cancer progression. Cancer-specific differences in citrulline, as molecular link between metabolic pathways and epigenetic programing, provide new option for the development and validation of anti-cancer therapies using inhibitors of enzymes catalyzing citrullination.