S100A9+CD14+ monocytes contribute to anti-PD-1 immunotherapy resistance in advanced hepatocellular carcinoma by attenuating T cell-mediated antitumor function.

BACKGROUND: The paucity of reliable biomarkers for predicting immunotherapy efficacy in patients with advanced hepatocellular carcinoma (HCC) has emerged as a burgeoning concern with the expanding use of immunotherapy. This study endeavors to delve into the potential peripheral biomarkers capable of prognosticating efficacy in HCC patients who are poised to receive anti-PD-1 monotherapy within the phase III clinical trial, KEYNOTE394. Additionally, we sought to elucidate the underlying molecular mechanisms for resistance to immune checkpoint blockade (ICB) and propose innovative combination immunotherapy strategies for future clinical application. METHODS: Patient blood samples were collected for single-cell RNA sequencing to evaluate the immune cell signature before receiving ICB therapy. Subsequently, in vitro assays and in vivo murine model experiments were conducted to validate the mechanism that S100A9+CD14+ monocytes play a role in ICB resistance. RESULTS: Our study demonstrates a notable enrichment of S100A9+CD14+ monocytes in the peripheral blood of patients exhibiting suboptimal responses to anti-PD-1 therapy. Moreover, we identified the Mono_S100A9 signature as a predictive biomarker, indicative of reduced efficacy in immunotherapy and decreased survival benefits across various tumor types. Mechanistically, S100A9 activates PD-L1 transcription by directly binding to the CD274 (PD-L1) gene promoter, thereby suppressing T-cell proliferation and cytotoxicity via the PD-1/PD-L1 axis, consequently diminishing the therapeutic effectiveness of subsequent anti-PD-1 treatments. Furthermore, our in vivo studies revealed that inhibiting S100A9 can synergistically enhance the efficacy of anti-PD-1 drugs in the eradication of hepatocellular carcinoma. CONCLUSIONS: Our study underscores the significance of S100A9+CD14+ monocytes in predicting inadequate response to ICB treatment and provides insights into the monocyte cell-intrinsic mechanisms of resistance to ICB therapy. We also propose a combined therapeutic approach to enhance ICB efficacy by targeting S100A9.

The upregulation of miR-3945 in CD14+ monocytes is the risk factor for cardiovascular disease.

Patients with type 1 diabetes (T1DM) show an increased risk of cardiovascular disease. Bioinformatics analysis revealed that it is characterized by changes in the function of CD14+ mononuclear macrophages. The current study was to explore the potential relationship between the miR-3845/TRIM35/PKM2 and abnormal polarization of mononuclear macrophages. Using bioinformatics to analyze the gene expression of mononuclear macrophages. The polarization of macrophages was analyzed using flow cytometry, and the expression changes of TRIM35/PKM2 were analyzed using Western blot, luciferase activity assay, and co-immunoprecipitation. Database analysis showed that T1DM patients showed an abnormal increase of miR-3945 in CD14+ monocyte macrophages. miR-3945 targets TRIM35 to release PKM2 to cytometry, and PKM2 causes M1-like polarization of mononuclear macrophages. Database analysis showed that miR-3945 was abnormally upregulated in CD14+ monocytes in T1DM patients. miR-3945 upregulates the expression of PKM2 in the cytoplasm by targeting TRIM35, which leads to M1 polarization of macrophages.

Circulating Monocytes Act as a Common Trigger for the Calcification Paradox of Osteoporosis and Carotid Atherosclerosis via TGFB1-SP1 and TNFSF10-NFKB1 Axis.

Background: Osteoporosis often occurs with carotid atherosclerosis and causes contradictory calcification across tissue in the same patient, which is called the "calcification paradox". Circulating monocytes may be responsible for this unbalanced ectopic calcification. Here, we aimed to show how CD14+ monocytes contribute to the pathophysiology of coexisting postmenopausal osteoporosis and carotid atherosclerosis. Methods: We comprehensively analyzed osteoporosis data from the mRNA array dataset GSE56814 and the scRNA-seq dataset GSM4423510. Carotid atherosclerosis data were obtained from the GSE23746 mRNA dataset and GSM4705591 scRNA-seq dataset. First, osteoblast and vascular SMC lineages were annotated based on their functional expression using gene set enrichment analysis and AUCell scoring. Next, pseudotime analysis was applied to draw their differentiated trajectory and identify the key gene expression changes in crossroads. Then, ligand-receptor interactions between CD14+ monocytes and osteoblast and vascular smooth muscle cell (SMC) lineages were annotated with iTALK. Finally, we selected calcification paradox-related expression in circulating monocytes with LASSO analysis. Results: First, we found a large proportion of delayed premature osteoblasts in osteoporosis and osteogenic SMCs in atherosclerosis. Second, CD14+ monocytes interacted with the intermediate cells of the premature osteoblast and osteogenic SMC lineage by delivering TGFB1 and TNFSF10. This interaction served as a trigger activating the transcription factors (TF) SP1 and NFKB1 to upregulate the inflammatory response and cell senescence and led to a retarded premature state in the osteoblast lineage and osteogenic transition in the SMC lineage. Then, 76.49% of common monocyte markers were upregulated in the circulating monocytes between the two diseases, which were related to chemotaxis and inflammatory responses. Finally, we identified 7 calcification paradox-related genes on circulating monocytes, which were upregulated in aging cells and downregulated in DNA repair cells, indicating that the aging monocytes contributed to the development of the two diseases. Conclusions: Our work provides a perspective for understanding the triggering roles of CD14+ monocytes in the development of the calcification paradox in osteoporosis- and atherosclerosis-related cells based on combined scRNA and mRNA data. This study provided us with an elucidation of the mechanisms underlying the calcification paradox and could help in developing preventive and therapeutic strategies.

Activin-A Induces Fewer, but Larger Osteoclasts From Monocytes in Both Healthy Controls and Fibrodysplasia Ossificans Progressiva Patients.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease characterized by heterotopic ossification (HO) that occurs in muscle tissue, tendons, and ligaments. The disease is caused by mutations in the Activin receptor type I (ACVR1) gene resulting in enhanced responsiveness to Activin-A. Binding of this molecule to the mutated receptor induces HO. Bone metabolism normally requires the coupled action of osteoblasts and osteoclasts, which seems to be disturbed during HO. We hypothesize that Activin-A may also counteract the formation of osteoclasts in FOP patients. In this study we investigated the effect of Activin-A on osteoclast differentiation of CD14+ monocytes from FOP patients and healthy controls. The lymphocytic and monocytic cell populations were determined by FACS analysis. Expression of the mutated R206H receptor was assessed and confirmed by allele specific PCR. The effect of Activin-A on osteoclastogenesis was assessed by counting the number and size of multinucleated cells. Osteoclast activity was determined by culturing the cells on Osteo Assay plates. The influence of Activin-A on expression of various osteoclast related genes was studied with QPCR. Blood from FOP patients contained similar percentages of classical, intermediate, or non-classical monocytes as healthy controls. Addition of Activin-A to the osteoclastogenesis cultures resulted in fewer osteoclasts in both control and FOP cultures. The osteoclasts formed in the presence of Activin-A were, however, much larger and more active compared to the cultures without Activin-A. This effect was tempered when the Activin-A inhibitor follistatin was added to the Activin-A containing cultures. Expression of osteoclast specific genes Cathepsin K and TRAcP was upregulated, gene expression of osteoclastogenesis related genes M-CSF and DC-STAMP was downregulated by Activin-A. Since Activin-A is a promising target for inhibiting the formation of HO in FOP, it is important to know its effects on both osteoblasts and osteoclasts. Our study shows that Activin-A induces fewer, but larger and more active osteoclasts independent of the presence of the mutated ACVR1 receptor. When considering FOP as an Activin-A driven disease that acts locally, our findings suggest that Activin-A could cause a more pronounced local resorption by larger osteoclasts. Thus, when targeting Activin-A in patients with neutralizing antibodies, HO formation could potentially be inhibited, and osteoclastic activity could be slightly reduced, but then performed dispersedly by more and smaller osteoclasts.

Response profiles of circulating leukocytes and metal ions in patients with a modular dual-mobility hip implant.

INTRODUCTION: The purpose of this study was to retrospectively evaluate blood metal ion levels and leukocyte profiles in patients with modular dual-mobility hip implant (MDM) during a postoperative follow-up up to 2 years. METHODS: We recruited 49 patients in a retrospective cohort study and had postoperative follow-up up to 2 years. Blood concentrations of chromium (Cr), cobalt (Co) and serum cytokines were measured. Flow cytometry was used to quantify the subpopulations of leukocytes, including CD14+ and CD16+ monocytes, CD3+ T lymphocytes, CD19+ B lymphocytes, CD4+ Helper T-cells and CD45+RA memory vs. naïve T-cells. RESULTS: Clinical performances of implants were good during 2 years of follow-up. Cr levels were normal in all patients and only detectable in 1 patient (1.4µg/L, ref < 5.0µg/L). Co levels were mildly elevated in 4 patients at 1 year (mean 1.375µg/L, range 1.2-1.7µg/L, ref < 1.0µg/L) and in 2 patients at 2-year follow-up (both 1.2µg/L). Interestingly, Co level observed in 3 patients at 1 year converted to undetectable at their 2-year follow-up. Percentages of B cells, T cells and their subpopulations were within normal levels. There was no increase of CD16+ inflammatory monocytes. DISCUSSION: With the recent introduction of MDM systems there is potential for metal ion release from the interface between the acetabular shell and CoCr liner. Clinical results have been good and metal levels undetectable or within acceptable ranges at 1-2 years. There was no evidence of activated immune response, as manifested by constant circulating leukocyte profiles and no increase of CD16+ inflammatory monocytes.

Increased Expression of T Cell Immunoglobulin and Mucin Domain 3 on CD14+ Monocytes Is Associated with Systemic Inflammatory Reaction and Brain Injury in Patients with Spontaneous Intracerebral Hemorrhage.

OBJECTIVE: To study the expression of T cell immunoglobulin and mucin domain 3 (Tim-3) on peripheral blood immunocytes, and the relationship between Tim-3 and the systemic inflammatory response or brain injury in patients with intracerebral hemorrhage (ICH). METHODS: According to the volume of hematoma at 12 hours after onset of ICH, 60 newly diagnosed patients with ICH were divided into the small (volume of hematoma <30 mL, 30 cases) and large (volume of hematoma ≥30 mL, 30 cases) ICH groups. The expression of Tim-3 on peripheral blood immunocytes was analyzed by flow cytometry. Real-time reverse transcriptase polymerase chain reaction was used to detect Tim-3 mRNA on peripheral blood mononuclear cells (PBMCs). Meanwhile, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and S-100B protein were measured by enzyme-linked immunosorbent assay. Glasgow outcome scale (GOS) score at Day 30 was used to estimate prognosis of patients. RESULTS: The leukocyte count, neutrophil count, monocyte count, TNF-α, IL-1ß, and S-100B protein increased remarkably after ICH. However, all of them in the large ICH group increased more obviously, and there were significant differences when compared with those in the small ICH group (P < .01). The expression of Tim-3 mRNA on PBMCs in the large ICH group increased remarkably, peaked at Day 3, and was positively associated with the concentrations of TNF-α, IL-1ß, and S-100B protein (P < .01). Tim-3 was predominantly expressed itself on CD14+ monocytes. There was a negative correlation between GOS score and Tim-3 mRNA, TNF-α, IL-1ß, or S-100B protein. CONCLUSIONS: The expression of Tim-3 on CD14 + monocytes involves in systemic inflammatory reaction after ICH and may be a novel treatment target.

Tobacco exposure-related alterations in DNA methylation and gene expression in human monocytes: the Multi-Ethnic Study of Atherosclerosis (MESA).

Alterations in DNA methylation and gene expression in blood leukocytes are potential biomarkers of harm and mediators of the deleterious effects of tobacco exposure. However, methodological issues, including the use of self-reported smoking status and mixed cell types have made previously identified alterations in DNA methylation and gene expression difficult to interpret. In this study, we examined associations of tobacco exposure with DNA methylation and gene expression, utilizing a biomarker of tobacco exposure (urine cotinine) and CD14+ purified monocyte samples from 934 participants of the community-based Multi-Ethnic Study of Atherosclerosis (MESA). Urine cotinine levels were measured using an immunoassay. DNA methylation and gene expression were measured with microarrays. Multivariate linear regression was used to test for associations adjusting for age, sex, race/ethnicity, education, and study site. Urine cotinine levels were associated with methylation of 176 CpGs [false discovery rate (FDR)<0.01]. Four CpGs not previously identified by studies of non-purified blood samples nominally replicated (P value<0.05) with plasma cotinine-associated methylation in 128 independent monocyte samples. Urine cotinine levels associated with expression of 12 genes (FDR<0.01), including increased expression of P2RY6 (Beta ± standard error = 0.078 ± 0.008, P = 1.99 × 10-22), a gene previously identified to be involved in the release of pro-inflammatory cytokines. No cotinine-associated (FDR<0.01) methylation profiles significantly (FDR<0.01) correlated with cotinine-associated (FDR<0.01) gene expression profiles. In conclusion, our findings i) identify potential monocyte-specific smoking-associated methylation patterns and ii) suggest that alterations in methylation may not be a main mechanism regulating gene expression in monocytes in response to cigarette smoking.

Acute high-intensity interval exercise reduces human monocyte Toll-like receptor 2 expression in type 2 diabetes.

Type 2 diabetes (T2D) is characterized by chronic low-grade inflammation that contributes to disease pathophysiology. Exercise has anti-inflammatory effects, but the impact of high-intensity interval training (HIIT) is not known. The purpose of this study was to determine the impact of a single session of HIIT on cellular, molecular, and circulating markers of inflammation in individuals with T2D. Participants with T2D (n = 10) and healthy age-matched controls (HC; n = 9) completed an acute bout of HIIT (7 × 1 min at ~85% maximal aerobic power output, separated by 1 min of recovery) on a cycle ergometer with blood samples obtained before (Pre), immediately after (Post), and at 1 h of recovery (1-h Post). Inflammatory markers on leukocytes were measured by flow cytometry, and TNF-α was assessed in both LPS-stimulated whole blood cultures and plasma. A single session of HIIT had an overall anti-inflammatory effect, as evidenced by 1) significantly lower levels of Toll-like receptor (TLR) 2 surface protein expression on both classical and CD16+ monocytes assessed at Post and 1-h Post compared with Pre (P < 0.05 for all); 2) significantly lower LPS-stimulated TNF-α release in whole blood cultures at 1-h Post (P < 0.05 vs. Pre); and 3) significantly lower levels of plasma TNF-α at 1-h Post (P < 0.05 vs. Pre). There were no differences between T2D and HC, except for a larger decrease in plasma TNF-α in HC vs. T2D (group × time interaction, P < 0.05). One session of low-volume HIIT has immunomodulatory effects and provides potential anti-inflammatory benefits to people with, and without, T2D.

TLR2 and TLR4 expression on CD14(++) and CD14(+) monocyte subtypes in adult-onset autoimmune diabetes.

BACKGROUND: Peripheral blood monocytes are key effectors of innate immunity. Dysfunction, changes in their counts or altered expression of cytokines and pattern-recognition receptors on monocytes may contribute to the development of the autoimmune type of diabetes mellitus (AD). AIMS: We aimed to analyze the counts and proportions of the two main subtypes of monocyte cells, CD14(++) and CD14(+), and to look for potential changes in the expression of toll-like receptors 2 (TLR2) and 4 (TLR4) as well as cytokine prolactin (PRL) in adult-onset AD, including diabetes mellitus type 1 (T1DM) and latent autoimmune diabetes in adults (LADA). METHODS: We examined 21 T1DM patients, 9 patients with LADA, 16 control patients with type 2 diabetes mellitus (T2DM) and 24 healthy individuals. All diabetic patients were diagnosed after the age of 18 years. Expression at the mRNA level was determined by quantitative PCR. Flow cytometry was used to ascertain membrane expression and cell counts. RESULTS: T1DM patients had fewer CD14(++) (P < 0.01) and CD14(+) (P < 0.0001) monocytes whereas T2DM subjects showed decreased counts of CD14(+) monocytes compared to healthy controls (P < 0.001). TLR2 protein expression was significantly increased in T1DM CD14(+) monocytes compared to healthy controls (P < 0.05), while TLR4 expression in T1DM CD14(++) cells was significantly lower (P < 0.0001). There was no significant difference between the groups in terms of PRL mRNA expression in monocytes. CONCLUSIONS: The observed changes in the proportions of both immune cell types and in the expression of functional pattern-recognition receptors on monocytes in the subjects examined may arise as a consequence of chronic inflammation that accompanies long-term diabetes.