Monocyte-derived Macrophages Of Patients Research Articles

Abstract 17720: The Role of Soluble Guanylyl Cyclase/NLRP3/RHOA/ROCK Axis in Coronary Artery Spasm: A Potential Biomarker and Therapeutic Target

Background: Coronary artery spasm (CAS) can lead to potentially life-threatening acute coronary syndrome. The second messenger cyclic guanosine 3',5'-monophosphate (cGMP), which is regulated by BNP-pGC-cGMP and nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP pathways, regulate not only vasodilation but also the functions of immune cells. However, the significance of sGC, as a primary receptor for NO, is unclear in CAS. Methods: We investigated sGC and downstream signaling in patient monocyte-derived macrophages (PMDMs) and human coronary artery smooth muscle cells (HCASMCs) using computational biology and bioinformatics to correlate macrophage polarization subsets, Lp(a), and inflammasome signaling axes with CAS. Results: Plasma sGC levels were significantly higher in 30 CAS patients compared to 20 non-CAS controls (P<0.001). RNA-sequencing analysis revealed a significant association of sGC expression with NLR Family Pyrin Domain Containing (NLRP)-3 in HCASMCs and induced expression of inflammatory cytokines-specific surface markers. Lp(a) induction promoted the sGC-NLRP3 mediated lipid metabolism axis in PMDMs at both mRNA and protein levels, with elevated M1 polarization. sGC inhibition in HCASMCs reduced RhoA-GTP activity and its downstream effector ROCK, mitigating the Lp(a) effect, while sGC-specific modulator 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) treatment decreased Lp(a)-induced expression of sGC-NLRP3-inflammasome mediators and induced caspase-1, as well as subsequent cleavage of proIL-1β and proIL-18 in VSMCs. Conclusion: sGC/GC molecule in conjunction with the RHOA/ROCK pathway is an early biomarker for CAS. The sGC-NLRP3-inflammasome mediators signaling axis plays an important role in Lp(a)-regulated macrophage polarization and CAS development.

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

Soluble PD-L1 improved direct ARDS by reducing monocyte-derived macrophages

Acute respiratory distress syndrome (ARDS) is common in intensive care units (ICUs), although it is associated with high mortality, no effective pharmacological treatments are currently available. Despite being poorly understood, the role of programmed cell death protein 1 (PD-1) and PD-ligand 1 (PD-L1) axis in ARDS may provide significant insights into the immunosuppressive mechanisms that occur after ARDS. In the present study, we observed that the level of soluble PD-L1 (sPD-L1), a potential activator of the PD-1 pathway, was upregulated in survivors of direct ARDS than in non-survivors. Administration of sPD-L1 in mice with direct ARDS relieved inflammatory lung injury and improved the survival rate, indicating the protective role of sPD-L1 in direct ARDS. Using high-throughput mass cytometry, we found a marked decrease in the number of lung monocyte-derived macrophages (MDMs) with proinflammatory markers, and the protective role of sPD-L1 diminished in ARDS mice with monocyte/macrophage depletion. Furthermore, PD-1 expression increased in the MDMs of patients and mice with direct ARDS. Finally, we showed that sPD-L1 induced MDM apoptosis in patients with direct ARDS. Taken together, our results demonstrated that the engagement of sPD-L1 on PD-1 expressing macrophages resulted in a decrease in pro-inflammatory macrophages and eventually improved direct ARDS. Our study identified a prognostic indicator for patients with direct ARDS and a potential target for therapeutic development in direct ARDS.

S100A9+ MDSC and TAM-mediated EGFR-TKI resistance in lung adenocarcinoma: the role of RELB.

BackgroundMonocytic myeloid-derived suppressor cells (MDSCs), particularly the S100A9+ subset, has been shown initial clinical relevance. However, its role in EGFR-mutated lung adenocarcinoma, especially to EGFR-tyrosine kinase inhibitor (EGFR-TKI) is not clear. In a clinical setting of EGFR mutated lung adenocarcinoma, a role of the MDSC apart from T cell suppression was also investigated.ResultsBlood monocytic S100A9+ MDSC counts were higher in lung cancer patients than healthy donors, and were associated with poor treatment response and shorter progression-free survival (PFS). S100A9+ MDSCs in PBMC were well correlated to tumor infiltrating CD68+ and S100A9+ cells, suggesting an origin of TAMs. Patient’s MDMs, mostly from S100A9+ MDSC, similar to primary alveolar macrophages from patients, both expressed S100A9 and CD206, attenuated EGFR-TKI cytotoxicity. Microarray analysis identified up-regulation of the RELB signaling genes, confirmed by Western blotting and functionally by RELB knockdown.ConclusionsIn conclusion, blood S100A9+ MDSC is a predictor of poor treatment response to EGFR-TKI, possibly via its derived TAMs through activation of the non-canonical NF-κB RELB pathway.MethodsPatients with activating EGFR mutation lung adenocarcinoma receiving first line EGFR TKIs were prospectively enrolled. Peripheral blood mononuclear cells (PBMCs) were collected for MDSCs analysis and for monocyte-derived macrophages (MDMs) and stored tissue for TAM analysis by IHC. A transwell co-culture system of MDMs/macrophages and H827 cells was used to detect the effect of macrophages on H827 and microarray analysis to explore the underlying molecular mechanisms, functionally confirmed by RNA interference.

MerTK-mediated regulation of myelin phagocytosis by macrophages generated from patients with MS.

Objective:To document functional differences between monocyte-derived macrophages (MDMs) of patients with MS and the ability of age/sex-matched healthy donor cells to phagocytose human myelin and to investigate the molecular mechanisms that underlie this.Methods:MDMs were derived from peripheral blood monocytes of 25 untreated patients with relapsing-remitting MS and secondary progressive MS and age/sex-matched healthy controls (HCs). Phagocytosis was assessed by flow cytometry using fluorescently labeled human myelin. Quantification of messenger RNA and protein expression of Tyro3, Axl, and MerTK family molecules was determined by quantitative PCR, Western blotting, and flow cytometry.Results:Cells of patients with MS display a reduced ability to phagocytose human myelin but not red blood cells as compared to matched HCs. These cells express significantly lower levels of the phagocytic tyrosine kinase receptor, MerTK, and its natural ligand, growth arrest-specific 6, independently of the activation state of the cells. Increased expression of interleukin 10 following myelin uptake by healthy donor cells is lost in MDMs of patients with MS; this effect is mediated through the MerTK pathway. Treatment of MS cells with transforming growth factor β (TGFβ) restored both phagocytosis and expression deficits.Conclusions:We describe a molecular mechanism that underlies a defect in myelin phagocytosis by macrophages generated from patients with MS. This abnormality involves decreased expression of MerTK and its ligands and can be rescued by treatment with TGFβ.

A novel missense mutation in the NADPH binding domain of CYBB abolishes the NADPH oxidase activity in a male patient with increased susceptibility to infections

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X-linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-γ). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91+CGD patient by rhIFN-γ.

Oxidation and uptake of LDL by monocyte-derived macrophages from blood of patients with IHD

The main goal of this study was to test our hypothesis that monocyte-derived macrophages of patients with ischemic heart disease (IHD, MPIHD) are in vivo prestimulated (primed) or stimulated cells. Their capacity for LDL oxidation and uptake exceeds that of macrophages from healthy donors (MPN). Monocytes were isolated from blood of 18 healthy donors and 25 IHD patients and LDL preparations were obtained from plasma of 16 healthy donors (LDLN) and 15 patients with familial hypercholesterolemia (LDLH). Aerobic incubation of LDLN or LDLH with MPIHD resulted in earlier accumulation (by 1 h) of TBARS in LDL, earlier aggregation of LDL (1 h vs 3 h in the case of MPN), more pronounced LDL apoB fragmentation as well as increased LDL uptake with the increase in accumulation of total cholesterol (TCh; by 1.8–2.1-fold, p < 0.05–0.01) and the decrease in cell viability compared with MPN (p < 0.01). MPIHD and MPN exhibited more effective oxidation and uptake of LDLH than LDLN and in most tests this capacity (to oxidize and uptake LDL) increased under hypoxic conditions. These results demonstrate that macrophages of IHD patients are in vivo stimulated cells and that this stimulation, especially in combination with hypercholesterolemic LDL and local or generalized hypoxia, represent serious predisposition for onset or progression of atherosclerosis in IHD patients. The express test model based on MPIHD may be used for estimation of monocyte/macrophage stimulation in IHD patients as well as for optimization of drug therapy and screening new antiatherosclerotic and antiischemic drugs.

Specific and scavenger low-density lipoprotein receptors involved in the disturbed lipid metabolism of patients with non—insulin-dependent diabetes mellitus are independent of obesity

Comparative studies were performed on monocyte-derived macrophages (MDMs), prepared by a 72-hour incubation of blood monocytes obtained from patients with non—insulin-dependent diabetes mellitus (NIDDM) and age-matched obese and non-obese controls. The MDMs, after a 72-hour culturing, expressed both specific and scavenger low-density lipoprotein (LDL) receptors on their surfaces. To study the binding capacity of both receptor types, [ 125I]LDL and [ 125I] acetylated LDL (acLDL) were applied to cells and the labeled ligands were then monitored to estimate the rate of intracellular degradations. The LDL-induced inhibition of endogenous cholesterol synthesis and the acLDL-triggered apolipoprotein (apo) E secretion were also studied, as the biological marker of receptor activation. The results indicate that the binding capacities of both specific and scavenger LDL receptors were not reduced in MDMs of diabetic patients. However, the intracellular degradation after LDL incorporation was decreased. The LDL-induced inhibition of cholesterol synthesis and the acLDL-transmitted apo E secretion were also found to be decreased in the MDMs of patients with NIDDM as compared with the obese and non-obese control groups. The NIDDM-induced impaired signal transduction of both specific and scavenger LDL receptors suggests an unclarified functional alteration of both receptor structures.

Adenovirus-mediated gene transfer into monocyte-derived macrophages of patients with X-linked chronic granulomatous disease: ex vivo correction of deficient respiratory burst.

The underlying cause of X-linked chronic granulomatous disease (X-CGD) is mutations in the gp91phox coding gene. Gp91phox is the larger subunit of the cytochrome b558, the membrane-bound terminal redox centre of the respiratory burst oxidase (NADPH oxidase). We have constructed a recombinant adenovirus which contains a functional copy of the human gp91phox cDNA under the control of the cytomegalovirus (CMV) enhancer/promoter region. This vector was used to infect monocyte-derived macrophages of gp91phox-deficient CGD patients. Expression of the gp91phox transgene resulted in ex vivo reconstitution of the respiratory burst activity. Nitroblue-tetrazolium staining indicated that 74% of the patient cells could be phenotypically corrected when compared with a corresponding control culture of normal monocyte-derived macrophages. Adenoviral gene transfer may become a promising tool and gain therapeutical potential by the targeting of autologous monocytes. Genetically corrected autologous monocytes may be used for supportive treatment of X-CGD patients to overcome acute life-threatening infections. Establishment of adenovirus-mediated reconstitution of gp91phox-deficient monocytes therefore represents a first step towards the development of a clinically applicable supportive transient somatic gene therapy in CGD.

Enhanced Low-Density Lipoprotein Degradation and Cholesterol Synthesis in Monocyte-Derived Macrophages of Patients with Adult Xanthogranulomatosis

Adult xanthogranulomatosis is an uncommon disorder in which dermal macrophages accumulate cholesterol intracellularly despite normal plasma cholesterol levels. In an attempt to elucidate an underlying biochemical abnormality in this disorder, we studied the rates of 125I-labeled low-density lipoprotein degradation, and intracellular cholesterol synthesis, in human monocyte-derived macrophages of three patients with adult xanthogranulomatosis. In all three patients, the rates of cellular 125I-low-density lipoprotein degradation and of cholesterol synthesis were 22-37% and 14-84% higher than those of the respective normal controls (p < 0.01). These findings suggest that in MDM of adult xanthogranulomatosis patients, the uptake and degradation of low-density lipoprotein-derived cholesterol and intracellular cholesterol biosynthesis are enhanced. Because dermal macrophages are derived from blood monocytes, it is possible that such an enhancement might play a role in the accumulation of cholesteryl esters in the macrophages that form the xanthogranulomatosis lesions.

Receptor Function of Low-Density Lipoproteins in Werner’s Syndrome

Abnormal lipoprotein metabolism and its cause were studied in 10 patients with Werner's syndrome. Seven of the 10 patients had hypercholesterolemia (greater than 250 mg dl-1). A significant positive correlation (p less than 0.01) was found between serum total cholesterol levels and LDL receptor activity. In monocyte-derived macrophages of patients with this syndrome, the uptake, lysosomal hydrolysis and re-esterification of free cholesterol are enhanced. This abnormal accumulation of cholesterol ester may cause accelerated conversion of macrophages to foam cells in Werner's syndrome.

Metabolism of acetylated low density lipoproteins by monocyte-derived macrophages from patients with Werner's syndrome.

Accumulation of lipid-laden foam cells of monocyte origin plays an important role in atherogenesis. Therefore, for determination of the mechanism of accelerated atherogenesis in Werner's syndrome, studies were carried out on the metabolism of acetylated low density lipoprotein (LDL) by monocyte-derived macrophages from patients with this syndrome. These macrophages showed abnormally high activities for degradation and uptake of 125I-acetylated LDL, incorporation of 14C-oleate into cellular cholesteryl ester in the presence of acetylated LDL, and accumulation of cholesteryl ester derived from internalized 3H-cholesteryl linoleate-labeled acetylated LDL. However, these macrophages showed normal binding of 125I-acetylated LDL. These results indicate that in monocyte-derived macrophages of patients with Werner's syndrome, the uptake, lysosomal hydrolysis, and re-esterification of free cholesterol are enhanced with no change in the receptor binding of acetylated LDL. As a result, these macrophages show increased accumulation of cholesteryl ester derived from acetylated LDL. This abnormal enhancement of cholesteryl ester accumulation may cause an accelerated conversion of macrophages to foam cells in Werner's syndrome.