Differentiation Of Monocytic THP-1 Cells Research Articles

The Promoter of the Immune-Modulating Gene TIR-Containing Protein C of the Uropathogenic Escherichia coli Strain CFT073 Reacts to the Pathogen's Environment.

The TIR-containing protein C (TcpC) of the uropathogenic Escherichia coli strain CFT073 modulates innate immunity by interfering with the Toll-like receptor and NALP3 inflammasome signaling cascade. During a urinary tract infection the pathogen encounters epithelial and innate immune cells and replicates by several orders of magnitude. We therefore analyzed whether these cell types and also the density of the pathogen would induce the recently defined promoter of the CFT073 tcpC gene to, in time, dampen innate immune responses. Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter. Differentiation of monocytic THP-1 cells to macrophages increased their potential to switch on the promoter. Cell-associated CFT073 displayed the highest promoter activity. Since potassium represents the most abundant intracellular ion and is secreted to induce the NLRP3 inflammasome, we tested its ability to activate the tcpC promoter. Potassium induced the promoter with high efficiency. Sodium, which is enriched in the renal cortex generating an antibacterial hypersalinity, also induced the tcpC promoter. Finally, the bacterial density modulated the tcpC promoter activity. In the search for promoter-regulating proteins, we found that the DNA-binding protein H-NS dampens the promoter activity. Taken together, different cell types and salts, present in the kidney, are able to induce the tcpC promoter and might explain the mechanism of TcpC induction during a kidney infection with uropathogenic E. coli strains.

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.

Intrinsically Disordered Regions/Proteins Compensate For Genomic Economization In Mycobacterium Tuberculosis

Central to the emergence of Mycobacterium tuberculosis (M. tb) as an extremely successful pathogen is its predilection to persist within the hostile host environment. In this context, the role of disordered proteins and linear motif rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection holds special importance. M.tb has a relatively high percentage of IDPs in its proteome among prokaryotes, the significance of which is not known. Moreover, M.tb has evolved through reductive evolution, with the exception of PE‐PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, we analyzed the M.tb proteome and its entire secretome. PE‐PGRS subfamily and the secretome were enriched in disorder and disordered binding sites, implying that structural disorder might be important for host‐pathogen interactions. We studied the PPE37 protein in more detail that carries an N‐terminal PPE domain capable of iron binding followed by two transmembrane domains and a disordered C‐terminal segment harboring number of eukaryotic linear motifs (ELMs). N‐terminal segment caused proliferation and differentiation of monocytic THP‐1 cells into CD11c, DC‐SIGN positive semi‐mature dendritic cells. The C‐terminal segment localizes to macrophage nucleus and induced caspase‐3 dependent apoptosis of host cells. We also studied the structural shift in PE35‐PPE68 and PE32‐PPE65 protein pairs of pathogenesis‐related RD1 and RD8 regions by analyzing their secondary structure through Fourier transformed infrared spectroscopy. These disordered proteins displayed a remarkable structural shift from disorder to order while engaging in the formation of complexes. These proteins are immunogenic individually and enhance pro‐pathogen response; however, their corresponding complexes enhanced the responses manifold in immunized mice. These results support the role of IDPs in performing contrasting activities to modulate the host processes possibly through molecular mimicry and cross talk. The results from our study further reinforce structural dynamics as a strategy to subvert host defence and to compensate genomic content loss due to reductional evolution.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein.

ABSTRACTPathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis.

단핵구세포주 THP-1의 대식세포로의 분화 및 활성화에서 CO의 억제 효과

Carbon monoxide (CO), a reaction product of cytoprotective enzyme heme oxygenase-1 (HO-1), is a gaseous messenger with anti-proliferative, anti-apoptotic, and anti-inflammatory actions in many cell types. Here, we investigated the role of CO on the process of monocyte differentiation induced by phorbol 12-myristate 13-acetate (PMA) in human monocytic THP-1 cells. CORM-2 (tricarbonyldichlororuthenium (II) dimer, Ru₂Cl₄ (CO) 6 ), a CO-releasing compound, decreased a marked cell adherence with a slight reduction of proliferation in monocytic THP-1 cells treated with PMA. And, CORM-2 significantly inhibited expression of differentiation markers such as CD14, CD11b plus CD18 (macrophage-1 antigen, Mac-1 or complement receptor 3, CR3) and phagocytosis of carboxylate-modified red fluorescent latex beads, in PMA-stimulated THP-1 cells. For the further experiments, differentiation of PMA-treated cells was enhanced after the initial 2 days stimulus by removing the PMA-containing media then incubating the cells in fresh media for a another 4 days. And, we observed the secretion of inflammatory cytokines and phagocytosis in differentiated macrophages. Treatment with CORM-2 significantly abolished the secretion of IL-6, TNF-α and phagocytosis using fluorescence-conjugated E. coli (K-12 strain) bioparticles in lipopolysaccharide (LPS)-stimulated differentiated macrophages. In conclusion, these results suggest that CO inhibits the differentiation of monocytic THP-1 cells as well as the activation of differentiated macrophages.

Considering autophagy, β-Catenin and E-Cadherin as innovative therapy aspects in AML.

Considering autophagy, β-Catenin and E-Cadherin as innovative therapy aspects in AML.

Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity

Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

Retinoid X receptor agonists inhibit phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 cells into macrophages

Monocyte/macrophage differentiation is an essential process during atherosclerosis development. The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily, which plays an important regulatory role in many metabolic disorders, including atherosclerosis. The purpose of this study was to investigate the effect of RXR agonist on monocyte/macrophage differentiation in vitro. The THP-1 cell line was differentiated into a macrophage-like phenotype by incubation with phorbol-12-myristate-13-acetate (PMA) in the presence or absence of RXR agonist. The viability of adherent differentiated THP-1 cells was determined by MTT assay. Macrophage surface marker CD11b and CD36 was analyzed by flow cytometry. Phagocytosis was measured by fluorescence-labeled latex beads. The production of Cytokine Tunlornecrosisfactor-alpha (TNF-alpha), Interlaken-12p70 (IL-12p70), and Matrix metalloproteinase-9 (MMP-9), each of which was analyzed by ELISA. In the presence of the RXR agonists 9-cis retinoic acid or SR11237, PMA-induced THP-1 cells became less adherent, showed decreased macrophage-like morphological changes, decreased cell surface antigen CD11b and CD36 expression, and down regulated the phagocytosis of latex beads and the production of TNF-alpha and MMP-9. These data suggest that RXR agonists inhibit PMA-induced THP-1 cell differentiation into macrophage-like cells, which may be helpful in understanding the anti-atherosclerotic effect of RXR and its agonists.

Local and Systemic Activation of the Mononuclear Phagocyte System in Aseptic Loosening of Total Hip Arthroplasty

Osteoarticular prosthesis loosening involves recruitment, activation, and differentiation of mononuclear phagocytes, with a complex and still unclear interplay of local and systemic inflammation. We hypothesized that aseptic hip prosthesis loosening is bound to a coordinated systemic and local activation of the mononuclear phagocyte system (MPS), which can be demonstrated by simultaneous assessment of both compartments. We, therefore, compared systemic and synovial inflammatory cytokines, circulating monocyte activation state, and synovial fluid (SF) ability to induce osteoclastic differentiation. Twenty-seven patients undergoing total hip replacement for aseptic loosening were compared to 30 patients receiving total hip prosthesis for primary osteoarthritis. SF from aseptic loosening patients induced a more effective osteoclast-like differentiation of monocytic THP-1 cells in vitro and a proinflammatory pattern of cytokine production in these osteoclast-like cultures. On the contrary, SF from osteoarthritis patients did not favor osteoclastogenesis and exerted an anti-inflammatory effect through IL-10 upregulation and TNF-alpha inhibition. Peripheral blood monocytes of aseptic loosening patients were primed for activation, with higher TNF-alpha responses than their counterparts in the osteoarthritis group. Finally, cytokine enrichment in SF versus serum was observed in both patient groups: a fivefold increase in synovial TNF-alpha in aseptic loosening patients and a 14-fold increase in synovial IL-10 in osteoarthritis patients. The TNF-alpha/IL-10 ratio was elevated in both systemic and synovial settings from aseptic loosening patients with respect to osteoarthritis patients. Taken together, our results demonstrate the integrated activation of the MPS and suggest the possible use of cytokines in the laboratory workup of prosthesis aseptic loosening.

Iron chelator induces THP-1 cell differentiation potentially by modulating intracellular glutathione levels

Iron chelators have been implicated to modulate certain inflammatory mediators and regulate inflammatory processes. Here we report that iron chelator deferoxamine (DFO) induces differentiation of monocytic THP-1 cells into functional macrophages. DFO rapidly phosphorylated both extracellular signal-regulated kinase (ERK) and p38 kinase. Blockade of ERK signaling by the MEK1/2 inhibitor PD098059 abolished DFO-induced class A scavenger receptor (SR-A) expression and phagocytic activity, indicating that ERK cascades mediate the induction of THP-1 differentiation. In contrast, in cells treated with the p38 inhibitor SB203580 or transfected with the dominant-negative variant of p38 kinase, DFO-mediated ERK activation became more prominent, and the induction of SR-A expression and phagocytic activity were significantly increased. Interestingly, differentiation by DFO was associated with decrease in cellular glutathione (GSH) level. Both MAPK inhibitors did not influence the GSH level; however, treatment with ferric citrate (Fe 3+) or N-acetyl-cysteine, a major precursor of GSH, markedly recovered GSH level to a normal extent, along with the significant decrease of differentiation. Collectively, these results indicate that oxidative stress by DFO and the resulting activation of ERK cascade play dominant roles in the process of THP-1 differentiation, while p38 acts as a negative signal transmitter.

Expression of nuclear receptors and apo E secretion during the differentiation of monocytic THP-1 cells into macrophages.

The human monocytic THP-1 cell line differentiates into macrophage-like cells that secrete apo E after addition of PMA. Using this model, we studied the time course of apo E transcriptional activation and secretion in relation with the expression of nuclear receptors. Upon treatment with PMA, apo E mRNA and protein secretion were triggered with the concomitant increase of LXRalpha, PPARgamma, and PPARbeta mRNA expression levels. PPARalpha was downregulated, RXRalpha expression was unchanged, and RARalpha and VDR showed only transient increases. FXR and SXR transcripts were not detectable. Specific agonists were used to investigate the functional role of these nuclear receptors upon apo E secretion. The LXRalpha ligands T0901317 and 22(R)-hydroxycholesterol were the most potent apo E inducers, followed by the PPARgamma agonist BRL49653. The PPARalpha agonist Wy14,643 was inactive and 1alpha,25-dihydroxyvitamin D3, 9-cis-retinoic acid and all-trans-retinoic acid decreased apo E secretion. Thus, during PMA-induced THP-1 differentiation, there is a sequential and coordinate regulation of apo E and nuclear receptor transcription.