Marc Daigneault

The Identification of Markers of Macrophage Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.

Distinct Cell Death Programs in Monocytes Regulate Innate Responses Following Challenge with Common Causes of Invasive Bacterial Disease

Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1–regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1α upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages.

Neutrophil Elastase Promotes Interleukin-1β Secretion from Human Coronary Endothelium

Background: The mechanism of IL-1 release from endothelial cells is not fully known. Results: Neutrophil elastase causes secretion of bioactive IL-1 from endothelial cells via microvesicles. Conclusion: A mechanistic link between IL-1 secretion from endothelial cells and neutrophil elastase in atherosclerotic plaques is revealed. Significance: Neutrophil elastase could be a potential target for preventing atherosclerosis. The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1β. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1β in the endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1β remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential “trigger” for IL-1β processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1β from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1β in ECs and causes significant secretion of bioactive IL-1β via extracellular vesicles. This release was attenuated significantly by inhibition of neutrophil elastase but not caspase-1. Transient increases in intracellular Ca2+ levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1β was detected within LAMP-1-positive multivesicular bodies. The released vesicles contained bioactive IL-1β. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1β. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1β secretion from ECs. This could reveal additional potential anti-IL-1β therapeutic targets and provide further insights into the inflammatory process by which vascular disease develops.

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3+ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3+ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3+ T-cells in PBMC cultures required ‘classical’ CD14+ monocytes, which enhanced T-cell activation. CD3+ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3+ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.

A decoy receptor 3 analogue reduces localised defects in phagocyte function in pneumococcal pneumonia

Background Therapeutic strategies to modulate the host response to bacterial pneumonia are needed to improve outcomes during community-acquired pneumonia. This study used mice with impaired Fas signalling to examine susceptibility to pneumococcal pneumonia and decoy receptor 3 analogue (DcR3-a) to correct factors associated with increased susceptibility. Methods Wild-type mice and those with varying degrees of impairment of Fas (lpr) or Fas ligand signalling (gld) were challenged with Streptococcus pneumoniae and microbiological and immunological outcomes measured in the presence or absence of DcR3-a. Results During established pneumonia, neutrophils became the predominant cell in the airway and gld mice were less able to clear bacteria from the lungs, demonstrating localised impairment of pulmonary neutrophil function in comparison to lpr or wild-type mice. T-cells from gld mice had enhanced activation and reduced apoptosis in comparison to wild-type and lpr mice during established pneumonia. Treatment with DcR3-a reduced T-cell activation and corrected the defect in pulmonary bacterial clearance in gld mice. Conclusions The results suggest that imbalance in tumour necrosis factor superfamily signalling and excessive T-cell activation can impair bacterial clearance in the lung but that DcR3-a treatment can reduce T-cell activation, restore optimal pulmonary neutrophil function and enhance bacterial clearance during S pneumoniae infection.

200 NEUTROPHIL-DERIVED MICROPARTICLES MODULATE MONOCYTE MIGRATION IN AN ENDOTHELIAL DEPENDENT MANNER

Introduction Neutrophil depletion can delay atherogenesis and conversely increasing circulating neutrophils enhances plaque progression in mice. Lack of evidence for the presence of neutrophils in atherosclerotic plaques makes their role in disease progression less clear. Neutrophils may have a greater role in facilitating the movement of other cells into the vessel wall, possibly through the release of microparticles. The ability of microparticles to deliver cargo to target cells may be an explanation for neutrophil-specific markers being co-localised with endothelial cells in atherosclerotic plaques, despite the absence of neutrophils themselves. Methods Neutrophils were incubated with PBS, fMLP (10–5 M), and acetylated LDL (AcLDL, 20 µg/ml) to stimulate microparticle formation. To remove residual fMLP, microparticle suspensions were dialysed. Microparticles were characterised and quantified using a standardised flow cytometry method. Their adhesion to human coronary artery endothelial cells (hCAEC) after incubation with anti-ICAM-1 or isotype control was quantified using flow cytometry. Internalisation was visualised using confocal microscopy. Microparticle binding to and activation of monocytes was also investigated using flow cytometry. Monocytes migration to CCL-2 in the presence of neutrophil-derived microparticles with and without hCAEC was also investigated. Results Neutrophil-derived microparticles adhered to hCAEC in an ICAM-1 dependent manner and were internalised. Microparticles bound to monocytes regardless of stimuli, but their adhesion was lower than observed with hCAEC. Only microparticles from neutrophils stimulated with fMLP induced monocyte L-selectin shedding and significantly enhanced monocyte-hCAEC adhesion. Microparticles increased monocyte migration to CCL2 in an endothelial cell and CD18-dependent manner. Conclusions Microparticles bind to ICAM-1 on the surface of hCAEC and are internalised. Monocyte migration is enhanced by neutrophil-derived microparticles only in the presence of endothelial cells. In conclusion, neutrophil-derived microparticles may play a role in activating hCAEC, potentially by their uptake, and thus amplify inflammatory responses at sites of atherosclerotic lesion development.

235 TRIBBLES-1 CONTRIBUTES TO MONOCYTE MIGRATION IN EXPERIMENTAL PERITONITIS

Introduction Mitogen activated protein kinase (MAPK) cascades are involved in the regulation of several cellular responses including migration or proliferation. We have shown previously that tribbles-1 (trb-1), a member of the tribbles family of adaptor/scaffold proteins controls vascular smooth muscle proliferation and chemotaxis via a direct interaction with the JNK activator kinase, MKK4. However, a regulatory role in monocyte/macrophage function for members of the tribbles family has also been reported. With the current work, we aimed to gain a better understanding of the early steps of monocyte recruitment under inflammatory conditions, and in particular, the contribution of tribbles-1 to these processes. Methods We induced experimental peritonitis in trb-1 KO and trb-1 transgenic mice, where the expression of this gene was specifically modulated in the monocyte/macrophage lineage, and their littermate controls by injecting 1ml 4% thioglycollate to the peritoneal cavity. The composition of the cells infiltrating the peritoneum was analysed at 0, 6 and 24 hours post-injection by multi-colour flow cytometry. We also measured the cytokine content of the lavage fluids via the BDTM Cytometric Bead Array (CBA). Results At 6 and 24 hours post-injection we found a significant increase in the infiltration of monocytes to the peritoneal cavity in trb-1 KO animals compared to littermate controls, while the trb-1 over-expressing monocytes refrained from entering the peritoneum. We quantified the amount of IL-1a/b, TNFa, MIP1a/b and KC in the samples and found no significant difference in the production of any of these cytokines. Conclusions Trb-1 is involved in the regulation of monocyte migration under inflammatory conditions and therefore may play a significant role in the early steps of monocyte recruitment. Via this mechanism, trb-1 may contribute to the development of chronic inflammation and atherosclerosis. Further investigations will determine whether differences in the expression of leukocyte cell adhesion molecules (CAM) or other signalling abnormalities are responsible for the altered migration pattern of trb-1 KO and trb-1 transgenic monocytes.

208 A NOVEL ROLE FOR THE GAS3/PMP22 FAMILY MEMBER EMP2 IN THE REGULATION OF INFLAMMATION

Introduction Cardiovascular disease such as atherosclerosis is currently the leading cause of death by noncommunicable diseases worldwide. Development of atherosclerosis which is considered a chronic inflammatory disease has been associated with a number of pro-inflammatory cytokines including interleukin-1 (IL-1). IL-1 has been associated with atherosclerotic plaque formation as well as plaque rupture. Previous reports using ApoE −/− /IL1-β−/− mice have described a significant decrease in atherosclerotic area further highlighting the importance of this apical cytokine. IL-1β is released by monocytes and macrophages following P2X7 receptor activation by ATP, however the exact mechanism by which release occurs is poorly understood. We have previously identified the GAS3/PMP22 family member, epithelial membrane protein-2 (EMP2), as a P2X7 C-terminus interacting protein. Blocking EMP2 has been shown to reduce early Chlamydia trachomatis infectivity and modify cytokine secretion; however the function of EMP2 in this role is not described. The purpose of this study was to establish the role of EMP2 in P2X7 receptor dependent IL-1β release. Methods THP1 monocytic cells were transfected with non-targeting control siRNA, siRNA specific for EMP2 or the fluorescent indicator SiGLO, using the reagent Dharmafect Duo with an optimised protocol for THP-1 cells. Transfection efficiency was determined by flow cytometry and the efficiency of the knock-down was assessed by real-time PCR. THP1 cells were then treated with PMA (500 nM) for 3 hours to promote differentiation to a more macrophage like phenotype. Differentiated THP1 cells were stimulated with 1 µg/ml of LPS with and without a P2X7 receptor antagonist (A438079 hydrochloride), followed by BzATP (300 µM, P2X7 agonist) for 20 minutes. Cell supernatants were collected and IL-1β release was measured by ELISA; cytotoxicity was determined by lactate dehydrogenase (LDH) release. Results BzATP treatment of THP1 cells significantly enhanced IL-1β release compared with LPS stimulation alone. P2X7 receptor dependent IL-1β release was almost completely inhibited by pre-treatment with the receptor antagonist. SiRNA knockdown of EMP2 was approximately 40%, as confirmed by real-time PCR, and significantly enhanced P2X7 receptor dependent IL-1β release compared with controls. There was no significant difference in LDH release following stimulation with LPS or BzATP, suggesting that the increase in IL-1β release was not due to cytotoxicity. Conclusions EMP2 contributes to the regulation of P2X7 receptor dependent IL-1β release by differentiated THP1 cells.