Activation Of Peripheral Monocytes Research Articles

A Phase 1 Clinical Trial of IL‐2 in Patients with Alzheimer’s Disease: A Regulatory T Cell Expansion Strategy Targeting Inflammation

AbstractBackgroundRegulatory T cells (Tregs) play a neuroprotective role by suppressing inflammation. We previously documented that Treg immunomodulatory mechanisms are compromised in people with Alzheimer’s disease (AD) and are associated with activation of peripheral monocytes and upregulation of inflammatory mediators. In this original study, we investigated the feasibility and potential impact of low‐dose IL‐2 immunotherapy on restoring Tregs and modifying inflammation in an AD clinical setting.MethodEight patients with mild‐to‐moderate AD dementia (MMSE:12‐25) were enrolled in a phase 1, open‐label, feasibility study of IL‐2 treatment. The presence of elevated brain amyloid, as demonstrated by either CSF or amyloid positron emission tomography (PET) scan, was confirmed in all participants. Enrolled individuals received monthly five‐day‐courses of subcutaneous IL‐2 (1 million units/day) for four months and were followed for an additional two months post‐treatment. Treg immunophenotype and functional analysis were obtained serially at screening, day 1 (before IL‐2 treatment) and day 8 (three days after the fifth dose) of each treatment cycle during therapy, and then at weeks 17 and 24. Two‐sided paired t‐tests were used to assess the statistical significance of changes in each analyte or clinical outcome at each timepoint.ResultOverall, low‐dose IL‐2 immunotherapy was well‐tolerated and all patients received the treatment as planned. The number of CD4+CD25highFoxP3+ Tregs increased two to three‐fold after each IL‐2 treatment cycle (p<0.01) and returned to baseline before the next cycle. Treg suppressive function on responder T cell (Tresp) proliferation also progressively increased through the four‐month IL‐2 treatment phase (p<0.01). IL‐2 treatment down‐regulated IL‐1β, IL‐6 and TNFa transcripts in circulating monocytes and modulated plasma pro‐inflammatory chemokines and cytokines (IL‐15, CCL2, CCL4, CCL11 and FLT3LG; p<0.01). Clinical benefits (mean change ± SE) were observed on the Mini‐Mental State Exam (+2.25 ± 0.59, p = 0.007) and the Clinical Dementia Rating Sum of Boxes (‐0.56 ± 0.24; p = 0.051).ConclusionIL‐2 immunotherapy restored peripheral Treg function and ameliorated systemic pro‐inflammatory mediators in AD patients. The results of this study warrant conducting a well‐controlled clinical study to further evaluate the safety and efficacy of low‐dose IL‐2 as a potential treatment for AD.

Hyperactivation of monocytes and macrophages in MCI patients contributes to the progression of Alzheimer's disease

BackgroundAlzheimer’s disease (AD) is the most common neurodegenerative disease ultimately manifesting as clinical dementia. Despite considerable effort and ample experimental data, the role of neuroinflammation related to systemic inflammation is still unsettled. While the implication of microglia is well recognized, the exact contribution of peripheral monocytes/macrophages is still largely unknown, especially concerning their role in the various stages of AD.ObjectivesAD develops over decades and its clinical manifestation is preceded by subjective memory complaints (SMC) and mild cognitive impairment (MCI); thus, the question arises how the peripheral innate immune response changes with the progression of the disease. Therefore, to further investigate the roles of monocytes/macrophages in the progression of AD we assessed their phenotypes and functions in patients at SMC, MCI and AD stages and compared them with cognitively healthy controls. We also conceptualised an idealised mathematical model to explain the functionality of monocytes/macrophages along the progression of the disease.ResultsWe show that there are distinct phenotypic and functional changes in monocyte and macrophage populations as the disease progresses. Higher free radical production upon stimulation could already be observed for the monocytes of SMC patients. The most striking results show that activation of peripheral monocytes (hyperactivation) is the strongest in the MCI group, at the prodromal stage of the disease. Monocytes exhibit significantly increased chemotaxis, free radical production, and cytokine production in response to TLR2 and TLR4 stimulation.ConclusionOur data suggest that the peripheral innate immune system is activated during the progression from SMC through MCI to AD, with the highest levels of activation being in MCI subjects and the lowest in AD patients. Some of these parameters may be used as biomarkers, but more holistic immune studies are needed to find the best period of the disease for clinical intervention.

Activation and transcriptional profile of monocytes and CD8+ T cells are altered in checkpoint inhibitor-related hepatitis

Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aimed to gain insights into the immunopathology of CPI-Hep by comprehensively characterising myeloid and lymphoid subsets. CPI-treated patients with or without related hepatitis (CPI-Hep; n=22 and CPI-noHep; n=7) were recruited. Phenotypic and transcriptional profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HCs). Invitro monocyte-derived macrophages (MoMFs) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n=4). A significant total monocyte depletion was observed in CPI-Hep compared with HCs (p=0.04), along with a proportionate increase in the classical monocyte population (p=0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HCs (p <0.0001). Invitro MoMFs from CPI-Hep showed enhanced production of pro-inflammatory cytokines. CD8+ T cells demonstrated increased perforin, granzyme B, ICOS and HLA-DR expression in CPI-Hep. Transcriptional profiling indicated the presence of activated monocyte and enhanced effector CD8+ T cell populations in CPI-Hep. Immunohistochemistry demonstrated co-localisation of CD8+/granzyme B+ T cells with CD68+CCR2+/CD68+CD163+ macrophages in CPI-Hep liver tissue. CPI-Hep is associated with activation of peripheral monocytes and an enhanced cytotoxic, effector CD8+ T cell phenotype. These changes were reflected by liver inflammation composed of CD163+/CCR2+ macrophages and CD8+ T cells. Some patients who receive immunotherapy for cancer develop liver inflammation, which requires cessation of cancer treatment. Herein, we describe ways in which the white blood cells of patients who develop liver inflammation differ from those of patients who receive the same immunotherapy but do not experience liver-related side effects. Targeting some of the pathways we identify may help to prevent or manage this side effect and facilitate cancer treatment.

507. Activation of Macrophages Enhances Susceptibility to SARS-CoV-2 Antibody-Dependent Enhancement and Promotes Damage to Downstream Epithelial Cells

BackgroundThe distinct shift in peripheral monocyte activation and infiltration of these cells into the respiratory tract observed in severe cases of COVID-19 suggests that like SARS-CoV-1, the acute respiratory distress syndrome (ARDs) observed in SARS-CoV-2 infections may result from damage to the respiratory epithelia by improperly activated macrophages (MPs). In this study, we examined the ability of non-neutralizing antibodies to sensitize MPs to killing by SARS-CoV-2, as well as the impact of these cells on downstream epithelial cells.MethodsRaw 264.7 cells were seeded into 96-well plates at a density of 1x104/well and incubated overnight in the presence or absence of heat-inactivated LPS derived from either E. coli (EC) or S. enteritidis (Sal). Cells were then treated with non-neutralizing antibodies or vehicle control at the time of infection with SARS-CoV-2. Viability was assessed 48 hours post-infection by luminescence following the addition of CellTiter-Glo® (Promega).ResultsWhile no decrease in cell viability was observed with SARS-CoV-2 alone, the presence of non-neutralizing antibodies against either the nucleocapsid or spike protein of SARS-CoV-2 decreased cell survival to 35.98% and 53.67% of the cell control, respectively (p< 0.0001 and p=0.0003). Activation of MPs with Sal-derived LPS sensitized MPs to viral killing, even in the absence of non-neutralizing antibody (20.12% viability, p< 0.0001). This was not observed in MPs activated by EC LPS. MP activation by both Sal and EC LPS further enhanced viral killing in the presence of anti-nucleocapsid, reducing cell viability to 12.21% (0.0001) and 6.46% (p< 0.0001). Finally, supernatants collected from naïve MPs subjected to ADE markedly increased the susceptibility of Vero E6 cells to SARS-CoV-2 nearly 9.8-fold (p< 0.0001).ConclusionHere we demonstrate that naïve MPs, normally resistant to infection by SARS-CoV-2, are rendered susceptible to viral killing by activation and the presence of non-neutralizing antibodies to SARS-CoV-2. Furthermore, MPs secrete an as yet, unknown factor that enhances the susceptibility of Vero E6 to SARS-CoV-2. Taken together, these data suggest that MPs play an important role in determining the severity of SARS-CoV-2 infection.Disclosures All Authors: No reported disclosures

Tu1823 Early Activation of Peripheral Monocytes With Hallmarks of M1 and M2 Monocytic Cells in Excessive Alcohol Drinkers

Tu1823 Early Activation of Peripheral Monocytes With Hallmarks of M1 and M2 Monocytic Cells in Excessive Alcohol Drinkers

Increased circulating CC chemokine levels in the metabolic syndrome are reduced by low‐dose atorvastatin treatment: evidence from a randomized controlled trial

Central obesity and insulin resistance are key components of the metabolic syndrome, which is associated with an increased risk of cardiovascular disease. In obesity, CC chemokines, such as monocyte chemotactic protein-1 (MCP-1), macrophage inhibitory protein-1β (MIP-1β) and eotaxin-1 and their respective receptors, are critically involved in peripheral monocyte activation and adipose tissue infiltration. The aim of the current study was to examine whether low-dose atorvastatin (10 mg/d) treatment modulated serum levels of CC chemokines in metabolic syndrome subjects. Serum levels of MCP-1, eotaxin-1, MIP-1β, C reactive protein (CRP) and interleukin-6 (IL-6) were measured in lean control and metabolic syndrome subjects at baseline, and following a 6-week randomized placebo-controlled clinical trial of atorvastatin (10 mg/d). Peripheral CD14(+) monocytes were isolated and mRNA levels of MCP-1, MIP-1 β and CCR5 determined. Serum MCP-1 (P = 0·02), eotaxin-1 (P = 0·02) and MIP-1β (P = 0·03), CRP (P < 0·001) and IL-6 (P = 0·006) were significantly increased in metabolic syndrome in comparison with lean controls. Furthermore, CD14(+) peripheral monocyte mRNA expression of the chemokine receptor, CCR5, of which MIP-1β and eotaxin-1 are ligands, was increased two-fold in the metabolic syndrome group (P = 0·03). In addition to the expected improvements in lipid profile, atorvastatin treatment significantly reduced circulating eotaxin-1 (P < 0·05), MIP-1β (P < 0·05) levels and CD14(+) peripheral monocyte CCR5 mRNA expression (P = 0·02). These results support a model whereby atorvastatin treatment, by inhibiting CD14(+) monocyte CCR5 expression, may inhibit monocyte trafficking, reduce chronic inflammation and, thus, lower circulating levels of CC chemokines.

Dyad of CD40/CD40 Ligand Fosters Neuroinflammation at the Blood-Brain Barrier and Is Regulated via JNK Signaling: Implications for HIV-1 Encephalitis

Human immunodeficiency virus 1 (HIV-1) infection may result in activation of peripheral monocytes followed by their infiltration into the CNS, where the release of proinflammatory mediators causes neurologic disease. Previously, we detected high levels of soluble CD40 ligand (CD40L) in CSF and plasma of HIV-infected patients with cognitive impairment. We now show that CD40, a receptor for CD40L, is highly expressed in brain endothelial cells of patients affected by HIV-1 encephalitis (HIVE), suggesting an important role for the CD40/CD40L dyad in regulating blood-brain barrier (BBB) functions. This concept was further supported by in vitro experiments. Exposure of primary human brain microvascular endothelial cells (BMVECs) to CD40L upregulated the expression of adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, which caused a fourfold increase in monocyte adhesion to BMVECs and stimulated migration across an in vitro BBB model. Investigations into the intracellular signaling pathways that govern these events revealed that cJUN-N-terminal kinase (JNK) is critical to CD40 activation in the BMVECs. CD40L induced activation of mixed-lineage-kinase-3 and JNK, leading to the subsequent activation of cJUN/AP-1 (activating-protein-1). JNK inhibition in the BMVECs prevented CD40L-mediated induction of adhesion molecules, monocyte adhesion, and transendothelial migration. These new findings support the concept that the CD40/CD40L dyad plays an important role in HIVE neuroinflammation.

Binding between azurocidin and calreticulin: its involvement in the activation of peripheral monocytes.

We found that azurocidin, a secretory protein in neutrophils, binds to calreticulin, a multifunctional chaperone of the endoplasmic reticulum. Azurocidin is known to induce cytokine production in monocytes, but the mechanism of monocyte activation by azurocidin remains unknown. On the other hand, an antibacterial peptide, KLKLLLLLKLK-NH(2) (L5), is known to bind to cell surface calreticulin of human neutrophils, resulting in their activation to produce O(2)(-). Therefore, we examined whether cell surface calreticulin is involved in the activation of human monocytes by azurocidin to produce IL-6. We found that carlreticulin is in fact located on the surface of monocytes and that the IL-6 production stimulated by an azurucidin is inhibited by anti-calreticulin antibody. Possibly, binding between cell surface calreticulin and azurocidin is prerequisite for the activation of monocytes by azurocidin to produce IL-6.

Recombinant human immunodeficiency virus type 1 (HIV-1) Tat protein inhibits phagolysosomal fusion in human peripheral blood monocytes.

The effect of recombinant HIV-1 Tat protein on different functions of peripheral blood monocytes, such as candidacidal activity and phagolysosomal fusion, was evaluated. HIV-1 Tat protein caused a significant impairment of phagolysosomal fusion at 100 ng/ml (p = 0.018). The inhibitory effect of Tat on phagolysosomal fusion was blocked by the addition of 1 microgram/ml monoclonal anti-Tat antibody. Candidacidal activity of peripheral monocytes was not altered by HIV-1 Tat protein at the concentration of 1 microgram/ml. These results indicate the HIV-1 Tat protein can affect the monocyte microbicidal mechanisms at the phagolysosomal fusion step with little or not effect upon the lytic activity.

Comparison of Activity of Peripheral Monocytes and Splenic Macrophages in the Monocyte Monolayer Assay

Comparison of Activity of Peripheral Monocytes and Splenic Macrophages in the Monocyte Monolayer Assay