AIM： To investigate the effect of short-chain fatty acids （SCFAs） on production of prostaglandin E2 （PGE2）, cytokines and chemokines in human monocytes.
METHODS： Human neutrophils and monocytes were ...isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative realtime polymerase chain reaction, The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells （PBMC） was studied by measuring PGE2, cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws.
RESULTS： Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE2 and that this effect can be enhanced in the presence of lipopolysaccharide （LPS）. In addition, we demonstrate that PGE2 production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 （MCP-1） production and LPS-induced interleukin-10 （IL-10） production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE2, MCP-1 and IL-10 after 5CFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-α and interferon-7 in human PBIVlC. Finally, we show that SCFAs and LPS can induce PGE2 production in vivo by intraplantar injection into rat paws （P 〈 0.01）.
CONCLUSION： SCFAs can have distinct antiinflammatory activities due to their regulation of PGE2, cytokine and chemokine release from human immune cells.
An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different ...size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5-10 μm in size displayed elevated cytokine release profiles compared with other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared with controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by microflow imaging, transmission electron microscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in vitro PBMC studies to rank-order the immunogenic potential of various types of mAb particles are discussed.
Pre-pregnancy (pregravid) obesity has been linked to several adverse health outcomes for both mother and offspring. Complications during pregnancy include increased risk for gestational diabetes, ...hypertension, preeclampsia, placental abruption, and difficulties during delivery. Several studies suggest that these negative outcomes are mediated by heightened systemic inflammation as well as changes in placental development and function. However, the molecular mechanisms by which pregravid obesity affects these processes are poorly understood. In this study, we aimed to address this question by carrying out a comprehensive analysis of the systemic maternal immune system coupled with placental gene expression and microbial profiling at term delivery (11 lean and 14 obese). Specifically, we examined the impact of pregravid obesity on circulating cytokines, chemokine, adipokines, and growth factors using multiplex Luminex assay. Innate and adaptive immune cell frequencies and their cytokine production in response to stimuli were measured using flow cytometry. Finally, changes in placental transcriptome and microbiome were profiled using RNA- and 16S-sequencing, respectively. Pregravid obesity is characterized by insulin and leptin resistance, high levels of circulating inflammatory markers IL-6 and CRP, in addition to chemokine IL-8 (
< 0.01). Moreover, pregravid obesity was associated with lower frequency of naïve CD4+ T-cells (
< 0.05), increased frequency of memory CD4+ T-cells (
< 0.01), and a shift towards Th2 cytokine production (
= 0.05). Myeloid cells from the obese cohort produced higher levels of pro-inflammatory cytokines but lower levels of chemokines following TLR stimulation (
< 0.05). Lastly, pregravid obesity is associated with increased abundance of Bacteroides and changes in the expression of genes important for nutrient transport and immunity (FDR < 0.05). Collectively, these data indicate that pregravid obesity is associated with heightened systemic inflammation and of dysregulated nutrient transport in the placenta and provide insight into the basis of fetal reprogramming.
Environmental pollution by microplastics (MPs) is a growing concern regarding their impact on aquatic and terrestrial systems and human health. Typical exposure routes of MPs are dermal contact, ...digestion, and inhalation. Recent in vitro and in vivo studies observed alterations in immunity after MPs exposure, but systemic studies using primary human immune cells are scarce. In our investigation, we addressed the effect of polystyrene (PS) and poly methyl methacrylate (PMMA) in three different sizes (50–1100 nm) as well as amino-modified PS (PS-NH2; 50 nm) on cells of the adaptive and innate immune system. T-cells isolated from human peripheral blood mononuclear cells (PBMCs) were least affected regarding the cytotoxicity but displayed increased activation marker expression after 72 h, and strongly modulated cytokine secretion patterns. Conversely, phagocytic dendritic cells and macrophages derived from isolated monocytes were highly sensitive to pristine MPs. Their marker expression suggested a downregulation of the inflammatory phenotypes indicative of M2 macrophage induction after MPs exposure for 24 h. Our results showed that even pristine MPs affected immune cell function and inflammatory phenotype dependent on MPs polymers, size, and immune cell type.
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•First comprehensive study on all major immune cell populations and their responses to plastic particles.•Aminated particles, but only if small, show greatest toxicity.•Macrophages are more sensitive to aminated particles than dendritic cells, with T-cells being most robust.•Plastic particle exposure affects immune checkpoint marker expression on all immune cell subpopulations tested.
•MIF promotes a differential Th1/Th2/Th17 inflammatory response in PBMC cultures.•PBMCs from healthy subjects showed a predominance of Th17 cytokine profile.•PBMCs from active SLE patients showed an ...inflammatory profile with IL-6 and TNF-α.
Macrophage migration Inhibitory Factor (MIF) is a cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive-feedback-loop with proinflammatory cytokines and could perpetuate the inflammatory process in Systemic Lupus Erythematosus (SLE).The aim of this study was to assess the effect of recombinant-human-MIF (rhMIF) on the expression of Th1, Th2 and Th17 cytokines in Peripheral Blood Mononuclear Cells (PBMC) from Healthy Subjects (HS) and SLE patients. The PBMC were isolated from SLE patients classified according to the 1997 SLE ACR criteria and HS donors; all subjects included were women from an unrelated Mexican-Mestizo population. The PBMC isolated were stimulated with rhMIF, LPS and ISO-1 in different combinations; Th1, Th2 and Th17cytokine profiles levels were determined by MAGPIX Bio-plex assay in supernatants from cell cultures. We observed in supernatants of PBMCs from HS treated with rhMIF a predominance of Th17 cytokine profile with an increase of IL-17A, IL-17F and IL-21 versus PBMCs from SLE patients, which showed an inflammatory profile represented by increase of IL-6 cytokine. According to SLE remission/activity presented at enrollment in the study (Mex-SLEDAI index), the PBMC from active SLE patients showed higher levels of TNF-α and IL-6 versus PBMC from remission SLE patients. In conclusion, our results suggest that MIF can induce a differential inflammatory response in physiological and pathological conditions with a predominance of a Th17 cytokine profile in PBMC from HS and an increase in TNF-α and IL-6 expression in PBMC from active SLE patients.
The use of nanoparticles as drug delivery carriers requires analysis of their safety, which among other tests, includes immunotoxicity. Nanoparticles are also increasingly used for applications ...intended to specifically activate, inhibit, or modify the immune system's responses to improve the treatment of inflammatory and autoimmune disorders, cancer immunotherapy, and vaccines targeting cancer cells and viral and bacterial pathogens. In addition to the safety, the analysis of nanoparticles intended for immune system targeting includes mechanistic immunology investigations. Immunophenotyping provides researchers with a tool to assess the immune cell viability and activation status. These results provide mechanistic insights into nanoparticle efficacy and toxicity and therefore are of interest to the biomedical nanotechnology field. However, no standardized approaches exist due to the breadth of methods and instruments available for this analysis. This chapter provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood. While this experimental strategy is specific to the NovoCyte 3005 flow cytometer, it can be adapted to other instruments. Instructions for instrument setup, calibration, and antibody qualification are described in this book's Chapter 24 , Immunophenotyping, part I.
Markers of extracellular mitochondria are present in giant cell arteritis (GCA) patients. However, their role in promoting inflammation and platelet activation is no known. To investigate this, ...isolated mitochondria were opsonized with plasma from GCA patients or healthy individuals and incubated with peripheral blood mononuclear cells (PBMCs) or platelets and assessed for inflammatory cytokine production and platelet activation.
Plasma from GCA patients promoted increased mitochondrial-mediated cytokine production by PBMCs as compared to healthy controls (p < 0.05). Mitochondria opsonized with plasma factors from patients with GCA induced higher platelet activation as compared to mitochondria opsonized with plasma factors from healthy individuals (p = 0.0015). Platelet levels of P-selectin were associated with disease activity in GCA (r = 0.34, p = 0.01).
GCA patients have impaired ability to regulate the clearance of extracellular mitochondria, possibly contributing to excessive inflammation and platelet activation. Targeting key drivers of mitochondrial extrusion and/or their clearance could lead to new therapeutic interventions in GCA.
•Extrusion of mitochondria can occur via several mechanisms, including platelet activation and neutrophil cell death•Extracellular mitochondria can be immunogenic and inflammatory.•Plasma factors from patients with GCA enhance the inflammatory properties of mitochondria.•Plasma factors from patients with GCA were insufficient in reducing mitochondrial-mediated platelet activation.•The ability of opsonized mitochondria to induce platelet activation was associated with disease activity in GCA patients.
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and ...prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.
Functional and quantitative Treg cell defects have been identified in a variety of autoimmune diseases. Therefore, Tregs are a major pharmaceutical target for these disorders. In the last decades, ...studies have been mainly focused on the identification and experimental understanding of the activity of Tregs and their mechanisms of action.
This study describes how overnight storage of isolated peripheral blood mononuclear cells in different media (PBS pH 7.3, PBS pH 7.3 containing 0.5% BSA, RPMI 1640 and RPMI 1640 containing 10% FBS) affects the viability and expression of the commonly used markers for Tregs identification: CD25, CD127, CTLA-4, GITR, PD-1, FoxP3 and Helios.
Incorrectly selected storage conditions (temperature, time, medium) may affect the expression of surface and intracellular markers, thus, compromising the quality of the obtained results.
Appropriate protocols of cell isolation and storage are important for providing appropriate conditions for cell growth. This is crucial when analyzing small cell populations like Tregs.
Inflammation has been involved in the pathophysiology and treatment response of major depressive disorder (MDD). Plasma cytokine profiles of 171 treatment-naive MDD patients (none of the MDD patients ...received an adequate trial of antidepressants or evidence-based psychotherapy) and 64 healthy controls (HCs) were obtained. MDD patients exhibited elevated concentrations of 18 anti- and proinflammatory markers and decreased concentrations of 6 cytokines. Increased inflammasome protein expression was observed in MDD patients, indicative of an activated inflammatory response. The plasma of MDD patients was immunosuppressive on healthy donor peripheral blood mononuclear cells, inducing reduced activation of monocytes/dendritic cells and B cells and reduced T cell memory. Comparison between 33 non-responders and 71 responders at baseline and 12 weeks revealed that after treatment, anti-inflammatory cytokine levels increase in both groups, whereas 5 proinflammatory cytokine levels were stabilized in responders, but continued to increase in non-responders. MDD patients exhibit remodeling of their inflammatory landscape.
•Treatment-naive MDD patients have elevated levels of inflammatory markers•Overall, plasma of treatment-naive MDD patients is immunosuppressive•Defective anti-inflammatory response occurs in non-responders
Treatment-naive MDD patients have increased levels of pro- and anti-inflammatory markers, but overall the balance shifts toward immunosuppression of immune cells. Consistent with these findings, absence of response to antidepressant treatments has been associated with defective anti-inflammatory response.
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