Immunology

Stem cell–based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies.

Key features

• The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells–derived macrophages.

• The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores.

• The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages.

• This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.

Graphical overview

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with ¹⁴C-serotonin release assay, another common assay for detecting antibody activity.

Key features

• This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT.

• Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies.

• Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist.

• For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

Chronic Kidney Disease (CKD) patients present a micro inflammation state due to failure renal function. The calcitriol has been described as an anti-inflammatory factor that might modulates the inflammatory response in CKD patients. However, these patients have deficiency of Calcitriol due to failure renal function. But, synthesis of this vitamin has been reported in extra renal production, as in monocytes. In this context, it has been reported that the supplementation with 25 vitamin D (calcidiol or inactive form of vitamin D) induces monocytes to downregulate inflammation, due to the intracellular 1α-hidroxilase that converts calcidiol to calcitriol in these cells. Besides some reports used RT-qPCR, Western Blot or immunofluorescence techniques to investigate the expression of inflammatory and vitamin D machinery biomarkers in several disease, in the present study we used flow cytometry technique to evaluate the effect of 25 vitamin D on CD14, Toll-like receptor 4 (TLR4), vitamin D receptor (VDR), 1-α hydroxylase (CYP27), 24 hydroxylase (CYP24) in monocytes lineage (U937). The U937 culture was incubated with healthy or CKD serum and treatment with/without 25-vitamin D (50 ng/ml for 24 h) to evaluate CD14, TRL4, VDR, CYP27 and CYP24 expression. This protocol showed the advantage to investigate the effect of treatment with 25 vitamin D on the intracellular and cell membrane biomarkers expression quickly and simultaneously. In addition, this technique is not laborious, but easy to perform and to interpret compared to RT-qPCR, western blot or immunofluorescence.

In malaria, rosetting phenomenon is a condition where a Plasmodium-infected erythrocyte stably adheres to at least an uninfected erythrocyte. This phenomenon that occurs in all species of human malaria parasite is likely to be an immune escape mechanism for the parasite. However, it has been associated with malaria pathogenesis, possibly by facilitating microvasculature occlusion along with direct endothelial cytoadherence by the infected erythrocytes. There are different microscopy-based techniques to visualize rosettes but neither of these techniques has yet to qualify as the official “gold standard” method. We have found that these techniques can be used interchangeably, provided that the conditions of the experiments are properly controlled. Here, we presented three methods as options for rosetting assay, i.e., the unstained wet mount technique, acridine orange based-fluorescence microscopy technique and Giemsa stained wet mount method, with preparation steps that enable consistent performance in rosetting experiments.

Leishmaniasis is a parasitic disease caused by the obligatory intracellular protozoa Leishmania spp. Current therapeutic options are limited and thus, drug discovery against leishmaniasis is very important. Nevertheless, there is a great difficulty to develop therapeutic strategies against the disease because the parasite deploys various mechanisms to evade the immune system and multiply inside the host. Among the main factors of the immunity that are recruited to confront the Leishmania infection are the macrophages (MΦs) that produce effector molecules such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS). Therefore, efficient drug agents should combine the antileishmanial effect of these gaseous transmitters along with the enhancement of the host’s adaptive immunity. In the quest of therapeutic alternatives, natural products have been extensively studied and are considered as candidate antileishmanial agents since they exhibit specific properties in modulating the host’s immune response towards an effective anti-leishmanial cell-mediated immunity capable to eliminate parasitic dissemination. In the current protocol, Leishmania-infected MΦs (J774A.1 cell line) that have been treated with various increasing concentrations of a natural compound, are tested for the production of the aforementioned molecules. In order to detect NO production, we employ the Griess colorimetric nitrite assay and quantification relies on the construction of an accurate standard curve using appropriate standards of known concentration. ROS detection and quantification is achieved by flow cytometry and relies on the use of carboxy-H₂DCFDA, an indicator that converts to a fluorescent form when interacts with ROS molecules.