Immunology

The tissue explant culture (histoculture) is a method that involves maintaining small pieces taken from an organ ex vivo or post mortem in a controlled laboratory setting. Such a technique has a number of advantages: unlike the 2D, organoid, or on-chip cultures, tissue explants preserve the whole complexity of the original tissue in vivo, its structure, extracellular matrix, and the diverse cell populations, including resident immune cells. The explant culture method can be applied to human tissue specimens obtained from biopsies or autopsies, provided that proper ethical protocols are followed. This avoids the difficulties that may arise in translating results obtained on animal models into biomedical research for humans. This advantage makes histocultures especially desirable for studying human pathogenesis in the course of infectious diseases. The disadvantage of the method is the limited lifespan of the cultured tissues; however, a number of approaches allow extending tissue viability to a period sufficient for observing the infection onset and development. Here, we provide a protocol for lung explant maintenance that allows tracing the local effects of infection with SARS-CoV-2 in humans. Further applications of the lung tissues cultured according to this protocol include, but are not limited to, histochemical and immunohistochemical studies and microscopy, FACS, qPCR, and ELISA-based analysis of the conditioned culture media.

Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite’s pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the singular advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments, which have not been reported in other published protocols to generate in vitro granulomas.

Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome screens. Here, we describe a CRISPR screening protocol that allows for efficient screening of the entire life cycle of HIV-1 through packaging of the HIV-CRISPR lentiviral genomes by infecting HIV-1 virus in trans.

Shigella flexneri is an intracellular bacterial pathogen that gains access to the gut epithelium using a specialized Type III Secretion System (T3SS). Various determinants mediating this invasive infection have been experimentally verified using the classical gentamicin protection assay presented here. In this assay epithelial cell lines are infected by bacteria in vitro and the extracellular bacteria are killed by gentamicin. The internalized bacteria, which are protected from the bactericidal action of gentamicin, are recovered by lysing the epithelial cells and enumerated by determining the colonies formed on solid medium. Various techniques based on light microscopy, such as immunofluorescence and bacteria expressing fluorescent proteins, are also used for studying intracellular bacteria. However, these techniques are not only labor intensive and require sophisticated equipment, but mostly are also not quantitative. Despite being an easy quantitative method to study invasiveness of bacteria, the gentamicin protection assay cannot distinguish between the survival and multiplication of the internalized bacteria over longer incubation periods. To alleviate the complications created by multiplication and dissemination of internalized bacteria, complementary assays like plaque formation assays are required. This protocol presents an easy and cost-effective method to determine the invasiveness and the capacity to establish an infection of Shigella under different conditions.

Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow.

Interferon regulatory transcription factor 3 (IRF3) is a transcription factor that upon activation by virus infection promotes the synthesis of antiviral genes, such as the interferons (Hiscott, 2007). In addition to inducing genes, IRF3 triggers antiviral apoptosis by RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA), which is independent of its transcriptional activity. RIPA protects against lethal virus infection in cells and mice (Chattopadhyay et al., 2016). In the absence of RIPA, caused by genetic ablation, chemical mutagenesis or inhibition of the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I), Sendai virus (SeV) infection does not trigger cellular apoptosis and become persistently infected (Peters et al., 2008; Chattopadhyay et al., 2013). IRF3-expressing wild type (WT) cells (U4C) undergo SeV-induced apoptosis; however, the P2.1 cells, which are deficient in IRF3 expression are not capable of triggering viral apoptosis (Figure 1). Ectopic expression of human IRF3 restores the apoptotic activity in P2.1 cells (P2.1/IRF3, Figure 1). SeV is used as a model for studying pathogenic human viruses, which are difficult to work with or require BSL3 facility. We have previously reported that both human and mouse cells can establish SeV persistence in the absence of IRF3’s apoptotic activity (Chattopadhyay et al., 2013). Here, we outline a detailed procedure for the development of a persistently SeV-infected human cell line (Figure 2), which continuously expresses viral protein and produces low levels of infectious viral particles.


Figure 1. SeV-induced apoptosis is IRF3-dependent. HT1080-derived cell lines (U4C, P2.1 and P2.1/IRF3) were infected with Sendai virus and three days post infection culture fields were photographed, scale bar represents 50 µm.

An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-Blue^TM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.

Exosomes are membranous extracellular nanovesicles of endocytic origin. Exosomes are known to carry host and pathogen-derived genomic, proteomic, lipidomic cargos and other extraneous molecules. Exosomes are secreted by diverse cell types into the extracellular milieu and are subsequently internalized by recipient neighboring or distal cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. Exosomes facilitate intercellular communication, modulate cellular phenotype, and regulate microbial pathogenesis. We have previously shown that semen exosomes (SE) inhibit HIV-1 replication in various cell types. Here, we describe detailed protocols for characterizing SE. This protocol can be adapted or modified and used for evaluation of other extracellular vesicles of interest.

The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.