Immunology

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4⁺ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au₁₃ nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

Both IgA nephropathy and IgA vasculitis, formerly known as Henoch-Schӧnlein purpura, are immune deposition diseases. IgA nephropathy is caused by the deposition of aberrantly formed poly-IgA complexes from blood circulation to the kidney glomerulus; IgA vasculitis is characterized by IgA-dominant immune deposits to small vessels of the skin and other organs, including the kidney. Therefore, measuring the disease-causing poly-IgA contents in the plasma is needed to study these conditions. However, while clinical tests for the level of total plasma IgA are routinely performed, methods for specific detection of poly-IgA contents are unavailable in clinical medicine. In this protocol, we describe a practical solution for measuring poly-IgA in patient samples. The new method is based on the biological selectivity of IgA Fcα receptor I (FcαRI/CD89) toward poly-IgA species, in contrast to its relatively low affinity for normal monomeric IgA. By devising recombinant CD89 ectodomain as the “capturing” probe, we validated the feasibility of the assay for measuring plasma poly-IgA levels in a 96-well format. The methodology was able to differentiate plasma samples of IgA nephropathy, or related IgA vasculitis, from those of other autoimmune kidney disease types or from healthy controls. Moreover, the measured poly-IgA indices not only correlated with the severity of IgA nephropathy, but the levels also trended lower following corticosteroid or immunosuppressant treatments of patients. Therefore, we anticipate the new assay will provide useful measurements of the IgA nephropathy disease activity index for stratifying disease severity or for evaluating treatment response.

Graphical abstract:

To identify causative substances for allergies to drugs or foods, the lymphocyte transformation test (LTT) is currently widely used as in vitro test, but its accuracy is not satisfactory. We have developed a novel method designated high-sensitivity allergy test (HiSAT) for determining allergy expression by measuring cell kinetics, using the chemotactic cells from non-allergic volunteers against a gradient field of cytokines released from immune cells when allergy develops. HiSAT requires a very small sample of 5 µL or less, and is applicable to three types of tests, depending on the situation in clinical practice: (i) diagnosis of the allergic expression, (ii) identification of the causative drug, and, in principle, (iii) pre-inspection.

Graphic abstract:

Schematic diagram of HiSAT. Serum from patients/subjects is used for rapid diagnosis in HiSAT. To identify the causative drug, the lymphocytes of interest are incubated with the candidate drug solution for 48 h to 72 h and then the culture supernatant is used in HiSAT. Before drug administration, it may possible to avoid the risk of allergies by performing pre-inspection, as well as the determination of the causative drug in HiSAT. A granulocyte-rich cell layer isolated from a non-allergic volunteer is used in HiSAT. Chemotactic cells migrate toward chemotactic factors in the test sample according to the concentration gradient. Cell kinetics (e.g., velocity or distance) are analyzed using sequential images of the test samples, and compared to the PHA-positive control.>

CD45 is a pan-leukocyte marker, and CD45 stain is widely used to determine the extent of inflammatory cell infiltration and its association with tissue injury. In this manuscript, we share a reliable immunohistochemistry (IHC) protocol for CD45 staining in sections of paraffin-embedded mouse kidney. A rat anti-CD45 antibody was used as primary antibody, and a mouse adsorbed biotin-conjugated goat anti-rat IgG was selected as secondary antibody. A horseradish peroxidase (HRP)-linked avidin/biotin detection system was used to amplify the signal, which was detected with 3,3′-Diaminobenzidine (DAB). With this protocol, we show that the CD45 antibody recognizes cells of hematolymphoid lineage in bone marrow, as well as monocyte/macrophages in liver and lung tissue. The utility of this protocol in pathology research was indicated by dramatically increased CD45-positive (CD45⁺) cells in the kidneys of a mouse model of diabetes. Double staining for CD45 and injury marker KIM-1 showed accumulated CD45⁺ cells around injured tubular cells. CD45 and F4/80 macrophage staining on adjacent tissue sections revealed overlap of CD45⁺ cells with other inflammatory cells.

In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte^® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuCyte^® Cell Migration Analysis Software. Thus, our protocol allows a time-lapse analysis of treatment effects on cell migration in a highly reliable, reproducible and re-analyzable manner.

The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with other alterations at nuclear and cellular level. These include nuclear decondensation and super-condensation, multi-lobulated nuclei versus 1-lobe nuclei and cell membrane damage. NETs markers can be quantified following variable periods of treatment with NETs inducers, prior to the formation of the specific extracellular DNA-scaffolded strands. Because these high throughput image-based cell analysis features can be performed with statistical rigor, this protocol is suited for both experimental and clinical applications as well as clinical evaluations of NETosis as a biomarker.

Pulmonary fibrosis is characterized by pathological scaring of the lung. Similar to other fibrotic diseases, scar formation is driven by excessive extracellular matrix deposition by activated, proliferative, and migratory fibroblasts.

Currently, the two most widely used chemotaxis and cell migration assays are the scratch assay and the transmembrane invasion assay. Here we present a gap closure assay that employs commercially available cell lines, equipment and reagents and is time efficient as well as straightforward. The protocol uses an Oris pro cell migration assay 96-well plate with a dissolvable plug in the center of each well to create a cell free area at the time of seeding. Cell repopulation of the empty zone is captured via light microscopy at different time points and quantified with free image analysis software. The clear advantages of this assay in comparison to similar protocols are the use of uncomplicated cell culture methods and the ability to image the experiment throughout.

The early life period represents a time of immunological plasticity whereby the functionally immature immune system is highly susceptible to environmental stimulation. Perennial aeroallergen and respiratory viral infection induced sporadic episodes of lung inflammation during this temporal window represent major risk factors for initiation of allergic asthmatic disease. Murine models are widely used as an investigative tool to examine the pathophysiology of allergic asthma; however, models in current usage typically do not encapsulate the early life period which represents the time of maximal risk for disease inception in humans. To address this issue, this protocol adapted an experimental animal model of disease for sensitization to ovalbumin during the immediate post-weaning period beginning at 21 days of age. By initially sensitizing mice during this early life post-weaning period, researchers can more closely align experimental allergic airway disease models with the human age group most at risk for asthma development.

We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.

Due to its particulate material, mono-sodium urate (MSU) crystals are potent activators of the NOD-like receptor NLRP3. Upon activation, NLRP3 induces the formation of inflammasome complexes, which lead to the production and release of mature IL-1β. Bioactive IL-1β is a potent activator of innate immune responses and promotes recruitment of inflammatory cells, including neutrophils from the blood into damaged/inflamed tissues. This protocol describes a method to study in vivo inflammasome activation via intraperitoneal injection of MSU crystals. MSU-injection results in a drastic increase of intraperitoneal IL-1β levels, promoting neutrophil infiltration. Early-stage neutrophil numbers correlate with the amount of released IL-1β and can be used as a read-out for the extent of in vivo inflammasome activation. In addition, this protocol might also be used as a sterile peritonitis model, to investigate mechanisms of neutrophil recruitment to the peritoneum, or as a means to obtain large numbers of in vivo activated neutrophils.