The immunomodulatory effect of oral NaHCO3 is mediated by the splenic nerve: multivariate impact revealed by artificial neural networks.

Stimulation of the inflammatory reflex (IR) is a promising strategy for treating systemic inflammatory disorders. Recent studies suggest oral sodium bicarbonate (NaHCO3) as a potential activator of the IR, offering a safe and cost-effective treatment approach. However, the mechanisms underlying NaHCO3-induced anti-inflammatory effects remain unclear. We investigated whether oral NaHCO3's immunomodulatory effects are mediated by the splenic nerve. Female rats received NaHCO3 or water (H2O) for four days, and splenic immune markers were assessed using flow cytometry. NaHCO3 led to a significant increase (p < 0.05, and/or partial eta squared > 0.06) in anti-inflammatory markers, including CD11bc + CD206 + (M2-like) macrophages, CD3 + CD4 + FoxP3 + cells (Tregs), and Tregs/M1-like ratio. Conversely, proinflammatory markers, such as CD11bc + CD38 + TNFα + (M1-like) macrophages, M1-like/M2-like ratio, and SSChigh/SSClow ratio of FSChighCD11bc + cells, decreased in the spleen following NaHCO3 administration. These effects were abolished in spleen-denervated rats, suggesting the necessity of the splenic nerve in mediating NaHCO3-induced immunomodulation. Artificial neural networks accurately classified NaHCO3 and H2O treatment in sham rats but failed in spleen-denervated rats, highlighting the splenic nerve's critical role. Additionally, spleen denervation independently influenced Tregs, M2-like macrophages, Tregs/M1-like ratio, and CD11bc + CD38 + cells, indicating distinct effects from both surgery and treatment. Principal component analysis (PCA) further supported the separate effects. Our findings suggest that the splenic nerve transmits oral NaHCO3-induced immunomodulatory changes to the spleen, emphasizing NaHCO3's potential as an IR activator with therapeutic implications for a wide spectrum of systemic inflammatory conditions.

Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Shared resource lab (SRL) strategies for supporting high-dimensional cytometry data analysis.

With the increase in the number of parameters that can be detected at the single-cell level using flow and mass cytometry, there has been a paradigm shift when handling and analyzing data sets. Cytometry Shared Resource Laboratories (SRLs) already take on the responsibility of ensuring users have resources and training in experimental design and operation of instruments to promote high-quality data acquisition. However, the role of SRLs downstream, during data handling and analysis, is not as well defined and agreed upon. Best practices dictate a central role for SRLs in this process as they are in a pivotal position to support research in this context, but key considerations about how to effectively fill this role need to be addressed. Two surveys and one workshop at CYTO 2022 in Philadelphia, PA, were performed to gain insight into what strategies SRLs are successfully employing to support high-dimensional data analysis and where SRLs and their users see limitations and long-term challenges in this area. Recommendations for high-dimensional data analysis support provided by SRLs will be offered and discussed.

Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles.

Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations - the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet-derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.

Unfermented ß-fructan Fibers Fuel Inflammation in Select Inflammatory Bowel Disease Patients.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBD) are affected by dietary factors, including nondigestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial, but not all fibers are alike, and some patients with IBD report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in patients with IBD, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation. METHODS: Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5 g/L), or fermentation supernatants (24-hour anaerobic fermentation) and immune responses (cytokine secretion [enzyme-linked immunosorbent assay/meso scale discovery] and expression [quantitative polymerase chain reaction]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software. RESULTS: Unfermented dietary ß-fructan fibers induced proinflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining ß-fructan supplementation. The proinflammatory response to intact ß-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of ß-fructans by human gut whole microbiota cultures reduced the proinflammatory response, but only when microbes were collected from patients without IBD or patients with inactive IBD. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance. CONCLUSION: Although fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities. The study is publicly accessible at the U.S. National Institutes of Health database (clinicaltrials.gov identification number NCT02865707).

Flow Cytometry and Cell Cycle Analysis: An Overview.

Cell cycle analysis is one of the earliest applications in flow cytometry and continues to be highly used to this day. Since the first reported method of Feulgen-DNA staining, cell cycle analysis has continued to grow and mature. With the recent advances in DNA dyes, understanding of additional cell cycle phase markers, and new technologies, cell cycle analysis continues to be a dynamic field within the flow cytometry community. This chapter will give an overview of the current state of cell cycle analysis by flow cytometry.

In-Cell Labeling Coupled to Direct Analysis of Extracellular Vesicles in the Conditioned Medium to Study Extracellular Vesicles Secretion with Minimum Sample Processing and Particle Loss.

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity.

Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and can also be activated by some Group 9/10 transition metals, which is believed to mediate immune hypersensitivity reactions. In this work, we test whether TLR4 can be activated by the Group 10 metal platinum and the platinum-based chemotherapeutic cisplatin. Cisplatin is invaluable in childhood cancer treatment but its use is limited due to a permanent hearing loss (cisplatin-induced ototoxicity, CIO) adverse effect. We demonstrate that platinum and cisplatin activate pathways downstream of TLR4 to a similar extent as the known TLR4 agonists LPS and nickel. We further show that TLR4 is required for cisplatin-induced inflammatory, oxidative, and cell death responses in hair cells in vitro and for hair cell damage in vivo. Finally, we identify a TLR4 small molecule inhibitor able to curtail cisplatin toxicity in vitro. Thus, our findings indicate that TLR4 is a promising therapeutic target to mitigate CIO.

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry.

Pseudomonas aeruginosa is a Gram-negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod-shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture-based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species-specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry.

Neutrophils exert protection in early Aeromonas veronii infections through the clearance of both bacteria and dying macrophages.

Aeromonas veronii is a gram-negative opportunistic pathogen capable of infecting both fish and mammals. Left untreated, natural infection in fish can prove fatal and result in irreparable damage to the aquaculture industry. Neutrophils are essential innate effector cells that play critical roles in pathogen defense. Our aim was to investigate the immunological roles of teleost neutrophils during infection with A. veronii. We began by examining the functional defenses of neutrophils in vitro, where neutrophils efficiently killed the pathogen. In addition, we developed an in vivo infection model to assess the roles of neutrophils during an infection in goldfish. This allowed us to explore the complex dynamics between immune cells and Aeromonas veronii. Interestingly, our studies found that neutrophils are capable of sensing a diverse range of dead and dying cells, resulting in varying downstream responses. Herein, we report that neutrophils internalized dead or dying macrophages previously infected with A. veronii. Moreover, once internalized, neutrophils went on to display classical pro-inflammatory ROS responses, in contrast to the more typical anti-inflammatory responses seen in cells following the uptake of a dead host cell. This led us to hypothesize that during infection, neutrophils are capable of simultaneously clearing dead and dying cells as well as A. veronii. This study provides additional insights into the complex mechanisms by which neutrophils operate within an inflammatory site and contribute to the induction and regulation of acute inflammatory responses.

Connected component masking accurately identifies the ratio of phagocytosed and cell-bound particles in individual cells by imaging flow cytometry.

Innate immune cell-mediated recognition, capture, and engulfment of large particulate targets such as bacteria is known as phagocytosis. This highly dynamic cellular process involves a series of steps including receptor-mediated target binding, phagocytic cup formation, pseudopod extension, and phagosome closure, which depend on distinct actin polymerization events. Using flow cytometry, precise determination of target locations relative to cell membranes (i.e., surface-bound vs. fully engulfed/internalized) during the phagocytic process is difficult to quantify. Here, we describe the application of new analysis features within the IDEAS® software to distinguish internalized and surface-bound particles on individual cells with a high degree of accuracy and reproducibility. Through the use of connected component masks, the accurate discrimination of surface-bound beads versus those internalized is clearly demonstrated. In addition, we were able to further analyze the ratio of beads that had been surface-bound or internalized within individual cells. This novel method of analyzing the phagocytic process provides more accurate determination of target-cell interactions that will assist in examination of the signalling events that occur during the various stages of phagocytosis. © 2017 International Society for Advancement of Cytometry.

Accurate Assessment of Cell Death by Imaging Flow Cytometry.

The number of investigators using cell death analysis applications has greatly expanded since the introduction of flow cytometry. The Annexin V/propidium iodide (PI) method is among the most commonly used procedures and allows users to determine if cells are viable, apoptotic, or necrotic, based on changes in membrane lipid composition, integrity, and permeability. Unfortunately, PI can intercalate into RNA, in addition to DNA, which contributes to a large number of events showing PI staining within the cytoplasmic compartment. We show that this occurs across a broad range of animal primary cells and commonly used cell lines, and is most prevalent in large cells (nuclear:cytoplasmic ratio <0.5). Any cellular system where RNA levels change throughout an experiment will be particularly affected, such as those that utilize virally infected cells. As two examples, we highlight our recent work on cells infected with vesicular stomatitis virus (VSV), an RNA virus, and herpes simplex virus-1 (HSV-1), a DNA virus. Similarly, these issues are relevant to experimental systems where cells have increased RNA content such as during genotoxic stress, following exposure to cell cycle arrest drugs such as thymidine or hydroxyurea, or where developmental progression promotes discrete changes in cellular RNA synthesis. This chapter outlines a modified Annexin V/PI method that addresses cytoplasmic RNA staining issues to allow for accurate assessment of cell death. This protocol takes advantage of an additional cellular permeability caused by fixation to promote RNase A entry into the cell. Based on our observations, cell morphological parameters are well maintained and less than 5 % of total cellular events exhibit cytoplasmic PI staining under this protocol.

Neutrophil contributions to the induction and regulation of the acute inflammatory response in teleost fish.

Neutrophils are essential to the acute inflammatory response, where they serve as the first line of defense against infiltrating pathogens. We report that, on receiving the necessary signals, teleost (Carassius auratus) neutrophils leave the hematopoietic kidney, enter into the circulation, and dominate the initial influx of cells into a site of inflammation. Unlike mammals, teleost neutrophils represent <5% of circulating leukocytes during periods of homeostasis. However, this increases to nearly 50% immediately after intraperitoneal challenge with zymosan, identifying a period of neutrophilia that precedes the peak influx of neutrophils into the challenge site at 18 h after injection). We demonstrate that neutrophils at the site of inflammation alter their phenotype throughout the acute inflammatory response, and contribute to both the induction and the resolution of inflammation. However, neutrophils isolated during the proinflammatory phase (18 h after injection) produced robust respiratory burst responses, released inflammation-associated leukotriene B(4), and induced macrophages to increase reactive oxygen species production. In contrast, neutrophils isolated at 48 h after infection (proresolving phase) displayed low levels of reactive oxygen species, released the proresolving lipid mediator lipoxin A(4), and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A(4) was a significant contributor to the uptake of apoptotic cells by teleost macrophages and also played a role, at least in part, in the downregulation of macrophage reactive oxygen species production. Our results highlight the contributions of neutrophils to both the promotion and the regulation of teleost fish inflammation and provide added context for the evolution of this hematopoietic lineage.

X-FISH: Analysis of cellular RNA expression patterns using flow cytometry.

Fluorescent in situ hybridization (FISH) is a powerful technique for the detection of RNA or DNA within cells and tissues, which provides a unique link between molecular and cell biology. This technique is broadly applicable across a range of biological systems. While FISH has been previously adapted to flow-based platforms, their use remains limited because of procedural challenges and costs associated with commercial kits. Herein we present a protocol that modifies existing techniques to sensitively and specifically detect and examine RNA expression patterns in primary cells and cell lines using flow cytometry (expression-FISH; X-FISH). As relevant examples, we show how this technique can be used to monitor changes in mRNA expression following activation, how it can be combined with antibody staining to study RNA and protein in the same sample, and how it can help distinguish among subsets in a mixed cell population. X-FISH can integrate multiple probes and can be performed in conjunction with other assays, allowing for informative multiparametric analyses and increased statistical robustness. For non-classical comparative animal models this procedure provides a time saving alternative to de novo production of antibody-based markers. Finally, X-FISH provides an economical solution that is applicable to conventional as well as multi-spectral imaging flow cytometry platforms.

Teleost soluble CSF-1R modulates cytokine profiles at an inflammatory site, and inhibits neutrophil chemotaxis, phagocytosis, and bacterial killing.

Soluble colony stimulating factor-1 receptor (sCSF-1R) is a novel bony fish protein that contributes to the regulation of macrophage proliferation. We recently showed that this soluble receptor is highly upregulated by teleost macrophages in the presence of apoptotic cells. Further, recombinant sCSF-1R inhibited leukocyte infiltration into a challenge site in vivo. Herein, we characterized the mechanisms underlying these changes as a platform to better understand the evolutionary origins of the CSF-1 immune-regulatory axis and inflammation control in teleosts. Using an in vivo model of self-resolving peritonitis, we show that sCSF-1R downregulates chemokine expression and inhibits neutrophil chemotaxis. Soluble CSF-1R also inhibited gene expression of several pro-inflammatory cytokines and promoted the expression of an anti-inflammatory mediator, IL-10. Finally, the phenotype of infiltrating neutrophils changed significantly in the presence of sCSF-1R. Both a reduced capacity for phagocytosis and pathogen killing were observed. Overall, our results implicate sCSF-1R as an important regulator of neutrophil responses in teleosts. It remains unclear whether this represents an inflammation regulatory factor that is unique to this animal group or one that may be evolutionarily conserved and continues to contribute to the regulation of antimicrobial processes at inflammatory sites in higher vertebrates.

Phagocytosis by Thrombocytes is a Conserved Innate Immune Mechanism in Lower Vertebrates.

Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5-3 µm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.

Expression of DP2 (CRTh2), a prostaglandin D2 receptor, in human mast cells.

PGD2 has long been implicated in allergic diseases. Recent cloning of a second PGD2 receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD2 in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD2 and 15R-15-methyl PGD2 induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.