These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Interleukin-1 beta (IL-1β) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer’s disease (AD); no conclusive data are nevertheless available in AD patients. mRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1β, IL-18) was up-regulated in severe and MILD AD. Monocytes co-expressing NLRP3 with caspase 1 or caspase 8 were significantly increased in severe AD alone, whereas those co-expressing NLRP1 and NLRP3 with PYCARD were augmented in both severe and MILD AD. Activation of the NLRP1 and NLRP3 inflammasomes in AD was confirmed by confocal microscopy proteins co-localization and by the significantly higher amounts of the pro-inflammatory cytokines IL-1β and IL-18 being produced by monocytes. In MCI, the expression of NLRP3, but not the one of PYCARD or caspase 1 was increased, indicating that functional inflammasomes are not assembled in these individuals: this was confirmed by lack of co-localization and of proinflammatory cytokines production. The activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD.

Pathogenesis of autoimmune disorders, including Multiple Sclerosis (MS), has been linked to an alteration of the resident microbial commensal community and of the interplay between the microbiota and the immune system. Dietary components such as fibers, acting on microbiota composition, could, in principle, result in immune modulation and, thus, could be used to obtain beneficial outcomes for patients. We verified this hypothesis in a pilot study involving two groups of clinically similar Relapsing-Remitting (RR) MS patients who had undergone either a high-vegetable/low-protein diet (HV/LP diet group; N=10) or a standard western diet (WD group; N=10) for at least 12 months. Gut microbiota composition, analyzed by 16S V4 rRNA gene sequencing and immunological profiles, were examined after a minimum of 12 months of diet. Results showed that, in the HV/LP diet group compared to the WD group: 1) Lachnospiraceae family was significantly more abundant; 2) IL-17-producing T CD4+ lymphocytes (p= 0.04) and PD-1 expressing T CD4+ lymphocytes (p= 0.0004) were significantly decreased; and 3) PD-L1 expressing monocytes (p= 0.009) were significantly increased. In the HV/LP diet group, positive correlations between Lachnospiraceae and both CD14+/IL-10+ and CD14+/TGFbeta+ monocytes (RSp=0.707, p=0.05 and RSp= 0.73, p=0.04; respectively), as well as between Lachnospiraceae and CD4+/CD25+/FoxP3 T lymphocytes (RSp= 0.68,p=0.02) were observed. Evaluation of clinical parameters showed that in the HV/LP diet group alone the relapse rate during the 12 months follow up period and the EDSS score at the end of the study period were significantly reduced.. Diet modulates dysbiosis and improves clinical parameters in MS patients by increasing anti-inflammatory circuits. Because Lachnospiraceae favour Treg differentiation as well as TGFbeta and IL-10 production this effect could be associated with an increase of these bacteria in the microbiota.

Regulatory T lymphocytes (Treg ) play a fundamental importance in modulating the relative balance between inflammation and immune tolerance, and alterations of these cells are observed in inflammatory diseases. To better characterize the neuroinflamm

Neurodegenerative diseases are chronic, progressive disorders that occur in the central nervous system (CNS). They are characterized by the loss of neuronal structure and function and are associated with inflammation. Inflammation of the CNS is called neuroinflammation, which has been implicated in most neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Much evidence indicates that these different conditions share a common inflammatory mechanism: the activation of the inflammasome complex in peripheral monocytes and in microglia, with the consequent production of high quantities of the pro-inflammatory cytokines IL-1β and IL-18. Inflammasomes are a group of multimeric signaling complexes that include a sensor Nod-like receptor (NLR) molecule, the adaptor protein ASC, and caspase-1. The NLRP3 inflammasome is currently the best-characterized inflammasome. Multiple signals, which are potentially provided in combination and include endogenous danger signals and pathogens, trigger the formation of an active inflammasome, which, in turn, will stimulate the cleavage and the release of bioactive cytokines including IL-1β and IL-18. In this review, we will summarize results implicating the inflammasome as a pivotal player in the pathogenesis of neurodegenerative diseases and discuss how compounds that hamper the activation of the NLRP3 inflammasome could offer novel therapeutic avenues for these diseases.