Inflammasome Assays In Vitro and in Mouse Models

This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation-mediated maturation of interleukin (IL)-1β and IL-18 and pyroptosis. Since the well-established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti-ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL-1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high-dose LPS challenge in a sepsis model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Priming and activation of inflammasomes in mouse macrophages. Basic Protocol 2: Activation of human NLRP1 inflammasome by DPP8/9 inhibitor talabostat. Basic Protocol 3: Purification and detection of ASC pyroptosome. Alternate Protocol 1: Detection of ASC speck by immunofluorescence staining. Basic Protocol 4: Activation of canonical NLRP3 inflammasome in mice by intraperitoneal delivery of MSU crystals. Alternate Protocol 2: Activation of noncanonical NLRP3 inflammasome in mice by intraperitoneal delivery of LPS

Influenza virus and sars-cov-2 vaccines

Seasonal influenza and the current COVID-19 pandemic represent looming global health challenges. Efficacious and safe vaccines remain the frontline tools for mitigating both influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced diseases. This review will discuss the existing strategies for influenza vaccines and how these strategies have informed SARS-CoV-2 vaccines. It will also discuss new vaccine platforms and potential challenges for both viruses

Re-Examination of the Exacerbating Effect of Inflammasome Components during Radiation Injury

Radiation can be applied for therapeutic benefit against cancer or may result in devastating harm due to accidental or intentional release of nuclear energy. In all cases, radiation exposure causes molecular and cellular damage, resulting in the production of inflammatory factors and danger signals. Several classes of innate immune receptors sense the released damage associated molecules and activate cellular response pathways, including the induction of inflammasome signaling that impacts IL-1β/IL-18 maturation and cell death. A previous report indicated inflammasomes aggravate acute radiation syndrome. In contrast, here we find that inflammasome components do not exacerbate gamma-radiation-induced injury by examining heterozygous and gene-deletion littermate controls in addition to wild-type mice. Absence of some inflammasome genes, such as caspase-1/11 and Nlrp3, enhance susceptibility of treated mice to acute radiation injury, indicating importance of the inflammasome pathway in radioprotection. Surprisingly, we discover that the survival outcome may be sex-dependent as more inflammasome-deficient male mice are susceptible to radiation-induced injury. We discuss parameters that may influence the role of inflammasomes as radioprotective or radioexacerbating factors in recovery from radiation injury including the use of littermate controls, the sex of the animals, differences in microbiota within the colonies and other experimental conditions. Under the conditions tested, inflammasome components do not exacerbate radiation injury, but rather provide protective benefit

Sputum inflammatory cells from patients with allergic rhinitis and asthma have decreased inflammasome gene expression

Deficits in inflammasomes, a key element of innate immunity, confer increased susceptibility to infection. We report that sputum cells from asthmatics have decreased expression of inflammasome factors, consistent with reports of increased infection risk in asthmatics

A microparticle platform for STING-targeted immunotherapy enhances natural killer cell- and CD8 + T cell-mediated anti-tumor immunity

Immunotherapies have significantly improved cancer patient survival, but response rates are still limited. Thus, novel formulations are needed to expand the breadth of immunotherapies. Pathogen associated molecular patterns (PAMPs) can be used to stimulate an immune response, but several pathogen recognition receptors are located within the cell, making delivery challenging. We have employed the biodegradable polymer acetalated dextran (Ace-DEX) to formulate PAMP microparticles (MPs) in order to enhance intracellular delivery. While treatment with four different PAMP MPs resulted in tumor growth inhibition, cyclic GMP-AMP (cGAMP) MPs were most effective. cGAMP MPs showed anti-tumor efficacy at doses 100-1000 fold lower than published doses of soluble cGAMP in two murine tumor models. Treatment with cGAMP MPs resulted in increased natural killer cell numbers in the tumor environment. Immune cell depletion studies confirmed that NK cells were responsible for the anti-tumor efficacy in an aggressive mouse melanoma model. NK cells and CD8 + T cells were both required for early anti-tumor function in a triple negative breast cancer model. In summary, cGAMP MP treatment results in NK and T cell-dependent anti-tumor immune response

Airway cells from atopic asthmatic patients exposed to ozone display an enhanced innate immune gene profile

This study identifies transcriptional phenotypes of sputum samples from normal volunteers and atopic asthmatics exposed to ozone. Network analyses suggest that asthmatics elevate immune signaling following oxidative stress, while nonasthmatics attempt to mitigate the ozone-induced response

TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice

Mice reconstituted with a human immune system (humanized mice) provide a robust model to study human immunology, vaccinology, and human infectious diseases. However, the development and function of B cells in humanized mice is impaired. B cells from humanized mice are immature and are impaired in IgM to IgG isotype switch in response to infection or vaccination. In the present study we report that Toll-like receptor 9 (TLR9) agonist CpG-B combined with CD40-targeting vaccination triggered human B cell immunoglobin class-switch from IgM+ to IgG+ B cells in humanized mice. Human B cells from mice vaccinated with CpG-B as adjuvant were more mature in phenotype and produced significant levels of both total IgG and antigen-specific IgG. We found that CpG-B treatment activated human pDCs (plasmacytoid dendritic cells) in vivo to induce interferon-alpha (IFN-α)expression in humanized mice. Pre-depletion of human pDC in vivo abrogated the adjuvant effect of CpG-B. Our results indicate that TLR9 and CD40-targeting vaccination triggers human B cell maturation and immunoglobulin class-switch in a pDC-dependent manner in humanized mice. The findings also shed light on induction of human IgG antibodies in humanized mouse models

The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity

NLRC3 limits inflammatory signaling in myeloid cells, but its role in T cells has been unclear. Uchimura et al. reveal that T cell expression of Nlrc3 restricts autoimmune and virus-specific CD4 + T cell responses by attenuating T cell signaling and metabolic pathways in CD4 + T cells. © 2018 Elsevier Inc.Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4 + T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3 −/− mice exhibited increased and prolonged CD4 + T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4 + T cell signaling and metabolism

Multi-omics analyses of radiation survivors identify radioprotective microbes and metabolites

Ionizing radiation causes acute radiation syndrome, which leads to hematopoietic, gastrointestinal, and cerebrovascular injuries. We investigated a population of mice that recovered from high-dose radiation to live normal life spans. These "elite-survivors" harbored distinct gut microbiota that developed after radiation and protected against radiation-induced damage and death in both germ-free and conventionally housed recipients. Elevated abundances of members of the bacterial taxa Lachnospiraceae and Enterococcaceae were associated with postradiation restoration of hematopoiesis and gastrointestinal repair. These bacteria were also found to be more abundant in leukemia patients undergoing radiotherapy, who also displayed milder gastrointestinal dysfunction. In our study in mice, metabolomics revealed increased fecal concentrations of microbially derived propionate and tryptophan metabolites in elite-survivors. The administration of these metabolites caused long-term radioprotection, mitigation of hematopoietic and gastrointestinal syndromes, and a reduction in proinflammatory responses

The global response to the COVID-19 pandemic: how have immunology societies contributed?

The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)