The septin modifier, forchlorfenuron, activates NLRP3 via a potassium-independent mitochondrial axis.

The Nod-like receptor protein 3 (NLRP3) inflammasome is activated by stimuli that induce perturbations in cell homeostasis, which commonly converge on cellular potassium efflux. NLRP3 has thus emerged as a sensor for ionic flux. Here, we identify forchlorfenuron (FCF) as an inflammasome activator that triggers NLRP3 signaling independently of potassium efflux. FCF triggers the rearrangement of septins, key cytoskeletal proteins that regulate mitochondrial function. We report that FCF triggered the rearrangement of SEPT2 into tubular aggregates and stimulated SEPT2-independent NLRP3 inflammasome signaling. Similar to imiquimod, FCF induced the collapse of the mitochondrial membrane potential and mitochondrial respiration. FCF thereby joins the imidazoquinolines as a structurally distinct class of molecules that triggers NLRP3 inflammasome signaling independent of potassium efflux, likely by inducing mitochondrial damage.

NLRP12 interacts with NLRP3 to block the activation of the human NLRP3 inflammasome.

Inflammasomes are multiprotein complexes that drive inflammation and contribute to protective immunity against pathogens and immune pathology in autoinflammatory diseases. Inflammasomes assemble when an inflammasome scaffold protein senses an activating signal and forms a signaling platform with the inflammasome adaptor protein ASC. The NLRP subfamily of NOD-like receptors (NLRs) includes inflammasome nucleators (such as NLRP3) and also NLRP12, which is genetically linked to familial autoinflammatory disorders that resemble diseases caused by gain-of-function NLRP3 mutants that generate a hyperactive NLRP3 inflammasome. We performed a screen to identify ASC inflammasome-nucleating proteins among NLRs that have the canonical pyrin-NACHT-LRR domain structure. Only NLRP3 and NLRP6 could initiate ASC polymerization to form "specks," and NLRP12 failed to nucleate ASC polymerization. However, wild-type NLRP12 inhibited ASC inflammasome assembly induced by wild-type and gain-of-function mutant NLRP3, an effect not seen with disease-associated NLRP12 mutants. The capacity of NLRP12 to suppress NLRP3 inflammasome assembly was limited to human NLRP3 and was not observed for wild-type murine NLRP3. Furthermore, peripheral blood mononuclear cells from patients with an NLRP12 mutant-associated inflammatory disorder produced increased amounts of the inflammatory cytokine IL-1ß in response to NLRP3 stimulation. Thus, our findings provide insights into NLRP12 biology and suggest that NLRP3 inhibitors in clinical trials for NLRP3-driven diseases may also be effective in treating NLRP12-associated autoinflammatory diseases.

Oxidized thioredoxin 1 places a leash on NLRP1 inflammasome activity.

The biology of the NACHT domain and leucine-rich repeat (NLR) and pyrin domain-containing 1 (NLRP1) inflammasome has perplexed researchers since this inflammasome was first described about two decades ago. The identification of oxidized thioredoxin 1 (TRX1) as a suppressor of NLRP1 recently linked cellular redox homeostasis to NLRP1 inflammasome signaling. Now, Zhang et al. present a molecular structure of TRX1-bound NLRP1 with unprecedented detail. This structure gives key insight into regulatory mechanisms governing NLRP1 activation and offers enormous potential for structure-based anti-inflammatory drug design.

Quantifying Cell Death Induced by the NLRC4 Inflammasome.

The Nod-like Receptor (NLR) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that sense cytosolic bacterial proteins. NAIP ligation induces its association with NLRC4, leading to the assembly of the NAIP/NLRC4 inflammasome, which induces the activation of the caspase-1 protease. Caspase-1 then cleaves pro-interleukin (IL)-1ß, pro-IL-18, and gasdermin D and induces a form of pro-inflammatory cell death, pyroptosis. These processes culminate in host defense against bacterial infection. Here we describe methods for activating NAIP/NLRC4 inflammasome signalling in human and murine macrophages and quantifying inflammasome-induced cell death.

Reversing the mitochondrial hex that bewitches NLRP3.

Hexokinase dissociation from mitochondria triggers calcium-induced oligomerization of VDAC within the outer mitochondrial membrane, leading to NLRP3 recruitment and inflammasome signaling (see related Research Article by Baik et al.).

Macrophage ACE2 is necessary for SARS-CoV-2 replication and subsequent cytokine responses that restrict continued virion release.

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Ex vivo microRNA and gene expression profiling of human Tr1-like cells suggests a role for miR-92a and -125a in the regulation of EOMES and IL-10R.

Ex vivo gene expression and miRNA profiling of Eomes+ Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4+ T-cells. Several microRNAs were downregulated in Eomes+ Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.

Vitamin D and IFN-ß Modulate the Inflammatory Gene Expression Program of Primary Human T Lymphocytes.

IFN-ß treatment is a commonly used therapy for relapsing-remitting multiple sclerosis (MS), while vitamin D deficiency correlates with an increased risk of MS and/or its activity. MS is a demyelinating chronic inflammatory disease of the central nervous system, in which activated T lymphocytes play a major role, and may represent direct targets of IFN-ß and vitamin D activities. However, the underlying mechanism of action of vitamin D and IFN-ß, alone or in combination, remains incompletely understood, especially when considering their direct effects on the ability of T lymphocytes to produce inflammatory cytokines. We profiled the expression of immune-related genes and microRNAs in primary human T lymphocytes in response to vitamin D and IFN-ß, and we dissected the impact of these treatments on cytokine production and T cell proliferation. We found that the treatments influenced primarily memory T cell plasticity, rather than polarization toward a stable phenotype. Moreover, our data revealed extensive reprogramming of the transcriptional output of primary T cells in response to vitamin D and IFN-ß and provide the bases for further mechanistic insights into these commonly used treatments.

A molecular network regulating the proinflammatory phenotype of human memory T lymphocytes.

Understanding the mechanisms that modulate helper T lymphocyte functions is crucial to decipher normal and pathogenic immune responses in humans. To identify molecular determinants influencing the pathogenicity of T cells, we separated ex vivo-isolated primary human memory T lymphocytes on the basis of their ability to produce high levels of inflammatory cytokines. We found that the inflammatory, cytokine-producing phenotype of memory T lymphocytes was defined by a specific core gene signature and was mechanistically regulated by the constitutive activation of the NF-κB pathway and by the expression of the transcriptional repressor BHLHE40. BHLHE40 attenuated the expression of anti-inflammatory factors, including miR-146a, a negative regulator of NF-κB activation and ZC3H12D, an RNase of the Regnase-1 family able to degrade inflammatory transcripts. Our data reveal a molecular network regulating the proinflammatory phenotype of human memory T lymphocytes, with the potential to contribute to disease.

Rebound of disease activity after fingolimod withdrawal: Immunological and gene expression profiling.

Discontinuation of disease-modifying therapy with fingolimod can lead to severe Multiple Sclerosis (MS) rebound activity; however, this phenomenon remains mechanistically incompletely understood, and the short-term impact of a therapy switch on inflammatory gene expression in T lymphocytes is unknown. We present the clinico-radiological and immunological description of a case of rebound activity after fingolimod discontinuation and switching to rituximab treatment in a relapsing-remitting MS patient. After severe rebound, a reduction in the expression of inflammatory cytokines and transcription factors was rapidly observed after administration of methylprednisolone and rituximab. Rituximab led to an effective suppression of inflammatory activity, and at least in this specific case it represented a valid switching approach after fingolimod discontinuation.

MicroRNAs as modulators of T cell functions in cancer.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression post-transcriptionally. They have emerged as important modulators of T lymphocyte biology, influencing cell activation, differentiation and proliferation in response to environmental signals. Here, we will discuss how miRNAs expressed by T cells can influence two key aspects of tumorigenesis, namely the direct, cell-intrinsic oncogenic transformation of T lymphocytes, as well as the indirect effects on tumor growth mediated by altered immune surveillance. We will specifically focus on three miRNAs that have been shown to regulate different aspects of T cell biology in both physiological and pathological conditions, namely miR-155, miR-146a and miR-181a. We aim at providing examples of the fundamental importance of miRNA-regulated networks in determining the fate of T lymphocytes during oncogenic transformation and in the control of tumor growth.

TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities.

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.

Epigenetics of T lymphocytes in health and disease.

The risk of developing autoimmune diseases depends on both genetic and environmental factors, with epigenetic mechanisms of regulation potentially translating environmental cues into stable modifications in gene expression. Such stable memory of a functional state has been deciphered into a number of molecular mechanisms that collectively define the epigenetic status of a cell. In recent years, it has become increasingly clear that epigenetic modifications are highly dynamic and are able to adapt to the changing environment, with important impact on the onset and development of a number of diseases. Here, we describe some of the epigenetic mechanisms of regulation of cellular functional states in T lymphocytes, with a particular focus on DNA methylation. We will also discuss current knowledge on the role of epigenetics in autoimmunity and consider open questions in the field.