Miao Tang

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1β (IL-1β) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses

Multivalent molecules with repetitive structures including bacterial capsular polysaccharides and viral capsids elicit antibody responses through B cell receptor (BCR) crosslinking in the absence of T cell help. We report that immunization with these T cell–independent type 2 (TI-2) antigens causes up-regulation of endogenous retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a mitochondrial antiviral signaling protein (MAVS)–dependent RNA sensing pathway or reverse-transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a second, sustained wave of signaling that promotes specific immunoglobulin M production. Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings suggest that ERV and two innate sensing pathways that detect them are integral components of the TI-2 B cell signaling apparatus. Endogenous retroviruses materially contribute to humoral immunity in mice. [Also see Perspective by Grasset and Cerutti] Endogenous retroviruses trigger B cells Scattered across our genome are endogenous retroviruses (ERVs), ancient “footprints” of previous viral infections. Scientists do not fully understand their functions, but Zeng et al. now report a role for ERVs in mobilizing a particular type of B cell–driven immune response in mice (T cell–independent, TID), which is usually mounted in response to viral capids or bacterial polysaccharides (see the Perspective by Grasset and Cerutti). Immunizing mice with a model TID antigen elicited an increase in ERV RNA and DNA in the cytoplasm of B cells. Innate immune receptors that recognize cytoplasmic nucleotides then triggered signaling cascades that resulted in the production of immunoglobulin M. Science, this issue p. 1486; see also p. 1454

Real-time resolution of point mutations that cause phenovariance in mice

Significance In forward genetics, a mutagen is used to randomly induce germline mutations that cause variant phenotypes. Forward genetics permits discovery of genes necessary for biological phenomena, but identifying the mutations that cause variant phenotypes is time-consuming and in the past usually occurred long after the phenotype was first recognized. Here we introduce a method and software tool, Linkage Analyzer, for identifying causative mutations present in the germline of mutant mice concurrent with recognition of variant phenotypes. It requires knowledge of genotype at all mutation sites in members of a pedigree prior to phenotypic assessment. Using this method and software, forward genetic studies in mice are limited only by the rates of mutant production and screening. With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled “superpedigrees” are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid tissue in mice.

Significance Immunoglobulins exist in several forms, or isotypes, that carry out distinct effector functions. During an antibody response, B cells can switch their immunoglobulin isotype through the process of class-switch recombination (CSR). CSR to IgD is a rare event compared with CSR to other isotypes, and its regulation is poorly understood. Here we report that mice lacking the DNA damage-response protein 53BP1 display a hyper-IgD syndrome despite deficiencies of other immunoglobulin classes. By studying these mice, we discovered that CSR to IgD in 53BP1 mutant mice and in wild-type mice depends on an intact microbiome and Toll-like receptor signaling, and is anatomically confined to B cells of mucosa-associated lymphoid tissues. Class-switch recombination (CSR) alters the Ig isotype to diversify antibody effector functions. IgD CSR is a rare event, and its regulation is poorly understood. We report that deficiency of 53BP1, a DNA damage-response protein, caused age-dependent overproduction of secreted IgD resulting from increased IgD CSR exclusively within B cells of mucosa-associated lymphoid tissues. IgD overproduction was dependent on activation-induced cytidine deaminase, hematopoietic MyD88 expression, and an intact microbiome, against which circulating IgD, but not IgM, was reactive. IgD CSR occurred via both alternative nonhomologous end-joining and homologous recombination pathways. Microbiota-dependent IgD CSR also was detected in nasal-associated lymphoid tissue of WT mice. These results identify a pathway, present in WT mice and hyperactivated in 53BP1-deficient mice, by which microbiota signal via Toll-like receptors to elicit IgD CSR.

Creatine maintains intestinal homeostasis and protects against colitis.

Significance The present study used a forward genetic approach to identify new causative mutations in an environmentally sensitized screen for colitis. A candidate mutation in Gatm, verified by CRISPR/Cas9 targeting, demonstrated the need for rapid replenishment of cytoplasmic ATP within colonic epithelial cells to restore barrier integrity. This environmentally specified requirement for energy to avert colitis suggests a new category of mutations with the potential to cause inflammatory bowel disease. Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea–mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9-targeted (Gatmc/c) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/c colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury.

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection.

Significance Chaperones in the endoplasmic reticulum (ER) are essential for protein folding and for the maintenance of an efficient secretory pathway. These chaperones can also accompany their substrates during transit from the ER to the Golgi. The prototypical mammalian KDEL receptor (KDELR1) functions by returning chaperones and other proteins to the ER. We show that a recessive missense mutation of Kdelr1 in mice is associated with low numbers of lymphocytes in the blood (lymphopenia), reduced expression of the T-cell receptor, and compromised antiviral immunity. Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrates C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.

Insulin resistance and diabetes caused by genetic or diet-induced KBTBD2 deficiency in mice

Significance We report an essential regulator of insulin sensitivity. Mutations affecting this protein, KBTBD2, cause severe insulin-resistant diabetes, lipodystrophy, hepatic steatosis, and growth retardation. KBTBD2 is the substrate recognition subunit of a ubiquitin ligase, and its essential molecular target is p85α, the regulatory subunit of phosphoinositol-3-kinase. KBTBD2 is highly conserved among vertebrates and expressed in liver, brain, muscle, and adipocytes. In the absence of KBTBD2, p85α levels rise 30-fold in adipocytes, interrupting the insulin signal, which is fully restored by p85α knockout. Transfer of KBTBD2-sufficient adipose tissue to KBTBD2-deficient animals rescues insulin-resistant hyperglycemia. Within adipocytes, KBTBD2 expression is markedly down-regulated in response to a high-fat diet. This appears to be an important cause of the insulin resistance caused by obesity. We describe a metabolic disorder characterized by lipodystrophy, hepatic steatosis, insulin resistance, severe diabetes, and growth retardation observed in mice carrying N-ethyl-N-nitrosourea (ENU)–induced mutations. The disorder was ascribed to a mutation of kelch repeat and BTB (POZ) domain containing 2 (Kbtbd2) and was mimicked by a CRISPR/Cas9-targeted null allele of the same gene. Kbtbd2 encodes a BTB-Kelch family substrate recognition subunit of the Cullin-3–based E3 ubiquitin ligase. KBTBD2 targeted p85α, the regulatory subunit of the phosphoinositol-3-kinase (PI3K) heterodimer, causing p85α ubiquitination and proteasome-mediated degradation. In the absence of KBTBD2, p85α accumulated to 30-fold greater levels than in wild-type adipocytes, and excessive p110-free p85α blocked the binding of p85α-p110 heterodimers to IRS1, interrupting the insulin signal. Both transplantation of wild-type adipose tissue and homozygous germ line inactivation of the p85α-encoding gene Pik3r1 rescued diabetes and hepatic steatosis phenotypes of Kbtbd2−/− mice. Kbtbd2 was down-regulated in diet-induced obese insulin-resistant mice in a leptin-dependent manner. KBTBD2 is an essential regulator of the insulin-signaling pathway, modulating insulin sensitivity by limiting p85α abundance.

Intracellular lipid metabolism impairs β cell compensation during diet-induced obesity

The compensatory proliferation of insulin-producing &bgr; cells is critical to maintaining glucose homeostasis at the early stage of type 2 diabetes. Failure of &bgr; cells to proliferate results in hyperglycemia and insulin dependence in patients. To understand the effect of the interplay between &bgr; cell compensation and lipid metabolism upon obesity and peripheral insulin resistance, we eliminated LDL receptor–related protein 1 (LRP1), a pleiotropic mediator of cholesterol, insulin, energy metabolism, and other cellular processes, in &bgr; cells. Upon high-fat diet exposure, LRP1 ablation significantly impaired insulin secretion and proliferation of &bgr; cells. The diminished insulin signaling was partly contributed to by the hypersensitivity to glucose-induced, Ca2+-dependent activation of Erk and the mTORC1 effector p85 S6K1. Surprisingly, in LRP1-deficient islets, lipotoxic sphingolipids were mitigated by improved lipid metabolism, mediated at least in part by the master transcriptional regulator PPAR&ggr;2. Acute overexpression of PPAR&ggr;2 in &bgr; cells impaired insulin signaling and insulin secretion. Elimination of Apbb2, a functional regulator of LRP1 cytoplasmic domain, also impaired &bgr; cell function in a similar fashion. In summary, our results uncover the double-edged effects of intracellular lipid metabolism on &bgr; cell function and viability in obesity and type 2 diabetes and highlight LRP1 as an essential regulator of these processes.

Adjuvant effect of the novel TLR1/TLR2 agonist Diprovocim synergizes with anti–PD-L1 to eliminate melanoma in mice

Significance Adjuvants enhance adaptive immune responses, sometimes through unknown mechanisms, and can be used to augment both humoral and cellular responses to cancer antigens. We report the immunological effects of the synthetic chemical adjuvant Diprovocim, which targets the innate immune receptor TLR1/TLR2 in mice and humans. Diprovocim displayed strong adjuvant activity in mice, particularly abetting cellular immune responses. Immunization against a genetically engineered tumor-specific antigen, ovalbumin, when adjuvanted with Diprovocim, inhibited growth of B16 melanoma and prolonged survival in the presence of immune checkpoint blockade by anti–PD-L1; 100% of mice responded to treatment. Our data suggest Diprovocim boosts the success of anti–PD-L1 treatment by increasing the number and activation of tumor-specific CTLs capable of responding to this checkpoint inhibitor. Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti–PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti–PD-L1 treatment.

HCFC2 is needed for IRF1- and IRF2-dependent Tlr3 transcription and for survival during viral infections

Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea–induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.