C1q restrains autoimmunity and viral infection by regulating CD8+ T cell metabolism.

Deficiency of C1q, the initiator of the complement classical pathway, is associated with the development of systemic lupus erythematosus (SLE). Explaining this association in terms of abnormalities in the classical pathway alone remains problematic because C3 deficiency does not predispose to SLE. Here, using a mouse model of SLE, we demonstrate that C1q, but not C3, restrains the response to self-antigens by modulating the mitochondrial metabolism of CD8+ T cells, which can themselves propagate autoimmunity. C1q deficiency also triggers an exuberant effector CD8+ T cell response to chronic viral infection leading to lethal immunopathology. These data establish a link between C1q and CD8+ T cell metabolism and may explain how C1q protects against lupus, with implications for the role of viral infections in the perpetuation of autoimmunity.

RETRACTED: T cell metabolism. The protein LEM promotes CD8⁺ T cell immunity through effects on mitochondrial respiration.

Protective CD8(+) T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named lymphocyte expansion molecule (LEM) that promoted antigen-dependent CD8(+) T cell proliferation, effector function, and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results. Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration, resulting in the production of pro-proliferative mitochondrial reactive oxygen species (mROS). LEM provides a link between immune activation and the expansion of protective CD8(+) T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified cytotoxic CD8(+) T cells.

N-Ethyl-N-nitrosourea mutagenesis in the mouse provides strong genetic and in vivo evidence for the role of the Caspase Recruitment Domain (CARD) of CARD-MAGUK1 in T regulatory cell development.

Natural regulatory T (nTreg) cells generated in the thymus are essential throughout life for the maintenance of T-cell homeostasis and the prevention of autoimmunity. T-cell receptor (TCR)/CD28-mediated activation of nuclear factor-κB and (J)un (N)-terminal kinase pathways is known to play a key role in nTreg cell development but many of the predicted molecular interactions are based on extrapolations from non-Treg cell TCR stimulation with non-physiological ligands. For the first time, we provide strong genetic evidence of a scaffold function for the Caspase Recruitment Domain (CARD) of the TCR signalling protein CARD-MAGUK1 (CARMA1) in nTreg cell development in vivo. We report two, new, N-ethyl-N-nitrosourea-derived mutant mice, Vulpo and Zerda, with a profound block in the development of nTreg cells in the thymus as well as impaired inducible Treg cell differentiation in the periphery. Despite independent heritage, both mutants harbour different point mutations in the CARD of the CARMA1 protein. Mutations in vulpo and zerda do not affect expression levels of CARMA1 but still impair signalling through the TCR due to defective downstream Bcl-10 recruitment by the mutated CARD of CARMA1. Phenotypic differences observed between Vulpo and Zerda mutants suggest a role for the CARD of CARMA1 independent of Bcl-10 activation of downstream pathways. We conclude that our forward genetic approach demonstrates a critical role for the CARD function of CARMA1 in Treg cell development in vivo.

ENU-induced phenovariance in mice: inferences from 587 mutations.

BACKGROUND: We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. FINDINGS: In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. CONCLUSIONS: Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Disruption of copper homeostasis due to a mutation of Atp7a delays the onset of prion disease.

Copper influences the pathogenesis of prion disease, but whether it is beneficial or detrimental remains controversial. Copper homeostasis is also essential for normal physiology, as highlighted by the spectrum of diseases caused by disruption of the copper transporting enzymes ATP7A and ATP7B. Here, by using a forward genetics approach in mice, we describe the isolation of three alleles of Atp7a, each with different phenotypic consequences. The mildest of the three, Atp7a(brown), was insufficient to cause lethality in hemizygotes or mottling of the coat in heterozygotes, but did lead to coat hypopigmentation and reduced copper content in the brains of hemizygous males. When challenged with Rocky Mountain Laboratory scrapie, the onset of prion disease was delayed in Atp7a(brown) mice, and significantly less proteinase-resistant prion protein was found in the brains of moribund Atp7a(brown) mice compared with WT littermates. Our results establish that ATP7A-mediated copper homeostasis is important for the formation of pathogenic proteinase-resistant prion protein.

Dissecting immunity by germline mutagenesis.

The last decades have seen numerous approaches being used to decipher biological phenomena, notably the strategies we employ to defend ourselves against pathogenic attacks. From microarrays to genetics to computing technologies, all have supported a better but not yet comprehensive understanding of the pathways regulating our immune system. Limitations are notably exemplified by cases of immune deficiencies in humans that often result in high susceptibility to infections or even death, without the genetic cause being evident. To provide further insight into the mechanisms by which pathogen detection and eradication occur, several in vivo strategies can be used. The current review focuses on one of them, namely germline mutagenesis in the mouse. After describing the main technical aspects of this forward genetic approach, we will discuss particular germline mutants that have all been instrumental in deciphering innate or adaptive immune responses. Mutations in previously uncharacterized genes in the mouse, like Unc93B or Themis, have demonstrated the impartiality of forward genetics and led to the identification of new crucial immunity actors. Some mutants, like PanR1, have informed us on particular protein domains and their specific functions. Finally, certain mutations identified by this non-hypothesis-driven method have revealed previously unknown gene functions, as recently illustrated by memi, which links a particular nucleoside salvage enzyme to cell proliferation and apoptosis.

Hypopigmentation and maternal-zygotic embryonic lethality caused by a hypomorphic mbtps1 mutation in mice.

The site 1 protease, encoded by Mbtps1, mediates the initial cleavage of site 2 protease substrates, including sterol regulatory element binding proteins and CREB/ATF transcription factors. We demonstrate that a hypomorphic mutation of Mbtps1 called woodrat (wrt) caused hypocholesterolemia, as well as progressive hypopigmentation of the coat, that appears to be mechanistically unrelated. Hypopigmentation was rescued by transgenic expression of wild-type Mbtps1, and reciprocal grafting studies showed that normal pigmentation depended upon both cell-intrinsic or paracrine factors, as well as factors that act systemically, both of which are lacking in wrt homozygotes. Mbtps1 exhibited a maternal-zygotic effect characterized by fully penetrant embryonic lethality of maternal-zygotic wrt mutant offspring and partial embryonic lethality (~40%) of zygotic wrt mutant offspring. Mbtps1 is one of two maternal-zygotic effect genes identified in mammals to date. It functions nonredundantly in pigmentation and embryogenesis.

A deficiency in nucleoside salvage impairs murine lymphocyte development, homeostasis, and survival.

The homeostasis of the immune system is tightly controlled by both cell-extrinsic and -intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function. In this article, we describe a deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway, which affects peripheral T cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosourea-induced mutation in the last α helix of DCK, a functionally null protein has been generated in the mouse and affects the composition of the hematopoietic system. Both B and T lymphocyte development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44(high)CD62L(low) memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notably the result of a cell-extrinsic-driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.

Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell-specific manner.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.

Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells.

Despite their low frequency, plasmacytoid dendritic cells (pDCs) produce most of the type I IFN that is detectable in the blood following viral infection. The endosomal Toll-like receptors (TLRs) TLR7 and TLR9 are required for pDCs, as well as other cell types, to sense viral nucleic acids, but the mechanism by which signaling through these shared receptors results in the prodigious production of type I IFN by pDCs is not understood. We designed a genetic screen to identify proteins required for the development and specialized function of pDCs. One phenovariant, which we named feeble, showed abrogation of both TLR-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving TLR responses intact in other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1) and has not previously been implicated in pDC function. The identification of the feeble mutation led to our subsequent observations that AP-3, as well as the BLOC-1 and BLOC-2 Hermansky-Pudlak syndrome proteins are essential for pDC signaling through TLR7 and TLR9. These proteins are not necessary for TLR7 or TLR9 signaling in conventional DCs and thus comprise a membrane trafficking pathway uniquely required for endosomal TLR signaling in pDCs.

Enhanced sensitivity to DSS colitis caused by a hypomorphic Mbtps1 mutation disrupting the ATF6-driven unfolded protein response.

Here, we describe an N-ethyl-N-nitrosourea (ENU)-induced missense error in the membrane-bound transcription factor peptidase site 1 (S1P)-encoding gene (Mbtps1) that causes enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. S1P cleaves and activates cAMP response element binding protein/ATF transcription factors, the sterol regulatory element-binding proteins (SREBPs), and other proteins of both endogenous and viral origin. Because S1P has a nonredundant function in the ATF6-dependent unfolded protein response (UPR), woodrat mice show diminished levels of major endoplasmic reticulum chaperones GRP78 (BiP) and GRP94 in the colon upon DSS administration. Experiments with bone marrow chimeric mice reveal a requirement for S1P in nonhematopoietic cells, without which a diminished UPR and colitis develop.

Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger.

A recessive phenotype called spin (spontaneous inflammation) was induced by N-ethyl-N-nitrosourea (ENU) mutagenesis in C57BL/6J mice. Homozygotes display chronic inflammatory lesions affecting the feet, salivary glands and lungs, and antichromatin antibodies. They are immunocompetent and show enhanced resistance to infection by Listeria monocytogenes. TLR-induced TNF and IL-1 production are normal in macrophages derived from spin mice. The autoinflammatory phenotype of spin mice is fully suppressed by compound homozygosity for Myd88(poc), Irak4(otiose), and Il1r1-null mutations, but not Ticam1(Lps2), Stat1(m1Btlr), or Tnf-null mutations. Both autoimmune and autoinflammatory phenotypes are suppressed when spin homozygotes are derived into a germ-free environment. The spin phenotype was ascribed to a viable hypomorphic allele of Ptpn6, which encodes the tyrosine phosphatase SHP1, mutated in mice with the classical motheaten alleles me and me-v. Inflammation and autoimmunity caused by SHP1 deficiency are thus conditional. The SHP1-deficient phenotype is driven by microbes, which activate TLR signaling pathways to elicit IL-1 production. IL-1 signaling via MyD88 elicits inflammatory disease.

Dissecting innate immunity by germline mutagenesis.

The innate arm of our immune system is the first line of defence against infections. In addition, it is believed to drive adaptive immune responses, which help fight pathogens and provide long-term memory. As such, the innate immune system is instrumental for protection against pathogens that would otherwise destroy their host. Although our understanding of the innate immune components involved in pathogen sensing and fighting is improving, it is still limited. This is particularly exemplified by increased documentation of innate immune deficiencies in humans that often result in high and recurrent susceptibility to infections or even death, without the genetic cause being evident. To provide further insight into the mechanisms by which pathogen sensing and eradication occur, several strategies can be used. The current review focuses on the forward genetic approaches that have been used to dissect innate immunity in the fruit fly and the mouse. For both animal models, forward genetics has been instrumental in the deciphering of innate immunity and has greatly improved our understanding of how we respond to invading pathogens.

Jinx, an MCMV susceptibility phenotype caused by disruption of Unc13d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis.

Mouse cytomegalovirus (MCMV) susceptibility often results from defects of natural killer (NK) cell function. Here we describe Jinx, an N-ethyl-N-nitrosourea-induced MCMV susceptibility mutation that permits unchecked proliferation of the virus, causing death. In Jinx homozygotes, activated NK cells and cytotoxic T lymphocytes (CTLs) fail to degranulate, although they retain the ability to produce cytokines, and cytokine levels are markedly elevated in the blood of infected mutant mice. Jinx was mapped to mouse chromosome 11 on a total of 246 meioses and confined to a 4.60-million basepair critical region encompassing 122 annotated genes. The phenotype was ascribed to the creation of a novel donor splice site in Unc13d, the mouse orthologue of human MUNC13-4, in which mutations cause type 3 familial hemophagocytic lymphohistiocytosis (FHL3), a fatal disease marked by massive hepatosplenomegaly, anemia, and thrombocytopenia. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus, exhibiting hyperactivation of CTLs and antigen-presenting cells, and inadequate restriction of viral proliferation. In contrast, neither Listeria monocytogenes nor MCMV induces the syndrome. In mice, the HLH phenotype is conditional, which suggests the existence of a specific infectious trigger of FHL3 in humans.

Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes.

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1-Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

Details of Toll-like receptor:adapter interaction revealed by germ-line mutagenesis.

The immunovariant N-ethyl-N-nitrosourea-induced mutations Pococurante (Poc) and Lackadaisical were found to alter MyD88, creating striking receptor-selective effects. Poc, in particular, prevented sensing of all MyD88-dependent Toll-like receptor (TLR) ligands except diacyl lipopeptides. Furthermore, Poc-site and classical BB loop mutations caused equivalent phenotypes when engrafted into any TLR/IL-1 receptor/resistance (TIR) domain. These observations, complemented by data from docking studies and site-directed mutagenesis, revealed that BB loops and Poc sites interact homotypically across the receptor:adapter signaling interface, whereas the C-terminal alpha(E)-helices support adapter:adapter and receptor:receptor oligomerization. We have thus defined the TIR domain surface that mediates association between TLRs and MyD88 and the surface required for MyD88 or TLR oligomerization. Moreover, MyD88 engages individual TLRs differently, suggesting the feasibility of selective pharmacologic TIR domain receptor blockade.

Efficient T cell activation via a Toll-Interleukin 1 Receptor-independent pathway.

Here, we describe a previously unrecognized pathway for activation of antigen-specific adaptive immune responses that was independent of Toll-Interleukin 1 Receptor signaling and directed toward detection of antigens expressed by apoptotic cells. This pathway is represented within Flt-3 Ligand-derived dendritic cells (DCs) that represent immature lymphoid DCs, but not within GM-CSF-treated bone marrow-derived dendritic cells. Exposure of these DCs to apoptotic cells resulted in production of type I interferon and favored the development of cytotoxic T cell responses. The N-Ethyl-N-Nitrosourea-induced germline mutation 3d (Unc3b1(3d/3d)) abolished both MHC class I and II responses elicited by this pathway, whereas a null allele of Cd36 selectively abolished class II responses. We propose that this mode of adaptive immune activation evolved to permit the sensitive detection of intracellular microbial infections, particularly viral infections, which frequently induce apoptotic cell death, but may also be important in transplantation, autoimmunity, and vaccine development.

PanR1, a dominant negative missense allele of the gene encoding TNF-alpha (Tnf), does not impair lymphoid development.

A dominant hypomorphic allele of Tnf, PanR1, was identified in a population of G(1) mice born to N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes produced no detectable TNF bioactivity, although normal quantities of immunoreactive TNF were secreted. The phenotype was confined to a critical region on mouse chromosome 17, and then ascribed to a C-->A transversion at position 3480 of the Tnf gene, corresponding to the amino acid substitution P138T. As a result of subunit exchange, the protein exerts a dominant-negative effect on normal TNF trimers, interfering with the trimer/receptor interaction. Homozygotes are highly susceptible to infection by Listeria monocytogenes, confirming the essential role of TNF in innate immune defense. However, PanR1 mutant mice show normal architecture of the spleen and Peyer's patches, suggesting that TNF is not essential for the formation of these lymphoid structures.

Analysis of the MCMV resistome by ENU mutagenesis.

The mouse cytomegalovirus (MCMV) resistome is the set of host genes with nonredundant functions in resistance to MCMV infection. By screening 3,500 G(3) germline mutant mice ( approximately 1,750 gamete equivalents), we have identified eight transmissible mutations that create MCMV susceptibility in C57BL/6 mice. Among these, a mutation called Domino was noted to cause macrophage susceptibility to vesicular stomatitis virus (VSV) in vitro. This accessory phenotype was not corrected by type I interferon (IFN), which suggested a defect of the type I IFN pathway. Domino corresponds to a point mutation that alters the DNA binding domain of STAT1, leading to a defect of STAT1 activation. Identification of the Domino mutation demonstrates that an in vivo MCMV susceptibility screen is feasible and illustrates how it can provide insight into the resistome. Moreover, some mutations are far more deleterious than Domino in MCMV-infected mice, consistent with the interpretation that certain protein(s) unrelated to IFN production or signaling are more important than IFNs with regard to their net antiviral effects.

Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large.

Classical genetic methods, driven by phenotype rather than hypotheses, generally permit the identification of all proteins that serve nonredundant functions in a defined biological process. Long before this goal is achieved, and sometimes at the very outset, genetics may cut to the heart of a biological puzzle. So it was in the field of mammalian innate immunity. The positional cloning of a spontaneous mutation that caused lipopolysaccharide resistance and susceptibility to Gram-negative infection led directly to the understanding that Toll-like receptors (TLRs) are essential sensors of microbial infection. Other mutations, induced by the random germ line mutagen ENU (N-ethyl-N-nitrosourea), have disclosed key molecules in the TLR signaling pathways and helped us to construct a reasonably sophisticated portrait of the afferent innate immune response. A still broader genetic screen--one that detects all mutations that compromise survival during infection--is permitting fresh insight into the number and types of proteins that mammals use to defend themselves against microbes.