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The primary goals of modern genetics are to identify disease-causing mutations and to define the functions of genes in biological processes. Two complementary approaches, reverse and forward genetics, can be used to achieve this goal. Reverse genetics is a gene-driven approach that comprises specific gene targeting followed by phenotypic assessment. Conversely, forward genetics is a phenotype-driven approach that involves the phenotypic screening of organisms with randomly induced mutations followed by subsequent identification of the causative mutations (i.e., those responsible for phenotype). In this article, we focus on how forward genetics in mice can be used to explore dermatologic disease. We outline mouse mutagenesis with the chemical N-ethyl-N-nitrosourea and the strategy used to instantaneously identify mutations that are causative of specific phenotypes. Furthermore, we summarize the types of phenotypic screens that can be performed to explore various aspects of dermatologic disease., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

As it is a hard-wired system for responses to microbes, innate immunity is particularly susceptible to classical genetic analysis. Mutations led the way to the discovery of many of the molecular elements of innate immune sensing and signaling pathways. In turn, the need for a faster way to find the molecular causes of mutation-induced phenotypes triggered a huge transformation in forward genetics. During the 1980s and 1990s, many heritable phenotypes were ascribed to mutations through positional cloning. In mice, this required three steps. First, a genetic mapping step was used to show that a given phenotype emanated from a circumscribed region of the genome. Second, a physical mapping step was undertaken, in which all of the region was cloned and its gene content determined. Finally, a concerted search for the mutation was performed. Such projects usually lasted for several years, but could produce breakthroughs in our understanding of biological processes. Publication of the annotated mouse genome sequence in 2002 made physical mapping unnecessary. More recently we devised a new technology for automated genetic mapping, which eliminated both genetic mapping and the search for mutations among candidate genes. The cause of phenotype can now be determined instantaneously. We have created more than 100,000 coding/splicing mutations. And by screening for defects of innate and adaptive immunity we have discovered many "new" proteins needed for innate immune function., Competing Interests: Statement The author declares no conflict of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The MYD88(L265P) mutation is found in 2% to 10% of chronic lymphocytic leukemia, 29% of activated B-cell type diffuse large B-cell lymphoma and 90% of Waldenström macroglobulinemia, making it conceptually attractive to treat these malignancies with inhibitors of endosomal Toll-like receptors (TLR9, TLR7) that activate MYD88. Here we show that genetic inhibition of endosomal TLRs has the opposite effect on accumulation of MYD88(L265P) B cells in vitro and in vivo. Activated mature B cells from wild-type, Unc93b1(3d/3d)-mutant, or Tlr9-deficient mice were transduced with retrovirus encoding MYD88(L265P) and analyzed either in vitro or after transplantation into Rag1(-/-) recipient mice. Unc93b1(3d/3d) mutation, which blocks TLR9 and TLR7 signaling, or Tlr9 deficiency suppressed MYD88(L265P) B-cell growth in vitro but paradoxically increased in vivo accumulation of MYD88(L265P) B cells as CD19(low) plasmablasts by 10- to 100-fold. These results reveal an unexpected, powerful inhibitory effect of TLR9 on MYD88(L265P) B-cell proliferation and differentiation that appears independent of TLR7, and they provide a preclinical indicator for caution in clinical trials of TLR7/9 inhibitors for MYD88(L265P) B-cell malignancies., (© 2016 by The American Society of Hematology.)

Forward genetic analysis is an unbiased approach for identifying genes essential to defined biological phenomena. When applied to mice, it is one of the most powerful methods to facilitate understanding of the genetic basis of human biology and disease. The speed at which disease-causing mutations can be identified in mutagenized mice has been markedly increased by recent advances in DNA sequencing technology. Creating and analyzing mutant phenotypes may therefore become rate-limiting in forward genetic experimentation. We review the forward genetic approach and its future in the context of recent technological advances, in particular massively parallel DNA sequencing, induced pluripotent stem cells, and haploid embryonic stem cells., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Inflammatory bowel diseases (IBD), including both ulcerative colitis and Crohn's disease, are extremely variable in severity and have strong genetic components. In mice, several mutations are known to favor or inhibit intestinal inflammation. But a comprehensive picture of the pathogenesis of IBD cannot be assembled based on the limited information so far available from mouse genetic analyses, nor can human IBD be stringently ascribed to mutations known to be influential in mice. This review highlights recent progress made using mouse models created through a forward genetic approach towards the understanding of genes that normally prevent intestinal inflammation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

TNFα is a powerful inflammatory stimulus, central both to the control of infection, and as an agent of inflammatory disease. The most potent inducers of TNFα secretion signal through the Toll-like receptors, and we describe here a chemically-induced mutation that impairs this response in macrophages. A missense mutation was revealed in the gene encoding the inactive rhomboid protease iRhom2, which was not complemented by a null allele of the same gene. Neither the missense nor the null allele affected TLR-induced secretion of IL-6. Moreover, unlike a mutation in TNFα, the iRhom2 missense mutation did not cause enhanced susceptibility to colitis induced by dextran sodium sulfate. These results establish a specific role for iRhom2 in the secretion of TNFα, and present a new target for the modulation of inflammation.

Iron is an essential component of heme and hemoglobin, and therefore restriction of iron availability directly limits erythropoiesis. In the present study, we report a defect in iron absorption that results in iron-deficiency anemia, as revealed by an N-ethyl-N-nitrosourea-induced mouse phenotype called sublytic. Homozygous sublytic mice develop hypochromic microcytic anemia with reduced osmotic fragility of RBCs. The sublytic phenotype stems from impaired gastrointestinal iron absorption caused by a point mutation of the gastric hydrogen-potassium ATPase α subunit encoded by Atp4a, which results in achlorhydria. The anemia of sublytic homozygotes can be corrected by feeding with a high-iron diet or by parenteral injection of iron dextran; rescue can also be achieved by providing acidified drinking water to sublytic homozygotes. These findings establish the necessity of the gastric proton pump for iron absorption and effective erythropoiesis.

This review focuses on genes required for resistance to mouse cytomegalovirus (MCMV), as identified through unbiased genetic screening. Components of the developmental, sensing, and effector pathways, functioning in multiple cell types, were detected by infecting 22,000 G3 mutant mice with MCMV at an inoculum easily contained by WT animals. Merging these findings with discoveries from hypothesis-based studies, we present a cohesive picture of the essential elements utilized by the mouse innate immune system to counter MCMV. We believe that many breakthrough discoveries will yet be made using a classical genetic approach., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory disorder with a poor prognosis for affected individuals. To find a means of suppressing the clinical phenotype, we investigated the cellular and molecular mechanisms leading to HLH in Unc13d(jinx/jinx) mice, in which cytolytic function of NK and CD8(+) T cells is impaired. Unc13d(jinx/jinx) mutants infected with lymphochoriomeningitis virus (LCMV) present typical clinical features of HLH, including splenomegaly, elevated serum IFNγ, and anemia. Proteins mediating cell-cell contact, cytokine signaling or Toll-like receptor (TLR) signaling were analyzed. We show that neither the integrin CD18, which is involved in adhesion between antigen-presenting cells and effector T cells, nor tumor necrosis factor (TNF) made nonredundant contributions to the disease phenotype. Disruption of IFNγ signaling reduced immune cell activation in Unc13d(jinx/jinx) mice, but also resulted in uncontrolled viral proliferation and exaggerated release of inflammatory cytokines. Abrogating the function of myeloid differentiation primary response gene 88 (MyD88) in Unc13d(jinx/jinx) mice suppressed immune cell activation and controlled cytokine production in an IL-1 receptor 1 (IL-1R1)-independent way. Our findings implicate MyD88 as the key initiator of myeloid and lymphoid proliferation in HLH, and suggest that blockade of this signaling molecule may reduce immunopathology in patients.

Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin-deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit(+) cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-beta induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-alpha and IFN-beta independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-beta promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-alpha and IFN-beta, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-alphabeta or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.

Previous work showed that administration of antigen-expressing apoptotic cells in vivo results in antigen-specific CD8+ T-cell responses independent of Toll-like receptor signaling. We report here that natural killer (NK) cells can serve a function directly upstream of this pathway and initiate robust adaptive immune responses via killing of antigen-expressing target cells. This pathway is highly sensitive, in that administration of as few as 10(4) target cells induced detectable antigen-specific CD8+ T-cell responses. Importantly, NK cell-mediated cytotoxicity of target cells could also induce robust antigen-specific CD4+ T-cell responses, which were critical for subsequent CD8+ T-cell priming and IgG responses. Unlike adaptive immune responses induced by gamma-irradiated cells, the NK-cell pathway required myeloid differentiating factor 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-beta (Trif) signaling. NK cells have previously been shown to detect and kill pathogen-infected host cells, as well as neoplastic cells and tissue allografts. The present data provide further evidence that they also discharge a strong tie with their relatives in the adaptive immune system. We think that the recognition and killing of target cells by NK cells represents an important pathway for the generation of robust CD8+ T and humoral responses that may be exploited for vaccine development.

In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.

Malaria pigment hemozoin was reported to activate the innate immunity by Toll-like receptor (TLR)-9 engagement. However, the role of TLR activation for the development of cerebral malaria (CM), a lethal complication of malaria infection in humans, is unknown. Using Plasmodium berghei ANKA (PbA) infection in mice as a model of CM, we report here that TLR9-deficient mice are not protected from CM. To exclude the role of other members of the TLR family in PbA recognition, we infected mice deficient for single TLR1, -2, -3, -4, -6, -7, or -9 and their adapter proteins MyD88, TIRAP, and TRIF. In contrast to lymphotoxin alpha-deficient mice, which are resistant to CM, all TLR-deficient mice were as sensitive to fatal CM development as wild-type control mice and developed typical microvascular damage with vascular leak and hemorrhage in the brain and lung, together with comparable parasitemia, thrombocytopenia, neutrophilia, and lymphopenia. In conclusion, the present data do not exclude the possibility that malarial molecular motifs may activate the innate immune system. However, TLR-dependent activation of innate immunity is unlikely to contribute significantly to the proinflammatory response to PbA infection and the development of fatal CM.

Resistance to infection is largely inherited rather than acquired, and is encoded by a definable set of host genes designated the 'resistome'. Logically speaking, piecemeal disruption of the resistome gives us the best chance to define it, and the most spectacular advances in understanding innate immunity have grown from spontaneous or induced germline mutations of the resistome. Mutations induced by random germline mutagenesis have now become so numerous that we are nearly in a position to define the size of the resistome, and both random and targeted mutations give us a fairly nice sketch of its components and how they interact. Our own N-ethyl-N-nitrosourea mutagenesis effort, which recently showed that components of Toll-like receptor signaling are essential constituents of the arsenal against MCMV infections, validated the forward genetic approach as a powerful tool to define the resistome.

The innate immune system senses pathogens largely through signals initiated by a collection of phylogenetically related proteins known as "Toll-like receptors" (TLRs), of which 10 representatives are encoded in the human genome. Our understanding of the sensing role played by the TLRs began with the positional cloning of a spontaneous mutation (Lps(d)) in the gene encoding the mammalian lipopolysaccharide (LPS) receptor. Other key innate immunity proteins have been disclosed by germline mutagenesis, and are discussed in the present review.

Mutations of the mouse Lps locus abolish responses to lipopolysaccharide (LPS). Positional cloning work has revealed that Lps encodes the Toll-like receptor 4 (Tlr4), which functions as the transmembrane component of the LPS receptor complex, an unduplicated pathway for the detection of endotoxin. The structurally related protein Tlr2 makes no contribution to LPS signal transduction.

To examine the effects of different wavelengths of ultraviolet (UV) radiation on tumor necrosis factor (TNF) production, we took advantage of mice carrying a chloramphenicol acetyl transferase (CAT) reporter transgene bearing the entire TNF promoter and 3'-untranslated region. Aside from constitutive expression in the thymus, CAT activity was detected only in locally UVB- or UVC-irradiated skin. After UVB irradiation, markedly greater amounts of CAT activity were traced to the dermis rather than the epidermis; by contrast, almost all CAT activity was localized to the epidermis after UVC irradiation. Fibroblasts have not been shown previously to express the TNF gene, i.e., the TNF gene is highly methylated and inaccessible to exogenous modulation in 3T3 fibroblasts. However, the present report reveals that cultured dermal fibroblasts are capable of producing both CAT and TNF in response to treatment in vitro with either UVB irradiation, UVC irradiation, or lipopolysaccharide. These findings indicate that dermal fibroblasts may serve not only as a target for but also as a source of TNF.

We have exploited the extremely high binding specificity of the 55 kDa human tumor necrosis factor (TNF) receptor in an assay designed to detect TNF with sensitivity limited only by limits in the detection of 131I. Bivalent derivatives of the 55 kDa TNF receptor (referred to here as the TNF binding protein), in which the extracellular domain is coupled to an IgG heavy chain, ordinarily bind TNF with very high affinity as a result of the fact that they interact with two separate sites on the trimer surface. The TNF binding protein is radioiodinated to a high specific activity and then added to plasma at a saturating concentration, so that it binds all active TNF present in the solution. Covalent adducts between molecules of TNF and molecules of the binding protein are then produced by crosslinking with disuccinimidyl suberate (DSS). The complexes are swept out of solution using sepharose beads to which polyclonal anti-TNF antibodies have been affixed. On electrophoresis, the complex presents itself as a band of M(r) = 200 kDa (as distinct from the uncomplexed binding protein, which has a size of 120 kDa and which in any case is removed by washing). As little as 50 fg of active TNF (600,000 trimers) can be detected in a 5 ml sample of plasma using this approach, corresponding to the detection of TNF at a 200 aM concentration. Notably, no TNF is detectable in normal plasma specimens, indicating that normal plasma contains active TNF at a concentration beneath 200 aM.

This study describes the isolation and characterization of human fetal Kupffer cells. We demonstrated that these cells have the potential to respond to cytokines and lipopolysaccharide with an increased production of tumor necrosis factor-alpha and interleukin-1 beta. Kupffer cells were characterized by: (1) morphologic characteristics after adherence to plastic, (2) staining for alpha-naphthyl acetate esterase, (3) immunofluorescence with monoclonal antibodies, and (4) phagocytosis of latex beads. More than 90% of the adherent cells were identified as macrophages. Kupffer cells cultured with lipopolysaccharide were able to produce interleukin-1 beta and tumor necrosis factor-alpha in a time- and dose-dependent fashion and maximal secretion was observed with the use of 10 micrograms of lipopolysaccharide per milliliter within 8 hours of treatment. We have demonstrated mature functional activity of human fetal Kupffer cells at an early gestational age (13 to 19 weeks) and discussed the roles that these cells may play in development and protection of the fetus.

By means of a sensitive competition assay, serum, urine, and CSF from patients with Huntington's disease (HD) and controls were tested for the presence of molecules capable of displacing radioactive kainic acid (KA) from rat brain receptors. No difference was found between HD and control samples. It is unlikely that systemic production of an endogenous toxin structurally related to KA occurs in HD.

A method for localization of glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate: NADP+ oxidoreductase; E.C. 1.1.1.49) activity has been applied to human nervous tissue. Intensely staining cells, not definable by conventional histologic techniques, have been identified in the human spinal cord, with highest numbers present in the substantia gelatinosa of the sacral region. The cells have a neuron-like morphology and express neuronal-specific antigen but are heterogeneous in size and shape. They are not detectable in infant spinal cord, but stain heavily in adults. We propose that these cells are homologous to the G-6-PD-active dorsal medullary cells first noted by Sakharova et al. (1979) and together with the latter group, may comprise a hitherto unrecognized system of neurons in the human central nervous system.

mRNA molecules encoding a number of inflammatory cytokines, as well as certain proto-oncogenes, contain a conserved UA-exclusive sequence in the 3' untranslated region that confers message instability in vivo. This sequence may comprise a critical regulatory element, governing the level of these mRNA molecules, and determining the efficiency with which they are translated. Through the use of a double-label RNAse assay, we have determined that lysates prepared from mouse macrophages selectively degrade mRNA molecules containing the 3' untranslated UA sequence found in the mRNA encoding human cachectin/TNF. The degree of instability is dependent upon the number of copies of inserted UA sequence present in the target mRNA molecule (a Xenopus beta-globin mRNA). mRNAs containing randomly generated UA sequences are more labile than unmodified globin mRNA, but less susceptible to degradation than mRNAs containing the authentic cachectin-derived sequence. mRNA molecules containing synthetic UG-exclusive sequences are normally stable or protected in vitro. The destruction of UA-containing mRNA is inhibited by random adenylate/uridilate copolymers, but not by guanylate/uridilate copolymers. Boiling or proteinase K treatment destroys the selective nucleolytic activity of macrophage lysates. We propose that the nuclease measured here may serve to regulate cellular levels of mRNA molecules encoding cachectin, other inflammatory cytokines, and certain proto-oncogene products.