Genetic dissection of host immune response

Genetic dissection of host immune response

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Infectious diseases are likely to be a major source of selective pressure on the evolution of the immune system.1 Unfortunately, the functional plasticity and redundancy of the immune system complicates the experimental study of each of its components. Because pathogens exhibit abilities to combat and avoid host immunity, they are excellent tools to dissect the function of each of the components of the immune system.2 In a paper published in this issue, Kamimura _et al_3 provide an example of how pathogens can be used to reveal specific immune response defects. The authors studied specific alterations introduced into the gp130 gene, which encodes a common subunit of IL-6 family cytokine receptors. Previously, the authors had identified molecular defects in the signals emanating from these mutant receptors.4 In the current study, the authors evaluated the role of mutant gp130 in resistance to infection with _Listeria monocytogenes_. The authors chose well in using _Listeria_ as a probe into the functioning of the immune system, since resistance to this pathogen relies on contributions from all the major components of innate and acquired immunity.5 In this article, we will to discuss the general significance of this strategy in the context of understanding the complexities of host immunity. In brief, there are a few mutually complementing genetic approaches that are used to study the host immune response. These approaches can be roughly split into two classes. Many investigators analyze alterations in the host response that are induced by directed genetic modification of an organism. Typical genetic modifications include mouse gene knock-outs, knock-ins, and introduction of transgenes. Because this approach is hypothesis driven, it is widely used, and the results reported by Kamimura _et al_3 fall in this category. In contrast, genetic mapping allows the identification of genes whose role in host immunity may not yet be suspected. Genetic mapping relies on an unbiased analysis of the immune response of progeny obtained from crosses. While this approach presents several experimental difficulties, it can, unlike the first one, simultaneously reveal both major and minor effects of several interacting immune components on the studied phenotype. ANALYSIS OF PHENOTYPES CREATED BY DIRECTED GENETIC MODIFICATION OF AN ORGANISM Targeted disruption of genes of interest is the most straightforward way to address the function of a particular molecule. Furthermore, a significant body of information has been derived from studies of mouse models disrupted for various immune genes. In particular, _Listeria_ is widely used to analyze such knock-outs, making it possible to compare and contrast the effects of these genetic alterations on immunity to this pathogen.6 There are drawbacks to knocking out genes as a means to understand their immune function. For example, while knock-out alleles can sometimes provide information about the role of an immunity gene in defense against specific pathogens, the norm is for such strains to exhibit wide ranging pleiotropic effects on the immune system. In addition, since null mutations represent an artificial situation rarely found in wild-type populations, the knock-out phenotype may not tell us very much about the effect of naturally occurring polymorphisms. Also, the immune system has several layers of redundancy and is often able to compensate for the absence of one of its components. This may obscure the effect of the deletion on any given immune response phenotype. As demonstrated by the work of Kamimura _et al_,3 a much more sophisticated approach takes advantage of hypothesis-based dissection of gene functionality. For example, one can use prior knowledge about the structural and functional domains of a protein to make specific modifications of the protein. This kind of approach is especially critical in studies of molecules with multiple shared functions, such as gp130. Gp130 is the common subunit of a family of IL-6 receptors.7 Upon receptor binding, gp130 appears to activate several Janus kinases, allowing the recruitment of SHP2 and STAT3.8 Unfortunately, deletion of gp130 leads to embryonic lethality, providing only limited information about its general importance in hematopoiesis and thymocyte development.9 In an effort to overcome the embryonic lethality, alleles designed to inducibly inactivate gp130 via Cre-loxP-mediated recombination _in vivo_ were constructed.10 Again, the resulting mice displayed pleiotropic defects, consistent with the requirement for gp130 in signaling through multiple cytokines. Ultimately, only mutational analysis of knock-in mice carrying a portion of the human version of gp130 allowed isolation of one of the branches of the intracellular signaling cascade. Interestingly, replacement of Tyr-759 with phenylalanine had no effect on viability of homozygous gp130F759 animals, but the gp130 mediated activation of SHP2 was inhibited in these animals. Among other T and B cell phenotypes, mutant gp130F759/F759 animals displayed a bias of naïve T cell development to Th1 and an IFN-γ mediated class switch to IgG2a.4 Kamimura _et al_3 continued studies of this F759 mutant of ‘humanized’ gp130 by evaluating its role in the immune response to bacterial infection. Interestingly, the authors found that gp130F759/F759 mice had a critical defect in resistance to _Listeria_. This suggests that intact signaling through gp130 is necessary for the ability of mice to resist this pathogen. A major molecular effect of the mutation was decreased production of IFN-γ in response to _Listeria_ infection. This observation is consistent with earlier studies that demonstrate a direct correlation between IFN-γ induction and _Listeria_ resistance of the animal.11 IFN-γ expression can be affected by many pathways and upregulation of IFN-γ has been noted to play a role in resistance to a number of infections.12 Therefore, it would be interesting to see the effect of the F759 mutation on resistance to other pathogens. Hopefully the authors will extend their studies of the gp130F759 mutants to other infectious agents in the future. The authors of this paper also addressed an issue often overlooked in studies of genetically modified mice. Since gene disruptions are done using ES cells derived from a limited set of inbred strains, the knockouts are frequently maintained in a mixed genetic background or, at best, as a congenic in an inbred background. Unfortunately, some of these mixed genetic backgrounds are formed by strains with very different susceptibility patterns to infectious agents, such as C57BL/6 and 129.13 In this study, the authors recognized that their knock-in mutation lies close to a previously identified _Listeria_ susceptibility QTL.14 To alleviate concerns about the potential for effect of a linked gene, the authors demonstrated that their congenic knock-in animals are homozygous for C57BL/6 alleles at the location of the QTL, proving that the phenotypic effect must be due to the gp130F759 mutation. We believe that the need for careful dissection of immune functions through the construction of specific knock-in alleles is perhaps greater than is widely appreciated. This assertion is supported recent data from a system with naturally occurring alleles that dissect the multiple functions of signaling molecules. IL-12Rβ2 is a signal transducing subunit of the IL-12 receptor and it is evolutionary closely related to gp130.15 IL-12 receptor appears to mediate signal transduction by activation of Janus kinases JAK2 and TYK2, together with STAT3 and 4. Similar to the IL-6 receptor, the function of the IL-12 receptor has been studied using traditional methods of gene deletion.16 Even though these studies revealed that IL-12Rβ2 is critical for Th1 responses, information about specific signaling pathways emanating from of IL-12Rβ2 came from an unexpected direction. It has been known for some time that the closely related inbred mouse strains C57BL/10ScCr (B10ScCr) and C57BL/10ScSn (B10ScSn) differ greatly in their ability to heal _Leishmania major_ infections. Following _Leishmania_ infection, the susceptible B10ScCr strain is not able to upregulate production of IFN-γ.17 In addition, splenocytes from B10ScCr fail to produce IFN-γ when treated with IL-12, suggesting that IL-12 mediated signaling could play a role in B10ScCr susceptibility to _Leishmania_.18 Subsequently, genetic linkage experiments implicated the _Ifnm_ locus on chromosome 6 as responsible for the defective IFN-γ production in response to IL-12.19 Since the _Ifnm_ locus co-localized to a region of the genome containing the IL-12Rβ2 gene, this gene was sequenced in B10ScCr and B10ScSn. Interestingly, the susceptible B10ScCr strain had a mutation which causes premature termination of IL-12Rβ2. The receptors containing this truncated version of IL-12Rβ2 are still partially functional and can activate JAK2 but fail to activate STAT4. Since STAT4 is a transcription factor required for IL-12 mediated production of IFN-γ, this specific aspect of impaired IL-12 signaling could explain the increased sensitivity of C57BL/10ScCr mice to _L. major_ infections. However, unlike IL-12Rβ2−/− mice, a normal mitogen stimulated Th1 response is retained in B10ScCr animals apparently due to the residual activity of truncated IL-12Rβ2. For the future, a source of predicted functional polymorphisms will be required for the productive study of any given immunity gene. Certainly, the recent rapid development of genomic sequencing and analysis tools is beginning to provide information about the existing diversity in known immunity genes, but it is difficult to sift through this data to identify likely deleterious alterations, as opposed to polymorphisms with little functional consequence.20 However, several association studies have implicated particular alleles of human genes in the pathogenesis of immune dysfunction. For example, examination of the aforementioned IL-12Rβ2 receptor subunit gene in the human population reveals that it has a high frequency of polymorphism. Association studies of several of these mutations have suggested that they contribute to development of atopy.21 Accordingly, these mutations are excellent candidates for functional evaluation by infectious agents using mouse knock-ins. As an alternative to the relatively complex process of constructing knock-in mice, these variant alleles could be introduced as transgenes. While there are several difficulties associated with this approach, such as poorly controlled construct integration and expression, it is attractive to consider studying such transgenes on genetic backgrounds that contain knockouts of the endogenous locus. ANALYSIS OF THE GENETIC MAKEUP OF ORGANISMS WITH KNOWN PHENOTYPES Over the years, extensive characterization of inbred mouse strains has revealed a wide range of phenotypic responses to many infectious agents. Recently, the advent of high resolution genetic mapping has led to the successful identification and characterization of polymorphisms defining several single gene host susceptibility phenotypes. For example, several key molecules, such as Slc11A1 (Nramp1), Tlr4, Ly49H and Ltxs1 have been identified in the last several years.22,23,24,25 To date, genes having a major phenotypic effect on immune quantitative traits have been the most likely candidates for identification by positional cloning. However, in many cases, the mapping and/or cloning of these genes has led to the subsequent observation of additional unexplained phenotypic variation.26,27 This is not surprising, since differences in host susceptibility are frequently a product of interactions of multiple allelic forms of different genes. Unfortunately, the mapping and cloning of the genes that underlie these minor quantitative effects require more specialized and difficult procedures. However, as should be clear from our previous discussion, naturally occurring, non-null alleles can be quite important in understanding host immunity. Therefore, we would argue that isolating as many host resistance genes as possible (even those with minor effects) will be crucial in our quest to understand susceptibility and resistance to microbial pathogenesis. Ultimately, the identification of the loci contributing to complex host response traits will require careful genetic study of experimental crosses. In an ideal situation with an unlimited number of cross animals and an easily scorable normally distributed phenotype, quantitative trait locus (QTL) analysis should provide exhaustive positional information about all the genes contributing to the differential phenotype.28 However, since the conditions are never ideal, the reliable identification of QTL has been difficult. Alternate statistical analysis, such as a nonparametric approach to study non-normally distributed traits, can sometimes maximize the information obtained from of non-ideal cross populations.29 In addition, analysis of subtraits mathematically derived from a non-ideal experimental dataset can help the identification of significant linkages and provide additional information about the role of the QTL.14 The ultimate and the most difficult task in a genetic mapping experiment is identification of the poly- morphism(s) underlying the studied immune phenotype. Recent technological advances promise significant acceleration of the QTL based gene discovery process. Improved access to array-based analysis of expression patterns of multiple immune components provides opportunities for parallel, gene-based genetic analysis of infection susceptibility traits. Furthermore, the development and dissemination of novel hybrid mouse strain panels, which carry defined combinations of the parental genomes, can be used to improve the reliability of quantitative infectious disease phenotypes while providing definitive positional information.30,31,32 Finally, the ultimate breakthrough in QTL identification will come with the emergence of complete sequences for genomes of model organisms. The task of identifying of genes underlying the phenotypic variation will be further simplified when sequences from a number of different strains become available which catalog all the polymorphisms in any given genomic region of interest. MERGING BOTH APPROACHES We have discussed a few mutually complementary approaches to study the genetics of immunity. However, other strategies are possible that rely on convergence in a single method of both genetic alteration of experimental animals and analysis of the resulting phenotypic data. For example, one can utilize selective breeding to simultaneously alter both the genome and the phenotype of the animal. The goal of selective breeding is to create, by intercrossing a number of strains, animals with an extreme, easily differentiated phenotype. If the selected phenotype is based on modification of immune function, it can also result in animals with altered responses to infectious agents. In a recent study, the immune response of two strains of mice genetically selected for extreme phenotypes of immunological tolerance to ovalbumin was evaluated using an experimental fungal infection.33 Interestingly, the ovalbumin resistant mice were 10-fold more resistant to infection with _Sporothrix schenckii_ then the ovalbumin sensitive mice.34 Similarly, the magnitude of the innate immune response and the host resistance to facultative intracellular pathogens were shown to be coupled in strains of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity.35 AIRmax mice were more resistant than AIRmin mice to _Salmonella typhimurium_ and _Listeria monocytogenes_ infection.36 Another approach suitable for the identification of components of the immune system involves the mutagenesis of experimental animals.37 Initially, random mutagenesis approaches have been directed towards identification of single gene traits with visible or developmental phenotypes. However, comprehensive phenotypic analysis of induced mouse mutant lines has been recently initiated in genome centers around the world.38,39 For example, the German GSF center performs dysmorphological, clinical chemistry, behavioral and immunological phenotypic screens. Undoubtedly, some of the animals identified in the immunological screens will be informative in studies of host-pathogen interaction. CONCLUSIONS The complexity of the immune system allows a significant level of tolerance to functional polymorphism in immune genes. Typically, only an unexpected outcome of an infection will reveal the existence of allelic combinations poorly suited to deal with that disease. Over a long period of time, subsets of pathogens and human hosts have been geographically compartmentalized. It will be interesting to see if changes in human society brought by technological advances, such as ease of travel and wide availability of antibiotics, are going to lead to increased danger of pathogenic infections. Regardless of the answer to this question, steps to customize prevention and treatment according to individual’s genetic makeup, often referred to as pharmacogenomics, will ultimately be extremely beneficial. Genetic studies of polymorphisms identified in human populations using model systems, such as inbred strains of mice, offers a direct way to evaluate their functional significance. In addition, novel opportunities to study function of the immune genes will be presented by the genetic mapping and discoveries of immunity gene polymorphisms already present in the genomes of various mouse strains. Recent advances in whole genome sequences of model organisms will provide a framework for such studies. In addition, a variety of new and reemerging pathogens will widen the repertoire of probes available to study function of immune molecules. REFERENCES * Levin BR, Lipsitch M, Bonhoeffer S . Population biology, evolution, and infectious disease: convergence and synthesis _Science_ 1999 283: 806–809 Article  CAS  Google Scholar  * Dietrich WF . Using mouse genetics to understand infectious disease pathogenesis _Genome Res_ 2001 11: 325–331 Article  CAS  Google Scholar  * Kamimura D _et al_. Tyrosine 759 of the cytokine receptor gp130 is involved in _Listeria monocytogenes_ susceptibility _Genes Immun_ 2002 3: 136–143 Article  CAS  Google Scholar  * Ohtani T _et al_. Dissection of signaling cascades through gp130 _in vivo_: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses _Immunity_ 2000 12: 95–105 Article  CAS  Google Scholar  * Milon G . _Listeria monocytogenes_ in laboratory mice: a model of short-term infectious and pathogenic processes controllable by regulated protective immune responses _Immunol Rev_ 1997 158: 37–46 Article  CAS  Google Scholar  * Lengeling A, Pfeffer K, Balling R . The battle of two genomes: genetics of bacterial host/pathogen interactions in mice _Mamm Genome_ 2001 12: 261–271 Article  CAS  Google Scholar  * Fukada T _et al_. Signaling through Gp130: toward a general scenario of cytokine action _Growth Factors_ 1999 17: 81–91 Article  CAS  Google Scholar  * Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L . Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway _Biochem J_ 1998 334: 297–314 Article  CAS  Google Scholar  * Yoshida K _et al_. Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders _Proc Natl Acad Sci USA_ 1996 93: 407–411 Article  CAS  Google Scholar  * Betz UA _et al_. Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects _J Exp Med_ 1998 188: 1955–1965 Article  CAS  Google Scholar  * Kiderlen AF, Kaufmann SH, Lohmann-Matthes ML . Protection of mice against the intracellular bacterium _Listeria monocytogenes_ by recombinant immune interferon _Eur J Immunol_ 1984 14: 964–967 Article  CAS  Google Scholar  * Shtrichman R, Samuel CE, Shtrichman R, Samuel CE . The role of gamma interferon in antimicrobial immunity _Curr Opin Microbiol_ 2001 4: 251–259 Article  CAS  Google Scholar  * Cheers C, McKenzie IF . Resistance and susceptibility of mice to bacterial infection: genetics of listeriosis _Infect Immun_ 1978 19: 755–762 CAS  PubMed  PubMed Central  Google Scholar  * Boyartchuk VL _et al_. Multigenic control of _Listeria monocytogenes_ susceptibility in mice _Nat Genet_ 2001 27: 259–260 Article  CAS  Google Scholar  * Presky DH _et al_. A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits _Proc Natl Acad Sci USA_ 1996 93: 14002–14007 Article  CAS  Google Scholar  * Wu C _et al_. IL-12 receptor beta 2 (IL-12R beta 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites _J Immunol_ 2000 165: 6221–6228 Article  CAS  Google Scholar  * Muller I, Freudenberg M, Kropf P, Kiderlen AF, Galanos C . _Leishmania major_ infection in C57BL/10 mice differing at the Lps locus: a new non-healing phenotype _Med Microbiol Immunol (Berl)_ 1997 186: 75–81 Article  CAS  Google Scholar  * Merlin T, Sing A, Nielsen PJ, Galanos C, Freudenberg MA . Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lps(d) C57BL/10ScCr mouse _J Immunol_ 2001 166: 566–573 Article  CAS  Google Scholar  * Poltorak A _et al_. A point mutation in the il-12rbeta2 gene underlies the il-12 unresponsiveness of lps-defective c57bl/10sccr mice _J Immunol_ 2001 167: 2106–2111 Article  CAS  Google Scholar  * Goodstadt L, Ponting CP . Sequence variation and disease in the wake of the draft human genome _Hum Mol Genet_ 2001 10: 2209–2214 Article  CAS  Google Scholar  * Kondo N _et al_. Atopy and mutations of IL-12 receptor beta 2 chain gene _Clin Exp Allergy_ 2001 31: 1189–1193 Article  CAS  Google Scholar  * Vidal S _et al_. The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene _J Exp Med_ 1995 182: 655–666 Article  CAS  Google Scholar  * Poltorak A _et al_. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene _Science_ 1998 282: 2085–2088 Article  CAS  Google Scholar  * Lee SH _et al_. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily _Nat Genet_ 2001 28: 42–45 CAS  PubMed  Google Scholar  * Watters JW, Dewar K, Lehoczky J, Boyartchuk V, Dietrich WF . Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor _Curr Biol_ 2001 11: 1503–1511 Article  CAS  Google Scholar  * Gervais F, Desforges C, Skamene E . The C5-sufficient A/Jcongenic mouse strain. Inflammatory response and resistance to _Listeria monocytogenes_ _J Immunol_ 1989 142: 2057–2060 CAS  PubMed  Google Scholar  * Kramnik I, Dietrich WF, Demant P, Bloom BR . Genetic control of resistance to experimental infection with virulent _Mycobacterium tuberculosis_ _Proc Natl Acad Sci USA_ 2000 97: 8560–8565 Article  CAS  Google Scholar  * Broman KW . Review of statistical methods for QTL mapping in experimental crosses _Lab Anim (NY)_ 2001 30: 44–52 CAS  Google Scholar  * Kruglyak L, Lander ES . A nonparametric approach for mapping quantitative trait loci _Genetics_ 1995 139: 1421–1428 CAS  PubMed  PubMed Central  Google Scholar  * Stassen AP, Groot PC, Eppig JT, Demant P . Genetic composition of the recombinant congenic strains _Mamm Genome_ 1996 7: 55–58 Article  CAS  Google Scholar  * Nadeau JH, Singer JB, Matin A, Lander ES . Analysing complex genetic traits with chromosome substitution strains _Nat Genet_ 2000 24: 221–225 Article  CAS  Google Scholar  * Fortin A _et al_. Recombinant congenic strains derived from A/J and C57BL/6J: a tool for genetic dissection of complex traits _Genomics_ 2001 74: 21–35 Article  CAS  Google Scholar  * da Silva AC, de Souza KW, Machado RC, da Silva MF, Sant’Anna OA . Genetics of immunological tolerance: I. Bidirectional selective breeding of mice for oral tolerance _Res Immunol_ 1998 149: 151–161 Article  CAS  Google Scholar  * da Silva AC _et al_. Effect of genetic modifications by selection for immunological tolerance on fungus infection in mice _Microbes Infect_ 2001 3: 215–222 Article  CAS  Google Scholar  * Ibanez OM _et al_. Genetics of nonspecific immunity: I. Bidirectional selective breeding of lines of mice endowed with maximal or minimal inflammatory responsiveness _Eur J Immunol_ 1992 22: 2555–2563 Article  CAS  Google Scholar  * Araujo LM _et al_. Innate resistance to infection by intracellular bacterial pathogens differs in mice selected for maximal or minimal acute inflammatory response _Eur J Immunol_ 1998 28: 2913–2920 Article  CAS  Google Scholar  * Nadeau JH, Frankel WN . The roads from phenotypic variation to gene discovery: mutagenesis versus QTLs _Nat Genet_ 2000 25: 381–384 Article  CAS  Google Scholar  * Brown SD, Balling R . Systematic approaches to mouse mutagenesis _Curr Opin Genet Dev_ 2001 11: 268–273 Article  CAS  Google Scholar  * Nelms KA, Goodnow CC, Nelms KA, Goodnow CC . Genome-wide ENU mutagenesis to reveal immune regulators _Immunity_ 2001 15: 409–418 Article  CAS  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Genetics, Harvard Medical School, Boston, MA, USA V Boyartchuk & W Dietrich * Howard Hughes Medical Institute, Harvard Medical School, Boston, USA W Dietrich Authors * V Boyartchuk View author publications You can also search for this author inPubMed Google Scholar * W Dietrich View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to W Dietrich. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Boyartchuk, V., Dietrich, W. Genetic dissection of host immune response. _Genes Immun_ 3, 119–122 (2002). https://doi.org/10.1038/sj.gene.6363843 Download citation * Published: 21 May 2002 * Issue Date: 01 May 2002 * DOI: https://doi.org/10.1038/sj.gene.6363843 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative

Infectious diseases are likely to be a major source of selective pressure on the evolution of the immune system.1 Unfortunately, the functional plasticity and redundancy of the immune system


complicates the experimental study of each of its components. Because pathogens exhibit abilities to combat and avoid host immunity, they are excellent tools to dissect the function of each


of the components of the immune system.2 In a paper published in this issue, Kamimura _et al_3 provide an example of how pathogens can be used to reveal specific immune response defects.


The authors studied specific alterations introduced into the gp130 gene, which encodes a common subunit of IL-6 family cytokine receptors. Previously, the authors had identified molecular


defects in the signals emanating from these mutant receptors.4 In the current study, the authors evaluated the role of mutant gp130 in resistance to infection with _Listeria monocytogenes_.


The authors chose well in using _Listeria_ as a probe into the functioning of the immune system, since resistance to this pathogen relies on contributions from all the major components of


innate and acquired immunity.5 In this article, we will to discuss the general significance of this strategy in the context of understanding the complexities of host immunity. In brief,


there are a few mutually complementing genetic approaches that are used to study the host immune response. These approaches can be roughly split into two classes. Many investigators analyze


alterations in the host response that are induced by directed genetic modification of an organism. Typical genetic modifications include mouse gene knock-outs, knock-ins, and introduction of


transgenes. Because this approach is hypothesis driven, it is widely used, and the results reported by Kamimura _et al_3 fall in this category. In contrast, genetic mapping allows the


identification of genes whose role in host immunity may not yet be suspected. Genetic mapping relies on an unbiased analysis of the immune response of progeny obtained from crosses. While


this approach presents several experimental difficulties, it can, unlike the first one, simultaneously reveal both major and minor effects of several interacting immune components on the


studied phenotype. ANALYSIS OF PHENOTYPES CREATED BY DIRECTED GENETIC MODIFICATION OF AN ORGANISM Targeted disruption of genes of interest is the most straightforward way to address the


function of a particular molecule. Furthermore, a significant body of information has been derived from studies of mouse models disrupted for various immune genes. In particular, _Listeria_


is widely used to analyze such knock-outs, making it possible to compare and contrast the effects of these genetic alterations on immunity to this pathogen.6 There are drawbacks to knocking


out genes as a means to understand their immune function. For example, while knock-out alleles can sometimes provide information about the role of an immunity gene in defense against


specific pathogens, the norm is for such strains to exhibit wide ranging pleiotropic effects on the immune system. In addition, since null mutations represent an artificial situation rarely


found in wild-type populations, the knock-out phenotype may not tell us very much about the effect of naturally occurring polymorphisms. Also, the immune system has several layers of


redundancy and is often able to compensate for the absence of one of its components. This may obscure the effect of the deletion on any given immune response phenotype. As demonstrated by


the work of Kamimura _et al_,3 a much more sophisticated approach takes advantage of hypothesis-based dissection of gene functionality. For example, one can use prior knowledge about the


structural and functional domains of a protein to make specific modifications of the protein. This kind of approach is especially critical in studies of molecules with multiple shared


functions, such as gp130. Gp130 is the common subunit of a family of IL-6 receptors.7 Upon receptor binding, gp130 appears to activate several Janus kinases, allowing the recruitment of SHP2


and STAT3.8 Unfortunately, deletion of gp130 leads to embryonic lethality, providing only limited information about its general importance in hematopoiesis and thymocyte development.9 In an


effort to overcome the embryonic lethality, alleles designed to inducibly inactivate gp130 via Cre-loxP-mediated recombination _in vivo_ were constructed.10 Again, the resulting mice


displayed pleiotropic defects, consistent with the requirement for gp130 in signaling through multiple cytokines. Ultimately, only mutational analysis of knock-in mice carrying a portion of


the human version of gp130 allowed isolation of one of the branches of the intracellular signaling cascade. Interestingly, replacement of Tyr-759 with phenylalanine had no effect on


viability of homozygous gp130F759 animals, but the gp130 mediated activation of SHP2 was inhibited in these animals. Among other T and B cell phenotypes, mutant gp130F759/F759 animals


displayed a bias of naïve T cell development to Th1 and an IFN-γ mediated class switch to IgG2a.4 Kamimura _et al_3 continued studies of this F759 mutant of ‘humanized’ gp130 by evaluating


its role in the immune response to bacterial infection. Interestingly, the authors found that gp130F759/F759 mice had a critical defect in resistance to _Listeria_. This suggests that intact


signaling through gp130 is necessary for the ability of mice to resist this pathogen. A major molecular effect of the mutation was decreased production of IFN-γ in response to _Listeria_


infection. This observation is consistent with earlier studies that demonstrate a direct correlation between IFN-γ induction and _Listeria_ resistance of the animal.11 IFN-γ expression can


be affected by many pathways and upregulation of IFN-γ has been noted to play a role in resistance to a number of infections.12 Therefore, it would be interesting to see the effect of the


F759 mutation on resistance to other pathogens. Hopefully the authors will extend their studies of the gp130F759 mutants to other infectious agents in the future. The authors of this paper


also addressed an issue often overlooked in studies of genetically modified mice. Since gene disruptions are done using ES cells derived from a limited set of inbred strains, the knockouts


are frequently maintained in a mixed genetic background or, at best, as a congenic in an inbred background. Unfortunately, some of these mixed genetic backgrounds are formed by strains with


very different susceptibility patterns to infectious agents, such as C57BL/6 and 129.13 In this study, the authors recognized that their knock-in mutation lies close to a previously


identified _Listeria_ susceptibility QTL.14 To alleviate concerns about the potential for effect of a linked gene, the authors demonstrated that their congenic knock-in animals are


homozygous for C57BL/6 alleles at the location of the QTL, proving that the phenotypic effect must be due to the gp130F759 mutation. We believe that the need for careful dissection of immune


functions through the construction of specific knock-in alleles is perhaps greater than is widely appreciated. This assertion is supported recent data from a system with naturally occurring


alleles that dissect the multiple functions of signaling molecules. IL-12Rβ2 is a signal transducing subunit of the IL-12 receptor and it is evolutionary closely related to gp130.15 IL-12


receptor appears to mediate signal transduction by activation of Janus kinases JAK2 and TYK2, together with STAT3 and 4. Similar to the IL-6 receptor, the function of the IL-12 receptor has


been studied using traditional methods of gene deletion.16 Even though these studies revealed that IL-12Rβ2 is critical for Th1 responses, information about specific signaling pathways


emanating from of IL-12Rβ2 came from an unexpected direction. It has been known for some time that the closely related inbred mouse strains C57BL/10ScCr (B10ScCr) and C57BL/10ScSn (B10ScSn)


differ greatly in their ability to heal _Leishmania major_ infections. Following _Leishmania_ infection, the susceptible B10ScCr strain is not able to upregulate production of IFN-γ.17 In


addition, splenocytes from B10ScCr fail to produce IFN-γ when treated with IL-12, suggesting that IL-12 mediated signaling could play a role in B10ScCr susceptibility to _Leishmania_.18


Subsequently, genetic linkage experiments implicated the _Ifnm_ locus on chromosome 6 as responsible for the defective IFN-γ production in response to IL-12.19 Since the _Ifnm_ locus


co-localized to a region of the genome containing the IL-12Rβ2 gene, this gene was sequenced in B10ScCr and B10ScSn. Interestingly, the susceptible B10ScCr strain had a mutation which causes


premature termination of IL-12Rβ2. The receptors containing this truncated version of IL-12Rβ2 are still partially functional and can activate JAK2 but fail to activate STAT4. Since STAT4


is a transcription factor required for IL-12 mediated production of IFN-γ, this specific aspect of impaired IL-12 signaling could explain the increased sensitivity of C57BL/10ScCr mice to


_L. major_ infections. However, unlike IL-12Rβ2−/− mice, a normal mitogen stimulated Th1 response is retained in B10ScCr animals apparently due to the residual activity of truncated


IL-12Rβ2. For the future, a source of predicted functional polymorphisms will be required for the productive study of any given immunity gene. Certainly, the recent rapid development of


genomic sequencing and analysis tools is beginning to provide information about the existing diversity in known immunity genes, but it is difficult to sift through this data to identify


likely deleterious alterations, as opposed to polymorphisms with little functional consequence.20 However, several association studies have implicated particular alleles of human genes in


the pathogenesis of immune dysfunction. For example, examination of the aforementioned IL-12Rβ2 receptor subunit gene in the human population reveals that it has a high frequency of


polymorphism. Association studies of several of these mutations have suggested that they contribute to development of atopy.21 Accordingly, these mutations are excellent candidates for


functional evaluation by infectious agents using mouse knock-ins. As an alternative to the relatively complex process of constructing knock-in mice, these variant alleles could be introduced


as transgenes. While there are several difficulties associated with this approach, such as poorly controlled construct integration and expression, it is attractive to consider studying such


transgenes on genetic backgrounds that contain knockouts of the endogenous locus. ANALYSIS OF THE GENETIC MAKEUP OF ORGANISMS WITH KNOWN PHENOTYPES Over the years, extensive


characterization of inbred mouse strains has revealed a wide range of phenotypic responses to many infectious agents. Recently, the advent of high resolution genetic mapping has led to the


successful identification and characterization of polymorphisms defining several single gene host susceptibility phenotypes. For example, several key molecules, such as Slc11A1 (Nramp1),


Tlr4, Ly49H and Ltxs1 have been identified in the last several years.22,23,24,25 To date, genes having a major phenotypic effect on immune quantitative traits have been the most likely


candidates for identification by positional cloning. However, in many cases, the mapping and/or cloning of these genes has led to the subsequent observation of additional unexplained


phenotypic variation.26,27 This is not surprising, since differences in host susceptibility are frequently a product of interactions of multiple allelic forms of different genes.


Unfortunately, the mapping and cloning of the genes that underlie these minor quantitative effects require more specialized and difficult procedures. However, as should be clear from our


previous discussion, naturally occurring, non-null alleles can be quite important in understanding host immunity. Therefore, we would argue that isolating as many host resistance genes as


possible (even those with minor effects) will be crucial in our quest to understand susceptibility and resistance to microbial pathogenesis. Ultimately, the identification of the loci


contributing to complex host response traits will require careful genetic study of experimental crosses. In an ideal situation with an unlimited number of cross animals and an easily


scorable normally distributed phenotype, quantitative trait locus (QTL) analysis should provide exhaustive positional information about all the genes contributing to the differential


phenotype.28 However, since the conditions are never ideal, the reliable identification of QTL has been difficult. Alternate statistical analysis, such as a nonparametric approach to study


non-normally distributed traits, can sometimes maximize the information obtained from of non-ideal cross populations.29 In addition, analysis of subtraits mathematically derived from a


non-ideal experimental dataset can help the identification of significant linkages and provide additional information about the role of the QTL.14 The ultimate and the most difficult task in


a genetic mapping experiment is identification of the poly- morphism(s) underlying the studied immune phenotype. Recent technological advances promise significant acceleration of the QTL


based gene discovery process. Improved access to array-based analysis of expression patterns of multiple immune components provides opportunities for parallel, gene-based genetic analysis of


infection susceptibility traits. Furthermore, the development and dissemination of novel hybrid mouse strain panels, which carry defined combinations of the parental genomes, can be used to


improve the reliability of quantitative infectious disease phenotypes while providing definitive positional information.30,31,32 Finally, the ultimate breakthrough in QTL identification


will come with the emergence of complete sequences for genomes of model organisms. The task of identifying of genes underlying the phenotypic variation will be further simplified when


sequences from a number of different strains become available which catalog all the polymorphisms in any given genomic region of interest. MERGING BOTH APPROACHES We have discussed a few


mutually complementary approaches to study the genetics of immunity. However, other strategies are possible that rely on convergence in a single method of both genetic alteration of


experimental animals and analysis of the resulting phenotypic data. For example, one can utilize selective breeding to simultaneously alter both the genome and the phenotype of the animal.


The goal of selective breeding is to create, by intercrossing a number of strains, animals with an extreme, easily differentiated phenotype. If the selected phenotype is based on


modification of immune function, it can also result in animals with altered responses to infectious agents. In a recent study, the immune response of two strains of mice genetically selected


for extreme phenotypes of immunological tolerance to ovalbumin was evaluated using an experimental fungal infection.33 Interestingly, the ovalbumin resistant mice were 10-fold more


resistant to infection with _Sporothrix schenckii_ then the ovalbumin sensitive mice.34 Similarly, the magnitude of the innate immune response and the host resistance to facultative


intracellular pathogens were shown to be coupled in strains of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity.35 AIRmax mice were more resistant than


AIRmin mice to _Salmonella typhimurium_ and _Listeria monocytogenes_ infection.36 Another approach suitable for the identification of components of the immune system involves the mutagenesis


of experimental animals.37 Initially, random mutagenesis approaches have been directed towards identification of single gene traits with visible or developmental phenotypes. However,


comprehensive phenotypic analysis of induced mouse mutant lines has been recently initiated in genome centers around the world.38,39 For example, the German GSF center performs


dysmorphological, clinical chemistry, behavioral and immunological phenotypic screens. Undoubtedly, some of the animals identified in the immunological screens will be informative in studies


of host-pathogen interaction. CONCLUSIONS The complexity of the immune system allows a significant level of tolerance to functional polymorphism in immune genes. Typically, only an


unexpected outcome of an infection will reveal the existence of allelic combinations poorly suited to deal with that disease. Over a long period of time, subsets of pathogens and human hosts


have been geographically compartmentalized. It will be interesting to see if changes in human society brought by technological advances, such as ease of travel and wide availability of


antibiotics, are going to lead to increased danger of pathogenic infections. Regardless of the answer to this question, steps to customize prevention and treatment according to individual’s


genetic makeup, often referred to as pharmacogenomics, will ultimately be extremely beneficial. Genetic studies of polymorphisms identified in human populations using model systems, such as


inbred strains of mice, offers a direct way to evaluate their functional significance. In addition, novel opportunities to study function of the immune genes will be presented by the genetic


mapping and discoveries of immunity gene polymorphisms already present in the genomes of various mouse strains. Recent advances in whole genome sequences of model organisms will provide a


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AFFILIATIONS * Department of Genetics, Harvard Medical School, Boston, MA, USA V Boyartchuk & W Dietrich * Howard Hughes Medical Institute, Harvard Medical School, Boston, USA W Dietrich


Authors * V Boyartchuk View author publications You can also search for this author inPubMed Google Scholar * W Dietrich View author publications You can also search for this author


inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to W Dietrich. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Boyartchuk, V., Dietrich, W.


Genetic dissection of host immune response. _Genes Immun_ 3, 119–122 (2002). https://doi.org/10.1038/sj.gene.6363843 Download citation * Published: 21 May 2002 * Issue Date: 01 May 2002 *


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