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

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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