key: cord-1034733-dbe5o8gl
authors: Shannon, M. Frances
title: Genomic Approaches to the Host Response to Pathogens
date: 2010-01-29
journal: Essentials of Genomic and Personalized Medicine
DOI: 10.1016/b978-0-12-374934-5.00057-x
sha: 653beef7ca2ab97f8f27c811a1c4543181e2a254
doc_id: 1034733
cord_uid: dbe5o8gl

The need to better understand host–pathogen interactions has risen with the expansion in genomics and related technologies. This chapter focuses on two aspects of the host response to pathogens where major advances are being made using genomic approaches. The availability of complete genomic sequences of an expanding number of pathogens, the human and mouse genome sequences, and the advent of genome-wide genotyping and gene expression profiling has opened up new avenues of investigation in the field. The genotype of the pathogen plays a major role in the response of the host to infection with more virulent pathogenic strains possessing the capability to interfere with the host immune response. In addition, different individuals in a population can have very different responses to a genetically identical pathogen. Part of the differential response is governed by the underlying genetic differences between individuals. The advent of genome-wide genotyping using single nucleotide polymorphisms or microsatellite markers is leading to major advances in molecular epidemiology. The future impact of genomic approaches on the development of diagnostics and therapeutics is discussed for infectious diseases. This includes defining the basis of genetic susceptibility to infection and system-wide molecular response to a pathogen.

Following a period of relative disinterest in infectious disease research due to the enormous impact of vaccines and antibiotics on the spread of and mortality from these diseases, there is now renewed and growing interest in this area of research. This has been driven by several recent worldwide developments including: (i) the rising incidence of diseases such as Acquired Immune Defi ciency Syndrome (AIDS) and antibiotic-resistant tuberculosis; (ii) antibiotic-resistant bacterial strains presenting a severe health threat in hospitals; (iii) the rapid spread of new pathogens such as Severe Acute Respiratory Syndrome (SARS); and (iv) the threat of bioterrorism. Indeed, nearly 25% of annual deaths worldwide are due to infectious disease ( Morens et al., 2004 ) . Thus, the need to develop new diagnostic methods, more effective vaccines and better therapeutic strategies is urgent.

In order to effectively deal with infectious disease threats, it is important to understand both the pathogen and the response of the host, since the outcome of infection is determined by complex host-pathogen interactions. Pathogens are initially detected by the surveillance cells of the innate immune system using cell surface receptors known as Toll-like receptors (TLRs) (reviewed in ). These TLRs recognize specifi c components of the pathogen, for example, bacterial lipopolysaccharide (LPS) or double-stranded (ds) RNA from viruses. While many cell types express TLRs, cells of the innate immune system such as dendritic cells (DCs) and macrophages play particularly important roles in detecting and responding to pathogens. The response of these cells to a pathogen is determined by the specifi c pathogen component that interacts with the TLR and the specifi c TLR family member that is activated. Widespread changes in gene expression are detected following TLR activation and the activated cells produce a plethora of cytokines and chemokines that then activate the adaptive arm of the immune system. The specifi c cytokines and chemokines produced by the TLR-activated cells tailor the response of the adaptive immune system to deal with the specifi c pathogen . Thus, the initial host response to a pathogen through the TLRs determines the outcome of the infection. Host response to infection can be a double-edged sword in that sometimes the response itself can create an adverse outcome for the host. In addition, the aberrant response of the host to self instead of foreign pathogens can create severe pathologies involving chronic infl ammatory and autoimmune diseases.

The urgent need to better understand host-pathogen interactions has come at a time when genomics and related technologies are expanding rapidly. The availability of complete genomic sequences of an expanding number of pathogens, the human and mouse genome sequences and the advent of genome-wide genotyping and gene expression profi ling has opened up new avenues of investigation in the fi eld.

The genotype of the pathogen plays a major role in the response of the host to infection with more virulent pathogenic 57 strains often possessing the capability to interfere with the host immune response ( Fitzgerald and Musser, 2001 ; Kato-Maeda et al., 2001 ; Schoolnik, 2002 ) . In addition, different individuals in a population can have very different responses to a genetically identical pathogen. While there are many complex reasons for this, it is clear that part of the differential response is governed by underlying genetic differences between individuals ( Clementi and Di Gianantonio, 2006 ; Zhang and Zhang, 2006 ) . Studies in mouse models of infection have clearly demonstrated that these genetic differences are complex and may involve more than one genetic locus for a given susceptibility or resistance trait (e.g., [ Delahaye et al., 2006 ] ). While there are some classic examples of genetic mutations affecting the response of the host to a pathogen (e.g., malaria and sickle cell mutations) there is much to be learned before the genetics of host susceptibility is fully understood. The advent of genome-wide genotyping using single nucleotide polymorphisms (SNPs) or microsatellite markers, leading to major advances in molecular epidemiology, will revolutionize our ability to determine the complexities of the genetic component of pathogen-host interactions ( Weiss and Terwilliger, 2000 ) .

It is well known that the cells of the host immune system are activated upon detection of a pathogen by TLRs as described above. This activation process includes widespread changes in the gene expression profi le of the cells with hundreds of genes being either switched on or off in response to signals generated from the pathogen-detecting TLRs. The response of individual genes has been studied in minute detail for a handful of genes and while this has produced an understanding of some aspects of host response to infection it by no means gives us the total picture. Understanding the molecular response of the host to infection has been greatly improved by using microarray-based technologies and these technologies are opening up new diagnostic possibilities as well as presenting new therapeutic options ( Aderem and Smith, 2004 ; Bryant et al., 2004 ; Feezor et al., 2005 ; Hedeler et al., 2006 ; Korth and Katze, 2002 ; Ng et al., 2006 ; Ricciardi-Castagnoli, 2005 ; Ricciardi-Castagnoli and Granucci, 2002 ; Smith and Bolouri, 2005 ) . This chapter will focus on two aspects of the host response to pathogens where major advances are being made using genomics approaches and will describe the future impact of these approaches on the development of diagnostics and therapeutics for infectious disease. These are (i) defi ning the basis of genetic susceptibility to infection and (ii) the defi nition of the system-wide molecular response to a pathogen.

It is now relatively easy to map genes associated with genetic diseases that show a Mendelian pattern of inheritance. However, these diseases account for only a very small proportion of the human disease burden and many of the more common and fatal diseases have a complex etiology with many genetic and environmental contributions. While it is clear that most complex disease has a genetic component, defi ning that genetic component has been diffi cult to date since many complex diseases such as coronary heart disease, diabetes and others are polygenic with different genetic loci contributing in major or minor ways to disease susceptibility. In addition, in different populations or under different environmental conditions, distinct but overlapping sets of genetic loci are likely to contribute. The sequencing of the human genome and the genome-wide genetic and functional mapping that has followed have raised hopes of mapping the genetic component of complex disease and there are many large efforts around the world with this aim.

Studies in both animal models and human populations have shown that infectious disease and the response of the host to a specifi c infection also has a complex genetic component ( Clementi and Di Gianantonio, 2006 ; Lipoldova and Demant, 2006 ; Marquet et al., 1996 ; Mira et al., 2004 ) . Thus, inbred mouse models have been developed that clearly show a genetic component to susceptibility for specifi c pathogens and in some cases at least part of the underlying genetic reason has been defi ned ( Beck et al., 2000 ; Mak et al., 2001 ; Rogner and Avner, 2003 ) . Mapping the genetic components of susceptibility to infection in human populations has been much more difficult due to the large natural variation in humans, the polygenic nature of this trait and the low penetrance of many of the susceptibility alleles. For infectious disease, this is complicated even more by the complex nature of the environmental infl uences particularly the fact that these diseases, unlike other complex diseases, are transmissible in populations. However, a combination of animal and human population studies, combined with the latest genomic technologies, is beginning to unravel the issues of genetic susceptibility to infection.

The use of inbred and congenic strains of mice are well established systems for identifying susceptibility loci ( Beck et al., 2000 ; Rogner and Avner, 2003 ) . In recent years genetic manipulation of specifi c loci by deletion or mutation has provided many mouse models for screening ( Mak et al., 2001 ) . The use of ethylnitrosourea (ENU) mutagenesis to randomly create point mutations in the mouse genome has opened up a new forward genetics approach to identifying susceptibility loci ( Papathanasiou and Goodnow, 2005 ) . This chemical mutagen, when used at appropriate doses and at the correct stage of development, can introduce single point mutations into the mouse genome. By screening libraries of mutant mice for susceptibility to specifi c pathogens, it should be possible to identify genetic loci that dictate susceptibility or resistance to a range of pathogens on a large scale. It is relatively straightforward to identify a chromosomal region involved in susceptibility in these mouse strains by genotyping with microsatellite markers, but identifying the specifi c gene that is mutated is still very time-consuming. The speed with which this can be achieved depends on the presence of candidate genes within the chromosomal interval or the ability to resequence large amounts of DNA. The latter is becoming achievable with the advent of new rapid sequencing technologies and is set to revolutionize forward genetic approaches to disease understanding ( Bennett et al., 2005 ; Serre and Hudson, 2006 ) .

The recent explosion in genetic information for the human genome including the complete genome sequence and detailed genetic and physical maps has increased our ability to fi nd variation in the human genome and correlate it with disease. Family studies, especially twin studies, and population studies have clearly shown a genetic component to susceptibility to infectious disease ( Frodsham and Hill, 2004 ; Lipoldova and Demant, 2006 ; Strunk and Burgner, 2006 ) . Susceptibility generally follows a complex pattern of inheritance and there are two main methods of mapping and identifying genetic loci involved in such complex heredity. These are either association studies or linkage studies. Association studies involve screening populations for specifi c mutations in a candidate gene(s) in case-control studies or in family studies. This type of approach identifi ed the link between Human Immunodefi ciency Virus (HIV) resistance and the chemokine receptor, CCR5 described below ( Dean et al., 1996 ; Samson et al., 1996 ) . Genome-wide association studies although still quite expensive are now becoming feasible and being used to defi ne linkage between specifi c markers and susceptibility. These linkage studies depend on the availability of markers and the density of these markers is rapidly increasing T A B L E 5 7 . 1 List of well-described susceptibility loci for resistance or susceptibility to infectious disease.

Genes References with the large scale identifi cation of new SNPs across the human genome. The selection of which SNPs to use and the large numbers of samples needed to generate statistically significant associations for low penetrance alleles are still challenges. The Haplotype MAP (HapMap) project is starting to identify haplotypes within different population groups and together with improvements in large scale genotyping technology and bioinformatics should be useful in studies of complex disease inheritance. Table 57 .1 summarizes the best studied genetic susceptibility loci for response to different infectious agents in both mouse models and human studies. One of the classical examples of genetic susceptibility to infection is the role of the hemaglobinopathies in the outcome of malaria infection ( Patrinos et al., 2005 ) . There are also certain chromosomal regions and families of genes that have attracted attention in terms of searching for susceptibility alleles or polymorphisms. Because the TLR family of receptors plays a major role in recognizing pathogens, it was speculated that genetic variation in these receptors or their signaling pathways might be responsible for some susceptibility phenotypes (reviewed in [ Schroder and Schumann, 2005 ] ). One of the best examples to date is the occurrence of a single polymorphism in the region of the human TLR4 gene encoding the extracellular domain of the receptor which confers reduced sensitivity to inhaled Escherichia coli ( E. coli ) LPS ( Arbour et al., 2000 ) . Interestingly, when septic shock patients were compared with a control group, these lower-responding alleles were found only in the septic shock group and these individuals had a higher incidence of Gramnegative bacterial infection ( Feterowski et al., 2003 ) . Such studies need further confi rmation since there are also a number of studies that failed to fi nd any linkage between TLR4 mutations and response to various infections ( Schroder and Schumann, 2005 ) . There is also enormous variation in the response of individuals to LPS even in the absence of TLR4 mutations implying that variation may occur in other components of the TLR4 signaling system. An example of this is the link between IRAK4 mutations and increased susceptibility to infection with pyogenic bacteria ( Picard et al., 2003 ) . Variation in other TLR genes has also been associated with disease susceptibility. For example, a mutation in the extracellular domain of TLR2 is linked to susceptibility to leprosy ( Alcais et al., 2005 ) and a mutation in TLR5 increases susceptibility to Legionella . Taken together these data support the idea that variation in the innate immune recognition of pathogens play an important part in governing susceptibility to an array of infectious diseases. However, caution needs to be exercised until larger population groups have been studied.

The extensive polymorphism at the chromosomal regions encoding major histocompatibility complex (MHC) proteins is thought to have arisen through natural selection in response to selective pressure from infectious disease. Although human leukocyte antigen (HLA) association with resistance or susceptibility to infectious disease has been diffi cult to identify because of the complex array of antigenic epitopes involved, a number of studies have implicated this locus in genetic susceptibility to infectious disease ( Ghodke et al., 2005 ; Little and Parham, 1999 ) . MHC molecules fall into two classes, Class I that present foreign antigens to CD8 ϩ cytotoxic T cells and Class II that play a similar role for CD4 ϩ helper T cells. Variation in specifi c Class I genes has been shown to confer susceptibility to pulmonary tuberculosis and to HIV whereas mutations in other Class I genes confer resistance to HIV and to severe malaria. Class II mutations that confer resistance to hepatitis B or hepatitis C have been identifi ed and susceptibility to typhoid fever and leprosy are also associated with specifi c Class II mutations. Further molecular analysis of these and other associations may in the future have an impact on the development of new vaccines and immunotherapeutics.

To date the most successful manner of identifying susceptibility genes in human populations has been the candidate gene approach. Candidate genes have emerged from many sources including mouse genetic studies as well as biochemical and function dissection of the immune system. Once a candidate gene is identifi ed, the chromosomal region spanning this gene in the human genome is then scanned for the occurrence of specifi c mutations or for functional polymorphisms in casecontrol studies across populations or in linkage studies in family groups. Such studies have identifi ed a number of well described susceptibility loci for infection with various pathogens. One of the most heralded example was the identifi cation of a deletion in the chemokine receptor, CCR5, which was shown to confer resistance to HIV infection ( Dean et al., 1996 ; Samson et al., 1996 ) . Biochemically, this can be explained by the fact that CCR5 is a co-receptor for HIV on the surface of T cells ( Dragic et al., 1996 ) . A mutation in another chemokine receptor, CCR2, has also been shown to confer HIV resistance in certain Caucasian populations ( O'Brien and Moore, 2000 ) .

Some genes have been associated with susceptibility or resistance to multiple pathogens. For example, variation in the NRAMP1/Slc11a1 gene is associated with susceptibility to Leishmania and to specifi c intracellular bacteria such as tuberculosis ( Barton et al., 1999 ; Govoni et al., 1996 ; Lipoldova and Demant, 2006 ; Sebastiani et al., 1998 ) . Mutations in the tumor necrosis factor (TNF) locus, mainly gene promoter mutations, have been linked with malaria and leprosy susceptibility ( Lipoldova and Demant, 2006 ) . Gene promoter or control region mutations have an impact on the level of protein produced from the gene rather than the function of the protein. This is an area of great interest but more diffi cult to study for several reasons, including the inability to identify control regions simply from sequence information and the complexity and fl exibility of transcriptional control. A recent review detailing the genes associated with Leishmania susceptibility describes a number of genes that can affect disease outcome including the interferon-gamma receptor type 1 ( IFNGR1 ), the interleukin-4 (IL-4 ) gene and the NRAMP-1/Slc11a1 gene ( Lipoldova and Demant, 2006 ) . These genes and others such as interleukin-12 ( IL-12 ) and its receptor are also linked with Salmonella and certain mycobacterial infections ( Lipoldova and Demant, 2006 ) . Thus, it is likely that variation in many genes can contribute to disturbing the fi nely balanced tuning of the immune system and lead to an altered response to a pathogen. It is clear from such studies that the same genes may be involved in susceptibility to an array of pathogens indicating a core immune response critical for any pathogen.

The identifi cation of susceptibility loci for infection with various pathogens will aid in developing new diagnostic screens based on the detection of genetic variants in these loci. It could be envisaged that a person's susceptibility or resistance to a pathogen could be defi ned by a simple genotyping screen either prior to exposure to any pathogen or upon presentation with an infection. It may also be possible to determine the likely outcome of the infection through a genotyping screen. The defi nition of susceptibility loci will also contribute to our ability to develop new vaccines and therapeutics.

The use of microarray technology to generate expression profi ling data is becoming common place in biomedical research (reviewed in [ Quackenbush, 2002 ; Sherlock, 2000 ] ). This technology allows the documentation of mRNA levels for thousands of genes from total RNA prepared from cells or tissue samples. The data obtained can be compared from sample to sample allowing the changes between samples to be documented and quantifi ed. The " expression profi le " for any cell or tissue is simply the list of genes whose expression can be detected using microarrays. The differences in the expression profi le from one cell or tissue to the next or in cells treated in a specifi c manner is a surrogate measure of the cell/tissue phenotype and shows how that phenotype responds to its environment. Expression profi ling is most useful when large datasets become available and when the data is combined with other data types and detailed bioinformatics studies. For example, using functional clustering of expression profi ling data can help identify pathways that are important for a particular process and co-expression clustering combined with other technologies can help defi ne regulatory networks within the cell.

Over the last 5-6 years, this technology has been applied to identifying the changes in gene expression that occur in response to infection by various pathogens ( Aderem and Smith, 2004 ; Boyce et al., 2004 ; Bryant et al., 2004 ; Feezor et al., 2005 ; Foti et al., 2006 ; Jenner and Young, 2005 ; Korth and Katze, 2002 ; Korth et al., 2005 ; Ricciardi-Castagnoli and Granucci, 2002 ) . To date there are more than 150 papers in the literature that describe gene expression changes that occur in response to infection with a plethora of pathogens and in many cell types (reviewed in [ Jenner and Young, 2005 ] ). Many of these are in vitro studies, taking specifi c cell types and infecting them with specifi c agents including bacteria, viruses, parasites and yeasts. In addition, cellular responses to bacterial components have also been documented, helping to identify pathogen-specifi c responses as well as determining the pathogenic component responsible for the major gene expression effects. Virulent or non-virulent strains of specifi c pathogens as well as mutant organisms have been used to determine the gene expression profi le associated with a negative or positive clinical outcome. Few in vivo studies have been carried out and have shed light on the more complex responses seen in whole animals and helped to validate the in vitro data.

Several pioneering studies demonstrated that microarrays could be used to determine changes in the gene expression profi le of cells in response to virus or bacterial infection ( Boldrick et al., 2002 ; Gao et al., 2002 ; Huang et al., 2001 ; Nau et al., 2002 ) . These studies paved the way for the analysis of the host response to a wide variety of infectious agents. The most signifi cant of these studies compared the response of macrophages or DCs to a variety of infectious agents in a single study. In these studies a strong shared response to all infections, be they bacterial, viral or parasitic in nature, was identifi ed. Not only was there commonality from one infectious agent to another but there was also some commonality across cell types. This expression signature has been interpreted as a general " alarm signal " for infection (reviewed in [ Jenner and Young, 2005 ] ). Studies of infection with Gram-positive and Gram-negative bacteria also revealed a common expression signature in peripheral blood mononuclear cells. Recently, the Young lab has interrogated all of the publicly available expression profi ling data related to the host response to infection ( Jenner and Young, 2005 ) . The dataset includes 785 experiments in cells ranging from macrophages and DCs to cells of the adaptive immune response, endothelial and epithelial cells and spanning a wide range of infecting agents. This meta-analysis revealed that a " common host response " can be detected across all of these cell types and infectious agents and show that although cells such as macrophages and DCs specialize in detecting infection, other cells of the body can mount the same " alarm response " as described above ( Figure 57.1 ) .

Not surprisingly this expression signature contains many genes associated with the immune system particularly those encoding infl ammatory cytokines, chemokines and their receptors. However, some more surprising patterns of expression were also detected. It has been long known that interferon-stimulated genes (ISGs) are regulated by virus infection, but it has only recently been recognized that bacteria and other infecting agents can also illicit the interferon response. This was borne out in these meta-analyses where upregulation of an ISG set is observed across a broad range of infecting agents and cell types.

Not only do these cells change the expression of secreted factors and their receptors during infection but the intracellular milieu is also modifi ed. Once again, there is a common pattern of change observed in all of these studies. The upregulation of signaling and transcription pathways that both augment and attenuate the immune response are observed leading to the interpretation that both positive and negative feedback loops operate within the cell to heighten or dampen the immune response. Temporal profi ling can reveal extra layers of complexity and in one study a pro-infl ammatory profi le followed by an anti-infl ammatory profi le was identifi ed in macrophages activated with LPS ( Wells et al., 2005 ) . Meta-analysis of temporal studies of activation through different TLRs also revealed that the infl ammatory chemokines/cytokine signature was an early response while the ISG response was later, presumably refl ecting the need for an indirect activation of the ISGs through interferon production ( Jenner and Young, 2005 ) . Changes in the expression level of genes involved in both activation and repression of apoptosis also fall under the " common signature " banner and this is interpreted as sending the cells into a state of high alert where apoptosis can be either initiated to eliminate infected cells or terminated if the infection resolves ( Jenner and Young, 2005 ) . Although these in vitro studies have provided an overview of the response of isolated cell types to pathogenic infection, in vivo studies are needed to validate any of these results before application to clinical medicine. A number of animal models have been used to profi le the host response to infection with a variety of agents. These studies are complicated especially if whole tissue samples are used in that changes in gene expression can result not only from genuine changes within the cells of the tissue but also from the recruitment especially of immune cells into the infected or infl amed tissue. Nevertheless, in vivo studies have, in some cases, shown good correlation with the expression profi les found from in vitro studies. For example, profi ling the brains of mice infected with virulent Sindbis virus revealed that ISGs as well as infl ammatory chemokines were upregulated ( Johnston et al., 2001 ) and in terms of diagnosis or treatment the exact reason behind these changes in expression signature may be irrelevant. In vivo studies also have some advantages in that the gene expression profi les detected in infected tissues will often make more sense when combined with other physiological or cell biology data from studies of the infected host. Thus an " infection signature " would not only describe the altered gene expression of the immune cells that are recruited to the sites of infection but also would include changes in the gene expression of the resident cells of the tissue and may provide a more robust profi le of the infection process for use in diagnostic applications.

What can be applied to clinical medicine from these studies? The ability to detect an " infection signature " using focused microarrays could potentially be used as a diagnostic tool. There would be an immediate need to identify a core set of genes with suffi ciently robust changes in gene expression to form the basis of a diagnostic array. Arraying technology would need to be priced for diagnostic use and the technology would have to be deemed suffi ciently robust to pass all the quality control requirements of a diagnostic laboratory. No doubt progress will be made toward these goals in the near future.

In addition to the " common host response " described above, microarray studies have revealed that pathogen-specifi c responses also exist. This is not surprising since it has long been known that different pathogens induce distinct arms of the adaptive immune response. For example, distinct types of helper T cells are activated in response to bacterial and viral infection compared to parasite infection and DC products such as cytokines and chemokines control the differentiation of T helper cell into the correct response type ( Mosmann et al., 2005 ) . Thus, the fact that cells of the innate immune system display a pathogenspecifi c transcriptional response as well as a general alarm signal helps to dictate the subsequent immune response.

Different TLRs are involved in recognizing and responding to different pathogens. For example, TLR2 is responsible for activation in response to Gram-positive bacteria while TLR4 responds to LPS, a component of Gram-negative bacteria ( Beutler and Rietschel, 2003 ; . TLR3 responds to dsRNA and thus dictates the viral immune response for many dsRNA viruses ( Beutler and Rietschel, 2003 ; . Studies using bacterial components such as LPS and fl agellin as well as dsRNA have revealed that each TLR induces a specifi c as well as general transcriptional response. The transcriptional response of macrophages and peripheral blood mononuclear cells is more robust in response to Gram-negative (TLR4) compared with Gram-positive (TLR2) bacteria and the ISG response is considerably reduced for the Gram-positive expression signature ( Boldrick et al., 2002 ; Nau et al., 2002 ) . These differences are further observed when bacterial components are used to activate cells. For example, LPS from Gram-negative bacteria, a specifi c ligand for TLR4, can activate the ISG profi le but TLR2 ligands such as LTA and MDP cannot ( Jenner and Young, 2005 ) . Not only the activation of specifi c gene sets but also the strength of the specifi c response signature may be important for the immune detection of the type of pathogen involved. E. coli infection of DCs strongly upregulates the chemokines/cytokine infl ammatory cluster whereas infection with infl uenza or other single stranded (ss) RNA viruses (through TLR7) has a weaker ability to regulate this cluster but a stronger ability to regulate the ISG signature ( Huang et al., 2001 ; Lund et al., 2004 ) .

These types of results raise the possibility that the diagnosis of the type of pathogen involved in an infection would be helped by the development of customized microarrays that could distinguish the gene expression profi les elicited by particular pathogens. Additionally, arrays that also detect RNAs produced by the pathogen may be even more signifi cant as a diagnostic tool.

An infecting agent can either be cleared from the body by the immune system mounting an appropriate response or cause severe or terminal pathology. The outcome depends on a multiplicity of events ranging from the genotype of the host, that is, whether the host displays a resistant or susceptible phenotype, the genotype of the infectious agent, that is, virulent or nonvirulent strains and many other less defi ned environmental factors. Can genomic approaches be used to determine the outcome of infection? Clearly, as discussed above, the technology is developing to defi ne susceptible and resistant host genotypes especially in animal models of infection but the ability to do this routinely in human populations is some way into the future.

Given the smaller genomes of pathogenic organisms, defi ning virulence genotypes is progressing at a faster rate ( Chan, 2003 ; Dorrell et al., 2005 ; Fitzgerald and Musser, 2001 ; Kato-Maeda et al., 2001 ; MacFarlane et al., 2005 ; Schoolnik, 2002 ; Zhang and Zhang, 2006 ) .

Expression profi ling studies have been used to investigate the differences in the host response to pathogenic and nonpathogenic strains of specifi c infectious agents. In one example, mice infected with a pathogenic strain of pneumonia virus upregulated the expected infl ammatory chemokines/cytokine profi le as well as the ISG profi le but an attenuated strain of the same virus could not, although the virus replicated in the lungs of these mice to the same degree ( Domachowske et al., 2001 ) . Temporal profi ling of the infection process in animals will also help to defi ne the expression signatures associated with the ability of the host to clear specifi c pathogens.

With the ongoing threat of new or reemerging pathogens, the use of genomic approaches to understand the host response to infectious agents continues to gain momentum. The sequencing of viral pathogens has become routine and there are now more than 800 bacterial genome sequences publicly available (http://www.ncbi.nlm.nih.gov/genomes). This combined with the availability of the human genome and that of an increasing number of model organisms is allowing deeper insight into genomic aspects of host-pathogen interactions.

Genetic susceptibility or resistance to pathogens is still an area of interest with new genetic associations being routinely identifi ed. A number of new studies have again focused on the TLR gene family and their role in susceptibility to infection as well as disease progression. An excellent analysis of all the previous genetic association and functional studies on TLR4 has recently been published (Ferwerda et al., 2008) . Some but not all of the association studies indicate a role for TLR4 polymorphisms in pathogen susceptibility (strongest for RSV infection), but many of these studies are small and lack statistical power and the larger studies tend not to fi nd strong associations (Ferwerda et al., 2008) . Similarly, functional studies do not always support an important role for TLR4 polymorphisms in controlling cellular responses such as cytokine production (Ferwerda et al., 2008) . There are a number of recent association studies that have shown a link between polymorphisms in other TLRs, including TLRs 7, 8 and 9, and HIV susceptibility or disease progression (Bochud et al., 2007; Oh et al., 2007; Soriano-Sarabia et al., 2008) . However, these studies will require further verifi cation with larger genetic association studies and more detailed functional studies. Similarly, polymorphisms in the TLR genes continue to emerge as potential players in susceptibility to other pathogens such as TB (Ma et al., 2007) . A relatively large association study (1312 individuals) combined with some functional studies revealed a correlation between polymorphisms in the TLR6, TLR1 and TLR10 genes, and the occurrence of TB in certain ethnic populations (Ma et al., 2007) . Recent studies of the TLR adaptor protein, TIRAP/MAL, suggest genetic associations between polymorphisms in this protein and TB susceptibility (Hawn et al., 2006; Khor et al., 2007) , again implicating the innate immune recognition system in controlling infectious disease responses. For a summary of the literature on genetic association and TB, the reader is referred to a recent review by Berrington and Hawn (Berrington and Hawn, 2007) and for a similar summary of TLR genetic association studies with various pathogens to Misch and Hawn (Misch and Hawn, 2008) . It will be very important to clarify the role of the TLR recognition system in pathogen susceptibility and disease progression before any of this information can be applied to clinical screening or drug/ vaccine development.

Of particular note, the fi rst whole genome association study to identify the host determinants of HIV-1 infection was recently published (Fellay et al., 2007) . Two polymorphisms associated with viral load during the asymptomatic period of infection were located near major histocompatibility allele human leukocyte antigen loci. A second component of the study, examining the time of HIV disease progression, implicated two genes one of which encoded an RNA polymerase I subunit (Fellay et al., 2007) . It will be important to follow up these genetic association studies with functional studies of the genes or loci implicated. Given the current availability of genomics resources, we are likely to see many more genomewide association studies published in this area, a welcome addition to the candidate gene approach.

The recently identifi ed SARS virus has already been the subject of several small scale genetic association studies, especially in relation to the HLA loci. While some associations have been identifi ed, they have not been replicated in different populations and so remain tentative (reviewed in Yang et al., 2008) . SNP associations in other genes such as that encoding low serum mannose binding lectin (MBL) and the promoter of the RANTES gene have been replicated in different populations and may stand the test of time (Ng et al., 2007; Zhang et al., 2005) .

Expression profi ling continues to be a useful tool in the analysis of the host-pathogen interaction. Some examples include a study of in vivo human rhinovirus infection (Proud et al., 2008) , innate host reponses to Ebola virus and the consequences of a mutation in the virus VP35 protein (Hartman et al., 2008 ) and Leishmania infection of human macrophages (Guerfali et al., 2008 ) . These and many other studies are leading to the identifi cation of general molecular consequences ACKNOWLEDGEMENT I would like to thank Stephanie Palmer for help with preparation and proof-reading of the manuscript. of infection as well as the specifi c consequences of individual pathogens. A recent review outlines the many insights gained into virus-host interaction using functional genomics as well as the use of these approaches in developing and evaluating vaccines (Katze et al., 2008) . Studies in model organisms such as C elegans support many of the fi ndings in humans (Wong et al., 2007) and provide a useful tool for detailed functional investigation. The "systems biology" approach to study infectious disease is being extended by the use of proteomic profi ling. Two recent articles illustrate the usefulness of this approach in examining the response of a human cell line to the avian H9N2 infl uenza virus (Liu et al., 2008) and of human macrophages to M tuberculosis lipids (Shui et al., 2009) . Systems biology approaches are moving rapidly in this and other biomedical fi elds and no doubt the next few years, given the advent of deep sequencing, will see enormous new strides in understanding host-pathogen interactions (for a recent review in relation to virus infection see Tan et al., 2007) . Other developments will undoubtedly lead to easier application of these technologies to the clinic in the foreseeable future.

Mycobacterium tuberculosis, macrophages, and the innate immune response: Does common variation matter?

Polymorphisms in Toll-like receptor 9 infl uence the clinical course of HIV-1 infection

A whole-genome association study of major determinants for host control of HIV-1

Functional consequences of toll-like receptor 4 polymorphisms

Simultaneous gene expression profi ling in human macrophages infected with Leishmania major parasites using SAGE

Whole-genome expression profi ling reveals that inhibition of host innate immune response pathways by Ebola virus can be reversed by a single amino acid change in the VP35 protein

A polymorphism in Toll-interleukin 1 receptor domain containing adaptor protein is associated with susceptibility to meningeal tuberculosis

Innate immune modulation by RNA viruses: Emerging insights from functional genomics

A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis

Proteomics analysis of differential expression of cellular proteins in response to avian H9N2 virus infection in human cells

Full-exon resequencing reveals toll-like receptor variants contribute to human susceptibility to tuberculosis disease

The association of RANTES polymorphism with severe acute respiratory syndrome in Hong Kong and Beijing Chinese

A functional toll-like receptor 8 variant is associated with HIV disease restriction

Gene expression profi les during in vivo human rhinovirus infection: Insights into the host response

Quantitative proteomic profi ling of host-pathogen interactions: The macrophage response to Mycobacterium tuberculosis lipids

Infl uence of the Toll-like receptor 9 1635A/G polymorphism on the CD4 count, HIV viral load, and clinical progression

Systems biology and the host response to viral infection

Genome-wide investigation reveals pathogenspecifi c and shared signatures in the response of Caenorhabditis elegans to infection

Infectious disease in the genomic era

Association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection

A systems approach to dissecting immunity and infl ammation

Genetic dissection of immunity in leprosy

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans

Nramp1: A link between intracellular iron transport and innate resistance to intracellular pathogens

Genealogies of mouse inbred strains

Toward the 1,000 dollars human genome

Innate immune sensing and its roots: The story of endotoxin

Cutting edge: A Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling

Stereotyped and specifi c gene expression programs in human innate immune responses to bacteria

Genomic-scale analysis of bacterial gene and protein expression in the host

Chips with everything: DNA microarrays in infectious diseases

Genetic control of visceral l eishmaniasis in a Sudanese population: Candidate gene testing indicates a linkage to the NRAMP1 region

Bacterial genomes and infectious diseases

Genetic susceptibility to infectious diseases

Toll-like receptors in the pathogenesis of human disease

Genetic regulation of endotoxininduced airway disease

Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study

Gene-expression profi ling discriminates between cerebral malaria (CM)-susceptible mice and CM-resistant mice

HLA-DRB1*12 is associated with protection against complicated typhoid fever, independent of tumour necrosis factor alpha

Glucocorticoid administration accelerates mortality of pneumovirus-infected mice

Comparative phylogenomics of pathogenic bacteria by microarray analysis

HIV-1 entry into CD4 ϩ cells is mediated by the chemokine receptor CC-CKR-5

Genes of the class II and class III major histocompatibility complex are associated with typhoid fever in Vietnam

Functional genomics and gene expression profi ling in sepsis: Beyond class prediction

Effects of functional Tolllike receptor-4 mutations on the immune response to human and experimental sepsis

Evolutionary genomics of pathogenic bacteria

Dendritic cells in pathogen recognition and induction of immune responses: A functional genomics approach

Genetics of infectious diseases

Regulation of gene expression in mouse macrophages stimulated with bacterial CpG-DNA and lipopolysaccharide

HLA and disease

The Bcg/Ity/Lsh locus: genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1 Gly169 allele

A common dominant TLR5 stop codon polymorphism abolishes fl agellin signaling and is associated with susceptibility to legionnaires ' disease

A classifi cation of tasks for the systematic study of immune response using functional genomics data

New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS

Genetic susceptibility to malaria and other infectious diseases: From the MHC to the whole genome

The immunogenetics of resistance to malaria

The plasticity of dendritic cell responses to pathogens and their components

Insights into host responses against pathogens from transcriptional profi ling

Identifi cation of genes involved in the host response to neurovirulent alphavirus infection

A polymorphism in the toll-like receptor 2 is associated with IL-12 production from monocyte in lepromatous leprosy

Microarray analysis of pathogens and their interaction with hosts

Virus infection and the interferon response: A global view through functional genomics

Unlocking the mysteries of virus-host interactions: Does functional genomics hold the key

Association of IL12RB1 polymorphisms with susceptibility to and severity of tuberculosis in Japanese: A gene-based association analysis of 21 candidate genes

SLC11A1 (formerly NRAMP1) gene polymorphisms and tuberculosis susceptibility: A meta-analysis

Human leukocyte antigen class I and class II allele frequencies and HIV-1 infection associations in a Chinese cohort

Genetic susceptibility to infectious disease: Lessons from mouse models of leishmaniasis

Polymorphism and evolution of HLA class I and II genes and molecules

Association of HLA-DR, -DQ, and vitamin D receptor alleles and haplotypes with tuberculosis in the Venda of South Africa

Recognition of singlestranded RNA viruses by Toll-like receptor 7

Genomic DNA microarrays for Entamoeba histolytica: Applications for use in expression profi ling and strain genotyping

Combined genotypes of CCR5, CCR2, SDF1, and HLA genes can predict the long-term nonprogressor status in human immunodefi ciency virus-1-infected individuals

Knockout mice: A paradigm shift in modern immunology

Genetic localization of a locus controlling the intensity of infection by Schistosoma mansoni on chromosome 5q31-q33

Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria

Variants of HLA-DR2/ DR51 group haplotypes and susceptibility to tuberculoid leprosy and pulmonary tuberculosis in Asian Indians

Characterization and functional investigation of single nucleotide polymorphisms (SNPs) in the human TLR5 gene

HLA alleles in relation to specifi c immunity to liver stage antigen-1 from plasmodium falciparum in Gabon

Susceptibility to leprosy is associated with PARK2 and PACRG

Genetic susceptibility to visceral leishmaniasis in The Sudan: Linkage and association with IL4 and IFNGR1

SLC11A1 (formerly NRAMP1) and susceptibility to visceral leishmaniasis in The Sudan

The challenge of emerging and re-emerging infectious diseases

Two types of murine helper T cell clone. I. Defi nition according to profi les of lymphokine activities and secreted proteins

Human macrophage activation programs induced by bacterial pathogens

pSTIING: A " systems " approach towards integrating signalling pathways, interaction and transcriptional regulatory networks in infl ammation and cancer

The effect of genetic variation in chemokines and their receptors on HIV transmission and progression to AIDS

Connecting mammalian genome with phenome by ENU mouse mutagenesis: Gene combinations specifying the immune system

Molecular diagnosis of inherited disorders: Lessons from hemoglobinopathies

Pyogenic bacterial infections in humans with IRAK-4 defi ciency

Microarray data normalization and transformation

Genomic signatures of immune and infl ammatory responses

Opinion: Interpretation of the complexity of innate immune responses by functional genomics

Congenic mice: Cutting tools for complex immune disorders

Tumor necrosis factor promoter polymorphism and susceptibility to lepromatous leprosy

Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene

Functional and comparative genomics of pathogenic bacteria

Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease

Mapping of genetic modulators of natural resistance to infection with Salmonella typhimurium in wildderived mice

Association of human leukocyte antigen-A11 with resistance and B40 and DR2 with susceptibility to HIV-1 infection in south India

Resources for genetic variation studies

Association and linkage of leprosy phenotypes with HLA class II and tumour necrosis factor genes

Analysis of large-scale gene expression data

Dissecting innate immune responses with the tools of systems biology

Genetic susceptibility to neonatal infection

Strong association of a tumor necrosis factor-alpha promoter allele with cerebral malaria in Myanmar

How many diseases does it take to map a gene with SNPs?

Infl ammation suppressor genes: Please switch out all the lights

Inverse associations of human leukocyte antigen and malaria parasite types in two West African populations

The impact of comparative genomics on infectious disease research

 Jenner, R.G., Young, R.A. (2005) . Insights into host responses against pathogens from transcriptional profi ling. Nat Rev Microbiol 3(4), 281-294. This review describes a meta-analysis of expression profi ling data from the literature of cells infected with different pathogens or treated with pathogenic components. Lipoldova, M., Demant, P. (2006) . Genetic susceptibility to infectious disease: Lessons from mouse models of leishmaniasis. Nat Rev Genet 7(4), 294-305. This paper reviews the literature on the genetic susceptibility to infection with Leishmania and compares susceptibility loci to those identifi ed for other infections. Cook, D.N., Pisetsky, D.S., Schwartz, D.A. (2004) . Toll-like receptors in the pathogenesis of human disease. Nat Immunol 5(10), 975-979. This review describes the role of Toll-like receptors in detection of pathogens and summarizes their involvement in infectious disease susceptibility. http://www.genmapp.org/ Gene Map Annotator and Pathway Profi ler is a computer application designed to visualize gene expression data on maps representing biological pathways and groupings of genes. http://www.ensembl.org/index.html Ensembl is a joint project between EMBL-EBI and the Sanger Institute to develop a software system which produces and maintains automatic annotation on selected eukaryotic genomes. http://www.informatics.jax.org/ MGD includes information on mouse genetic markers, molecular clones (probes, primers and YACs), phenotypes, sequences, comparative mapping data, graphical displays of linkage, cytogenetic and physical maps, experimental mapping data, as well as strain distribution patterns for recombinant inbred strains (RIs) and cross haplotypes. http://www.geneontology.org/ The Gene Ontology project provides a controlled vocabulary to describe gene and gene product attributes in any organism. http://www.ncbi.nlm.nih.gov/geo/ Gene Expression Omnibus is a gene expression/molecular abundance repository supporting MIAME compliant microarray data submissions, and a curated, online resource for gene expression data browsing, query and retrieval. http://www.genome-www5.stanford.edu/ The Stanford MicroArray Database stores raw and normalized data from microarray experiments, and provides data retrieval, analysis and visualization. http://www.expression.microslu.washington.edu/expression/index. html Public Microarray Data Download Site powered by Expres-sion Array Manager. http://www.ncbi.nlm.nih.gov/projects/SNP/ The National Center for Biotechnology Information has established the Single Nucleotide Polymorphism (dbSNP) database to serve as a central repository for both single base nucleotide substitutions and short deletion and insertion polymorphisms.Recommended Resources ■ 743