Refinement of the Genetics of the Host Response to Salmonella Infection in MOLF/Ei: Regulation of Type 1 IFN and TRP3 Pathways by Ity2

ORIGINAL ARTICLE Refinement of the genetics of the host response to Salmonella infection in MOLF/Ei: regulation of type 1 IFN and TRP3 pathways by Ity2

R Khan1, V Sancho-Shimizu2, C Prendergast3, M-F Roy4, J-C Loredo-Osti5 and D Malo1,3 1Department of Human Genetics, McGill University, Montre´al, Que´bec, Canada; 2Universite´ Paris Rene´ Descartes, Necker Medical School, Paris, France; 3Department of Medicine, McGill University, Montre´al, Que´bec, Canada; 4Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada and 5Memorial University of Newfoundland, St John’s, Newfoundland, Canada

Typhoid fever, which is caused by Salmonella typhi and paratyphi, is a severe systemic disease that remains a major public health issue in several areas of the world. We can model the human disease using mice infected with a related bacterium, Salmonella typhimurium. This model recapitulates several clinical aspects of the human disease and allows for the study of the host response to Salmonella typhimurium infection in vivo. Previous work in our laboratory has identified three Immunity to typhimurium loci (Ity, Ity2 and Ity3) in the wild-derived MOLF/Ei mice, influencing survival after infection with Salmonella typhimurium. The MOLF/Ei alleles at Ity and Ity2 are protective, while the MOLF/Ei allele at Ity3 confers susceptibility. In this paper, we have generated a novel cross combination between the highly susceptible strain, MOLF/Ei, and the resistant strain, 129S6, to better define the genetic architecture of susceptibility to infection in MOLF/Ei. Using this cross, we have replicated the locus on chr 11 (Ity2) and identified a novel locus on chr 13 (Ity13). Using microarrays and transcriptional profiling, we examined the response of uninfected and infected Ity2 congenic mice. These analyses demonstrate a role for both type-1- interferon (IFN) and TRP53 signaling in the pathogenesis of Salmonella infection. Genes and Immunity (2012) 13, 175–183; doi:10.1038/gene.2011.69; published online 29 September 2011

Keywords: Ity2; Ity13; infection; Trp53; type 1 IFN; innate immunity

Introduction results mainly in a self-limiting acute gastroenteritis (salmonellosis), which is one of the most common and Salmonella enterica is a Gram-negative facultative intra- widely distributed food-borne disease.1 cellular bacteria with more than 2500 serovars identified, Scientific research over the past few decades has although only a limited number of serovars are of public proven that both bacterial and host genetic factors health importance. In humans, Salmonella enterica are the influence the outcome of several types of infection.2 cause of a broad spectrum of diseases varying from Our current understanding of the host immune response asymptomatic carriage to systemic febrile infection or to Salmonella infection has been achieved mainly through sepsis. The outcome of infection is determined by the the use of mouse models mimicking the various stages serovar causing the infection as well as the genetic of infection. As Salmonella typhi infects only humans, predisposition of the infected individual. The human- typhoid fever has been modeled in the mouse using specific serovars Salmonella enterica typhi and paratyphi Salmonella typhimurium, a natural pathogen of the mouse. are the causative agent of typhoid fever, a serious Administration of a sublethal dose (either per os, systemic often fatal infection. Typhoid fever remains a intraperitoneally or intravenously) of Salmonella typhi- major public health problem in areas of the world with murium in mice results in a clinical disease that resembles poor sanitation and lack of access to clean water. It is human typhoid fever. The infection results in dissemina- estimated that globally there are 21.5 million cases tion of the bacteria to the spleen and liver, the main sites of typhoid annually resulting in 200 000 deaths.1 of Salmonella replication, resulting in systemic disease In humans, infection with several non-host-specific and activation of both the innate and adaptive immune serovar, including Salmonella typhimurium and enteritidis, system. After oral infection, Salmonella typhimurium does not replicate in the intestine of mice, but invades the M cells, which are specialized epithelial cells of the small Correspondence: Dr D Malo, Department of Human Genetics, intestine and colonizes the Peyer’s patches and mesen- McGill University, McGill Life Sciences Complex, 3649 Promenade teric lymph nodes before spreading into the liver and Sir-William-Osler, Room 369, Montre´al, Que´bec, Canada H3G 0B1. 3 E-mail: [email protected] spleen. Several mouse strains (C57BL/6J, C3H/HeJ, Received 20 May 2011; revised 23 August 2011; accepted 29 August AcB61 and ITY9) are unable to control bacterial replica- 2011; published online 29 September 2011 tion and succumb early during the infection.4–7 Other Ity2 induces type 1 interferon and Trp53 responses R Khan et al 176 strains of mice, such as several 129 substrains, are 129S6/F1 capable of controlling early bacterial replication and survive the infection.4,8 The wild-derived MOLF/Ei mice

are highly susceptible to infection despite the fact that tion they are capable of controlling bacterial replication in the target organs.9 An initial genome scan in an F2 cross

between the two susceptible strains, C57BL/6J and l Post Infec Post l MOLF/Ei, identified three regions linked to the clinical F2 phenotype: Ity and Ity3 on chromosome 1 and Ity2 on

chromosome 11. The MOLF/Ei alleles at Ity and Ity3 Surviva confer susceptibility to infection, whereas the MOLF/Ei MOLF/Ei allele at Ity2 confers resistance. The molecular basis of Ity was elucidated previously, and the gene conferring resistance to infection in MOLF/Ei is Slc11a1, initially Days Post Infection named Nramp1.10,11 The existence of Ity2 and Ity3 was confirmed by creating congenic mice carrying each of the 40 locus in isolation.12,13 In the current paper, we have used a novel cross between the highly resistant 129S6 and susceptible 30 MOLF/Ei for linkage analysis. In this particular cross, a functional Slc11a1 allele (Ity) was present in all F2 20 progeny. Surprisingly, the Ity2 locus was detected despite

the resistant background contributed from the 129S6 Frequency mice. An additional locus named Ity13 was identified on 10 chromosome 13 and MOLF/Ei alleles at this locus result in an increased susceptibility to infection. In order to further study the biological contribution of Ity2 to 0 resistance to infection, we used Ity2 congenic mice and a 5 101520253035 genome-wide microarray analysis to identify important pathways and networks regulated by the Ity2 interval. Days Post Infection Figure 1 Survival analysis of parental 129/S6 (n ¼ 5) and MOLF/Ei (n ¼ 5) and F1 (n ¼ 10) and F2 (n ¼ 181) progeny after infection with 103 colony-forming units of Salmonella typimurium (a). The F1 Results behave exactly like the parental 129S6 strain, while the F2 have an intermediate survival. The frequency distribution of the survival Identification of a novel locus (Ity13) controlling susceptibility times of the mice after infection (b). About 25% of the mice survived to Salmonella infection in wild-derived MOLF/Ei mice until the end point and were considered resistant, while the We have previously reported, using a cross between the susceptible mice can be subdivided into three groups, the first C57BL/6J and MOLF/Ei mice, the identification of three succumbing to infection by day 15, the second by day 24 and the last loci linked to the Salmonella host response in MOLF/ by day 30. Ei mice: Ity on proximal chromosome 1, Ity2 on chromosome 11 and Ity3 on distal chromosome 1.13 In the current study, we have generated a novel cross using (22.5%) survived towards the end point of the experi- 129S6 mice and wild-derived MOLF/Ei to identify novel ment (Figure 1b). The F2 distribution is consistent with susceptibility loci. The choice of the strain combination the results obtained from our previous cross and was based on the observation that 129S6 mice are highly illustrates the complex nature of the host response to resistant to Salmonella infection (100% survival 30 days Salmonella infection.13 The F2 frequency distribution post-infection; Figure 1a), and also because this parti- suggests that the host response is under the influence cular strain does not carry any known Salmonella- of more than one gene. There was no effect of sex on the susceptibility genes/loci. As observed before, MOLF/Ei survival of the F2 population, therefore data from males were extremely susceptible and developed clinical and females were pooled for further analyses. disease within the first week of infection (Figure 1a). A genome scan was done in the F2 population using Strain 129S6 and wild-derived MOLF/Ei were out- 121 custom-made single-nucleotide polymorphisms crossed to generate F1 and F2 progeny. Both F1 and F2 and microsatellite markers, spaced approximately at mice were infected with Salmonella typhimurium and the 20 cm interval (Supplementary Table S1). Through a mice were monitored for a period of 34 days. The F1 Cox regression model, two loci, one on chromosome population showed a survival phenotype identical to the 11 and the other on chromosome 13, were found to be resistant 129S6 parent, while the F2 population showed significantly associated with survival time (Figure 2). The partial survival with variable time to death (Figure 1a). most significant peak was seen on chromosome 11 at Upon closer examination, the F2 population appears D11Mit141 (position 52.9 Mb; P ¼ 0.0007). This region to segregate into distinct groups. Mice susceptible to overlaps with the identified proximal region of the Ity2 infection did not show a normal distribution of time interval. A second locus was identified on chromosome to death. Instead, they could be subclassified into three 13 with the peak value at D13Mit19 (position 43.9 Mb; groups: a group developing clinical disease before day P ¼ 0.0046). This locus was named Ity13 (Figure 2). 17, and two intermediate groups with observed mortality Consistent with the previous findings,13 the mode of between days 18–22 and days 23–29. A group of mice inheritance for chromosome 11 is additive, with the

Genes and Immunity Ity2 induces type 1 interferon and Trp53 responses R Khan et al 177 4 4 4 Chr 1 Chr 11 Chr 13

3 3 3 p p p 10 10 10 2 2 2 − log − log − log

1 1 1

0 20406080100 0 20 40 60 80 100 0 20406080100 Notice: x-axis units are cM. Figure 2 Cox Proportional Hazards survival analysis in 181 (129S6 Â MOLF/Ei) F2 infected with 103 colony-forming units of Salmonella typhimurium IV. The -log10 P values are shown for each position on chromosome 1 (a), chromosome 11 (b) and chromosome 13 (c). Two significant peaks were detected on chromosome 11 (b) and chromosome 13 (c).

Splenic transcriptional signature in Ity2 congenic mice during Salmonella infection We have previously reported that the Ity2 region harbored at least two loci: Ity2A, a proximal region of 22 Mb between D11Mit109 and D11Mit26,andIty2B consisting of a 13-Mb distal interval extending from D11Mit5 to D11Mit8.13 In order to better understand the contribution of the Ity2 locus during Salmonella infection, we examined global gene expression in two congenic mouse strains: B6.MOLF-Ity/ Ity2 mice, which carry the entire Ity2 interval and B6.MOLF-Ity/Ity2.RecD mice, which harbor only the distal Ity2.B portion of the Ity2 segment. These two congenic strains present significantly different survival times following Salmonella infection. With the aim of identifying genes differentially regulated upon infection in B6.MOLF-Ity/Ity2 and B6.MOLF-Ity/Ity2.RecD, the relative abundance of transcripts at day 3 post-infection to their levels in the control uninfected tissue was compared. Using a combination of statistical filters and hierarchical clustering, the gene list was refined and a total of 361 genes were differentially regulated upon infection in the susceptible B6.MOLF-Ity/ Ity2.RecD strain and 173 genes in the more resistant strain, B6.MOLF-Ity/Ity2. Among those differentially regulated genes during infection, 109 genes were commonly expressed in both the strains (Supplementary Table S2). We examined their gene ontogeny classifications based on PANTHER (Protein ANalysis THrough Evolutionary Relationships) to identify biological functions that were regulated during the infection.14 The transcriptional signature common to both the strains consisted of genes known to be involved in the immune response to infection, with a Figure 3 Cumulative survival curves after infection with Salmo- significant number of genes that were regulated by nella typhimurium in (129S6 Â MOLF/Ei) F2 population based on interferon (IFN)-g (Cebpb, Cxcl10, Gbp1, Gbp2, Gbp3, Gbp4, their genotypes at the peak marker for Ity2 (D11Mit141)(a) and Gbp5,Gbp6,Gbp10,Ifit2,Ifit3,Ifng,Irf7and Irg1)(Supple- Ity13 (D13Mit19)(b). MOLF/Ei represents mice homozygous for the mentary Figure S1A and Supplementary Table S3). Other MOLF/Ei allele and 129S6 represents mice homozygous for the clusters identified were involved in the early response to 129S6 allele. Het represents mice heterozygous at the peak markers. Both loci are inherited under an additive model with the protective infection, including acute-phase proteins (Saa3), pro-in- effect associated with the MOLF/Ei allele for Ity2 and with 129S6 flammatory mediators (Il1b and Il1rn) and genes implicated for Ity13. in cellular recruitment (Ccl3, Ccl4, Ccl7, Ccl12, Ccl24, Cxcl9 and Cxcl10). Some additional genes that were identified have already been shown to have a critical role during the MOLF/Ei allele contributing to resistance (Figure 3a). Salmonella infection in vitro and/or in vivo (Ifng, Cebpb, This result confirms the notable effect of the MOLF/Ei Gbp5, Slc11a1 and Usp18).4 allele at Ity2, as it contributes a resistance allele despite In susceptible B6.MOLF-Ity/Ity2.RecD strain, there was the genetic background contribution from a highly an over-representation of genes belonging to additional resistant 126S6 strain. The Ity13 locus showed an biological function categories including apoptosis (Birc5, additive mode of inheritance with the MOLF/Ei allele Caps4, and Ppp1r15a) and cell cycle (Ccnb1, Cdc2, Cdc6, conferring susceptibility (Figure 3b). Cdkn1a, and Cdkn3) (Supplementary Figure S1B and

Genes and Immunity Ity2 induces type 1 interferon and Trp53 responses R Khan et al 178 Supplementary Table S3). Several genes involved in of IFN-related gene expression significantly increased in erythropoiesis and iron metabolism (Ank1, Klf1, Nfe2, both mouse strains during infection (Figure 4a). Ad- Rhag and Tfrc) were also upregulated in the B6.MOLF- ditionally, we noticed that the genes involved in Ity/Ity2.RecD strain (Supplementary Figure S1). All these erythropoiesis and those classified within the cell-cycle genes were known to be expressed in the mouse bone category and the TRP53 pathway, were predominantly marrow and human early erythrocytes expressing the up-regulated within the B6.MOLF-Ity/Ity2.RecD suscep- transferring receptor.15 In addition, disruption of several tible strain (Figure 4b and Supplementary Figure S1). The of these genes (Ank1, Klf1, Tfrc and Nfe2) has been shown transcripts for these genes were unaffected in the to cause severe anemia.16–21 Another group of genes resistant strain, while they were significantly more involved in type 1 IFN signaling (Ifitm1, Irf7, Oas1, Oas2, abundant in the susceptible strain during the infection. Oasl, Isg15 and Usp18) were upregulated in the B6.MOLF-Ity/Ity2.RecD strain. A striking difference in Type I IFN and Trp53 pathways are regulated by Ity2 the transcriptional response of B6.MOLF-Ity/Ity2.RecD in the spleen and liver during infection compared with B6.MOLF-Ity/Ity2 is the To validate the microarray data, we used quantitative presence of a distinct TRP53 pathway signature (Fig- RT-PCR (qRT-PCR) on 13 selected genes including the ure 4). In fact several genes regulated by TRP53 were IFN-regulated genes (Isg15, Isg20, Stat1, Irf7, Irf9 and overexpressed in the susceptible B6.MOLF-Ity/Ity2.RecD. Jmjd3) and genes known to be involved in cell cycle, The temporal changes of a subset of these genes in the apoptosis and Trp53 regulation (Cdc2, E2f2, Birc5, Ccng1, two congenic strains are shown in Figure 4. The level Ppp2a, Pttg1 and Trp53). The genes located within the

Gbp1 Gbp2 Gbp5 Epb4.9 Nfe2 Epb4.1 11.0 11.0 11.0 12.0 11.0 10.5 10.0 10.0 10.0 11.0 10.0 9.5 9.0 9.0 9.0 10.0 8.0 9.0 8.0 8.5 8.0 Day 0 Day 0 Day 0 Day 3 Day 0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 3 Day 0 Day 3 Day 0 Day 3 Day 0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 0 Day 3 Day 3

Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2

Ifi47 Ifit3 Oas2 E2f2 Ccnb1 Cdc2a

11.0 14.0 13.0 10.5 12.0 10.5

10.0 13.0 10.0 12.0 9.5 11.0 9.5

12.0 9.0 11.0 8.5 10.0 9.0 Day 0 Day 0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 3 Day 3 Day 0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 3 Day 3 Day 3 Day 0 Day 3 Day 0 Day 0 Day 0 Day 0 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2

Irf7 Irg1 Isg15 Cdkn3 Hist1h2ag Dyrk3 10.5 11.5 13 10.5 11.0 11.5

10.0 12 9.5 10.0 10.5 11.0 11 9.5 9.0 10.5 8.5 10 9.5 9.0 8.0 10.0 Day 0 Day 0 Day 3 Day 3 Day 0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 0 Day 3 Day 0 Day 0 Day 0 Day 3 Day 3 Day 3 Day 0 Day 3 Day 0 Day 3 Day 0 Day 3

Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2

Isg20 Stat1 Stat2 Hist2h2ab Oip5 Birc5 9.5 12.0 12.5 10.2 12.0 10.5 9.0 12.0 9.8 11.0 10.0 11.0 8.5 11.5 9.4 9.5 10.0 10.0 11.0 9.0 8.0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 0 Day 0 Day 0 Day 3 Day 0 Day 3 Day 3 Day 0 Day 3 Day 0 Day 3 Day 0 Day 0 Day 0 Day 3 Day 3 Day 0 Day 3 Day 3 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Ity2.RecD Ity2 Figure 4 Box plots showing the expression of a subset of genes from the microarray analysis. Genes within the type 1 IFN pathway are shown on the left (a), while genes within the cell-cycle pathway are represented on the right (b). All of the genes in panel a are significantly upregulated in the resistant and susceptible strain upon infection (P-value o0.05), with the exception of Isg15 and Isg20 which were only significantly upregulated in the resistant strain. In contrast to this, all of the genes, except Hist1h2ag and Hist2h2ab in panel b were significantly downregulated during infection in the susceptible stain (P-value o0.05), but there was no significant changes seen in the resistant strain. Furthermore, there were no significant differences between B6.MOLF-Ity/Ity2 and B6.MOLF-Ity/Ity2.RecD at day 0, but all the genes were significantly lower in the susceptible strain as compared with the resistant strain, 3 days post-infection. These data illustrate that a subset of genes within the cell-cycle pathway showed differential expression following infection in the susceptible strain B6.MOLF-Ity/ Ity2.RecD as compared with the resistant strain B6.MOLF-Ity/Ity2.

Genes and Immunity Ity2 induces type 1 interferon and Trp53 responses R Khan et al 179 congenic interval on chromosome 11 (Ccng1, Ppp2a, were also significantly upregulated only in the suscep- Pttg1, Trp53 and Jmjd3) were sequenced in MOLF/Ei to tible strain (Figure 5). Expression of genes within the help the design of sequence-specific primers. Transcript Ity2A region known to regulate Trp53 expression (Pttg1, abundance was evaluated in the spleen of uninfected Ccng1 and Ppp2ca) was also studied. Pttg1 (P-value: mice and during infection in both congenic strains 0.00052) and Ppp2ca (P-value: 0.03407) were significantly (Figures 5a and b). The selected genes were detectable upregulated post-infection in the susceptible strain at very low levels in uninfected mice although we could B6.MOLF-Ity/Ity2.RecD, while they were mildly down- observe a tendency for lower mRNA expression for most regulated in the resistant strain. These analyses con- genes studied in the B6.MOLF-Ity/Ity2.RecD compared firmed the pattern of up or downregulation observed in with the B6.MOLF-Ity/Ity2 (Figure 5a). During infection, the spleen of congenic mice after infection as observed in the transcription factors, Stat1 and Irf7, were significantly the microarray analysis (Figure 5b). upregulated in both congenic mice (P-values o0.05)), We also studied the expression of the same panel of whereas Irf9 and the IFN-regulated gene, Isg15,were genes in the liver, which is a major site for Salmonella significantly upregulated only in susceptible congenic mice replication and growth during infection (Figures 5c and d). (P-value: 0.00013 and 0.00006, respectively). The transcrip- As observed in the spleen, several IFN-regulated genes tion factors STAT1 and IRF9 are part of the transcriptional (Stat1, Irf7, Irf9 and Isg15) were significantly upregulated in activator complex ISGF3, activated by type 1 IFN. The both B6.MOLF-Ity/Ity2.RecD mice and B6.MOLF-Ity/Ity2 increased transcription of Irf7 and Isg15 in the susceptible following infection. A small but significant increase in strain is most likely a reflection of increased translocation Trp53 expression was detected in the liver in B6.MOLF-Ity/ of ISGF3 to the nucleus and binding to the IFN-stimulated Ity2.RecD (P-value: 0.04810), which was not observed in the regulatory element present in these two target genes. This resistant strain. The activity of genes regulated by or confirms the involvement of the type 1 IFN signaling regulating TRP53 activity (E2f2 and Ptgg1) was also pathway in the host response of susceptible B6.MOLF-Ity/ affected during infection in the liver (Figure 5c). Overall, Ity2.RecD mice during infection. the transcriptional activity inthespleenandintheliver The qRT-PCR data also confirmed that during Salmo- during infection in B6.MOLF-Ity/Ity2 and B6.MOLF-Ity/ nella infection in vivo,theTrp53 pathway is dysregulated. Ity2.RecD present common signatures. The expression of Trp53 (P-value: 0.00301) was significantly increased only in the susceptible B6.MOLF-Ity/ Ity2.RecD mice. In addition, genes/proteins acting up- stream to regulate Trp53 activity, such as E2f2 (P-value: Discussion 0.000), and others acting downstream to mediate Trp53 Our study reports the identification of loci and pathways effects (Birc5, P-value: 0.0216; Cdc2a, P-value: 0.0315) involved in the host immune response to Salmonella

Figure 5 Real-time PCR validation of genes identified using microarray analysis as differentially regulated in the spleen (a, b) and liver (c, d) of B6.MOLF-Ity/Ity2 compared with B6.MOLF-Ity/Ity2.RecD. Relative expression ratios for 13 genes that were differentially regulated according to the microarray analysis are shown. Panels a and c show the relative change in expression of each gene for B6.MOLF-Ity/Ity2 (white) and B6.MOLF-Ity/Ity2.RecD (black), when comparing day 3 with the reference group, day 0, in the spleen and liver, respectively. Panels b and d illustrate the expression of B6.MOLF-Ity/Ity2.RecD compared with the reference group (B6.MOLF-Ity/Ity2) at day 0 (white) and day 3 (black) in the spleen and liver, respectively. An * represents a P-value of less than 0.05.

Genes and Immunity Ity2 induces type 1 interferon and Trp53 responses R Khan et al 180 infection contributed by the wild-derived MOLF/Ei abundance of erythropoiesis-related transcripts in strain background. The study design was structured susceptible B6.MOLF-Ity/Ity2.RecD mice was most likely to control for the effect of mutant Slc11a1 that over- related to an increase in the reticulocyte population of shadowed minor QTLs (quantitative trait loci) identified the spleen in these mice. We have previously observed in the previous crosses using MOLF/Ei.11 We replicated that highly susceptible mice infected with Salmonella Ity2, a locus that was identified using an intercross typhimurium develop anemia concomitantly with extra- between two strains susceptible to Salmonella infection. medullary hematopoiesis in the liver and the spleen.6,29 Consistent with the previous findings, our study showed In humans, the presence of anemia during Salmonella that the Ity2 MOLF/Ei allele confers resistance to typhi infection is well documented and correlates with Salmonella infection even in linkage analysis using a reduced abundance of genes associated with erythroid cross between MOLF/Ei and the most Salmonella- differentiation in the peripheral blood.28 The second resistant mouse strain identified to date. Additionally, specific signature observed in B6.MOLF-Ity/Ity2.RecD this study identified a locus on chromosome 13 (Ity13) involved upregulation of type 1 IFN signaling. We have where the MOLF/Ei allele contributes to increased shown recently that increased type 1 IFN signaling is susceptibility to infection in an additive model of detrimental during Salmonella infection in the mouse.4 In transmission. A suggestive Salmonella-susceptibility lo- addition, a correlation between the increase in blood type cus was previously reported in this location that 1 IFN-inducible transcripts and disease severity has been explained 3% of the phenotypic variance and presented recently reported in patients with active tuberculosis a recessive mode of transmission.11 We did not know at supporting an important role for type 1 IFN in that time whether the two loci are identical or not, but it the pathogenesis of intracellular bacterial infections in is clear that the impact of chromosome 13 MOLF/Ei mouse models of human diseases and in humans.30 alleles on susceptibility to infection is much stronger in Finally, the third cluster showing transcriptional the current cross. An additional Salmonella-related locus, dysregulation in B6.MOLF-Ity/Ity2.RecD involves the modifier of Salmonella typhimurium susceptibility 4, has TRP53 pathway (Supplementary Figure S2). Trp53 is a been mapped to this region and modulates acute tumor suppressor gene known to balance cell survival inflammatory reactions.22 and death in response to a variety of intrinsic and We chose to further characterize the Ity2 locus because extrinsic stress signals. In response to stress signal, TRP53 of the availability of congenic and subcongenic mice. We protein is activated by post-translational modification showed previously that the two regions within the Ity2 leading to either cell-cycle arrest or cellular apoptosis.31,32 interval (Ity2A and Ity2B) were necessary for the Viral infections have also been shown to affect TRP53 resistance of B6.MOLF-Ity/Ity2 mice to infection on an activity by inducing type 1 IFN-mediated Trp53 mRNA Slc11a1-corrected C57BL/6J background.13 This led us to upregulation.33–35 On the other hand, TRP53 activation hypothesize that gene(s) within one segment must enhances IFN signaling.36 During Salmonella infection, we regulate the effect of gene(s) within the other segment. observed both an increase in type 1 IFN expression and We have used microarray analysis to study the major enhanced Trp53 transcription. It is not possible at the biological pathways involved in disease progression in moment to determine if Trp53 upregulation was a direct, Ity2 congenic strains, with the hypothesis that biological or indirect (through type 1 IFN signaling) consequence of knowledge about pathways regulated by Ity2 may help infection. Interestingly, Trp53 is located within the Ity2B the identification of the underlying genes and mechan- region and we observed a difference in the expression of isms involved in Salmonella pathogenesis. We focused on Trp53 between the two congenic strains during the major pathways, which showed differential regulation infection, despite the fact that both strains carry the same between two Ity2 congenic strains showing resistance MOLF/Ei allele at the Trp53 gene, suggesting that gene(s) (B6.MOLF-Ity/Ity2) or susceptibility (B6.MOLF-Ity/Ity2.RecD) located within the most Ity2 proximal region (Ity2A)is to infection. A dominant transcriptional signature pre- (are) affecting the expression of Trp53 (Figure 6). There sent in both the congenic strains involved transcripts that are candidate genes within the Ity2A region that may mediate inflammation including Il1 and several influence directly or indirectly Trp53 expression and/or IFN-g-inducible genes. This type of signature appears activity, including Irf1, Ccng1, Ppp2ca and Pttg1. Irf1 has to be common among different strains of mice during an important role in type 1 IFN signaling and together Salmonella typhimurium infection in vivo.4,6,9 The set with Trp53, it regulates Cdkn1a (p21) gene expression to of genes defined in our analysis was also shown to be promote the cell-cycle arrest.37 During Salmonella infec- induced in several human cell types in response to tion, increased Trp53 expression is paralleled by upregu- different pathogens in vitro, and were suggested to lation of genes within the Ity2A region, including Ccng1 belong to a common host response signature.23 In addition, and Pttg1, two genes known to serve as negative the importance of the interleukin-12/IFN-g axis in the regulators of TRP53 activity. CCNG1 interacts with host response to Salmonella-induced systemic disease in PPP2CA and dephosphorylates MDM2, resulting in mice24–26 and salmonellosis in humans27 is well known. degradation of TRP53,38 and PPTG1 binds to Trp53 to Additional data to support these observations came from inhibit Trp53 transcriptional activity37 and Trp53- recent publications reporting the induction of large mediated cell death.39 Despite increased expression of proportion of genes induced by IFN-g in the peripheral these two Trp53 negatively regulating genes, levels of blood of patients during the acute phase of typhoid fever.28 Trp53 remain elevated in the Salmonella-susceptible In the most susceptible B6.MOLF-Ity/Ity2.RecD con- congenic mice. Additionally, Trp53 can direct E2f2- genic strain, three clusters of genes showed significant mediated growth arrest involving the target gene specific profiles. They include genes involved in ery- Gadd45.40 E2f2 and Gadd45 are two genes that were thropoiesis and iron metabolism, type 1 IFN-inducible upregulated in the Salmonella-susceptible B6.MOLF-Ity/ genes and genes regulated by TRP53. The increased Ity2.RecD congenic strain, which further supports the

Genes and Immunity Ity2 induces type 1 interferon and Trp53 responses R Khan et al 181 infection. Subcongenic mice were previously developed in our laboratory by transferring the entire MOLF/Ei Ity 0 Mbp and Ity2 region onto a C57BL/6J background. The resulting congenic mice (B6.MOLF-Ity/Ity2) carry a functional MOLF/Ei allele at the Slc11a1 gene in addition to the MOLF/Ei genomic interval at the Ity2 locus. Subcongenic B6.MOLF-Ity/Ity2.RecD mice were also D11Mit109 developed carrying the distal region of Ity2.13 After Ccng1 40 infection, the B6.MOLF-Ity/Ity2 mice were more resistant Pttg1 to infection with a mean survival time of 11.1±1.1 days Ppp2ca while B6.MOLF-Ity/Ity2.RecD had a reduced survival 13 D11Mit141 with a mean survival time of 7.9±0.4 days. All the mice were housed at the Montreal General Hospital D11Mit208 60 Research Institute Animal Facility in accordance D11Mit26 with the conditions specified by the Canadian Council D11Mit5 on Animal Care, and the Animal Use Protocol Trp53 was approved by the McGill University Animal Care

B6.MOLF/Ei-Ity.Ity2 Committee. D11Mit8 80 In-vivo Salmonella infection D11Mit326 Mice aged 6–16 weeks were infected with Salmonella 13 B6.MOLF/Ei-Ity.Ity2.RecD typhimurium strain Keller as described previously. Briefly, mice were inoculated with 0.2 ml of physiological saline containing 103 colony-forming units of the bacteria 120 through the caudal vein. The infectious dose was verified by serial dilutions on trypticase soy agar. Mice that survived until the end point of 34 days were considered resistant.

Resistant Susceptible Genotyping Figure 6 Schematic representation of mouse chromosome DNA was isolated from tail biopsies using overnight 11 illustrating the position of the congenic intervals in B6.MOLF- digestion in a lysis buffer containing proteinase K (10 nM Ity/Ity2 and B6.MOLF-Ity/Ity2.RecD strains. The box represents Tris, 2 mM EDTA, 0.5%SDS, 0.4 M NaCl and 0.2 mg mlÀ1 regions inherited from MOLF/Ei. Positions and location (in Mb) of proteinase K), followed by a phenol/chloroform extrac- the candidate genes are indicated along the chromosome. Both Pttg1 tion. The concentration of the DNA was measured using and Ccng1 are located within the Ity2A region, and are known to be downstream targets of Trp53. the Quant-iT DNA Assay Kit (Molecular Probes, Invitro- gen, Burlington, ON, Canada) and was adjusted to 30 ng mlÀ1. Genotyping was done using a custom panel hypothesis that in B6.MOLF-Ity/Ity2.RecD, cells are consisting of 60 single-nucleotide polymorphisms and 61 maintained in a cell-cycle arrest state. It has been microsatellite markers (Supplementary Table S1), which documented that other bacterial pathogens have elabo- were identified using publicly available databases.44,45 rated sophisticated mechanisms to block cell-cycle pro- The single-nucleotide polymorphism genotyping was gression to favor conditions for colonization and/or performed at the McGill University and Ge´nome Que´bec dissemination.41–43 We showed here that Trp53 expression Innovation Centre using the Sequenom iPlex Gold is affected by specific Ity2A genomic context, and we Technology, while conventional polymerase chain reac- speculate that its impact on resistance to Salmonella tion was used for microsatellite amplification in F2 infection is explained by favoring a cell-cycle arrest state progeny. The PCR products were resolved on a 3-% high- allowing the bacteria to proliferate more successfully. resolution agarose gel (BioShop Canada Inc., Burlington, Therefore, we conclude that the MOLF/Ei allele at Ity2 ON, Canada). maintains the levels of Trp53 expression within a range that allows control of bacterial growth during infection, a Genetic analysis situation that is beneficial to the host. In order to test for the association between survival time after infection and the different chromosomal regions, we used Cox proportional hazards regression at the Materials and methods markers. Genome-wide empirical P-values were obtained by bootstrapping using 10 000 resamples. A peak Animals used region with P-value lower than 0.01 (which corresponds

Classical inbred 129S6/SVEvTac (129S6) and wild- to a Àlog10 value of 2) was considered to be significantly derived inbred MOLF/Ei mice were obtained from linked to survival. Computations were carried out in the Taconic (Germantown, NY, USA) and the Jackson software package R.46 Laboratories (Bar Harbor, ME, USA), respectively. These parental inbred mouse strains were outcrossed Microarray analysis to produce the F1 generation, which were further RNA was extracted from the spleen of uninfected controls, intercrossed to produce the F2. C57BL/6J mice (Jackson as well as B6.MOLF-Ity/Ity2. (Salmonella-resistant) and Laboratories) were used as susceptible controls for B6.MOLF-Ity/Ity2.RecD strains (Salmonella-susceptible) 3

Genes and Immunity Ity2 induces type 1 interferon and Trp53 responses R Khan et al 182 days post-infection. The RNA extraction was carried Acknowledgements out using TRIzol reagent (Invitrogen Canada Inc., Bur- lington, ON, Canada). Three age-matched mice were We wish to acknowledge the technical assistance of used per group. The concentration of the RNA Nadia Prud’homme, Line Larivie`re and Melissa Herman was determined using a NanoDrop spectrophotometer and the contribution of the McGill University and (ThermoFisher Scientific, Waltham, MA, USA) and integ- Ge´nome Que´bec Functional Genomics platform person- rity of the RNA was confirmed using denaturing agarose nel and the Innovation Centre. We thank Dr Shauna gel electrophoresis. All hybridization and scanning of mice Dauphinee for critical reading of this manuscript. This microarrays were carried out at the McGill University and work was supported by the Canadian Institutes of Ge´nome Que´bec Innovation Centre using the Illumina Health Research (to DM). RK is a recipient of a Fonds BeadArray technology (Illumina Inc., San Diego, CA, de la Recherche en Sante´ du Que´bec Studentship. DM is USA). a McGill Dawson Scholar. The data from the microarray were analyzed using FlexArray.47 They were normalized using a Lumi algorithm (Illumina) with Robust Multichip Average References background correction. The data were further refined by carrying out a Cyber t-test comparing the expression 1CDC. Typhoid Fever. In: Centres for Disease Control and data from the infected samples with the uninfected Prevention, 2005. samples.48 This approach allowed us to generate a profile 2 Roy MF, Malo D. Genetic regulation of host responses of the genes differentially regulated in each mouse to Salmonella infection in mice. 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