Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA,

Beltsville, MD)

Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes

Transcriptional Response of Pigs to Salmonella infection: Comparison of responses to infection with Salmonella enterica serotype Typhimurium as that observed in S. Choleraesuis infection JJ Uthe1,2, SMD Bearson2, YF Wang 1, L Qu 1, D Kuhar3, TJ Stabel2, SH Zhao4, OP Couture1, D Nettleton5, JC Dekkers1, CK Tuggle1, JK Lunney3
1Dept.

APDL, BARC, ARS, USDA, Beltsville, MD Thursday 7th June 2007 E. coli and Salmonella workshop Utrecht, The Netherlands

Joan Lunney

of Animal Science, Iowa State University, Ames, IA USA Animal Disease Center, USDA-ARS, Ames, IA USA 3Animal Parasitic Diseases Lab, USDA-ARS, Beltsville, MD USA 4Lab of Molecular Biology and Animal Breeding, Huazhong Agricultural Univ., Wuhan, PR China 5Dept. of Statistics, Iowa State University, Ames, IA
2National

Funding: USDA ARS funds and CSREES grants

Research Goals
General: Understand immune and genetic basis of swine infectious disease responses to pathogens Specific: Compare effects of Salmonella enterica serotypes Choleraesuis (SC) and Typhimurium (ST) on gene and protein expression in infected pig tissues and in monolayer cultures of porcine epithelial cells (IPEC J2 cells) Targets: Predict host protective responses to bacteria Improve strategies for vaccination to prevent infection Discover new targets for antibacterial agents for disease control Identify differences in host responses between Salmonella pathogens Long term goal: Prevent bacterial persistence
E. Thacker. The Pig Journal 54: 55 (2004) www.thepigsite.com/FeaturedArticle/Default.asp?Display=1284

Innate Immunity

Differentiation Swine Immune Gene Expression: T helper 1 (Th1) versus Th2

Definition of Th1 (Toxoplasma gondii) and Th2 (Ascaris suum) immune responses in swine.
Use of real-time RT-PCR technology to measure targeted gene expression at multiple tissues sites Dawson HD, Beshah E, Nishi S, Solano-Aguilar G, Morimoto M, Zhao A, Madden KB, Ledbetter TK, Dubey JP, Shea-Donohue T, Lunney JK, Urban JF Jr. 2005. Infection and Immunity 73: 1116. PIN (Porcine Immunology and Nutrition) database http://www.ars.usda.gov/Services/docs.htm?docid=6065 Curated by Harry Dawson, NRFL, BHNRC, BARC

Link between Innate and Adaptive Immune Systems

IFNA?

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD)
Toll-Like Receptor and NFB Pathways [Tuggle]

www.biocarta.com

Swine Salmonella infections Comparative gene expression response studies

Salmonella enterica serovar Typhimurium [S. Typhimurium, ST] pig and human pathogen - broad host range clinical disease - usually enterocolitis food safety issue Salmonella enterica serovar Choleraesuis [S. Choleraesuis, SC] pig pathogen - narrow host range clinical disease - septicemia, enterocolitis, pneumonia and/or hepatitis References: Zhao et al, Mamm Genome. 17: 777, 2006 Uthe et al, Vet Micro 114: 60, 2006; Molec Immunol 44: 2900, 2007 Wang et al, Genomics In Press, 2007 (ST); In preparation (SC). Tuggle et al, next talk

Swine Gene Arrays

swine long oligo microarrays
NRSP8-Qiagen 12,500 probes, 2003 design

Zhao et al. Genomics. 86: 618, 2005; Mamm Genome 17: 777, 2006 updated NRSP8-Illumina 20,000 probes, 2006 design Testing underway

Affymetrix arrays
20,000 probes, 2004

Wang et al. Genomics. 2007 May 11 epub Arrays enable broad tissue specific gene expression analyses; data affirmed with real time PCR analyses and protein assays [when assay and relevant sample are available.]

Quantitative bacteriology of the ileocecal lymph nodes in swine inoculated with Salmonella
[Most probable number method used for Salmonella cfu; # statistically different at p<0.05.]

Salmonella immunity studies

Infection: Salmonella enterica serovar Choleraesuis (pig pathogen; clinical disease) Animals: 3 pigs/necropsy at 24, 48 hr pi Tissue: lung Arrays: NRSP8-Qiagen 12,357 probes Result: 57 genes showed differential expression
(p < 0.001; false discovery rate = 12%).
Zhao et al. Mammalian Genome. 17: 777, 2006

S . Choleraesuis

S . Typhim urium

Non-infected

A
1000000

cfu/g ICLN

100000 10000 1000 100 10 1 8h 24h

48h

7d

21d

time post-inoculation

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD)
Gene expression profiling in Salmonella serovar Choleraesuis infected lung using a long oligonucleotide Qiagen-NRSP-8 swine array

Real-time immune gene expression in S. Choleraesuis infected porcine lung

Function classification of increased genes at 48 hour infection with p values < 0.001

Use real time assays to target sets of genes involved in pathways identified with arrays and with anti-bacterial immune responses.

Zhao et al. 2006. Mammalian Genome. In Press.

Summary of immune gene expression in S. Choleraesuis infected porcine lung

qRT-PCR of 61 differentially expressed (DE) genes confirmed the microarray results. [23/33 DE genes confirmed] Two transglutaminase family genes (TGM1 and TGM3), associated with apoptosis, showed dramatic increases post inoculation; affirmed by qRT-PCR for other genes, indicating induction of apoptotic pathways Predominant T helper 1 (Th1)-type immune response, with interferon gamma (IFNG) significantly increased at 48 hpi along with genes induced by IFNs (GBP1, GBP2, C1S, C1R, MHC2TA, PSMB8, TAP1, TAP2) in porcine lung infection. Limited changes in innate and Th2 associated genes and general immune associated genes.
Zhao et al. Mammalian Genome. 17: 777, 2006
www.biocarta.com

Swine Salmonella infections comparative response studies

Infections: Salmonella enterica serovar Typhimurium (ST) S. Typhimurium; pig and human pathogen; clinical disease and food safety issue S. Choleraesuis (SC) pig pathogen; clinical disease Animals: 3 pigs/necropsy at 8, 24, 48 hr pi; 7 and 21 dpi Tissues collected: lung, mesenteric lymph node (MLN), liver, spleen Analyses: suppression subtractive hybridization (SSH)
http://users.path.ox.ac.uk/~ciu/FionaPowrieGroup1.htm

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD)
Differentially expressed genes identified by SSH in MLN from swine experimentally inoculated with S. Choleraesuis
Uthe et al. Vet Micro 114: 60, 2006

MLN Differential gene expression in response to S. Choleraesuis and S. Typhimurium

Suppression subtractive hybridization (SSH) identified 7 genes as up-regulated in MLN at 24 hpi in S. Choleraesuisinfected pigs (ARPC2, CCT7, HSPH1, LCP1, PTMA, SDCBP, VCP) 3 genes in S. Typhimurium-infected pigs (CD47/IAP, CXCL10, SCARB2) Experimental design: These genes were then targeted for comparative analyses of gene expression on MLN RNA collected at 8, 24, 48 hr pi; and at 7 and 21 dpi
Uthe et al., Molec. Immunol. 44: 2900, 2007

ARPC2
Fold change in
Fold change in

SDCBP
8 expression 4
#

8 expression 4 2 1 0 .5 8h 24h 48h 7d 21d * * * * * *

Fold change in

Fold change in

expression

expression

Differential expression of the SSH-enriched genes from the Salmonellainfected swine using realtime PCR.
The results are expressed as the fold change in gene expression in the S. Choleraesuis- (solid) or S. Typhimurium- (open) infected pigs compared to the non-infected controls.
Statistical differences (* = P<0.05) between control and infected pigs are represented by an asterisk (*) and between the S. Choleraesuis- and S. Typhimurium-infected pigs are represented by a number sign (#). Uthe et al., Molec. Immunol. 44: 2900, 2007

2 1 0 .5

* *

* *

Differential gene expression (SSH) in response to S. Choleraesuis and S. Typhimurium SSH enriched genes were up-regulated in both infections. SSH enriched genes from S. Choleraesuis-infected pigs that were up-regulated in both infections, except for HSPH1 which was differentially activated early in the S. Choleraesuis infection . SSH enriched genes from S. Typhimurium-infected pigs showed significant differences in transcriptional induction early in the infection (824 h) and were differentially expressed in the S. Typhimurium-infected pigs
Uthe et al., Molec. Immunol. 44: 2900, 2007

8h

24h

48h

7d

21d

C CT7
8 4 2 1 0 .5 8h 24h 48h 7d 21d * * * * * * *
8 4 2 1 0 .5 8h 24h
#

VCP

* *

* *

* *

48h

7d

21d

HSPH1
Fold change in

C D 4 7 /IA P
Fold change in 8 expression 4 2 1 0 .5 8h 24h 48h 7d
# #

ST
* * *

16 expression 8 4 2 1 0 .5 8h 24h *
#

*#
#

* *

* *

48h

7d

21d

21d

LC P1
Fold change in
Fold change in

C XCL10
64 32 16 8 4 2 1 0 .5 expression *
# # #

ST

8 expression 4 2 1 0 .5 8h 24h 48h 7d 21d

#

* *

* *

* *

*#

8h

24h

48h

7d

21d

PTM A
Fold change in
Fold change in

SC AR B2
16 expression 8 4 2 1 0 .5 8h 24h 48h 7d
# #

ST
#

8 expression 4 2 1 0 .5 8h 24h 48h 7d 21d * * * * * * * *

* *

* *

21d

time post-inoculation

N o n - in f e c te d

S . C h o le r a e s u i s

S . T y p h im u r iu m

Swine Salmonella infections comparative responses Serum Cytokines

2000

IFNG pg/ml

1750 1250 1000 750 500 250 0 8h

500

Effect of Salmonella infection on the expression of swine immune response genes

* *
# 48h

* *
24h

* *

* (S C)

7d

21d

TNF pg/ml

400 300 200 100 0 8h 24h 48h 7d 21d

* ** *

*
#

1000000

cfu/g ICLN

Bacteria in ICLN

100000 10000 1000 100 10 1

Uthe et al., Molec. Immunol. 44: 2900, 2007

8h

24h

48h

7d

21d

time post-inoculation

Uthe et al., Molec. Immunol. 44: 2900, 2007

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD)

Conclusions: Salmonella immunity studies

For both pathogens, immune response initiated within 24 hr; intensity, nature and persistence differed between pathogens. S. Choleraesuis infection stimulated classic innate and inflammatory response lasting to 7, even 21, dpi S. Typhimurium infection stimulated a mild and transient immune response, potentially evading (suppressing?) pigs immune system. This may aid progression into persistent infection, and carrier state. Immune gene expression correlates well with the clinical signs (fever) in infected animals, indicating the expression profiles will reveal pathways important to disease resistance and prevention Gene expression kinetics may reveal genes responsible for the variation in disease progression observed in swine infected with S. Typhimurium compared to S. Choleraesuis.
Uthe et al., Molec. Immunol. 44: 2900, 2007

Salmonella response studies

Compare early response [0, 8, 24, 48 hpi] to both pathogens, S. Typhimurium (ST) and S. Choleraesuis (SC) infections Affymetrix swine array [23,256 transcripts from 20,201 genes] 16,229 and 16,046 probe sets (~70 %) showed MLN expression during the ST infection and the SC infection, respectively 848 and 1,949 genes showed differential expression across different times after ST and SC inoculation or when compared to non-inoculated controls.
Wang, Qu, et al Genomics 2007 epub; in preparation

Gene Ontology (GO) categorization of porcine ST infected MLN transcriptome Transcriptional profile of genes selected as cell-type markers or immune response pathway genes shows no evidence that transcriptional changes are due to cell migration into ST infected MLN.
The fold change were calculated using Genecluster. Statistical differences (P<0.05) between control and infected pigs (*). Expression patterns for specific marker genes for T cell, macrophage, granulocyte and dendritic cells Genes in pathways that respond to ST infection

Wang, Qu, et al Genomics 2007

All probe sets from analysis of infected animals showing a present call for all three replicates for at least one time point post-inoculation) and 848 differentially expressed genes (p<0.01, fc>2, q<0.24) by using ISU specific GO slim. Statistical significance of p<0.05 or p<0.01 are denoted with an asterisk (* or **).

Wang, Qu, et al Genomics 2007

Effect of Salmonella infection on the expression of swine chemokines

S. Typhimurium infection stimulated an earlier and milder chemokine response S. Choleraesuis infection stimulated a later and stronger chemokine response Does this limited chemokine response facilitate the persistence of S. Typhimurium infection and development of the carrier state? Note: differences from IPEC J2 cell line studies
Wang, et al unpublished http://users.path.ox.ac.uk/~ciu/FionaPowrieGroup1.htm

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD)

Early Take Home Lessons from Gene Expression Studies

Developed improved understanding of immune pathways (T helper 1, apoptosis, NFB) regulating anti-Salmonella responses Verified complexity of host specific factors Identified potential genes which encode genomic controls Highlighted target responses and pathways for new vaccination and drug treatment strategies Test use of cell lines (IPEC J2) for in vitro studies Pathogen specific patterns [dependence on pathogen isolate, local tissue, cellular interactions, time after infection] could be enhanced with microbial arrays
Combine 24h/0, 48h/0, 24h/8h, 48h/24h up regulated genes in SC.

Salmonella-cell culture invasion assay using IPEC-J2 cells Question: Can selected cell cultures reveal bacterial infection effects? Model: Porcine intestinal epithelial cells (IPEC-J2) grown in transwell cultures are enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. Proven to be a relevant in vitro model system for porcine intestinal pathogen-host cell interactions; can be infected with Salmonella
(Schierack et al. Histochem Cell Biol. 125: 293, 2006.)

Samples collected: Cells for RNA by adding Trizol, freezing at 80C and shipping to BARC for gene expression assays Supernatant from the Transwell insert and bottom of the well for protein expression assays. [not yet tested] Salmonella invasion analysis: Triton x-100 to Transwell; plate lysate dilutions

Salmonella-cell culture invasion assay using IPEC-J2 cells

I. Preparation of polarized IPEC-J2 cells: Polarize IPEC-J2 cells in 6 well plates-permeable supports (Transwell polyester membranes with 0.4 m pore size). Seed IPEC-J2 cells at density 3.5-2.5x105 cells/well. Polarize cells for 7-9 days. II. Preparation of bacterial inoculum: Start bacterial cultures from a single colony. Grow overnight. III. IPEC-J2 invasion-gene expression experiment: Prewash IPEC-J2 cells; Add inoculum at MOI=300; control medium only. At 2 hr after inoculation, add Gentamicin bacteria killing media Incubate cells at 37C, 5% CO2 for 2 hr, 4 hr and 8 hr

Effect of S. Choleraesuis (SC) and S. Typhimurium (ST) infection on gene expression in monolayer cultures of porcine IPEC J2 epithelial cells
In vivo earlier, but limited, immune response to S. Typhimurium infection In vitro earlier, but higher, up regulation of chemokines, innate cytokines and TLRs with S. Typhimurium infection Similar kinetics of IL8 and CCL20, not TNF, RNA upregulation in response to SC and ST infections at lower MOI (Skjolaas Vet Im Immunopathol. 115:299, 2007, 108 ST/well). Broader array of chemokines (CCL2, CCL3) and apoptosis (TGM3) markers shown to be up-regulated. NFkB targets upregulated with ST infections. Note: gene not protein expression results. Protein expression planned; certain reagents not available.
Unpublished data

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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Genomics of interaction of Salmonella with porcine lymph nodes and enterocytes Joan Lunney (APDL, BARC, ARS, USDA, Beltsville, MD)

US Veterinary Immune Reagents Network

Plan to begin to systematically address the immunological reagent gap for the veterinary immunology research community Goal: express immune proteins (cytokines, chemokines, acute phase reactants) and produce specific monoclonal antibodies to immune markers [T cell receptors (TCRs), toll-like receptors (TLRs), cell surface differentiation (CD) markers] Develop panel of markers for immune proteomic studies Coordination + mAb Production: Baldwin, Univ. MA
Protein Expression: LaBresh, Kingfisher Biotech. Ig and TCR Expression: Wagner, Cornell Univ. Species: Ruminants, concentrating on cattle - Baldwin, Univ. MA Swine - Lunney, ARS BARC Poultry, primarily chickens Lillehoj, ARS BARC Horses Horohov, Univ. KY; Wagner, Cornell Univ. Aquaculture species: channel catfish Miller, MSU; trout Hansen, USGS

Swine Toolkit
Priorities
TCR, reagents TLRs chemokines CD45RO Cytokines, e.g., IFNA more sensitive reagents IgGs note separate NPB grant with J Butler, U Iowa

Goals: Produce large scale quantities of bioactive protein needed with accurate measure of bioactivity; Provide full length cDNAs Produce mAb useful for cytokine and chemokine protein quantitation using ELISAs and ELISpot assays as well as alternate formats, e.g., Luminex, intracellular and fixed tissue staining Repository What do you need? Send me suggestions email: jlunney@anri.barc.usda.gov

USDA CSREES Toolkit Network grant 2006-2010

Colleagues

Thanks!
Nishi
Nishi Samon Royaee Lunney Kuhar BARC

Lunney Dawson

email: jlunney@anri.barc.usda.gov soon to be: joan.lunney@ars.usda.gov websites: www.anri.barc.usda.gov/pbel/sy_lunney.asp

Wang Tuggle Iowa State Univ. Couture Uthe Bearson NADC

www.ars.usda.gov/pandp/people/people.htm?personid=3471

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NL This presentation is the property of Joan Lunney of the USDA. This presentation should not be reproduced without the author's permission.

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