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Genomics of interaction ofSalmonella withporcine lymph nodes and enterocytes

Joan LunneyAPDL, BARC, ARS, USDA, Beltsville, MD

Thursday 7th June 2007

E. coli and Salmonella workshopUtrecht, The Netherlands

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. of Animal Science, Iowa State University, Ames, IA USA2National Animal Disease Center, USDA-ARS, Ames, IA USA3Animal Parasitic Diseases Lab, USDA-ARS, Beltsville, MD USA4Lab of Molecular Biology and Animal Breeding, Huazhong

Agricultural Univ., Wuhan, PR China5Dept. of Statistics, Iowa State University, Ames, IA

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 ofSalmonella 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 hostprotective 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/FeaturedA rticle/Default.asp?Display=1284

InnateImmunity

Differentiation Swine Immune GeneExpression: 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?

?

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

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NLThis presentation is the property of Joan Lunney of the USDA.

This presentation should not be reproduced without the author's permission. 1/7

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www.biocarta.com

Toll-Like Receptor and NFB Pathways [Tuggle]

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/orhepatitis

References:

Zhao et al, Mamm Genome. 17: 777, 2006

Uthe et al, Vet Micro 114: 60, 2006; Molec Immunol 44: 2900, 2007Wang et al, Genomics In Press, 2007 (ST); In preparation (SC).

Tuggle et al, next talk

Swine Salmonella infectionsComparative gene expression response studies 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 proteinassays [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 Salmonellacfu;

# statistically different at p

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

Gene expression profiling in Salmonella serovar Choleraesuis

infected lung using a long oligonucleotide Qiagen-NRSP-8 swine array

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

Real-time

immune gene

expression in

S. Choleraesuis

infected porcine

lung

Use real time assays to

target sets of genes

involved in pathways

identified with arrays

and with anti-bacterial

immune responses.

Summary of immune gene expression in

S. Choleraesuis infected porcine lung

qRT-PCR of 61 differentially expressed (DE) genes confirmedthe microarray results. [23/33 DE genes confirmed]

Two transglutaminase family genes (TGM1 and TGM3),associated with apoptosis, showed dramatic increases postinoculation; affirmed by qRT-PCR for other genes, indicatinginduction of apoptotic pathways

Predominant T helper 1 (Th1)-type immune response, withinterferon gamma (IFNG) significantly increased at 48 hpialong 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 andgeneral immune associated genes.

Zhao et al. Mammalian Genome. 17: 777, 2006www.biocarta.com

http://users.path.ox.ac.uk/~ciu/FionaPowrieGroup1.htm

Infections:

Salmonella enterica serovar Typhimurium (ST)

S. Typhimurium; pig and human pathogen; clinical

disease and food safety issueS. 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)

Swine Salmonella infections comparative response studies

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

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NLThis presentation is the property of Joan Lunney of the USDA.

This presentation should not be reproduced without the author's permission. 3/7

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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 responseto S. Choleraesuis and S. Typhimurium

Suppression subtractive hybridization (SSH) identified

7 genes as up-regulated in MLN at 24 hpi in S. Choleraesuis-infected 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 forcomparative analyses of gene expression on MLN RNAcollected at 8, 24, 48 hr pi; and at 7 and 21 dpi

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

A R P C 2

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

** ** *

*

P T M A

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

* **

*****

C C T 7

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

*

**

**

* *

H S P H 1

8h 24h 48h 7d 21d0.5

1

2

4

8

16

Foldchangein

expression

*

*

* * * *

*##

#

L C P 1

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

**

*** *#

S D C B P

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

* **

**

*#

#

#

S C A R B 2

8h 24h 48h 7d 21d0.5

1

2

4

816

Foldchangein

expression

***** *

*#

#

#

VC P

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

* *** ***

#

C D 4 7 / IA P

8h 24h 48h 7d 21d0.5

1

2

4

8

Foldchangein

expression

* * * *

*

**

# #

C X C L 1 0

8h 24h 48h 7d 21d0.5

1248

163264

Foldchangein

expression

* *

**

*

*

*#

#

#

#

Differential expression of

the SSH-enriched genes

from the Salmonella-

infected swine using real-

time 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

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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 andinflammatory response lasting to 7, even 21, dpi

S. Typhimurium infection stimulated a mild and transient immune

response, potentially evading (suppressing?) pigs immunesystem. This may aid progression into persistent infection, andcarrier state.

Immune gene expression correlates well with the clinical signs(fever) in infected animals, indicating the expression profiles willreveal pathways important to disease resistance and prevention

Gene expression kinetics may reveal genes responsible for thevariation in disease progression observed in swine infected withS. 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

All probe sets from analysis of infected animals showing a present call for allthree replicates for at least one time point post-inoculation) and 848 differentiallyexpressed genes (p2, q

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Combine 24h/0, 48h/0, 24h/8h, 48h/24h up regulated genes in SC.

Early Take Home Lessons from

Gene Expression Studies

Developed improved understanding of immunepathways (T helper 1, apoptosis, NFB) regulatinganti-Salmonella responses

Verified complexity of host specific factors Identified potential genes which encode genomiccontrols

Highlighted target responses and pathways for newvaccination and drug treatment strategies

Test use of cell lines (IPEC J2) for in vitro studies

Pathogen specific patterns [dependence on pathogenisolate, local tissue, cellular interactions, time afterinfection] could be enhanced with microbial arrays

Salmonella-cell culture invasion assay using IPEC-J2 cells

Question: Can selected cell cultures reveal bacterial infectioneffects?

Model: Porcine intestinal epithelial cells (IPEC-J2) grown intranswell cultures are enterocyte-like with microvilli, tight

junctions and glycocalyx-bound mucin. Proven to be a relevantin vitro model system for porcine intestinal pathogen-host cellinteractions; 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; controlmedium 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 ofS. 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 up-regulation 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 expressionplanned; certain reagents not available.

Unpublished data

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

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NLThis presentation is the property of Joan Lunney of the USDA.

This presentation should not be reproduced without the author's permission. 6/7

8/3/2019 09 Salmonella Joan Lunney

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US Veterinary Immune Reagents Network

Plan to begin to systematically address the immunological reagentgap for the veterinary immunology research community

Goal: express immune proteins (cytokines, chemokines, acutephase reactants) and produce specific monoclonal antibodies toimmune markers [T cell receptors (TCRs), toll-like receptors

(TLRs), cell surface differentiation (CD) markers]Develop panel of markers for immune proteomicstudies

Coordination + mAb Production: Baldwin, Univ. MAProtein 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

USDA CSREES Toolkit Network grant 2006-2010

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 accuratemeasure 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

Colleagues

Nishi Samon Royaee Lunney Kuhar

BARC

Nishi Lunney Dawson

Wang Tuggle Couture

Iowa State Univ.Uthe BearsonBearson

NADC

email:jlunney@anri.barc.usda.gov

soon to be:joan.lunney@ars.usda.gov

websites: www.anri.barc.usda.gov/pbel/sy_lunney.aspwww.ars.usda.gov/pandp/people/people.htm?personid=3471

Thanks!

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

EADGENE E. coli and Salmonellla Workshop, 7 June 2007, Utrecht, NLThis presentation is the property of Joan Lunney of the USDA.

This presentation should not be reproduced without the author's permission. 7/7