HOST GENE EXPRESSION RESPONSE TO SPECIFIC BRD PATHOGENS

Transcription

1 HOST GENE EXPRESSION RESPONSE TO SPECIFIC BRD PATHOGENS

2 Bovine Respiratory Disease Complex Major cause or morbidity and mortality in beef cattle and dairy calves Multiple pathogens interact with stress to cause pneumonia Quiescent bacteria in URT move to LRT when virus infection occurs

3 Dehorning Weaning Shipping Castration Mannheimnia hemolytica Pasteurella multocida Mycoplasma bovis Histophilus somni Bovine Herpes Virus -2 (IBR) BRSV BVDV

4 Pathogens of the BRDC Bovine Herpes Virus/ Infectious Bovine Rhinotracheitis (IBR) Bovine Respiratory Syncytial Virus (BRSV) syncytial cells Bovine Virus Diarrhea Virus Mannheimnia hemoytica Pasteurella multocida Mycoplasma bovis Histophilus somni

5 Hypothesis and Rationale Each pathogen triggers a unique immune response in the host. Determining which genes were transcribed, by RNA sequencing, would provide an expression pattern of genes that could then be used to predict which molecular pathways were activated to combat that particular pathogen. This information has direct application to new therapeutic options i.e. potential drug effectiveness.

6 Aims of the study: To create single pathogen infections with each of six pathogens of the BRDC for the purpose of determining differential gene expression for each host-pathogen interaction. Characterization of clinical signs, lung pathology and pathogen isolation Collection of tissues at necropsy for RNA sequencing and transcript analysis Comparison of data between challenged steers and control steers for identification of specific immune pathways activated in each infection.

7 The Experiments: Six steers infected with each of the six BRD pathogens (separately), necropsied at the time of maximal clinical signs. Four control steers were sham infected 6 to 8 month old steers, Angus-Hereford cross Antibody negative for the viral pathogens Virus infection by aerosol with face mask Bacterial infections by endotracheal tube Bacterial cultures taken at intervals Daily clinical signs tabulated by DVM s & vet students

8 Experimental Challenge with BRD Agents Infection with viral pathogens Infection with bacterial pathogens

9 Scoring of Clinical Signs

10 Clinical Sign Scores

11 BRSV IBR IBR Larynx BVDV

12 BVDV Immunostaining and histology BVDV ileum showing Peyer s Patches with lymphoid hypoplasia Immunoperoxidase staining for BVDV in ileum of infected steer brown stain indicates presence of virus

13 Mannheimnia haemolytica Mannheimnia haemolytica Pasteurella multocida Mycoplasma bovis

14 Procedure for gene expression studies

15 MDS Plot of expressed genes to evaluate clustering of biological replicates Clustering of genes from control, bacterially and virally challenged steers

16 Numbers of Up- and Down-Regulated Differentially Expressed Genes and Isoforms for each Challenge Group in Contrast to Controls.

17 BRSV Most Significant Pathways (IPA) Oxidative phosphorylation (p<8.97x10-22 ) Mitochondrial dysfunction (p<4.15x10-20 ) Protein ubiquitation (p<3.06x10-11 ) Glucocorticoid receptor signaling (p<2.33x10-8 ) Upstream Regulators Activated LPS TNF TGFβ1 β-estradiol Needed for activated T cells to kill virus infected cells, B cell activation for antibody formation

18 BVDV Most significant pathways (IPA) Hepatic fibrosis, hepatic stellate cell activation (p<5.41x10-17 ) Acute phase response signaling (p<5.83x10-11 ) IGF1 signaling (p<5.83x10-11 ) Upstream regulators activated PPBP (TGB1) TNF IRF6 (LPS) TP53 Dexamethasone Leukocyte extravasation signaling (p<4.47x10-9 ) Liver cells make acute phase proteins, which facilitate inflammatory responses

19 IBR Virus Most significant pathways (IPA) B cell receptor signaling (p<3.81x10-9 ) Acute phase response signaling (p<4.65x10-9 ) PI3k signaling in lymphocytes (p<1.7x10-8 ) Upstream regulators activated Same as BVDV B cell receptor signaling is important for formation of an antibody response

20 Mannheimnia haemolytica Most significant pathways Granulocyte adhesion and diapedesis (p<1.65x10-17 ) Cell adhesion molecules Chemokine signaling Upstream regulators activated TGFβ1 activated IL-4 IFNΥ- inhibited Neutrophils must adhere to endothelial cells lining blood vessels to accomplish diapedesis, move between cells, and follow a chemotactic gradient towards the offending bacteria. These 3 features are highly up-regulated to facilitate the needed inflammatory response.

21 Mycoplasma bovis & Pasteurella multocida Most significant pathways Similar to Mannheimnia haemolytica Upstream regulators activated IL1β(M. b) TGF1β(P. m) Dexamethasone (P. m)

22 RNA sequence data showed: Both common and pathogen specific differences in the bronchial lymph node transcriptome of viruses and bacteria that are causes of BRDC. Differential expression of genes in common to all pathogens were primarily related to innate immune responses. Pattern recognition receptors (Toll like receptors, C-type lectins, etc.), MAPK activation, type 1 interferons. TLR1and 6 for M. haemolytica and TLR 2 and 4 for IBR virus Cytokine and chemokine pathways were up-regulated in all challenged animals Viral versus bacterial pathogens showed different gene usage: IL- 12 and IFNγfor viruses and IL-4 and IL-17 for bacteria.

23 So what does this mean? We now know which immune pathways are preferentially used in response to these pathogens. We identified molecular pathways are used in response to each pathogen; viral pathogens differ from bacterial pathogens. T and B cell signaling pathways were up-regulated in viral infections. These are adaptive immune responses required for effective immune responses to viral infection.

24 Cells of innate & acquired immunity

25 Toll like receptors and ligands

26 Gene usage in TLR pathways Both common and pathogen-specific differences were found in the bronchial lymph node transcriptome Differentially expressed (DE) genes in common to all pathogens were up-regulated and were mainly related to the innate immune response. Common pathways across pathogens, but different DE genes were used in pathway activation, e.g. for M. hemolytica TLR1 & TLR6 but TLR2 & TLR4 for IBR

27 Fig 6. Differentially expressed genes enriched within the toll-like receptor pathway. IBR challenge Mannheimnia haemolytica challenge Pathways are Similar but gene usage is different between the bacterial and viral pathogens Tizioto PC, Kim J, Seabury CM, Schnabel RD, Gershwin LJ, et al. (2015) Immunological Response to Single Pathogen Challenge with Agents of the Bovine Respiratory Disease Complex: An RNA-Sequence Analysis of the Bronchial Lymph Node Transcriptome. PLoS ONE 10(6): e doi: /journal.pone

28 Application 200 most variable of the differential expressed (DE) genes were able to classify each replicate steer into its challenge group. This data will be used for prediction of disease immunopathogenesis by each pathogen and from there to determine the potential effects of genetic manipulation on disease outcome. 142 of the DE genes were located in previously identified loci (GWAS study) associated with risk of BRD. Key upstream regulators were identified; these may be targets for molecular therapies and studies on modulation of the bovine immune system.

29 Acknowledgements Mark Anderson DVM PhD Dipl. ACVP (UCD & CAHFS) Rachel Toaff-Rosenstein DVM, (graduate student, UCD Animal Science) Heather McEligot MS (UCD PMI) The late Matt Shao MD PhD (UCD PMI) Student helpers CAP Research Team led by James Womack PhD (Texas A&M) Alison van Eenennaam PhD (UCD) Jeremy Taylor PhD (Univ. of Missouri, animal science) USDA/AFRI #