Genome-wide analysis of TNF-alpha response in pigs challenged with porcine circovirus 2b

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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Papers and Publications in Animal Science Animal Science Department 4-2015 Genome-wide analysis of TNF-alpha

1 University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Papers and Publications in Animal Science Animal Science Department Genome-wide analysis of TNF-alpha response in pigs challenged with porcine circovirus 2b C. A. Kreikemeier University of Nebraska-Lincoln T. B. University of Nebraska-Lincoln K. L. Lucot University of Nebraska-Lincoln Thomas E. Burkey University of Nebraska-Lincoln, tburkey2@unl.edu Daniel C. Ciobanu University of Nebraska-Lincoln, dciobanu2@unl.edu Follow this and additional works at: Part of the Genetics and Genomics Commons, and the Meat Science Commons Kreikemeier, C. A.; T. B.; Lucot, K. L.; Burkey, Thomas E.; and Ciobanu, Daniel C., "Genome-wide analysis of TNF-alpha response in pigs challenged with porcine circovirus 2b" (2015). Faculty Papers and Publications in Animal Science This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.

2 Published in Animal Genetics 46:2 (April 2015), pp doi: /age Copyright 2015 Stichting International Foundation for Animal Genetics; published by John Wiley & Sons. Used by permission. Accepted for publication 14 November digitalcommons.unl.edu Genome-wide analysis of TNF-alpha response in pigs challenged with porcine circovirus 2b C. A. Kreikemeier, T. B. Engle, K. L. Lucot, S. D. Kachman, T. E. Burkey and D. C. Ciobanu Animal Science Department, University of Nebraska Lincoln, Lincoln, NE 68583, USA. Corresponding author D. C. Ciobanu, Animal Science Department, University of Nebraska Lincoln, Lincoln, NE , USA; Abstract Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine with a role in activating adaptive immunity to viral infections. By inhibiting the capacity of plasmacytoid dendritic cells to produce interferon-α and TNF-α, porcine circovirus 2 (PCV2) limits the maturation of myeloid dendritic cells and impairs their ability to recognize viral and bacterial antigens. Previously, we reported QTL for viremia and immune response in PCV2- infected pigs. In this study, we analyzed phenotypic and genetic relationships between TNFα protein levels, a potential indicator of predisposition to PCV2 co-infection, and PCV2 susceptibility. Following experimental challenge with PCV2b, TNF-α reached the peak at 21 days post-infection (dpi), at which time a difference was observed between pigs that expressed extreme variation in viremia and growth (P < 0.10). A genome-wide association study (n = 297) revealed that genotypes of SNPs explained 73.9% of the variation in TNF-α at 21 dpi. Major SNPs were identified on SSC8, SSC10 and SSC14. Haplotypes based on SNPs from a SSC8 (9 Mb) 1-Mb window were associated with variation in TNF-α (P < 0.02), IgG (P = 0.05) and IgM (P < 0.13) levels at 21 dpi. Potential overlap of regulatory mechanisms was supported by the correlations between genomic prediction values of TNF-α and PCV2 antibodies (21 dpi, r > 0.22), viremia (14 21 dpi, P > 0.29) and viral load (r = 0.31, P < ). Characterization of the QTL regions uncovered genes that could influence variation in TNF-α levels as well as T- and B-cell development, which can affect disease susceptibility. Keywords: immunity, porcine circovirus 2, swine, tumor necrosis factor alpha Porcine circovirus 2 (PCV2) is the primary cause of a group of associated diseases (PCVAD) that includes postweaning multisystemic wasting syndrome (PMWS), characterized by severe weight loss, jaundice, dyspnea, diarrhea, lymphadenopathy and interstitial pneumonia (Carman et al. 2008). In natural outbreaks, 99% of the PMWS cases are the result of PCV2 co-infection with other pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, influenza or parvovirus. PCV2 infection of cells involved in the innate defense impairs the recognition of viral and bacterial antigens and favors secondary infections (Vincent et al. 2005, 2007). Specifically, PCV2 reduces the capacity of plasmacytoid dendritic cells to induce interferon-α and tumor necrosis factor alpha (TNF-α), limiting the maturation of associated myeloid dendritic cells. TNF-α is an innate pro-inflammatory cytokine with a role in activating adaptive immunity to viral challenges (Trevejo et al. 2001). Variation in TNF-α following PCV2 infection could affect the ability of innate host defense to trigger the adaptive response, clear the pathogen and respond to secondary infections. We showed recently that highdensity SNP genotypes influence variation in viremia (40 61%) and immune response (10 60%) in a population of crossbred pigs (n = 384) experimentally infected with PCV2b (McKnite et al. 2014). One of the major QTL regions that influenced viremia was mapped on SSC7 from 29 to 34 Mb, overlapping the SLAII complex of genes and also close to the location of the TNF gene (27.6 Mb). Taking into account the potential role of TNF-α, directly or indirectly, in the activation of immune response following PCV2 infection, in this study we explored the genetic factors that modulate protein expression of TNF-α and estimate phenotypic and genetic relationships with PCV2 viremia and specific immune response. We hypothesized that TNF-α could represent a potential indicator of predisposition to co-infection in PCV2-infected pigs. 205

3 206 Kreikemeier et al. in Animal Genetics 46 (2015) Experimental infection with a PCV2b strain was initiated at 43 days of age. The pigs were infected intranasally and intramuscularly with a titer of TCID50/ ml as described in McKnite et al. (2014); average daily gain (ADG), PCV2-specific antibodies (IgM and IgG) and viremia were measured at 0, 7, 14, 21 and 28 days postinfection (dpi). PCV2-specific antibodies were measured using ELISA (Ingenasa) and viremia was measured by real-time quantitative PCR (McKnite et al. 2014). Viral load was calculated as the area under the curve based on viremia profiled over time. High-density genotypes were obtained using the Porcine 60K SNP BeadArray (Illumina). The genome-wide association study (GWAS) included SNPs and was performed using a Bayes B model (Kizilkaya et al. 2010) with litter, pen and batch as class variables and passive IgG and age at infection as covariates. The protein serum level of TNF-α was quantified initially in pigs expressing high viral load and low ADG (n = 6) and low viral load and high ADG (n = 6) using ELISA (R&D Systems). A slight increase in TNF-α level was observed over time, reaching the peak at 21 dpi, where a difference was shown between the two groups (P < 0.1) (Fig. 1). At this time point, the rest of the resource population was profiled for TNF-α (n = 297). The increase in TNF-α level during viremic states may be characteristic of PCV2; previous studies showed a slight, non-significant increase in TNF-α level in swine influenza virus infections and inhibition of TNF-α expression to evade immune response in PRRSV infections (Murtaugh et al. 2002; Khatri et al. 2010). Chronic overexpression or inhibition of TNF-α has been shown to be involved in the pathogenesis of several diseases (Clancy et al. 2004; Mc- Cullough et al. 2009). Pairwise correlations between TNF-α at 21 dpi, viremia and antibody profile across time points were based on residuals obtained from a model that included batch as a fixed effect, litter and pen as random, and age at infection and level of passive IgG as covariates. TNF-α level was positively correlated with viral load (r = 0.25) and with viremia during the last 3 weeks of the challenge (r = ) (P < , Table 1). The relationship between TNFα (21 dpi) and PCV2-specific antibodies was detected following their surge, starting at 14 dpi for IgM (r = ) and at 21 dpi for IgG (r = 0.17) (P < 0.005). Small negative estimates were obtained between TNF-α and overall ADG (r = 0.15) and also with weekly ADG, with the largest estimate observed during the last week of challenge (r = 0.17) (P < 0.01). Table 1. Phenotypic and genomic correlations between TNF-α levels at 21 days post-infection (dpi) and indicator traits of PCVAD susceptibility following an experimental infection with PCV2b. Correlations with TNF-α (log 10) Traits Phenotypic Genomic Viral load 0.248** 0.319** Viremia 7 dpi Viremia 14 dpi 0.273** 0.315** Viremia 21 dpi 0.15* 0.276** Viremia 28 dpi 0.231** IgM 7 dpi IgM 14 dpi 0.199** IgM 21 dpi 0.284** 0.302** IgM 28 dpi 0.338** 0.433** IgG 7 dpi 0.19** 0.260** IgG 14 dpi 0.254** 0.198** IgG 21 dpi 0.19** 0.226** IgG 28 dpi 0.11* 0.225** ADG 0 to 28 days 0.157* 0.137* ADG week ADG week ADG week * ADG week * Significance of the correlation estimates: * P < 0.05 ; ** P < Figure 1. Means and standard errors of TNF-α levels in pigs expressing high viral load and low ADG (HL) and low viral load and high ADG (LH) following an experimental infection with PCV2b.

4 TNF-alpha response in pigs with porcine circovirus 2b 207 Figure 2. Genome-wide association study between 56,33 SNPs and TNF-α at 21 days post-infection (dpi) following an experimental infection with PCV2b. Each dot represents the proportion of genetic variance explained by an individual SNP. The x-axis represents the position of each SNP in the swine genome based on build 10.2 of the reference swine genome. The y-axis represents the contribution of each SNP to the genetic variance. Alternate colors represent autosomes, from SSC1 to 18, and chromosome X followed by a set of SNPs without a precise location. Combined genotypes of the SNPs explained 73.9% of the phenotypic variation in TNF-α. Although the proportion of the variation explained by SNPs is substantial, none of the mapped regions explained the majority of the effect. Regions characterized by clusters of SNPs associated with major effects were mapped on build 10.2 of the reference swine genome on SSC8 (9 Mb), SSC10 (73 Mb) and SSC14 (120 and 127 Mb) (Fig. 2). Functional characterization of the genes located in these regions uncovered potential candidates that modulate variation in TNF-α levels. Two transcription factors, KLF6 (SSC10, 73 Mb) and NKX3 (SSC8, 8.7 Mb), are involved in immune system development, with KLF6 specifically involved in B-cell development and differentiation ( Absence of a major association at the TNF locus (SSC7, 27 Mb) indicated a trans-modulation mechanism that influenced its variation in response to PCV2 infection. A 1-Mb window located at the proximal end of SSC8 (9 Mb) explained the largest additive genetic variation of TNF-α (1.27%), having a model frequency of 40%, although the variation explained by this window was not significantly greater than the average (P < 0.33) (Table S1). SNP ASGA (SSC8, 9.4 Mb), located in this window, was associated with the largest genetic variance. Genotype AA was associated with highest level of TNF-α (1.44 pg/ml) compared to AG (1.37 pg/ ml, P < 0.18) and GG genotypes (1.28 pg/ml, P < 0.05). Potential overlap of the genetic control or pathways that influence the variation of multiple traits in response to infection were evidenced by correlations between genomic prediction values of TNF-α and IgM (r > 0.30) and IgG (r > 0.22) from 21 dpi, viremia from 14 to 21 dpi (P > 0.27) and viral load (r = 0.32) (P < ) (Table 1). The 1-Mb window located on SSC8 (9 Mb) ranked in the top 2% in the GWAS with IgG at 21 dpi and with IgM at 21 and 28 dpi (McKnite et al. 2014). Haplotypes of the top four SNPs in the respective window on SSC8, including ASGA , MARC , MARC and MARC , were associated with variation in IgG (P = 0.05) and IgM (P < 0.15) at 21 dpi. Haplotype 4, the only haplotype that carries the G allele from ASGA and the A allele from MARC , was associated with the lowest substitution effect for IgG (P < 0.10), IgM (P < 0.25) and TNF-α (P < 0.02) at 21 dpi (Table S2). Haplotype 2, which varies from haplotype 4 across all four sites and from haplotypes 1 (MARC ) and 3 (MARC ) at a single site, was associated with the highest substitution effect and was significantly different from the average for both PCV2 antibodies at 21 dpi (P < 0.05). No significant relationships were detected between haplotypes and measures of viremia. One of the SNPs (ASGA , SSC9, 0.70 Mb) that captured the negative relationship between viral load and ADG, reported by McKnite et al. (2014), was also associated with variation in TNF-α (P < 0.05). The AA genotype was associated with lower levels of TNF-α (P < 0.05), lower viral load (P < 0.01) and higher ADG (P < 0.10) compared to the GG genotype. These results suggest that individuals with a genetic profile that expressed high viral load during challenge experienced reduced growth, high IgM and IgG response (McKnite et al. 2014) and high levels of TNF-α. Based on previous reports (Murtaugh et al. 2002), a lower or lack of TNF-α surge is not desired due to the ability of the certain pathogens (e.g., PRRSV) to trigger an effective immune response. Future functional characterization of genomic regions that influence variation in TNF-α could lead to better understanding of T- and B-cell development, immune response and the ability of the host to reduce viral replication and susceptibility to PCV2-associated diseases including secondary infections.

5 208 Kreikemeier et al. in Animal Genetics 46 (2015) Acknowledgments This work was supported by a Strategic Investment Program Grant from University of Nebraska-Lincoln and by Merial s Veterinary Summer Scholar Research Program. References Carman S., Cai H.Y., Delay J. et al. (2008) The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease Canadian Journal of Veterinary Research (Revue Canadienne de Recherche Veterinaire), 72, Clancy R.M., Backer C.B., Yin X., Chang M.W., Cohen S.R., Lee L.A. & Buyon J.P. (2004) Genetic association of cutaneous neonatal lupus with HLA class II and tumor necrosis factor alpha: implications for pathogenesis. Arthritis and Rheumatism, 50, Khatri M., Dwivedi V., Krakowka S., Manickam C., Ali A., Wang L., Qin Z., Renukaradhya G.J. & Lee C.W. (2010) Swine influenza H1N1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human H1N1 influenza virus. Journal of Virology, 84, Kizilkaya K., Fernando R. L. & Garrick D. J. (2010) Genomic prediction of simulated multibreed and purebred performance using observed fifty thousand single nucleotide polymorphism genotypes. Journal of Animal Science, 88, McCullough K.C., Ruggli N. & Summerfield A. (2009) Dendritic cells at the front-line of pathogen attack. Veterinary Immunology and Immunopathology, 128, McKnite A.M., Bundy J.W., Moural T.M. et al. (2014) Genomic analysis of the differential response to experimental infection with porcine circovirus 2b. Animal Genetics, 45, Murtaugh M.P., Xiao Z. & Zuckermann F. (2002) Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunology, 15, Trevejo J.M., Marino M.W., Philpott N., Josien R., Richards E.C., Elkon K.B. & Falck-Pedersen E. (2001) TNF-alpha-dependent maturation of local dendritic cells is critical for activating the adaptive immune response to virus infection. Proceedings of the National Academy of Sciences of the United States of America, 98, Vincent I.E., Carrasco C.P., Guzylack-Piriou L., Herrmann B., Mcneilly F., Allan G.M., Summerfield A. & Mccullough K.C. (2005) Subsetdependent modulation of dendritic cell activity by circovirus type 2. Immunology, 115, Vincent I.E., Balmelli C., Meehan B., Allan G., Summerfield A. & Mccullough K.C. (2007) Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA. Immunology, 120, I Supporting information Additional supporting information follows. Table S1. GWAS between 1 Mb genome windows and TNFα at 21 dpi following an experimental infection with PCV2b. (75 pages) Table S2. Haplotype substitution effects for TNF-α (21 dpi), PCV2-specific antibodies and viremia based on SNPs ASGA , MARC , MARC and MARC located on SSC8 (9 10 Mb). (1 page)

6 Table S1. GWAS between 1 Mb genome windows and TNF-α at 21 dpi following an experimental infection with PCV2b. Window start end #SNPs %Var Cum%Var p>0 p>average map_pos0 map_posn chr_mb var(ghat) corr regr _0 21_0 21_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

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Genome Analysis In Domestic Animals By H. Geldermann

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