1. Genetic variation and regulation of TLR4 in relation to bovine mastitis.
Filiz T. Korkmaza,b & David E. Kerra,b
a Cellular, Molecular & Biomedical Sciences Program University of Vermont, Burlington VT
b Department of Veterinary & Animal Science University of Vermont, Burlington VT
Introduction
Methods
Results
On Irish dairy farms, it has been estimated that an increase in average milk SCC, typically as a result of mastitis, decreases farm profitability by €19,504 from €31,252/yr at <100,000 cells/ml to €11,748/yr at >400,000 cells/ml.1
Severity of experimental mastitis is highly variable between animals.2
In contrast to Gram positive infections, the severity of mastitis caused by coliform species of bacteria is heavily dependent on the intensity of the host inflammatory response.3
Overall
Animals: Adult multiparous cows (n=60) were randomly selected from a commercial dairy farm and ear notched in early lactation (118 ± 3 & 78 ± 10 DIM)
Cell Isolation: Primary dermal fibroblasts (DFs) from ear notches were isolated by collagenase digestion and cryo-preserved.
LPS Treatment: DFs were revived & treated with 100 ng/ml LPS (E. coli O11:B4) or media only for 24 hours.
Protein production: Protein was measured by IL-8 (Mabtech) & IL-6 (R&D) ELISAs in LPS challenge & control media.
Gene expression: Expression was measured by RT-qPCR with β-actin as housekeeping gene control.
Experiment One
Animal Selection: RT-qPCR analysis of a 190 bp region in the 3rd exon of TLR4 was performed on tissue RNA to identify PCR(-) and PCR(high) animals (n=6 per group).
Mastitis Challenge: Selected animals were challenged with 200 cfu of E. coli (P4) in their right hind quarter.
RNA-seq: Single-end, 100 bp mRNA sequencing was performed on unstimulated DFs from 5 PCR(-) and 5 PCR(high) cultures using the Illumina HiSeq 2000.
Experiment Two
Animal Selection: Animals were ranked using a multi- variable approach based on DF expression of TLR4 & LPS- induced production of IL-8 & IL-6 protein.
Statistics
Protein & expression differences were measured by unpaired Mann-Whitney U test.
Milk bacteria counts were measured using a linear mixed model assuming a first-order auto-regressive covariance structure. Time post infusion was used as the repeated measure.
RNA-sequencing
Raw reads were filtered on a median quality score >20. Reads with greater than 3 uncalled bases or with a length <25 bp post trimming were removed.
Alignment to the UMD 3.1.1/bosTau8 genome was performed with the NextGENe software package v Mapping to the reference of >85% was required for successful alignment.
Raw read counts were filtered using R statistical software (edgeR) for >1 CPM in at least 50% of samples. A generalized linear model likelihood ratio test was then performed using limma and multiple comparisons adjustment using the Benjamini-Hochberg method.
Experiment One: * *
Figure 6: Cellular IL-6 vs. IL-8 production in response to 24 h LPS treatment in a cohort of early lactation cows. Responses are ranked by cow from low to high IL-6 (blue) values and graphed against the same animal’s IL-8 (red) values. Values are normalized to both cell number and LPS challenge batch mean & standard deviation [(fg/cell – batch mean)/batch std dev.]
Figure 2: Cellular Response to 24 h LPS and TLR4 Expression in PCR(-) vs. PCR (high) animals. IL-6 (pg/ml) was measured in response to 24 h stimulation with LPS. Unstimulated cells were used to measured TLR4 expression. Averages values (± SEM) are shown for pg/ml & change in cycles to threshold (dCT) in comparison to β-actin and *=P<0.05.
IL-6
IL-8
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Caption for your photo ✝
Figure 7: Cellular Response to LPS and TLR4 Expression in High Vs. Low Responding animals. Presented are average protein responses and gene expression values from 8 high and 8 low responding animals. Values are presented as normalized fg/cell (based on batch mean and deviation) and change in cycles to threshold (dCT) in comparison to β-actin ± SEM and ***=P<0.001.
Conclusions
Figure 3: In vivo response to E. coli in PCR(-) vs PCR(high) animals. Bacterial counts (CFU/ml) were measured at 12 or 24 h increments for 10 days post infection. Values are presented in average CFU/ml (log10) and ± SEM and ✝=P<0.10.
Genetic differences exist in exon 3 of TLR4 that result in minor differences in TLR4 expression and a differential IL-6 response to LPS.
In experiment one, animals with higher TLR4 expression and IL-6 production tended to have less severe E. coli mastitis in response to experimental infection as measured by lower bacterial burden and greater milk production (data not shown).
RNA-seq analysis revealed several immune response genes that differed between PCR(-) and PCR(high) animals, most notably CD36, SAA3, FOS and M-SAA3.2.
In experiment two, animals have been selected on high and low cellular TLR4 expression and LPS-induced IL-8 and IL-6 production. Selected animals have an ~10-fold difference in TLR4 expression and significant differences in LPS-induced IL-8 and IL-6 protein production.
Figure 4: RNA-seq analysis in PCR(-) vs PCR(high) DFs. Unstimulated cells were used to measure baseline differences in RNA expression. Significantly different genes are listed by gene name, fold change and FDR values. Negative fold change indicates higher expression in PCR(-) cells.
Gene Name
Fold Change
FDR
CD36
-3.1
0.030
EGR1
1.5
ERAP2
2.0
M-SAA3.2
3.4
FOS
1.9
0.031
MXRA5
LOC512486
1.8
0.042
PLA2G3
2.6
0.064
SAA3
2.9
0.076
LOC509808
1.6
0.097
Figure 1: Inflammatory Response to E. coli in Mammary Gland Alveoli. Bacteria that gain access to the mammary gland activate epithelial cells through LPS/TLR4 signaling leading to PMN influx & increased milk SCC and inflammation. [photo courtesy of the Hebrew University of Jerusalem, 2009]
Objectives
Experiment Two:
What causes between-animal variation in host immune response to E. coli?
Objective 1: Identify genes that are expressed in high versus low responding animals  increased susceptibility to severe mastitis.
Objective 2: How are susceptibility genes regulated? Identify genetic and/or epigenetic mechanisms of regulation.
References & Funding
Geary U, et al. Estimating the effect of mastitis on the profitability of Irish dairy farms. Journal of dairy science 2012, 95(7):
Burvenich, C., et al. Severity of E. coli mastitis is mainly determined by cow factors. Vet Res 2003, 34(5):
Petzl W, et al. Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge. Innate Immun 2016, 22(4):
Funding for this project was provided by the USDA-AFRI-NIFA pre-doctoral fellowships grant program, grant number VT-0076CG.
This project was awarded a graduate student travel bursary provided by the International Society of Animal Genetics to present at ISAG 2017.
Figure 5: Cellular TLR4 Expression in a cohort of early lactation cows. Unstimulated cells were used to measure the range of TLR4 expression in 55 cows. Values are expressed as change in cycles to threshold (dCT) in comparison to β-actin.