Leukocyte gene expression of dairy cattle in response to flooring type and additional substance P

Authors: Eicher, Susan; Silva, Ediane; Marchant, Jeremy; SCHUTZ, MICHAEL - Purdue University

Submitted to: Society for Leukocyte Biology Meetings Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 9/4/2018
Publication Date: 10/14/2018
Citation: Eicher, S.D., Silva, E.B., Marchant-Forde, J.N., Schutz, M.M. 2018. Leukocyte gene expression of dairy cattle in response to flooring type and additional substance P. IN: Proceedings of the Society for Leukocyte Biology, October 14-16, 2018, Chandler, Arizona. p. 30.

Interpretive Summary:

Technical Abstract: Cows on concrete flooring have classical immunological indicators of chronic pain compared with those housed on rubber flooring; increased peripheral blood mononuclear cell numbers, and more leukocyte RNA expression of IL-1ß and less expression of IL-1Ra. Previous results showed that the leukocyte populations and their phenotypes that enable functions (CD18 and CD14) are altered by concrete flooring and that responses to additional substance P (a neurotransmitter associated with pain; SP) are altered in cows under those conditions. We then hypothesized that certain gene expressions may be modulated by the flooring, day of the study, and additional SP. The model was that 1st-calf heifers were assigned to rubber or concrete flooring. On day -3, 7, 21, 45 and 90 of the 2nd lactation, 5-part differential counts of whole blood and buffy coat leukocyte marker expressions were analyzed. Buffy coat cells were further stimulated with SP in vitro. We evaluated RNA expression of 2 cell populations (adherent or those in the supernatant) that were from cows on the concrete or rubber flooring that has been incubated with or without additional SP. Cells for RNA analysis were incubated in 6-well plates for 30 min at 37° incubator with 5% CO2. Cells in the supernatant were removed and treated as lymphocytes. The remaining cells were scraped into additional RPMI and considered the monocyte/macrophage population. The 4 treatments were lymphocyte +/- SP and monocytes +/- SP; (L, LP, M, MP). We used a Trizol extraction protocol, amplified cDNA, and tested gene expression of GRK (known to be modified with chronic pain and an indicator of the switch to chronic pain), and Toll Like Receptors (TLR) 2, 4, and 5 (to determine if recognition of pathogens may be attenuated with chronic pain or additional SP) using Taqman gene expression assays with 18S as the internal standard. We only used RNA extracted from d 7 and 21 from the second lactation, since that is where we saw the greatest difference in cell phenotype and differential counts. Expression of TLR2 was not different on d 7 or between d 7 and 21, but on d 21 TLR2 was less (P = 0.05) for LP than for either of the monocyte treatments (M or MP). Additionally, L expression of TLR2 was less (P = 0.03) than MP on that day. No other treatment differences were detected for TLR4, TLR5, or GRK. However, a day effect (P = 0.01) was found for GRK, such that all treatments expressed less GRK on d 21 than on d 7. These data suggest that the older rubber flooring in this study may not be having as great an effect as we saw in the first study with the new flooring. But, the data still showed some modulation by SP for lymphocytes and by time after calving in the second lactation. These results suggest that rubber flooring may need to be monitored for resilience to maintain the advantages gained by its use.