Boulougouris, Xanthippe

Burvenich, Christian

De Spiegeleer, Bart

Delesalle, Catherine

Duchateau, Luc

Peelman, Luc

Rogiers, Carolien

Van Poucke, Mario

English

Burvenich, Christian Paul G.

Ghent University Academic Bibliography

TWO PHENOTYPES OF BOVINE NEUTROPHILS DURING LACTATION: FICTION OR FACT? Xanthippe Boulougouris1, Carolien Rogiers1, Catherine Delesalle1, Luc Duchateau1, Luc Peelman2, Mario Van Poucke2, Bart De Spiegeleer3, Christian Burvenich1  1Department of Comparative Physiology & Biometrics; 2Department of Animal Nutrition, Genetics and Ethology; 3Department of Pharmaceutical Analysis, Ghent University, Belgium  Periparturient (E) E.coli mastitis in cows is accompanied by a large variation in systemic symptoms varying from mild and moderate, to severe. Severely affected cows suffer from unbalanced inflammation. During mid lactation (M), intramammary infections with E. coli cause less variable and less severe symptoms because of a well-regulated inflammatory reaction. A few years ago we demonstrated that pre-infection capacity of blood neutrophils (PMN) to produce reactive oxygen species (ROS) is negatively correlated with the severity of systemic symptoms during experimental E.coli mastitis in E-cows, indicating that pre-infection PMN may play a pivotal role in variation of severity of periparturient E.coli mastitis. This correlation lasts for 24 h and subsides after a few days. According to the cows' stage of lactation and parity, we observed two PMN chemiluminescence (CL) phenotypes, after stimulation with Zymosan (Zym) and phorbol-myristate-acetate (PMA). In a first step we wanted to know if typical kinetics of PMN CL activation could be explained through the differential expression of isoforms of genes that are known to regulate ROS production in PMN. If this would be the case the high sensitivity of some cows to develop severe E.coli mastitis could be related to one or more isotypes. Therefore we selected a small group of genes : CAT (catalase), SOD2 (superoxide dismutase 2), CYBA and CYBB (cytochrome b245, alpha and beta polypeptide), NCF1 and NCF4 (neutrophil cytosolic factor 1 and 4) and RAC1 and RAC2 (ras related C3 botulinum toxin substrate 1 and 2). In contrast to the afore-mentioned studies, each cow was sampled two times over time: first sample within 24 h after parturition (E), and second sample 135 days later (M). Thirteen primi- and twelve multiparous lactating dairy Holstein cows were used. Four groups of observations can thus be distinguished: primiparous cows sampled around parturition (PrE) and established lactation (PrM), and multiparous cows (2-5 calvings) at parturition (MuE) and established lactation (MuM). PMN were isolated and ROS-production was measured after stimulation with Zym and PMA. We herewith present some preliminary results. In the current study similar differences between ROS-kinetics (phenotypes) were detected in E- and M-cows as previously described with the different populations of cows. Isoforms of the afore-mentioned genes were not detected. A particular PMN phenotype and probably also the high sensitivity of some cows to develop severe systemic symptoms during E.coli mastitis can therefore not be explained by the afore-mentioned gene isoforms. In a second step of this study, we are studying the gene expression and its epigenetic regulation during lactation.   !"10000	  20000	  30000	  40000	  50000	  60000	  70000	  1	   6	   11	   16	   21	   26	   31	   36	   41	   46	   51	   56	   61	   66	   71	   76	   81	   86	   91	  PMA	  -­‐	  Primi	  Early	  PMA	  -­‐	  Primi	  Mid	  PMA	  -­‐	  Mul9	  Early	  PMA	  -­‐	  Mul9	  Mid	  0	  5000	  10000	  15000	  20000	  25000	  30000	  35000	  40000	  45000	  50000	  1	   6	   11	   16	   21	   26	   31	   36	   41	   46	   51	   56	   61	   66	   71	   76	   81	   86	   91	  ZYM	  -­‐	  Primi	  Early	  ZYM	  -­‐	  Primi	  Mid	  ZYM	  -­‐	  Mul9	  Early	  ZYM	  -­‐	  Mul9	  Mid	  FACULTY OF VETERINARY MEDICINEapproved by EAEVETwenty-­‐ﬁve	  lacta9ng	  dairy	  Holstein-­‐Friesian	  cows	  were	  used	  as	  blood	  donors.	  The	  ﬁrst	  samples	  were	  taken	  <	  24	  h	  post	  partum	  	  and	   the	   second	   samples	   during	  mid	  lacta9on	  at	  135	  DIM	  (range	  126	  to	  141	  days).	  	  We	   selected	   a	   small	   group	   of	  genes	   involved	   in	   ROS	   pro-­‐duc9on.	   Exon-­‐overlapping	   DNA	  primers	   were	   designed	   using	  Primer3Plus	   and	   taking	   the	  predicted	  isoforms	  into	  acount.	  PMNs	  were	   isolated	  and	  ROS-­‐produc9on	  was	  measured	  aZer	  s9mula9on	   with	   Zymosan	   (Zym)	   and	   phorbol-­‐myristate-­‐acetate	  (PMA).	  Four	  cows	  were	  selected	  on	  the	  basis	  of	  their	  divergent	  ROS	  phenotypic	  results.	  CAT	  	   catalase	  SOD2	   superoxide	  dismutase	  2	   	  CYBA	  	   cytochrome	  b245	  alpha	  polypep>de	  alpha	   	  CYBB	   cytochrome	  b245	  beta	  polypep>de	   	  NCF1	  	   neutrophil	  cytosolic	  factor	  1	  	  NCF4	  	   neutrophil	  cytosolic	  factor	  4	  	  RAC1	  	   ras	  related	  C3	  botulinum	  toxin	  substrate	  1	  	   	  RAC2	  	   ras	  related	  C3	  botulinum	  toxin	  substrate	  2	  Fig	  2.	  ROS	  produc0on	  of	  bovine	  neutrophils	  a;er	  s0mula0on	  with	  Zym	   Fig	  3.	  ROS	  produc0on	  of	  bovine	  neutrophils	  a;er	  s0mula0on	  with	  PMA	  Fig	  1.	  Diﬀerent	  subpopula0ons	  used	  in	  this	  study	  	   Table	  1.	  Genes	  of	  interest	  involved	  in	  ROS	  produc0on	  Primiparous	  	  d=0	  Primi	  Early	  Mul9parous	  	  d=0	  Mul0	  Early	  Primiparous	  	  d=135	  Primi	  Mid	  Mul9parous	  	  d=135	  Mul0	  Mid	  25	  cows	  

Two phenotypes of bovine neutrophils during lactation: fiction or fact?

https://biblio.ugent.be/publication/5878828/file/5885799

Two phenotypes of bovine neutrophils during lactation: fiction or fact?

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