Rol van microbiota bij de ontwikkeling van immuun competentie in biggen
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1 Rol van microbiota bij de ontwikkeling van immuun competentie in biggen D. Schokker, J. Zhang, H. Smidt, J.M.J. Rebel, M.A. Smits Livestock Research, Central Veterinary Institute, and Laboratory of Microbiology Background (literature) Gut microbiota influence gut physiology and health from birth to old age. Environmental factors (diet, stress, disease, injury, antibiotics) can alter the diversity and composition of gut microbiota. Disruption of the gut microbiota (dysbiosis) can lead to a variety of immune-related diseases. Early maternal and environmental factors (delivery, nutrition) affect the microbial colonization of the gut. Changes in early life microbial colonization of the gut has an effect on the risks for developing disease later in life. 1
2 Objectives of the study Do early-life factors, as experienced by piglets under normal husbandry conditions, affect gut microbial colonization? If yes, does this also affects immune competence later in life? Exposure to early life factors (Treatment) Treatment Antibiotic* Stressor T1 - - T2 + - T3 + + Treatment: At day 4 after birth. * Tulathromycine Antibiotic: Stressor: Regularly used in intensive farming systems to prevent respiratory diseases. Common in intensive farming systems: weighing, numbering, and tail docking. 2
3 Experimental design Sow 1 Sow 2 Sow 3 Sow 4 Sow... Sow 16 Day 4 T1 T2 T3 Day 8 Day 25 Day 55 weaning Collection of intestinal tissue (jejunum, ileum), blood, intestinal content Day 176 Transcriptomics and Microbiota data 4 pools of 4 animals per treatment T1 T2 T3 3
4 Diversity microbiota T1 T2 T3 Day 8 Diversity Diversity Day 55 Day 176 T1 T2 T3 T1 T2 T3 Composition microbiota (RDA plots) Day 8 Day 55 Day 176 Day 176 4
5 Gene expression jejunum and ileum (PCA) Day 8: Clustering of treatments(colors) Day 55: Clustering of treatments(colors) and tissues (symbols) Ileum Jejunum Day 176: Clustering of tissues (symbols) Number of regulated genes in jejunum and ileum ANOVA p<0.05 & FC >1.5 Day / Tissue T2 vst1 T3vsT1 T3vsT2 Down UP Down Up Down Up 8 / Jejunum 8 / Ileum 55 / Jejunum 55 / Ileum 176 / Jejunum 176 / Ileum
6 Functional analysis of regulated genes. Day 8: T2 vs T1 Tissue Jejunum Down Up # Name # Name 1 chemotaxis 1-2 cytokine activity 2-3 chemokine activity 3-4 reg. of secretion /immune effector 4 - process 5 cell migration/motion (leukocyte) 5 - Ileum #Name # Name 1 cytokine activity 1 cell fraction 2 chemotaxis 2-3 second-messenger-mediated 3 - signaling (camp) 4 chemokine activity 4-5 response to bacterium/reg. systemic 5 - process Decrease in activity of immune related processes in piglets treated with antibiotics Functional analysis of regulated genes. Day 8: T3 vs T1 Tissue Jejunum Ileum Down Up # Name # Name 1 chemotaxis 1 nucleotide binding 2 cytokine activity 2 membrane fraction 3 extracellular region 3 ATP binding 4 chemokine activity 4-5 second-messenger-mediated 5 - signaling (camp) # Name # Name 1 response to wounding/defense 1 nucleotide binding response 2 cytokine activity 2 positive reg. of catalytic activity/signaling cascade 3 chemotaxis 3 plasma membrane 4 extracellular region 4-5 chemokine activity 5 - Decrease in activity of immune related processes in piglets treated with antibiotics + stressors 6
7 Functional analysis of regulated genes. Day 8: T3 vs T2 Tissue Down Up Jejunum # Name # Name 1-1 chemotaxis 2-2 nucleotide binding Ileum # Name # Name 1-1 Immunoglobulin 2-2 nucleotide binding 3-3 plasma membrane 4-4 nucleotide/atp binding 5-5 positive reg. of catalytic activity/signaling cascade Increase in activity of some immune related processes in piglets treated with antibiotics + stressor (compared to antibiotic alone) Development chemokine signalling network: day 8 T1 T2 T3 Similar observations for TLR network 7
8 Development TLR network: day 8 T1 T2 T3 TLR network Ileum Day 8 8
9 Functional analysis Day 55 T2vsT1 T3vsT1 T3vsT2 Differences in activity of several processes including immune related processes Functional analysis Day 176 T3vsT1 T2vsT1 T3vsT2 Differences in activity of a small number of processes in T2, some of which are immune related 9
10 blood ANOVA p<0.05 & FC >1.5 Day / Tissue T2 vst1 T3vsT1 T3vsT2 Down UP Down Up Down Up 8 /blood 55 / blood 176 / blood nd nd nd nd nd nd T3vsT1 T3v st1 T2vs T1 T2vs T1 T3vsT2 T3vsT2 ion (potassium) transport T3 T1 T2 T1 T3 T2 cell cycle metabole (tryptophan) response to bacterium lumen TLR (9 / 4) / innate mitochondrion mrna mature Day 55 Conclusies Toedienenvan antibioticaop vroegeleeftijdheefteffect op de microbiëlekolonisatievan de darm(samenstellingen diversiteit). de ontwikkeling van het immuunsysteem. de activiteit van het immuunsysteem op latere leeftijd. Toedienen van antibiotica op vroege leeftijd heeft mogelijk effect op het immunologischevermogenvan biggenomlater te kunnen reageren op verstoringen waaraan ze blootgesteld worden. Day 8 10
11 Betekenis Vroege kolonisatie van de darm met microbiota is van belang voor de ontwikkeling en status van het immuunsysteem. Veranderingenin microbiota gaangepaardmet veranderingenin de immuun status. Omdatdiervoedingde samenstellingen diversiteitvan microbiota moduleertis het eenbelangrijkefactor voor(de ontwikkeling van) immuun competentie. Day 8 Dank voor uw aandacht mari.smits@wur.nl 11
12 Functional analysis of regulated genes. Blood, day 55: Down Up #Name #Name T2vs. T1 1pos. reg. trancription/met. process 1plasma membrane 2reg. phosporylation / met. Process 2blood circulation/pressure 3pos. reg. transcription 3nucleotide biosynthetic process 4pos. reg. synaptic transmission 4binding; retinoid, isoprenoid, retinol 5kinase acvitity 5melanocortin receptor #Name #Name T3vs. T1 1pos. reg. trancription/met. process 1plasma membrane 2pos. reg. transcription 2nucleotide biosynthetic process 3pos. reg. synaptic transmission 3binding; retinoid, isoprenoid, retinol 4reg. kinase acvitity / met. process 4melanocortin receptor 5reg. synaptic transmission 5neg. reg. (immune) cell activation #Name #Name 1reg. phosporylation / met. process 1reg. apoptosis 2reg. caspase/(endo)peptidase 2pos. reg. apoptosis 3(neg.) reg. (hormone) secretion 3 4reg. apoptosis 4 5pos. reg. hydrolase/catalytic activity 5 T3vs. T2 12
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