Impaired capacity of neutrophils to producereactive oxygen species, release extracellular    traps and express genes encod...
Neutrophils are key immune cells                          Chemotaxis                                 Killing
Neutrophil killing mechanisms        Intracellular                     Extracellular                               Reactiv...
Compromised immune defenses: periparturition                                   Shuster et al., 1996
ObjectiveInvestigate changes in gene expression associated withneutrophil dysfunction during the periparturient period ind...
Research Methods• Collection of bloodNeutrophil function (n = 35)Late pregnancy: -58 ± 1.7 dPre-partum: -7 ± 0 dPostpartum...
Research Methods• Reactive oxygen species (ROS)• Extracellular trap formation (NETs)• Chemotaxis towards IL-8• Phagocytosi...
Altered neutrophil function in periparturient cows                                 ROS Production   ChemotaxisEffect of pe...
Altered neutrophil function in periparturient cows                                 Phagocytosis   NETs releaseEffect of pe...
Reduced cytokine and ROS gene expression      Postpartum    Late pregnancy   Postpartum    Late pregnancy                 ...
Transcriptome analysis of bovine neutrophils                   RNA-seq quality control (average of 14 libraries)Total read...
Functional clustering: ↓NADPH/oxido-reductase                                                           ROS               ...
Pathway analysis: ↓ROS Gene Expression                           Down-regulated, P < 0.01                                K...
Functional clustering: ↓ChemokinesChemotaxis                      DAVID Bioinformatics Resources 6.7
Transcriptome analysis: future direction          Gene functional ontology term   Number of down-regulated genes Cation bi...
Summary• Periparturient neutrophil function and mRNA expression   •   ↓ Reactive oxygen species   •   ↓ Chemotaxis   •   ↓...
Take home message
Questions
Transcriptome analysis of bovine neutrophilsAdapter ligation   Cluster generation    Sequencing by synthesis              ...
Transcriptome analysis of bovine neutrophils                               Illumina® Sequencing protocol
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Crwad 2011 presentation revelo

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  • One of the key cellular components of the innate immune system, which is critical during the first stages of the infection, is the PMN or neutrophil. In a healthy animal, neutrophils are found in circulation whereas macrophages are patrolling for invading pathogens. In the case of an E coli infection, the lipopolisacharide component of bacterial cell wall also known as LPS will form a complex with LBP and cD14 and will be recognized by those macrophages that will secrete several cytokines and chemoattractants such as IL-8, TNF, and these cytokines will enhance the expression of adhesion molecules by the endothelial cell lining. Adhesion molecules will then bind the neutrophils which will slow down, adhere to the epithelial cell lining and finally migrate through the intercellular spaces. Once the neutrophil is in the infected tissue, it will migrate towards the pathogen in a process known as chemotaxis. Then the neutrophil will find the bacteria and attempt to destroy them using several killing mechanisms. Mature neutrophils leave the bone marrow, enter the circulation and are recruited to the site of infection by inflammatory mediators that induce the expression of E- or P- selectin on the surface of endothelial cells, which bind to mucins on the neutrophil cell surface. Once bound, the neutrophils start to roll along the endothelial surface. Here, chemokines, such as interleukin (IL)-8 , complement factors, such as C5a , and N- formyl-peptides from bacteria can activate the neutrophils, which start to express integrins, such as lymphocyte-function-associated antigen 1 (LFA-1). The integrins bind to cell adhesion molecules belonging to the immunoglobulin superfamily (intercellular adhesion molecules, or ICAMs) on the surface of endothelial cells and establish a tight adhesion that arrests neutrophil migration. Following a chemotactic gradient, neutrophils then traverse through endothelial gaps and migrate towards infected tissue 5
  • Neutrophils can kill bacteria and other pathogens by intracellular or extracellular means. First, the PMN recognizes the invading pathogen and engulf it into a membrane-bound compartment known as the phagosome. This phagosome is fused with specific and azurophilic granules cointaining antimicrobial proteins as well as ROS generated by the enzyme NADPH. Therefore the microbe is exposed to these antimicrobial molecules in the phagolisosome where the pathogen is destroyed. Also the activated PMN can release some of these ROS to the extracellular space or mix its own nuclear material mixed with antimicrobial molecules, released these structures known as nets to physically trap and destroy the pathogen in a process known as extracellular traps formation.
  • There is substantial evidence suggesting that the incidence and severity of intrammary infections is highest during the period between 3 wk prior to calving until 3 wk after calving. As an example, Shuster and coworkers infused 2 mL of E. Coli at a concentration of 15 CFU/mL into one gland of 6 midlactating cows with more than 30 days in milk or periparturient cows between their 6 and 10 days of lactation. They collected milk samples from infected glands to determine bacterial growth as ilustrated in this graph. They reported that by 10 hours after infusion the number of cfus of E coli was 36 fold greater in periparuturient cows compared to midlactating cows. Interestengly, SCC were no different among mid and periparturient cows, suggesting that the same number of PMN might have reached the gland, but perphaps those PMNs in periparturient cows did not have the same ability to kill bact. Iowa State - Compared susceptibility to coliform mastitis between periparturient and midlactating cows. - 6 mid-lactating (&gt;30DIM) paired with 6 periparturient (6-10 DIM) challenged with 2 mL (15CFU/mL) of E. Coli. (Strain 487): one gland - Milk samples collected for electronic cell count analysis to determine bacterial growth - By general observation, periparturient cows had more severe mastitis (1 died) - Bacteria grew 36-fold (5.3 vs 3.7) faster by 10 hours in periparturient cows They were able to recruit leukocytes (no differences in SCC) but may be due to a defect on PMN
  • To increase the understanding of PMNL activity in dairy cows and its relation to clinical mastitis, the first objective of this study was to investigate the effect of LPS on neutrophil functionality. More specifically on….
  • We collected blood from 20 healthy cows on days 49, 28, 19 and 9 prior to calving and on days 1,7,14 and 30 post partum. We then purified neutrophils blood by dgc and measured the ROS, NET, Chemotaxis and killing of S aureus using previously published assays validated in our lab.
  • Explain how AUC are obtained Combine ros and nets First, we collected PMN from the blood of 7 cows 12 days before parturition, 7 days after parturition and in midlactation which is around 150 days after parturition and measured the ROS produced by the PMN collected from different physiological states. ROS production decreased by about 25% between prepartum and posrpartum and then recovered by day 150. I also measured the ability of the PMN to release extracellular traps in the different physiological states. Surprisingly, the release of these Nets increased during the postpartum period, perhaps to compensate for the dimished ROS generation.
  • Difference in cytb when the cells are stimulated. Do t-test effect of LPS on Mid. IL8 not needed I also extracted mRNA from the purified cells and measure the expression of genes encoding for cytokines, enzymes involved in ROS generation using real time PCR. Expression levels relative to the housekeeping gene bactin were measured in non-stimulated and LPS-activated states. First, the expression of the cytokine IL8 was lower in early lactation, compared with midlactation suggesting that these cells have reduced capacity to secrete this cytokine and communicate with other components of the immune system. In the case of TNF-alpha, another inflammatory mediator, there was a dramatic increase in TNF expression by activated PMNL from ML but no significant change in EL.
  • Results of sequencing RNA from neutrophils extracted from 8 cows in prepartum and 8 cows in postpartum. In average 27M reads were generated, 25M passed the quality filter, 22M aligned to the genome. Interestengly the cow’s genome is about 2.5 Billion bases, and the bases from our sequenced covered about 10% of the genome, which makes sense as this cells are effector cells and thus carry limited genetic material. Bottom line we found 147 upregulated genes and about 261 downregulated genes. Now, I am still working with this data, and so far I have focused on the downregulated genes.
  • To understand the biological meaning of our list of genes with low expression in the posrtpartum period, we used a bioinformatics tool to group the genes in functional clusters. Using DAVID we can generate 2-dimentional graphs showing genes with overlapping functions on the y axis and their corresponding functional terms in the x axis. When the intersection between both axis is green we know that this gene supports that specific biological function. In this specific map, low expression of the 11 genes highlighted in red indicate that overall, PMN have reduced NAPDH and oxido-reductase activities in the postpartum period. Also, we found at least 3 downregulated genes that have NADPH activity. Therefore, since the enzyme-complex NADPH oxidase is involved in the production of ROS, we decided to take a closer look into the pathway responsible for ROS production.
  • When we looked deeper in the ROS pathway depicted in this illustration we further ID 8 genes with low expression in postpartum. 2 of them, the Fc phagocytic promoting receptors, also TLR4/CD14 complex which senses the endotoxin in gram negative bacteria and initiates the inflammatory response. As described earlier, the pathogen is engulfed in a membrane bound compartment known as the phagosome, there are 3 types of phagosomes, early, mature and phagolysosome. In the phagolysosome, the pathogen is exposed to potent antomicrobial compounds. Now, 4 genes, NOS, MPO, and 2 subunits of NADPH oxidase were downregulated in early lactation cows. So, lower expression of these genes during the postpartum period may explain why PMN produced less ROS, and its only only example of how next genreation seq can be used to discover the underlying mechanisms of immune dysfunction.
  • In agreement with our findings that in-vitro chemotaxis is reduced postpartum, we found that the expression of leukotrine receptor-1, chemokine receptor 3 and G-coupled receptor 44, involved in the migration and chemotaxis of granulocytes was downregulated in postpartum cows.
  • For the future, we want to use a similar approach to make sense of groups of genes with similar function that were downregulated in the postpartum period. As you can see, the amount of information that can be obtained is impressive.
  • Emphasize new findings Nutritional intervetnions to follow up (signicance of findings
  • What’s the take home message from this presentation? As of 2007, 16.5% of dairy cows in the US had clinical mastatis. Increasing the understanding of the cellular and molecular mechanisms involved in periparturient immuno-alteration offers the possibility to develop effective TRTs to improve the health of transition cows.
  • After identifying a few genes that are potentially involved in reduced performance by the neutrophils, we decided to use 2 nd generation sequencing to investigate differences in neutrophil gene expression profiling between early lactation and midlactation periods. We extracted RNA from 7 cows 60 days prior to parturition and then again 7 days after parturition. To summarize RNA seq technology, purified poly(a) RNA is fragmented and reverse transcribed to cDNA, adapters are ligated on both ends and amplified using PCR. Then, these fragments are allowed to bind the surface of a flow cell, nucleotides and enzyme are added to initiate amplification which results in the formation of millions of clusters of double stranded DNA in each channel of the flow cell. Each fragment is sequenced by incorporation of single fluorescent nucleotides. The sequence of each fragment (25-100 bp) can be aligned to a reference genome. The numerical frequency of a fragment aligning to an anottated sequence is used to estimate the abundance of mRNA for that gene.
  • After identifying a few genes that are potentially involved in reduced performance by the neutrophils, we decided to use 2 nd generation sequencing to investigate differences in neutrophil gene expression profiling between early lactation and midlactation periods. We extracted RNA from 7 cows 60 days prior to parturition and then again 7 days after parturition. In summary, purified RNA is reverse transcribed to cDNA, adapters are ligated on both ends and Sequencing-based RNA analysis records the numerical frequency in the library population .
  • Crwad 2011 presentation revelo

    1. 1. Impaired capacity of neutrophils to producereactive oxygen species, release extracellular traps and express genes encoding forcytokines may contribute to altered immune function in periparturient dairy cows X. Revelo*, A. Kenny, N. Barkley, and M. Waldron Division of Animal Sciences University of Missouri, Columbia
    2. 2. Neutrophils are key immune cells Chemotaxis Killing
    3. 3. Neutrophil killing mechanisms Intracellular Extracellular Reactive oxygen Neutrophil extracellular species (ROS) traps (NETs)Kennedy 2009 Brinkmann 2004
    4. 4. Compromised immune defenses: periparturition Shuster et al., 1996
    5. 5. ObjectiveInvestigate changes in gene expression associated withneutrophil dysfunction during the periparturient period indairy cows- Neutrophil function:- Reactive oxygen species (ROS) production- Extracellular trap formation (NET)- Chemotaxis towards interleukin-8- Phagocytosis of E. coli-Gene expression:-Tumor necrosis factor-α mRNA-Interleukin-8 mRNA-Cytochrome-b mRNA-Bactericidal/permeability increasing protein (BPI) mRNA-Transcriptome (mRNA) shotgun sequencing
    6. 6. Research Methods• Collection of bloodNeutrophil function (n = 35)Late pregnancy: -58 ± 1.7 dPre-partum: -7 ± 0 dPostpartum: 6 dGene expression (n = 7)Late pregnancy: -58 ± 1.7 dPostpartum: 7 d• Isolation of neutrophils Density gradient centrifugation
    7. 7. Research Methods• Reactive oxygen species (ROS)• Extracellular trap formation (NETs)• Chemotaxis towards IL-8• Phagocytosis of E. coli• Gene expressionRT-PCR: - Tumor necrosis factor-α - Interleukin-8 - Cytochrome b - BPIIllumina® RNA Sequencing
    8. 8. Altered neutrophil function in periparturient cows ROS Production ChemotaxisEffect of period, P < 0.001 (n = 35) Revelo et al., 2011 (in preparation)
    9. 9. Altered neutrophil function in periparturient cows Phagocytosis NETs releaseEffect of period, P < 0.001 (n = 35) Revelo et al., 2011 (in preparation)
    10. 10. Reduced cytokine and ROS gene expression Postpartum Late pregnancy Postpartum Late pregnancy Postpartum Late pregnancy Postpartum Postpartum Late pregnancy Revelo and Waldron, 2011 (JDS, accepted)
    11. 11. Transcriptome analysis of bovine neutrophils RNA-seq quality control (average of 14 libraries)Total reads 27,210,968Reads converted 25,252,266Perfect match 18,516,011Matched reads 22,315,658Unmatched reads 2,936,423Matched bases 1,955,924,633Unmatched bases mutations 2,640,876Average read length 48Reference length 2,670,102,999Covered bases 259,076,285Percent coverage, % 9.70Percent mapped, % 88.37Genes up-regulated postpartum (> 1.5 fold, P < 0.01) 43Genes down-regulated postpartum (< 0.5 fold, P < 0.01) 423 Revelo et al., 2011 (in preparation)
    12. 12. Functional clustering: ↓NADPH/oxido-reductase ROS pathway NADPH Oxido-reductase activity activity DAVID Bioinformatics Resources 6.7
    13. 13. Pathway analysis: ↓ROS Gene Expression Down-regulated, P < 0.01 KEGG pathway database
    14. 14. Functional clustering: ↓ChemokinesChemotaxis DAVID Bioinformatics Resources 6.7
    15. 15. Transcriptome analysis: future direction Gene functional ontology term Number of down-regulated genes Cation binding 67 Ion binding 67 Metal ion binding 66 Transition metal ion binding 52 Zinc ion binding 40 Endoplasmic reticulum 24 Mitochondrion 24 Intracellular organelle lumen 21 Organelle lumen 21 Membrane-enclosed lumen 21 Oxidation reduction 20 Lipid biosynthetic process 19 Transcription 18 Organelle membrane 18 Steroid metabolic process 17 Nuclear lumen 17 Sterol metabolic process 16 Endomembrane system 16 Cholesterol metabolic process 15 Regulation of apoptosis 13 Regulation of programmed cell death 13 Regulation of cell death 13 Steroid biosynthetic process 12 RNA processing 12 Cholesterol biosynthetic process 11 Sterol biosynthetic process 11 Revelo et al., 2011 (in preparation)
    16. 16. Summary• Periparturient neutrophil function and mRNA expression • ↓ Reactive oxygen species • ↓ Chemotaxis • ↓ Phagocytosis • ↓ Interleukin-8 mRNA • ↓ Tumor necrosis factor-α mRNA • ↓ Cytochrome-b, alpha polypeptide mRNA • ↓ Bactericidal/permeability increasing protein mRNA• Transcriptome analysis reveals clusters of genes with reduced expression in neutrophils during postpartum
    17. 17. Take home message
    18. 18. Questions
    19. 19. Transcriptome analysis of bovine neutrophilsAdapter ligation Cluster generation Sequencing by synthesis Illumina® Sequencing protocol
    20. 20. Transcriptome analysis of bovine neutrophils Illumina® Sequencing protocol

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