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(November 30, 2022) Webinar: Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses

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(November 30, 2022) Webinar: Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses

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Today, poultry meat is at the top of consumers’ lists as an affordable, nutritious, and healthy protein source. The modern meat-type chicken’s highly efficient growth rate and outstanding meat yield account for much of the poultry industry’s success, but many of today’s consumers care more than the price tag. As concerns for animal welfare, health, and sustainability grow along with the global population’s craving for animal protein, effectively addressing lameness incidence in meat birds is of key interest. A major obstacle in tackling lameness is the lack of understanding of the mechanistic underpinnings involved in a common cause of lameness, femur head necrosis (FHN), also called bacterial chondronecrosis with osteomyelitis (BCO).

The work of Dr. Alison Ramer’s lab has been to identify mechanisms by which bacteria induce the symptoms of FHN/BCO, bone attrition, inflammation, and infection through both in vivo and in vitro studies. This work has resulted in several key molecular pathways implicated in FHN/BCO, including DICER dysregulation and dsRNA accumulation, mitochondrial dysfunction, and autophagy dysregulation. In addition to identifying causative pathways, the lab has also sought to identify potential non-invasive biomarkers for the presence of FHN/BCO, which led to the identification of a unique cytokine/chemokine signature both in circulation and at the local bone level of affected birds. Their quest to better define and diagnose this consequential skeletal disease also means seeking new means of measuring and visualizing bone health in meat-type birds to feed the future healthily and sustainably.

Today, poultry meat is at the top of consumers’ lists as an affordable, nutritious, and healthy protein source. The modern meat-type chicken’s highly efficient growth rate and outstanding meat yield account for much of the poultry industry’s success, but many of today’s consumers care more than the price tag. As concerns for animal welfare, health, and sustainability grow along with the global population’s craving for animal protein, effectively addressing lameness incidence in meat birds is of key interest. A major obstacle in tackling lameness is the lack of understanding of the mechanistic underpinnings involved in a common cause of lameness, femur head necrosis (FHN), also called bacterial chondronecrosis with osteomyelitis (BCO).

The work of Dr. Alison Ramer’s lab has been to identify mechanisms by which bacteria induce the symptoms of FHN/BCO, bone attrition, inflammation, and infection through both in vivo and in vitro studies. This work has resulted in several key molecular pathways implicated in FHN/BCO, including DICER dysregulation and dsRNA accumulation, mitochondrial dysfunction, and autophagy dysregulation. In addition to identifying causative pathways, the lab has also sought to identify potential non-invasive biomarkers for the presence of FHN/BCO, which led to the identification of a unique cytokine/chemokine signature both in circulation and at the local bone level of affected birds. Their quest to better define and diagnose this consequential skeletal disease also means seeking new means of measuring and visualizing bone health in meat-type birds to feed the future healthily and sustainably.

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(November 30, 2022) Webinar: Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses

  1. 1. Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses Dr. Alison Ramser Post-Doctoral Researcher at Center for Excellence in Poultry Science, University of Arkansas
  2. 2. Efficient Affordable Popular Sustainability Welfare Robustness
  3. 3. Day 1 Day 42 Dynamic Molecular Reorganization Fast Growth and the Leg Bones of the Modern Broiler Courtesy of Dr. Wideman
  4. 4. The Modern Broiler and BCO Wideman, R. 2016. Poult Sci (95)2:325-344
  5. 5. The Avian Growth Plate and BCO Wideman, R. 2016. Poult Sci (95)2:325-344
  6. 6. Wideman, R. 2016. Poult Sci (95)2:325-344 The Avian Growth Plate and BCO
  7. 7. Lameness, BCO, and the Broiler BCO Welfare Food Safety Economic https://www.allaboutfeed.net/animal-feed/feed-additives/research-probiotics-reduces- lameness-in-broilers/
  8. 8. How do Bacteria Cause BCO?
  9. 9. dsRNA NLRP3 Inflammasome IL-1β ↓ Cell Viability Dicer1 Mitochondria Dysfunction Autophagy Dysregulation
  10. 10. Experimental Design – in vivo Wideman, R. F., K. R. Hamal, J. M. Stark, J. Blankenship, H. Lester, K. N. Mitchell, G. Lorenzoni, and I. Pevzner. Poult. Sci. 91:870-883, 2012 • Healthy and BCO affected • Sampling – Femur and tibia – Blood
  11. 11. Experimental Design – in vitro • hFOB 1.19 (ATCC) – S. agnetis 908 (Courtesy of Dr. Rhoads); MOI 50:1 – Cellular pathway manipulation – Cyto(chemo)kine exposure • Primary chondrocytes*
  12. 12. Sample Preparation and Analysis Gene expression • Trizol method • rtPCR • Real-time qPCR • Student t-test or one-way ANOVA – p-value < 0.05 – GraphPad Protein expression • Protein lysis buffer • Western Blot analysis – AlphaView software quantification • Student t-test or one- way ANOVA – p-value < 0.05 – GraphPad Immunofluorescence • Primary chondrocyte cells • Primary; fluorescent secondary antibodies – Vectashield with DAPI • Imaged using Zeiss Imager M2 and AxioVision software version LE2019 MTT cell viability assay
  13. 13. Double-stranded RNA in BCO
  14. 14. dsRNA and DICER1 Dysregulation in BCO Greene E, et al. Am J Pathol. 2019. doi: 10.1016/j.ajpath.2019.06.013
  15. 15. dsRNA NLRP3 Inflammasome IL-1β ↓ Cell Viability Dicer1 Mitochondria Dysfunction Autophagy Dysregulation
  16. 16. Bacterial infection effect on mitochondria Varnesh Tiku, Man-Wah Tan, Ivan Dikic. Mitochondrial Functions in Infection and Immunity. February 11, 2020DOI:https://doi.org/10.1016/j.tcb.2020.01.006
  17. 17. Mitochondrial Biogenesis C o n tro l B C O 0 1 2 3 D -lo o p m R N A e x p r e s s io n * C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 S k i m R N A e x p r e s s io n C o n t r o l B C O 0 1 2 3 4 5 P G C - 1  m R N A e x p r e s s io n * C o n t r o l B C O 0 1 2 3 4 5 P G C - 1  m R N A e x p r e s s io n * C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 T F A M m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 S S B P 1 m R N A e x p r e s s io n
  18. 18. Mitochondrial Dynamics C o n tro l B C O 0 .0 0 .5 1 .0 1 .5 O P A 1 m R N A e x p r e s s io n * C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 O M A 1 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 M F N 1 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 M T F P 1 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 D N M 1 m R N A e x p r e s s io n C o n t r o l B C O 0 1 2 3 4 M F N 2 m R N A e x p r e s s io n * C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 M T F R 1 m R N A e x p r e s s io n C o n t r o l B C O 0 1 2 3 4 5 M F F 1 m R N A e x p r e s s io n
  19. 19. Mitochondrial Dynamics OPA1 processing in cell death and disease – the long and short of it Thomas MacVicar, Thomas Langer Journal of Cell Science 2016 129: 2297-2306; doi: 10.1242/jcs.159186
  20. 20. Mitochondrial Function C o n tro l B C O 0 .0 0 .5 1 .0 1 .5 A N T m R N A e x p r e s s io n * C o n tro l B C O 0 .0 0 .5 1 .0 1 .5 C O X 5 A m R N A e x p r e s s io n * C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 C O X IV m R N A e x p r e s s io n * C o n t r o l B C O 0 1 2 3 4 5 a v - U C P m R N A e x p r e s s io n * C o n tro l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 N R F 1 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 F O X O 1 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 F O X O 3 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 F O X O 4 m R N A e x p r e s s io n C o n t r o l B C O 0 1 2 3 4 5 K e a p 1 m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 P P A R  m R N A e x p r e s s io n C o n t r o l B C O 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 P P A R  m R N A e x p r e s s io n
  21. 21. Conclusion • Implicates mitochondrial dysfunction – Potential: • ↑ cell death (chondronecrosis) • Alteration of cellular processes • Contribution to previously found mechanisms • Further research needed
  22. 22. dsRNA NLRP3 Inflammasome IL-1β ↓ Cell Viability Dicer1 Mitochondria Dysfunction Autophagy Dysregulation
  23. 23. Bacteria, Bone, and Autophagy Campoy, E, Colombo, M. 2009. BBA – Mol. Cell Res. (9)1465-1477. Xiao, L, Xiao, Y. 2019. Front. Endocrinol. (10)490.
  24. 24. 1. in vivo study Initiation Normal BCO GAPDH Beclin1 ←37 kDa ←60 kDa Beclin1 N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 9 A m R N A e x p r e s s io n N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 1 3 m R N A e x p r e s s io n * N o r m a l B C O 0 5 1 0 1 5 2 0 2 5 B e c lin 1 /G A P D H *
  25. 25. Nucleation Normal BCO GAPDH ATG5 ←37 kDa ←55 kDa N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 U V R A G m R N A e x p r e s s io n N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 1 4 m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 5 /G A P D H
  26. 26. Elongation – Protein Expression ATG16L ATG3 LC3A/B GAPDH ATG7 ATG12 Normal BCO ←37 kDa ←14,16 kDa ←40 kDa ←53 kDa ←66 kDa ←78 kDa N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 L C 3 /G A P D H *
  27. 27. Elongation – mRNA Expression ATG7; ATG3; ATG4A; LC3A; LC3B; N o rm a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 1 2 m R N A e x p r e s s io n * N o rm a l B C O 0 .0 0 .5 1 .0 1 .5 L C 3 C m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 1 6 L m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 2 B m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 1 0 m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 9 B m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 A T G 4 B m R N A e x p r e s s io n *
  28. 28. Fusion Normal BCO GAPDH Rab7 ←37 kDa ←22 kDa LAMP2 N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 S Q S T M 1 ( p 6 2 ) m R N A e x p r e s s io n * N o rm a l B C O 0 .0 0 .5 1 .0 1 .5 R A B 7 A m R N A e x p r e s s io n * N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 R A B 7 /G A P D H
  29. 29. 2. in vitro study Effect of S. agentis 908 on hFOB cell viability - + 0 5 0 1 0 0 1 5 0 S . a g n e tis 9 0 8 % o f c o n t r o l v ia b ility *
  30. 30. BCO isolate and Autophagy – in vitro ATG16L ATG3 LC3A/B GAPDH ATG7 ATG12 ATG5 Beclin1 Rab7 S. agnetis 908 - - - + + + ←37 kDa ←14 type I ←16 type II ←40 kDa ←53 kDa ←66 kDa ←78 kDa ←55 kDa ←60 kDa ←22 kDa A T G 7 A T G 1 6 L B e c l i n 1 A T G 5 A T G 1 2 A T G 3 R a b 7 L C 3 I I / I r a t i o 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 p r o te in /G A P D H - + * S . a g n e tis 9 0 8
  31. 31. Mechanisms for Autophagy Inhibition InvivoGen http://www.invivogen.com/autophagy-inhibitors
  32. 32. Effect of Autophagy Inhibition – in vitro C o n t r o l 3 - M A C Q 0 5 0 1 0 0 1 5 0 % o f c o n t r o l v ia b ility a b c
  33. 33. Effect of Autophagy Inhibition on Machinery in vitro ATG3 LC3A/B GAPDH ATG7 ATG12 Beclin1 Control 3-MA(5mM) CQ (10µM) ←37 kDa ←14 type I ←16 type II ←40 kDa ←53 kDa ←78 kDa ←60 kDa C o n t r o l 3 - M A C Q 0 1 2 3 L C 3 II:L C 3 I a b c C o n t r o l 3 - M A C Q 0 .0 0 .5 1 .0 1 .5 B e c lin 1 /G A P D H a b b C o n t r o l 3 - M A C Q 0 .0 0 .5 1 .0 1 .5 A T G 7 /G A P D H b b a C o n t r o l 3 - M A C Q 0 .0 0 .5 1 .0 1 .5 A T G 1 2 /G A P D H a a b b C o n t r o l 3 - M A C Q 0 .0 0 .5 1 .0 1 .5 A T G 3 /G A P D H a a b
  34. 34. Summary Killian, S. 2012. AIDS Research and Therapy. 9(1):16
  35. 35. Conclusions • Dysregulation of autophagy in BCO could be caused by bacterial manipulation and contribute to BCO pathology by affecting cell survival and homeostasis
  36. 36. dsRNA NLRP3 Inflammasome IL-1β ↓ Cell Viability Dicer1 Mitochondria Dysfunction Autophagy Dysregulation
  37. 37. Effect of plasma from normal and BCO broilers on hFOB cell viability N o r m a l B C O 0 .0 0 .5 1 .0 1 .5 % v ia b ility o f c o n t r o l *
  38. 38. Cytokines Bone Blood T N F  I L - 4 I L - 1 8 I L - 3 I L - 6 I L - 1 0 I L - 1 2 B I L - 1 7 0 1 2 3 4 5 m R N A e x p r e s s io n N o rm a l B C O * * T N F  I L - 4 I L - 1 8 I L - 1  I L - 6 I L - 1 0 I L - 1 2 B I L - 1 7 0 1 2 3 4 5 m R N A e x p r e s s io n N o rm a l B C O *
  39. 39. Chemokines Bone Blood C C L - 4 C C L - 2 0 C C L L - 4 C X C L - 1 4 C C L - 5 C R P I L - 8 L 1 0 2 4 6 m R N A e x p r e s s io n N o rm a l B C O * * C C L -4 C C L -2 0 C C L L -4 C X C L -1 4 C C L -5 C R P IL -8 L 1 IL -8 L 2 0 1 2 3 4 m R N A e x p r e s s io n N o rm a l B C O *
  40. 40. Inflammasomes Bone Blood N L R C 3 N L R C 5 N L R X 1 N L R P 3 0 1 2 3 4 5 m R N A e x p r e s s io n N o rm a l B C O * N L R C 3 N L R C 5 N L R X 1 0 .0 0 .5 1 .0 1 .5 2 .0 m R N A e x p r e s s io n N o rm a l B C O
  41. 41. FGF23 Pathway Bone Blood F G F 2 3 F G F R 1 K l o t h o 0 1 2 3 4 m R N A e x p r e s s io n N o rm a l B C O F G F 2 3 F G F R 1 K l o t h o 0 .0 0 .5 1 .0 1 .5 2 .0 m R N A e x p r e s s io n N o rm a l B C O *
  42. 42. Effect of recombinant cyto(chemo)kines on hFOB cell viability c o n t r o l I L - 1  I L - 8 T N F  6 0 8 0 1 0 0 1 2 0 V ia b ilit y ( % o f c o n t r o l) * *
  43. 43. Conclusions • BCO has systemic effects on pro-inflammatory factors • These factors form a unique, detectable signature of BCO and could contribute to BCO etiology
  44. 44. dsRNA NLRP3 Inflammasome IL-1β ↓ Cell Viability Dicer1 Mitochondria Dysfunction Autophagy Dysregulation
  45. 45. Materials and Methods Animal Rearing •Day1 chicks → Day10 •Ad libitum food and water •Weighed •Humanely euthanized Chondrocyte Isolation •Tibia proximal head •Cross cut and shavings from growth plate •Serum-free media •Digestion media Chondrocyte Culture •2 x 105 cells/cm2 •Complete media •DMEM; sodium pyruvate; ascorbic acid; FBS; P/S •Cytation3 imaging •Protein and RNA
  46. 46. https://musculoskeletalkey.com/cartilage-and-chondrocytes/ Jul 3, 2016 | Posted by admin in RHEUMATOLOGY
  47. 47. Primary Chondrocytes within Culture Day 3 7 11 14 18 21
  48. 48. Gene Expression Changes in Culture d 3 d 7 d 1 1 d 1 4 d 1 8 d 2 1 0 1 2 3 4 A C A N m R N A e x p r e s s io n a ab b b b b d 3 d 7 d 1 1 d 1 4 d 1 8 d 2 1 0 1 2 3 4 5 C O L IA 1 m R N A e x p r e s s io n a ab ab ab b b d 3 d 7 d 1 1 d 1 4 d 1 8 d 2 1 0 2 0 4 0 6 0 8 0 1 0 0 C O L IA 2 m R N A e x p r e s s io n a a c b b b c b c d 3 d 7 d 1 1 d 1 4 d 1 8 d 2 1 0 .0 0 .5 1 .0 1 .5 C O L II m R N A e x p r e s s io n a ab ab b b c d 3 d 7 d 1 1 d 1 4 d 1 8 d 2 1 0 .0 0 .5 1 .0 1 .5 S o x 9 m R N A e x p r e s s io n a b c b c c ab a b c d 3 d 7 d 1 1 d 1 4 d 1 8 d 2 1 0 .0 0 .5 1 .0 1 .5 C O L X m R N A e x p r e s s io n a b c c c c
  49. 49. Protein Expression Changes in Culture COL I COL II GAPDH Cell Lysate Media Day 3 7 11 14 18 21 Day 3 7 11 14 18 21 COLXA1 ACAN Sox9 COL I COL II COLXA1 ACAN 150 83 66 250 56 37 Ponceau S kDa 3 7 1 1 1 4 1 8 2 1 0 1 2 3 4 5 T im e (d a y s ) p r o te in e x p r e s s io n /G A P D H C O L I S ox9 C O L II A C A N C O L X A 1 $ # 3 7 1 1 1 4 1 8 2 1 0 1 2 3 4 5 T im e (d a y s ) p r o te in e x p r e s s io n /P o n c e a u S C O L I C O L II A C A N C O L X A 1 + *
  50. 50. Protein Expression Changes in Culture DAPI COL II DAPI COL II DAPI COL I DAPI COL I Day 7 Day 18
  51. 51. BFA treatment on COLII secretion COL II Cell Lysate Media BFA - - + + BFA - - + + (1 µg/mL) (1 µg/mL) GAPDH COL II 150 37 Ponceau S 0 .0 0 .5 1 .0 1 .5 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 p r o te in e x p r e s s io n /G A P D H p r o te in e x p r e s s io n /P o n c e a u S * B F A - + - + ly s a te m e dia ( 1  g /m L ) kDa
  52. 52. Conclusion Chondroprogenitor Proliferating chondrocyte Hypertrophic chondrocyte Dedifferentiated chondrocyte Early-culture d3 – d7 Mid-culture d11 – d14 Late-culture d18 – d21 Cell Secreted COLII Sox9 COLII COLXA1 ACAN Cell Secreted COLII COLI COLXA1 COLII COLI Cell Secreted COLI COLXA1 ACAN COLI
  53. 53. dsRNA NLRP3 Inflammasome IL-1β ↓ Cell Viability Dicer1 Mitochondria Dysfunction Autophagy Dysregulation
  54. 54. Audience Poll
  55. 55. What to DO about BCO? Primary Breeder Grower Feed and Nutrition Processor 1. Prevention 2. Detection • Phenotyping • Diagnosing 3. Treatment or Mitigation
  56. 56. Improved Mechanisms of Measurement • DXA vs. other means
  57. 57. DXA as a Tool in Avian Physiology Research What we tested • Live bird imaging • Ex vivo leg quarter analysis • Breast myopathies • In ovo imaging What we found • Skeletal diagnostics and imaging • BMD and BMC analysis across treatment groups* • Embryonic positioning *data not shown
  58. 58. Live bird imaging
  59. 59. Live bird imaging
  60. 60. In ovo and Newly Hatched Imaging
  61. 61. The Takeaway • Understanding the molecular mechanisms provides the foundation for non-invasive biomarkers and therapeutic or selection targets for BCO • Developing a reliable and relevant in vitro model of avian growth plate diseases is key for mechanistic studies • The fast screen time and accuracy make DXA imaging a promising tool in developing more robust phenotypes for bone health, skeletal disorders, and in ovo imaging • Streamlining the process and further research is key!
  62. 62. Q&A Session WWW.SCINTICA.COM INFO@SCINTICA.COM Please enter your questions in the Q&A section. Thank You!

Editor's Notes

  • Leg Bones Increase in 4X in Length & 7X in Diameter
  • cell utilizes the yellow tetrazolium salt which is metabolized by mitochondrial succinic dehydrogenase activity of proliferating cells to yield a purple formazan product by the mitochondria of viable cell .
  • Dysregulation of autophagy could be involved in the pathology of the bacterial component of BCO
  • Dysregulation of autophagy machinery is present in BCO affected femur heads which is indicative of decreased autophagy
    Challenge with a known BCO isolate also induces dysregulation of the autophagy machinery
    Inhibition of autophagy via separate mechanisms results in not only dysregulated autophagy machinery as seen in tissue and bacterially challenged cells, but also decreased cell viability
  • Courtesy of Dr. Orlowski
  • While BCO is bacterially derived, the processes involved point to metabolic, immune, and boen health and also giv ethe foundation for developing non-invasive biomarkers and therapeutic or selection targets for BCO

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