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The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry, Lioubov M. Pletneva, Christopher L. Karp, Jaclyn McAlees, Theresa L. Gioannini, Jerrold Weiss, Wilbur H. Chen, Robert K. Ernst, Daniel P. Rossignol, Fabian Gusovsky, Jorge C. G. Blanco & Stefanie N. Vogel. Nature 23 May 2013 volume 497 p498-502.

The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

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Page 1: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

The TLR4 antagonist Eritoran protects mice from lethal influenza infection

 Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry, Lioubov M. Pletneva, Christopher L. Karp,

Jaclyn McAlees, Theresa L. Gioannini, Jerrold Weiss, Wilbur H. Chen, Robert K. Ernst, Daniel P. Rossignol, Fabian Gusovsky,

Jorge C. G. Blanco & Stefanie N. Vogel.

Nature 23 May 2013 volume 497 p498-502.

Page 2: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 8

Model

Page 3: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 1a

Infection Protocol

Page 4: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 1b

Survival of PR8 (Influenza virus) –infected C57BL mice.

Page 5: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Nhu QM et al. (2010) Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo. Mucosal Immunity 3, 29-30.

Figure 7c

Page 6: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 1b

Page 7: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 1a

Page 8: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suupl. 1b

Eritoran-mediated protection Is overcome by increased PR8 challenge doses.

Page 9: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 1c

Infection with human pandemic influenza A/California/07/2009 H1N.

Page 10: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 1c

Survival when Eritoran treatment starts 2, 4, and 6 days p.i.

Page 11: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 2a

Mock PR8 untreated

PR8 + E5564

(80% of sections) (20% of sections)

Page 12: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 2b

Pathology score of PR8 infection in C57Bl mice.

Page 13: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Puls-Oxymetry mouse pup.

Page 14: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 2c

Pulse-Oxymetry of PR8 infected C57Bl mice.

Page 15: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Sigmodon hispididus or Cotton rat.

Page 16: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 2d

Uninfected + E5564 H3N2 H3N2 + E5564

Lung pathology of H3N2 in cotton rats.

Page 17: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 2e

Lung Pathology of H3N2 infection in cotton rats.

Page 18: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 3

Lung viral titers after PR8 infection.

Page 19: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Calculate TCID50.

Page 20: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Suppl. Table 1

Page 21: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 2

Page 22: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Measuring DNA amplification:Every cycle releases 2 molecules of the Reporter Fluorophore from their quencher;Fluorescence measures the number of amplification cycles.

qRT- PCR

Page 23: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,
Page 24: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,
Page 25: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 4a

Tumor Necrosis Factor a Interleukin-1b

Prostaglandin endoperoxide synthase 2= Cyclo-oxygenase 2

Chemotaxin CXCL1 = MIP1

PR8-induced cytokine production in mice lungs.

Page 26: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 3a

PR8-induced cytokine production in mice lungs.

Page 27: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 3b

Page 28: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 4b

H3N2 induced cytokine production in cotton rat lungs.

Page 29: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,
Page 30: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 4

Page 31: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,
Page 32: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 5a

Page 33: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,
Page 34: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,
Page 35: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. 5b

Page 36: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 5.

Page 37: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 6

Page 38: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

(Palmitoic Acid)3 Cysteine Serine (lysine)4 is a specific agonist of TLR-2

Page 39: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Figure. OxPLs of diverse origin competitively inhibit the binding of LPS to LBP, CD14, and MD2, the soluble and cell-associated proteins that confer LPS sensitivity on the

transmembrane signaling component TLR4.

Erridge C Arterioscler Thromb Vasc Biol 2009;29:337-338Copyright © American Heart Association

Page 40: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 7

1-palmitoyl- 2- linoleyl phospho choline

1- palmitoyl- 2- arachidonoyl phospho choline

1-palmitoyl-2 (9-oxo) nonanoyl phosphocholine

1-palmitoyl- 2- expoxy-sisoprostane E2 phospho choline

Page 41: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 7

Page 42: The TLR4 antagonist Eritoran protects mice from lethal influenza infection Kari Ann Shirey, Wendy Lai, Alison J. Scott, Michael Lipsky, Pragnesh Mistry,

Shirey Kari Ann et al. (2013) Fig. Suppl. 8

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Shirey Kari Ann et al. (2013) Fig. 1a