1) Influenza infection exerts prominent inflammatory and thrombotic effects on atherosclerotic plaques in aged Apo E-deficient mice. Approximately one third of the mice showed increased plaque inflammation and some died 4-10 days after influenza inoculation. 2) A protocol was tested using cytokines and SPIO nanoparticles to enhance macrophage homing to plaques in Apo E-deficient mice. Mice that received cytokines prior to SPIO injection showed more iron-labeled macrophages in plaques compared to controls. 3) The results suggest that influenza infection exacerbates plaque inflammation in mice and cytokines may account for some of the effects of influenza on atherosclerosis. Longer studies are needed to determine if plaque

1. Influenza Infection Exerts Prominent
Inflammatory and Thrombotic Effects
on Atherosclerotic Plaques of Apo
E-Deficient Mice
Silvio Litovsky, Philip Wyde, Mohammad Madjid, Adeeba Akhtar, Sameh Naguib,
Said Siadaty, Susan Sanati, Ward Casscells, Morteza Naghavi, Center for
Vulnerable Plaque Research, University of Texas-Houston, and Texas Heart
Institute, Houston, Texas.

2. Outline:
Infection/ inflammation and atherosclerosis
Influenza and complications of atherosclerosis
Influenza on aged Apo E-deficient mice

3. Am J Epidermiol. 1998;148,10:937-948
Introduction of antibiotics and reduction in CAD (hypothetical)

4. Am J Epidermiol. 1998;148,10:937-948
Aorta of rabbits experimentally inoculated with infectious agents
Streptococcus C. pneumoniae

6. Effect of INF-γ on the development of
atherosclerosis in young Apo E-
deficient mice.

7. Exogenous interferon-gamma enhances atherosclerosis in
apolipoprotein E-/- mice.
Whitman SC, Ravisankar P, Elam H, Daugherty A.
A role for interferon-gamma (IFN-gamma) has been implied in the atherogenic process. To determine
whether exogenously administered IFN-gamma exerts an effect on the development of
atherosclerosis, we intraperitoneally administered either recombinant IFN-gamma (100 U/g body
weight) or phosphate buffered saline daily for 30 days to atherosclerosis-susceptible apolipoprotein
E-/- mice (16-week-old male mice, n = 11 per group) fed a normal diet. Atherosclerotic lesion size
was quantified in the ascending aorta. The number of T lymphocytes and major histocompatibility
complex (MHC) class II-positive cells within lesions were also quantified in this region. IFN-gamma
administration reduced serum cholesterol concentrations by 15% (P = 0.02). For both groups, the
majority of cholesterol was present in very low density lipoproteins, which were modestly reduced in
mice receiving IFN-gamma. Despite the decrease in serum cholesterol concentrations, IFN-gamma
injections significantly increased lesion size twofold compared to controls (119,980 +/- 18, 536 vs.
59,396 +/- 20,017 micrometer(2); P = 0.038). IFN-gamma also significantly increased the mean
number of T lymphocytes (19 +/- 4 vs. 7 +/- 1 cells; P = 0.03) and MHC class II-positive cells (10 +/-
3 vs. 3 +/- 1 cells; P = 0.04) within lesions. These data lend further support to a pro-atherogenic role
of IFN-gamma.

8. Outline
Influenza and complications of atherosclerosis

14. Cir
Circulation. 2000;102:3039-30045

17. Outline
Influenza on aged Apo E-deficient mice

18. Background
• Influenza infection is associated with elevated C-reactive
protein and serum amyloid A, especially in the elderly
• HDL loses its anti-inflammatory properties and LDL
becomes more susceptible to oxidation during influenza
infection*
• Mouse is a standard model for influenza and the apo E K/O
mouse is a model for atherosclerosis
• LD50 in apo E K/O mouse is comparable to LD50 of wild
type
• Maximal viral titers in the lung occur on day 4 after
inoculation; animals usually die between days 4 - 10.
(Van Lenten, Circulation 2001;103:2283-8)

19. Methods
• 33 apo E K/O mice of either sex 2-2.5 years old and 10 age-
matched C57 BL were intranasally injected with 1LD50 of
influenza A virus
• Body weight, heart rate and O2 saturation determined at
inoculation, day 3, day 5 and at time of sacrifice
• Sacrificed at days 3, 5, 10 and 15. In case of spontaneous
death, autopsy was performed within 12 hours
• Virus quantification on homogenized lungs determined on
day 4 by hemagglutination
• RT-PCR for the presence of influenza mRNA in the aorta of
2 animals
• Aorta up to the level of the renal arteries were excised, fixed
in formalin and processed

20. Results
• Hemagglutinating virus isolated from every virus-
inoculated mouse.
• No clear evidence of influenza RNA on aortic samples
• 13 animals died between days 4 and 10. All inoculated
animals lost weight. Eleven infected
(7 sacrificed and 4 fatalities) showed striking intimal
infiltrates. Nine out of the 11 with intimal infiltrates
died or were sacrificed on day 10 +/- 1 day.

24. Smooth muscle actin
Mac-1
CD4

26. Cytokine-SPIO Protocol
•Purpose: To enhance macrophage homing to plaque and monitor it by
SPIO. Compare the iron particles present in macrophages of apo E-deficient
atherosclerotic plaques under baseline conditions (control group) and after the
administration of TNF-α, IL-1β and IFN-γ (influenza simulated group).

27. Cytokine-SPIO Protocol
•Protocol: Eight retired female breeders, approximately 12-month old
were divided in 2 groups. Five received I.P. 0.2 µg each of mouse
recombinant TNF- and IL-1ß; and 100 units/g INF-γ for six days; the 3 control
received 0.5 mL saline containing 1% BSA. Three hours later, all the animals
were I.V. injected with Feridex 1 mMol/kg of iron. Seven days later, the
recipients were euthanized with CO2 and perfused under physiological pressure.
The entire aorta up to the iliac bifurcation was formalin-fixed, serially sectioned
transversally and stained for H&E and iron.

28. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
No cytokines

29. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
No cytokines

30. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
Cytokines added

31. Iron Staining H&E Staining
Apo E-deficient mouse injected with SPIO
Cytokines added

32. Intramural coronary
artery involvement
Myocardial injury
Apo E-deficient mouse injected with SPIO
Cytokines added

33. Conclusion
• Influenza A virus exerts prominent pro-inflammatory
and pro-thrombotic effects in about one third of aged
apo E K/O mice
• Studies with longer follow-up periods are necessary to
determine whether increased plaque burden and/or
aneurysm formation occur
• Significance of LD 50 dose is unclear for
atherosclerosis. Other doses are being planned
• Plaque inflammation as seen in this model has not been,
to our knowledge, previously reported in experimental
models of atherosclerosis

34. Conclusion
Cytokine effect likely accounts, at least partially,
for the effects of influenza infection on the
atherosclerotic plaques

35. Acknowledgement
James T Willerson, MD
S Ward Casscells, MD
Morteza Naghavi, MD
Philip Wyde, PhD
Mohammad Madjid, MD
Adeeba Akhtar, MD
Said Siadaty, MD