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.

Outline:

Infection/ inflammation and atherosclerosisInfluenza and complications of atherosclerosisInfluenza on aged Apo E-deficient mice

Am J Epidermiol. 1998;148,10:937-948

Introduction of antibiotics and reduction in CAD (hypothetical)

Am J Epidermiol. 1998;148,10:937-948

Aorta of rabbits experimentally inoculated with infectious agents

Streptococcus C. pneumoniae

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

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.

Outline

Influenza and complications of atherosclerosis

Cir

Circulation. 2000;102:3039-30045

Outline

Influenza on aged Apo E-deficient mice

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)

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

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.

Smooth muscle actin

Mac-1

CD4

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).

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.

Iron Staining H&E Staining

Apo E-deficient mouse injected with SPIO No cytokines

Iron Staining H&E Staining

Apo E-deficient mouse injected with SPIO No cytokines

Iron Staining H&E Staining

Apo E-deficient mouse injected with SPIO Cytokines added

Iron Staining H&E Staining

Apo E-deficient mouse injected with SPIO Cytokines added

Intramural coronary artery involvement

Myocardial injury

Apo E-deficient mouse injected with SPIO Cytokines added

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

Conclusion

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

Acknowledgement

James T Willerson, MDS Ward Casscells, MDMorteza Naghavi, MDPhilip Wyde, PhDMohammad Madjid, MDAdeeba Akhtar, MDSaid Siadaty, MD