WO2012068355A2 - Treating aortic aneurysm by modulating toll-like receptors - Google Patents

Abstract

Methods for treating aortic aneurysm, such as abdominal aortic aneurysm (AAA), by either activating TLR2 signaling or suppressing TLR4 signaling and methods for diagnosing aortic aneurysm and assessing aortic aneurysm treatment efficacy in, e.g., a laboratory animal, based on the amount of an immune cell population such as the regulatory T cell population and/or the M1 and M2 macrophage populations. Also disclosed herein are pharmaceutical compositions for use in treating aortic aneurysm, the composition comprising a TLR2 agonist, a TLR4 antagonist, or a combination thereof.

Description

Treating Aortic Aneurysm by Modulating Toll-like Receptors

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 61/415,039, filed on November 18, 2010, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Aortic aneurysm is associated with intense inflammation of the media characterized by infiltration of macrophages and T cells and release of matrix metalloproteinases (MMPs), leading to progressive degradation of the extra cellular matrix and weakening, dissection and rupture of the aortic wall. See Schermerhorn et al., N Engl J Med. 358(5):464-474; 2008. Abdominal aortic aneurysm (AAA) is localized to the abdominal aorta. It is a potential life- threatening degenerative vascular disease that affects 6-9% of men over the age of 65 years. To date, surgery is the only therapeutic approach to AAA and is associated with a significant morbidity and mortality.

Previously, AAA had been attributed to atherosclerotic degeneration of the vessel wall. However, recent studies indicate that AAA differs from occlusive vascular disease (most commonly caused by atherosclerosis) in at least two aspects. First, degenerative changes in the media and the elastin layer occur in AAA, but not in occlusive vascular disease. Second, the distribution of T cells and the cytokines secreted in occlusive atherosclerotic and aneurysmal lesions are very different. More specifically, the former are predominantly populated by Thl cells and interferon gamma stimulating cytokines IL-12 and IL-2, while the latter contain predominantly Th2 cells and demonstrate increased levels of IL-4 and IL-10, which are not detected in stenotic lesions. See Cowan et al., Ann N Y Acad Sci. 1085: 1-10; 2006 and Brewster et al., Am J Med Sci. 326(1): 15-24; 2003.

Toll-like receptors (TLRs) are a class of molecular pattern recognition receptors that mediate the innate immune response, resulting in rapid recruitment of inflammatory cells to the site of a range of pathogen-associated molecular patterns. TLRs are found to be involved in atherosclerosis and have been implicated in the remodeling of blood vessel walls in atherosclerotic plaques. See Longo et al., J Clin Invest. 110(5):625-632, 2002; Pyo et al., J Clin Invest. 105(11): 1641-1649, 2000; and Malle et al, Cell Mol Life Sci. 66(l):9-26, 2009. For example, TLR2 and TLR4 have been found to play an important role in the development of atherosclerosis, while TLR4 has been implicated in protection of the myocardium against ischemia/reperfusion injury. See Erridge et al., Trends Cardiovasc. Med. 18(2): 52-56, 2008; and Guzik et al., J Exp Med. 204(10):2449-2460, 2007. However, it is unknown whether TLRs, such as TLR2 and TLR4, are involved in AAA development.

SUMMARY OF THE INVENTION

The present invention provides a method of treating aortic aneurysm, e.g., thoracic aortic aneurysm (TAA) or abdominal aortic aneurysm (AAA), by either activating TLR2 signaling or suppressing TLR4 signaling. It also provides methods for monitoring aortic aneurysm development and/or progress in a subject (e.g., a laboratory animal) based on the amount of an immune cell population, such as the regulatory T cell population and/or the macrophage population (including Ml and M2 macrophages).

In one aspect, this invention features a method for treating aortic aneurysm (e.g., AAA). The method includes administering to a subject in need thereof (e.g., a human patient suffering from the disease, at risk for the disease, or suspected of having the disease) an effective amount of a TLR2 agonist, a TLR4 antagonist, or a combination thereof. In one example, the subject in need is a human male at the age of 60 or over (e.g., 65 or over). The term "treating" as used herein refers to the application or administration of a composition including one or more active agents to a subject, who has aortic aneurysm, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition toward the disease.

The TLR2 agonist to be used in the method described above can be a heat-killed bacterium, an insoluble preparation of a microorganism, a liposaccharide, a lipoteichoic acid, a peptidoglycan, or a lipoprotein. Examples are, but are not limited to, PAM3CSK4 (PAM3), PAM2CSK4 (PAM2), MALP-2, and FSL-1.

The TLR4 antagonist can be a lipid A mimetic (e.g., CRX-526), a soluble TLR4 (optionally fused with the Fc fragment of an immunoglobulin), an anti-TLR4 antibody, an antisense oligonucleotide specific to TLR4 (e.g., an interfering RNA against TLR4), or an under- acylated lipopolysaccharide (e.g., cynobacterial CyP or Eritoran). It also can be a small molecule such as TAK-242.

In another aspect, the invention features a method for diagnosing aortic aneurysm, the method including determining the amount of at least one immune cell, such as at least a macrophage (Ml, M2, or both for determining, e.g., the ratio of M1/M2) in a sample obtained from a subject suspected of having aortic aneurysm (e.g., a human patient or a laboratory animal subjected to induction of aortic aneurysm), and assessing disease occurrence in the subject or predisposition to the disease of the subject based on the amount of the at least one immune cell. In one example, the amounts of Ml and M2 macrophages are determined and an elevated ratio of Ml to M2 in the sample as compared to that in an aortic aneurysm- free sample indicates that the subject has the disease or is at risk for the disease.

In yet another aspect, the invention features a method for assessing the efficacy of an aortic aneurysm treatment in a subject, such as a laboratory animal. This method includes obtaining samples from the subject either before and after an aortic aneurysm treatment, or during the course of the treatment, determining the amounts of at least one immune cell (e.g., at least a T_reg cell or at least a macrophage such as Ml and/or M2 macrophages) in the samples; and assessing whether the treatment is effective in the subject based on changes in the amounts of the at least one immune cells. When the amount of the T_reg cells is determined, an increase in the amount of the T_reg cells after the treatment or along the course of the treatment indicates that the treatment is effective. When the amounts of the Ml and M2 macrophages are determined, a decreased M1/M2 ratio after the treatment or along the course of the treatment indicates that the treatment is effective.

In any of the foregoing methods, if applicable, the T_reg cell can be CD4⁺Foxp3⁺ positive, the Ml macrophages can be CD1 lc positive, and the M2 macrophages can be CD206 positive.

Also within the scope of this invention are (a) a pharmaceutical composition for use in treating aortic aneurysm, the pharmaceutical composition containing a TLR2 agonist, a TLR4 antagonist, or a combination thereof; and (b) the use of the just-described pharmaceutical composition in manufacturing a medicament for the treatment of the disease. In some embodiments, the composition contains a TLR2 agonist or a TLR4 antagonist in an amount effective in treating aortic aneurysm (e.g., AAA).

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several examples, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

Figure 1 is a diagram showing the effects of PAM3 pretreatment on angiotensin II (Angll)-induced ApoE^_/~ mice. Panel A: The effect of PAM3 pretreatment on AAA formation in Angll-induced ApoE^_/~ mice. Panel B: The effect of PAM3 pretreatment on serum amyloid levels in Angll-induced ApoE^_/~ mice.

Figure 2 is a bar graph showing homing of CFSE-labeled CD4⁺ T cells derived form control mice and mice treated with Angll to the suprarenal aorta in Angll-induced ApoE^_/~ mice via flow cytometric analysis. Y-axis values represent the mean numbers of CFSE- positive CD4⁺ T cells in the aortas of the mice.

Figure 3 is a bar graph showing Angll-induced AAA formation and severity in

ApoE^_/~ mice and ApoE^_/~ TLR2^_/~ double knock-out mice.

Figure 4 is a chart showing the therapeutic effect of PAM3 given 7 days post- treatment on Angll-induced AAA in ApoE^_/~ mice.

Figure 5 is a diagram showing the effect of Angll and PAM3 on the distribution of CD4⁺ (Pane A), CD8⁺ (Panel B), and Foxp3⁺ (Pane C) T cell populations in iliac lymph nodes of ApoE^_/~ mice treated with saline (a vehicle control), Angll alone, and

Angll + PAM3.

Figure 6 is a bar graph showing Angll-induced AAA formation and severity in ApoE^_/~ mice and ApoE^"/ LR4^"/" double knock-out mice.

Figure 7 is a diagram showing the effect of Angll on M1/M2 macrophage levels in the supra renal aorta in ApoE-/- mice. Panel A: a diagram showing the levels of CD206- positive (M2 macrophages) and CDl lc-positive lymphocytes (Ml macrophages) in control and angiotensin (Angll) -treated mice as determined by FACS. Panel B: a chart showing the M1/M2 ratios in control and Angll- treated mice.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on unexpected discoveries that (a) PAM3, a TLR2 agonist, reduces AAA formation and severity in Angll-induced ApoE^_/~mice, a well- recognized AAA mouse model; (b) Ang II induced AAA is markedly more severe in ApoE^_/~ TLR2^_/~ double knock-out mice as compared to ApoE^_/~ mice, indicating a protective role of TLR2 from AAA formation (c) Angll-induced AAA is less severe in ApoE^_/" TLR4^_/" double knock-out mice as compared with ApoE^_/~ mice, indicating that TLR4 potentiates AAA; and (d) Angll-induced AAA formation results in a decrease of CD4⁺Foxp3⁺ T_reg cells or an increase in CD4⁺ and/or CD8⁺ T-lymphocytes in iliac lymph nodes and PAM3 rescues this AAA-induced T_reg cell decrease, and (e) Angll induction in ApoE^_/~ mice is associated with an increase in the ratio of Ml macrophages to M2 macrophages.

Accordingly, this invention features a method for treating aortic aneurysm (e.g., AAA) by either activating TLR2 signaling or suppressing TLR4 signaling. It also features methods of diagnosing aortic aneurysm and assessing therapeutic efficacy of an aortic aneurysm treatment in, e.g., a laboratory animal or a human patient, based on the amount of T_reg, CD4⁺, or CD8⁺ T-lymphocytes, and/or the amount of macrophages (including Ml and M2).

Treating Aortic Aneurysm

The aortic aneurysm treatment described herein can be effected by administering to a subject in need (e.g., a human patient having, suspected of having, or at risk of aortic aneurysm) an effective amount of a TLR2 agonist, which activates the TLR2 signaling pathway, an effective amount of TLR4 antagonist, which suppresses the TLR4 signaling pathway, or a combination thereof.

TLR2 agonists

A TLR2 agonist binds, directly or indirectly, to TLR2 or a TLR2-containing heterodimer, leading to activation of the TLR2-mediated signaling pathway. It can be a naturally- occurring molecule or a synthetic molecule (e.g., a functional variant of a naturally- occurring molecule). Exemplary TLR2 agonists include, but are not limited to, heat-killed bacteria, liposaccharides (including lipoglycans and lipopolysaccharides), lipoteichoic acids, peptidoglycans, lipoproteins, glycoproteins, lysophospholipids (e.g., lysophosphatidylserine), and insoluble microbial preparations (e.g., a cell-wall or cell-membrane preparation from a microorganism).

Heat-killed bacteria to be used as TLR2 agonists can be freeze-dried preparations of various bacteria, including cell-wall less bacteria (e.g., Acholeplasma laidlawii), Gram- positive bacteria (e.g., Listeria monocytogenes, Lactobacillus rhamnosus, Streptococcus pneumoniae, Staphylococcus aureus), and Gram-negative bacteria (e.g., Legionella pneumophila, Porphyromonas gingivalis, Pseudomonas aeruginosa, Helicobacter pylori).

Lipoglycans, including lipoarabinomannans and glycolipids, are unique components of cell membranes and bacterial envelopes. Suitable lipoglycan-based TLR2 agonists include lipoarabinomannans (LAM) and lipomannans (LM) from the cell wall of a mycobacterium (e.g., Mycobacterium smegmatis), glycophosphatidylinositol (GPI) or GPI anchor (also known as glycophosphatidylinositol) from, e.g., Trypanosoma cruzi or Plasmodium falciparum, and lipophosphoglycan (LPG) from, e.g., Leishmania major. Optionally, the LPS is not from an enterobacterium.

Lipopolysaccharides (LPS), typical or atypical, are large molecules consisting of a lipid moiety and a polysaccharide moiety joined by a covalent bond. Such molecules are found in the outer membrane of both Gram-positive and Gram-negative bacteria (e.g., Porphyromonas gingivalis). Examples of LPS molecules that can be used in the therapeutic methods described herein include, but are not limited to, LPS from Leptospira interrogans, Legionella pneumophila, Rhizobium species Sin-1, Porphyromonas gingivalis, Bacteroides fragilis (e.g., NCTC-9343), Chlamydia trachomatis LGV-1, or Pseudomonas aeruginosa (e.g., PAC-611).

Lipoteichoic acid (LTA) is a major immuno stimulatory component of Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, Leptospirosis, and Porphyromonas gingivalis. It is an amphiphile formed by a hydrophilic polyphosphate polymer linked to a neutral glycolipid. LTA stimulates immune cells through TLR2 to produce TNFa and other inflammatory cytokines. See Schwandner et al., J Biol Chem, 274(25): 17406-9; 1999;

Schroder NW. et al., J Biol Chem, 278(18): 15587-94; 2003; and Han SH. et al., Infect Immun. 71(10):5541-8; 2003.

Peptidoglycan (PGN) is a major surface component of Gram-positive bacteria (e.g., Bacillus subtilis and Staphylococcus aureus). In addition to Gram-positive bacteria, PGN also exists in certain Gram-negative bacteria, such as E. coli. PGN is embedded in a relatively thick cell wall and is usually covalently attached to other polymers, such as lipoproteins and LTAs. PGN is known to be a potent activator of NF-κΒ and TNF-a through TLR2. See Takeuchi et al, Immunity, 11(4):443-51; 1999.

Lipoproteins and glycoproteins, either naturally- occurring or synthetic, can also serve as TLR2 agonists. Examples are MALP-2 or MALP-404 from Mycoplasma, OspA from Borrelia burgdorferi, porin from Neiseria meningitides or Haemophilus influenzae, LcrV from Yersinia pestis, hemagglutinin from measles, influenza, parainfluenza, or mumps, FSL1 (a diacylated synthetic lipoprotein derived from Mycoplasma salivarium similar to MALP-2), Pam3CSK4 (also known as PAM3; a synthetic tripalmitoylated lipopeptide that mimics the acylated amino terminus of bacterial lipopeptides), and Pam2CSK4 (also known as PAM2, a synthetic diacylated lipopeptide).

Moreover, TLR2 agonists include insoluble microbial cell preparations, such as Zymosan (a cell wall preparation from S. cerevisiae) and antigen mixtures from

Propionibacterium acnes, Aspergillus fumigatus, or Candida albicans, as well as small molecules, which can be identified by compound library screening.

TLR4 antagonists

Compounds that inhibit TLR4 activity, i.e., TLR4 antagonists, are well known in the art. See, e.g., US Patent 7,592,003 (e.g., an siRNA, ribozyme, morpholino oligo, single-chain Fv, or single chain antibody specific for TLR4). Some examples are provided below: a soluble TLR4 receptor, an anti-TLR4 antibody, an antisense oligonucleotide specific to TLR4, an interfering RNA against TLR4, a lipid A mimetic, or an under-acylated

lipopolysaccharide.

A soluble TLR4 receptor is a fragment of TLR4 that does not have a functional transmembrane domain. It directly binds to a TLR4 microbial ligand, thereby blocking the TLR4 signaling pathway triggered by the ligand. When necessary, a soluble TLR4 can be fused with a protein partner, such as the Fc fragment of an immunoglobulin, to improve its stability, bioavailability, and/or therapeutic effectiveness.

An anti-TLR4 antibody useful in the AAA treatment described herein is an antibody that specifically binds to TLR4 and neutralizes its activity. Such an antibody can be a naturally- occurring antibody, an antigen-binding fragment thereof, e.g., F(ab')₂, Fab, or Fv, or a genetically-engineered antibody derived therefrom, e.g., a humanized antibody, a chimeric antibody, a single-chain antibody, or a domain antibody. A naturally- occurring antibody can be obtained from any suitable species, such as human, rabbit, mouse, guinea pig, and rat, and can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

Anti-TLR4 antibodies can be prepared via a conventional method, e.g., hybridoma technology or recombinant technology. See, e.g., Kohler et al. Nature 256:495; 1975;

Kosbor et al. Immunol Today 4:72, 1983; Cole et al. Proc. Natl. Acad. Sci. USA 80:2026, 1983; Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983; Green et al., Nature Genetics 7: 13, 1994, Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851, 1984; Neuberger et al. Nature 312:604, 1984; and Takeda et al. Nature 314:452, 1984; and U.S. Patent Nos. 4,376,110, 5,545,806, and 5,569,825, 4,946,778, and 4,704,692.

An antisense oligonucleotide specific to TLR4 is an oligonucleotide (DNA or RNA) at least a portion of which is complementary (i.e., completely or partially) to a fragment of the nucleic acid coding for TLR4 (either the sense chain or the antisense chain), i.e., capable of forming a double-strand duplex via base-pairing according to the standard Watson-Crick complementarity rules. See, e.g., US Patent Application Publication 20100111936. In one example, it is an interfering RNA (e.g., a small inhibitory RNA or siRNA) that blocks TLR4 expression via RNA interference. When necessary, the antisense oligonucleotide to be used in the aortic aneurysm treatment described herein can be modified, e.g., containing a modified nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or any combination thereof, or a non-phosphodiester linkage between two of its nucleotides.

A lipid A mimetic is structurally similar to the lipid A moiety of a microbial lipopolysaccharide (LPS) and competes against that LPS for binding to the same site on MD-2, resulting in blockage of LPS-dependent TLR4 activation. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. Kobayashi et al., J. Immonol. 176:6211-6218; 2006. Lipid A mimetics that inhibit TLR4 activity are well-known in the art. Examples are under- acylated lipid A (i.e., having 5 or less acyl groups), CRX-526 (aminoalkyl-glycosaminide phosphate), and those disclosed in US Patent No. 7,727,974. A LPS that contains a lipid A mimetic (e.g., an under- acylated lipid A) can also serve as a TLR4 antagonist. Under-acylated LPS molecules include, but are not limited to, cyanobacterial LPS CyP (see Macagno et al., J. Experimental Medicine 203: 1481-1492; 2006), R. sphaeroides LPS provided by InvivoGen, San Diego, CA; and eritoran (also known as E5564). Eritoran is a commercially available drug provided by Eisai Co. for treating severe sepsis.

A TLR4 antagonist can also be a small molecule such as resatorvid (also known as TAK-242). Other small molecule TLR4 antagonists can be identified by compound library screening.

Treatment

Any of the TLR2 agonists and TLR4 antagonists described above include the compounds themselves, as well as their salts and prodrugs, if applicable. The salts, for example, can be formed between a positively charged substituent (e.g., amino) on a compound and an anion. Suitable anions include, but are not limited to, chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a negatively charged substituent (e.g., carboxylate) on a compound can form a salt with a cation. Suitable cations include, but are not limited to, sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as teteramethylammonium ion. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing the compounds described above. See Goodman and Gilman's The Pharmacological basis of Therapeutics, 10^th ed., McGraw-Hill, Int. Ed. 2001, "Biotransformation of Drugs".

The TLR2 agonists and TLR4 antagonists described herein are either commercially available, e.g., provided by InvivoGen, San Diego, CA, or can be prepared by routine technology, such as isolation from natural sources or chemical synthesis. Their activity to trigger TLR2 signaling or suppress TLR4 signaling, thereby preventing or ameliorating aortic aneurysm, can be verified by either in vitro or in vivo assays known in the art, for example, the Angll- stimulated ApoE^_/~ mouse model described in the Examples below.

To perform the aortic aneurysm treatment of this invention, a TLR2 agonist and/or a TLR4 antagonist can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for administration to a subject in need of the treatment. A pharmaceutically acceptable carrier is compatible with the active ingredient(s) in the composition (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents such as cyclodextrins, which form more soluble complexes with certain TLR2 agonist/TLR4 antagonist, or more solubilizing agents, can be utilized as pharmaceutical carriers for delivery of the agonist/antagonist. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, sodium lauryl sulfate, and D&C Yellow # 10. See, e.g., Remington's Pharmaceutical Sciences, Edition 16, Mack Publishing Co., Easton, Pa (1980); and Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Tenth Edition, Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001.

The pharmaceutical composition mentioned above, containing an effective amount of a TLR2 agonist, a TLR4 antagonist, or a combination thereof, can be administered to a subject in need of the treatment via a suitable route, e.g., oral, parenteral, by inhalation spray, topical, rectal, nasal, buccal, vaginal, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

A sterile injectable composition, e.g., a sterile injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation. See, e.g., Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing Co., Easton, Pa (1980); and Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Tenth Edition, Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001.

A composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added. A nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation. The pharmaceutical composition described herein can also be administered in the form of suppositories for rectal administration.

"An effective amount" as used herein refers to the amount of a TLR2 agonist, a TLR4 antagonist, or a combination thereof that alone, or together with further doses or one or more other active agents, produces the desired response, e.g. activating TLR2 signaling or suppressing TLR4 signaling. In the case of treating a particular disease or condition associated with TLR2 or TLR4 signaling, such as aortic aneurysm, the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods, such as physical examination, X-ray analysis, ultrasound analysis, computed tomography (CT), and magnetic resonance imaging (MRI), or can be monitored according to the diagnostic/efficacy assessing methods described herein, which are particularly useful in assessing therapeutic efficacy of an aortic aneurysm treatment in animal models. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

The interrelationship of dosages between animals and humans (e.g., based on milligrams per meter squared of body surface or milligrams per body weight) is well known in the art. See, e.g., Freireich et al., (1966) Cancer Chemother Rep 50: 219. Body surface area may be approximately determined from height and weight of the patient.

In one example, the amount of the TLR2 agonist or TLR4 antagonist is effective in increasing a regulatory T cell population (e.g., CD4⁺Foxp3⁺ T cells, CD4⁺CD25⁺ T cells, or CD4⁺CD25⁺Foxp3⁺ T cells) at an aorta site where aneurysm occurs or may occur. In another example, the amount of the TLR2 agonist or TLR4 antagonist is effective in decreasing CD4⁺ and/or CD8⁺ T cell infiltration. In yet another example, the amount of the TLR2 agonist or TLR4 antagonist is effective in increasing the amount of M2 macrophages, decreasing the amount of Ml macrophages, or decreasing the ratio of the Ml macrophage to M2

macrophage. Human dosage of the TLR2 agonist or TLR4 antagonist for achieving either goal can be extrapolated from data obtained from animal models.

A subject to be treated by the method described above can be a human patient suffering from aortic aneurysm (e.g., AAA), at risk for, or suspected of having the disease. A patient having preexisting aneurysm can be identified by a routine medical procedure, e.g., physical examination, X-ray analysis, ultrasound analysis, computed tomography (CT), and magnetic resonance imaging (MRI). A subject at risk for developing the disease can be a smoker, a diabetic, a human patient having high blood pressure or serum cholesterol. In addition, males at the age of 60 or above and individuals suffering from certain genetic diseases (e.g., Ehlers-Danlos syndrome and Marfan' s syndrome) or with first-degree relatives having aortic aneurysm are more likely to develop aortic aneurysm, particularly AAA, as compared with the general population. In some embodiments, males at the age 60 or above are identified as human subjects at risk for developing AAA. In others, human subjects who are at risk for AAA are identified based on their family history of AAA and/or occurrence of one of more of the above-noted genetic diseases. A subject suspected of having aortic aneurysm might be asymptomatic or show one or more symptoms of the disease, including belly, chest, or back pain and discomfort. Such subjects can be identified via routine medical procedures.

Diagnosing Aortic Aneurysm

Aortic aneurysm (e.g., AAA) can be diagnosed based on the amount of a particular immune cell population, such as macrophages (including both Ml and M2 macrophages), a particular T cell population, for example, CD4⁺, CD8⁺, and/or regulatory T (T_reg) cells (e.g., CD4⁺Foxp3⁺ T cells, CD4⁺CD25⁺ T cells, or CD4⁺Foxp3⁺CD25⁺ T cells), or a combination of two or more immune cell populations. For example, AAA can be diagnosed based on the amount(s) of Ml and/or M2 macrophages or the M1/M2 ratio in a subject. This diagnostic approach is particularly useful in monitoring aortic aneurysm development in an animal model.

To practice this diagnostic method, a sample can be taken from a candidate subject, i.e., a subject suspected of having aortic aneurysm as those described above, or a laboratory animal subjected to induction of the disease. The sample can be a lymph node biopsy sample, which can be obtained by, e.g., fine-needle aspiration or core needle aspiration. Alternatively, it can be a tissue sample from a site of aorta, such as a suspected aortic aneurismal lesion or the suprarenal aorta. The amount of a target immune cell population, such as a T cell population, (preferably T_reg, CD4⁺, and/or CD8⁺ T cells) or macrophage populations (Ml and/or M2), in the sample is determined by a routine method, e.g., FACS analysis or in situ immuno staining. This level is then compared with a reference point to determine whether the subject has the disease. Typically, this reference point is the representative amount of the same immune cell population in an aneurysm-free sample, which can be derived from an aneurysm-free subject or from an aneurysm-free region of the same subject. An increased amount of CD4⁺ or CD8⁺ T cells or a decreased level of the T_reg cells in the candidate subject as compared with the reference point is indicative of aortic aneurysm occurrence. When the amount of macrophages is used as a biomarker for diagnosing aortic aneurysm, an increased amount of Ml or a decreased amount of M2 in a subject relative to that in an aortic aneurysm-free subject indicates that the subject has the disease. Alternatively, an increased M1/M2 ratio in a candidate subject as compared with the reference point is indicative of aortic aneurysm occurrence.

In some embodiments, the diagnostic method described above is carried out by determining both the amount of T_reg cells and the amount of macrophages (Ml, M2, or both) in a suitable sample from a subject (e.g., a human patient suspected of having AAA or a laboratory animal) and assessing whether the subject has the disease based on both the amount of T_reg cells and the amount of macrophages. For example, a decreased level of T_reg cells, an increased level of CD4⁺ and/or CD8⁺ T cells, and/or an increased M1/M2 ratio indicate that the subject has AAA.

Regulatory T cells (also known as suppressor T cells) are a subpopulation of T cells that regulate (e.g., suppress) the activation of the immune system, thereby maintaining tolerance to self-antigens. Typically, regulatory T cells express, e.g., CD4, CD25, and/or Foxp3, which are cell surface markers of T regulatory cells. Thus, such T cells can be detected by one or more of these surface markers.

Macrophages are a type of white blood cells that ingest foreign materials. This type of immune cell plays an important role in immune responses against foreign invaders such as infectious microorganisms. Macrophages include two subtypes, Ml macrophages and M2 macrophages. Ml macrophages, typically characterized as CD1 lc-positive, produces proinflammatory cytokines and acts as a cell-killing effector. M2 acts to dampen inflammatory responses via production of scavenger receptors and IL- 1 receptor antagonist, and decreased production of proinflammatory cytokines such as IL-Ιβ. Gough, Aci. Signal 2(81):ec252 (2009).

Assessing Treatment Efficacy

The amount of any of the immune cell populations described above can be used as a biomarker to assess efficacy of an aortic aneurysm treatment in a subject in need, particularly in a laboratory animal bearing aortic aneurysm. In this method, biosamples as described above can be collected from the subject before, during, and/or after a treatment. In one example, a pre-treatment sample and a post-treatment sample are obtained. In another example, samples at multiple time intervals during a treatment can be obtained. The amounts of one or more of the immune cell populations in the samples are determined by a routine method. The efficacy of the treatment can be determined based on the amounts of the immune cell population(s) before, during, and after the treatment. For example, if the amount of CD4⁺ or CD8⁺ T cells remains the same or decreases along the course of the treatment (that is, the amounts of the T cells remain the same or are lower in samples taken at intervals later in the course of the treatment as compared to those in samples taken at intervals earlier in the course of the treatment), it indicates that the treatment is effective. On the other hand, if the amount of T_reg increases along the course of the treatment, it indicates that the treatment is effective. In another example, if the amount of Ml macrophages remains the same or decreases, or if the amount of M2 macrophages remains the same or increases, it indicates that the treatment is effective. Efficacy of an aortic aneurysm treatment can also be assessed based on the M1/M2 ratio. That is, if this ratio remains the same or decreases along the course of the treatment, it is indicative of the treatment being effective.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLE 1: Treating abdominal aortic aneurysm with TLR2 agonist PAM3

(a) Induction and characterization of abdominal aortic aneurysm development in ApoE^'A mice induced with angiotensin II

ApoE^_/~ mice, obtained from Jackson Laboratories (see Piedrahita et al., Proc Natl Acad Sci U SA. 89(10):4471-4475, 1992) were implanted subcutaneously with Alzet pumps calibrated to deliver 600 ng/kg/min angiotensin II (Angll) or saline. Four weeks after the implantation, the mice were examined by ultrasonographic analysis to determine AAA formation, which typically appears as para-aortic structures. These structures were

characterized as those which communicate directly with the aortic lumen, may or may not demonstrate blood flow, and in some cases contain densities consistent with the presence of clot. Views in both the transverse and longitudinal plane helps establish the continuity of the aneurysms with the aorta and visualize the areas of structural independence of the 2 lumens. To study the time course of Angll-induced AAA formation, mice were followed by ultrasonography every 8-10 days after the Angll induction and the development of aneurysmal structures determined.

After 28 days, both the Angll-treated and the saline-treated mice were perfused with 4% paraformaldehyde or PBS, the aorta exposed under a dissecting microscope and the periadventitial tissue carefully removed from the aortic wall. Aneurysm severity was rated from Type I to Type IV detailed below according to the method described in Daugherty et al, Br. J. Pharmacol. 134(4):865-870; 2001 ("the Daugherty classification").

Type I: dilated lumen without thrombus;

Type II: remodeled aneurysmal tissue with little thrombus;

Type III: a pronounced bulbous form of Type II with thrombus;

Type IV: multiple, often overlapping aneurysms containing thrombus; multiple overlapping aneurysms in the supra renal region as well as

involvement of the aortic arch and the thoracic aorta.

The outer diameter of the suprarenal aorta was measured with a caliper. Neovascularization was determined by staining of aortas in situ with anti CD31.

Histological analysis was also performed to evaluate AAA formation in the treated mice. Briefly, perfusion-fixed aortas were embedded in paraffin, cut in cross-section (5-10 μιη) and stained with hematoxylin and eosin. These serial cryostat sections taken from equivalent regions of the suprarenal aorta can be stained with specific antibodies for immunohistochemical analysis. See Gotsman et al., J Clin Invest. 117(10):2974-2982, 2007. More specifically, these tissue samples were stained with rat anti-mouse Mac3, anti-CD8, anti-CD4 (all from Pharmingen), and antibody FJK-16s (an anti-Foxp3 antibody from eBioscience) for determining presence of macrophages and T cells. They can also be stained with Verhoeff Van Geison (VVG) to determine the levels of elastin and an anti-CD31 antibody to detect endothelial cells. Quantification of inflammatory infiltration degree (i.e., counting individual positively stained lesion cells) can be performed using IP Lab software (Scanalytics, Billerica, MA), which would permit identification of regions with differences in the distribution of color. The region of brown Mac-3 staining in each section is to be determined and normalized to the total area of the aorta in that low power field. In situ CD31 staining can be carried out as described in Zhang et al Arterioscler Thromb Vase Biol. 2009 Nov;29(l 1): 1764-71 and quantitated by counting microvessels per high power field.

When necessary, blood pressure can be monitored in the treated mice as follows. Systolic blood pressures is measured in conscious mice using a computerized tail-cuff system (Kent Scientific Corporation). To avoid procedure-induced anxiety, mice are initially acclimated to the instrument for 3 consecutive days before the actual measurement. On the fourth day, pressures are measured in both the morning and afternoon.

Moreover the first 5 of 30 blood pressure values at each session are disregarded, and the remaining 25 values averaged and used for analysis. Total serum cholesterol is determined enzymatically.

The results obtained from this study show that 12 out of 16 (75%) ApoE^_/~ mice treated with Angll infusion developed AAAs. Of these, 9 demonstrated either Type III or TypelV aneurysms See Figure 1, Panel A. In a study of the time course of AAA formation in response to ANG II , at day 3 following ANG II treatment none of the mice demonstrated signs of AAA formation as determined by the Dougherty classification described above; 2 out of the 14 mice demonstrated Type III AAAs at day 7, and at day 28, 10 out of the 14 mice showed signs of Type III and Type IV aneurysms. In addition, infiltration of both CD8⁺ and CD4⁺ T cells was observed in the media of aneurysms of ApoE^_/~ mice treated for 28 days with Angll.

Additionally, in a homing experiment CD4⁺ cells from 8 mice treated with Angll or placebo for 14 days as described above were pooled and stained with CFSE. Six recipient mice were injected via the retro-orbital vein with 5xl0⁶ CFSE labeled CD4⁺ cells isolated from the Angll-treated mice and 3 recipients were injected with cells from placebo-treated mice. Aortas were harvested after 48 hours, digested with a mixture of collagenase type XI, hyaluronidase, DNAsel and collagenase I. Three aortas were pooled for digestion in order to obtain sufficient cells for FACS analysis. Cell suspensions of splenocytes and aortic digests were analyzed by flow cytometry. FACS analysis demonstrated about 3 times more CD4⁺ cells in the suprarenal aorta from Ang-II treated donors compared to placebo and no difference in the spleen. See Figure 2. Furthermore, 4 of 6 recipients who received cells from Angll-treated donors demonstrated severe Type IV aneurysms with involvement of the thoracic aorta, which were markedly more severe than AAAs observed in other mice at this dose of Angll. These data demonstrated that CD4+ cells from ANG II treated ApoE-/- mice migrated preferentially to the area of aneurysm formation in the aortas of recipient mice compared to CD4+ cells form placebo treated mice and that these mice developed more severe aneurysms.

(b) PAM3 treatment prior to Angll induction

To study the protective effect of PAM3 on AAA development, ApoE^"7" mice were pretreated for 24 hours with 50 μg/mouse PAM3 injected LP., prior to Angll infusion. After 28 days, only 5 of 16 mice pretreated with PAM3 (32%) developed AAAs, all of which were either Type I or Type II. By contrast, 12 out of 16 (75%) of mice treated with Angll alone developed aneurysms. See Figure 1, Panel A. The distribution of aneurysms between no aneurysms (none), Type I, Type II, Type III, and Type IV was significantly different between the ApoE^"7"mice treated with PAM3 prior to the Angll induction and the mice induced with Angll alone, as determined by the Likelihood Ratio χ2 20.2 (P = 0.00045), the Goodman and Kruskal Tau 0.483 (P = 0.005), and the Cramer's V 0.695 (P = 0.004). These data demonstrate that PAM3 decreases the incidence and severity of Angll- mediated AAA formation in ApoE^"7" mice. In addition, none of the 17 mice treated with PAM3 alone developed aneurysms.

The levels of an acute-phase protein serum amyloid A (SAA) were examined in the PAM3 -pretreated ApoE^"7" mice to determine whether PAM3 treatment was associated with an appropriate inflammatory response. SAA serves as a marker for acute and chronic inflammatory diseases. See Malle et al., Cell Mol Life Sci. 66(l):9-26, 2009. As shown in Figure 1, Panel B, both PAM3 and Angll stimulated SAA release, despite the fact that PAM3 markedly decreased AAA severity (see Figure 1 Panel A). This result indicates that the therapeutic effect of PAM3 on AAA did not reflect an inhibition of the systemic

inflammatory process.

ApoE^"7" TLR2^"7" double knock-out mice were used to determine whether the protective effect of PAM3 is dependent upon the TLR2 signaling pathway. ApoE^"7" mice and TLR2^"7" mice were obtained from Jackson Laboratories. See Piedrahita et al., Pwc Natl Acad Sci U S A. 89(10):4471-4475, 1992; and Spiller et al., J Biol Chem. 282(18): 13190-13198, 2007. These two types of knock-out mice were crossed to produce the double knock-out mice.

Their phenotype was determined by the absence of MALP-2 stimulation of MCP-1 mRNA in smooth muscle cells derived from ApoE^{" "} double knock-out mice.

ApoE^_/~ TLR2^_/~ double knock-out mice were subjected to Angll infusion to induce AAA formation following the procedures described above. The results thus obtained indicate that the effect of Angll infusion on AAA formation was markedly increased in the double knock-out mice as compared to Angll treated ApoE^_/~ controls. More specifically, of the 19 ApoE^~/ LR2^~/~ double knock-out mice studied, 16 or 84% developed aneurysms compared to 69% of the ApoE^_/~ mice. Most importantly, the severity of the aneurysms was significantly more marked in ApoE^_/~ TLR2^_/~ double knock-out mice. Seven of the 19 ApoE^_/" TLR2^_/" mice demonstrated a severe form of the Type IV lesion that extended into the thoracic aorta, compared with only 1 of 16 ApoE^_/~ mice treated with ANG II who developed a Type IV aneurysm. See Figure 3. These differences were statistically significant: Likelihood Ratio χ2 13.1 (P = 0.011), the Goodman and Kruskal 0.320 (P = 0.028), and the Cramer's V 0.566 (P = 0.024).

Alternatively, the bone marrow transplantation assay described below can be employed to confirm that PAM3 protects against AAA development through TLR2 signaling.

Bone marrow transplantation is carried out as described in Zhong et al., Proc Natl Acad Sci U SA. 106(11):4372-4377, 2009. Briefly, twenty-eight 4-month old male ApoE^_/" TLR2^_/~ double knock-out mice, as recipients, are subjected to 1,000 rads of total body irradiation. Among them, 4 are treated with saline loaded Alzet pumps, 6 with Alzet pumps releasing Angll at 600 ng/kg/min, 6 with PAM3 alone (50 μg/mouse i.p./week), 6 pretreated with PAM3 followed by installation of ANGII loaded pumps. The 6 control ApoE^_/" TLR2^_/" double knock-out mice that receive marrow transplants from ApoE^_/~ TLR2^_/~ double knockout mice are treated with Angll releasing pumps and 3 of these mice pre-treated with PAM3.

Bone marrow cells for transplantation into the irradiated mice are prepared by flushing both femurs of 28 male ApoE^_/~ donor mice of the same age. Irradiated mice are injected via the tail vein with 2 x 10⁶ bone marrow cells. As a control, six of the mice receive bone marrow cells from ApoE^_/~ TLR2^_/~ double knock-out mice. To determine whether the bone marrow of the recipient is fully populated by the donor's bone marrow cells, after 28 days, bone marrow cells from the recipient femur are subjected to PCR genotyping for the presence of the TLR2 gene.

Aortas are harvested from the recipient mice and the incidence and severity of AAA were determined. A decrease in AAA formation/severity in the mice receiving TLR2- positive bone marrow cells indicates that TLR2 plays an essential role in the protective effect of PAM3.

Without being bound by theory, PAM3 might inhibit antigen presenting dendritic cells (DCs) from activating T cells. This can be confirmed by comparing the ability of DCs in iliac lymph nodes from Angll-treated ApoE^_/~ mice in the presence and absence of PAM3 pretreatment to stimulate the proliferation and activation of naive CD4⁺ T cells.

DCs can be isolated from minced and collagenase-treated iliac lymph nodes from saline-treated ApoE^_/" mice, PAM3 treated mice, ANGII treated mice, and PAM3 + ANGII treated mice, using a commercially available kit that relies on anti-CD 1 lc-coated magnetic beads (Miltenyi Biotec), as described in Packard et al, Circ Res. 103(9):965-973, 2008. The DCs are pretreated with mitomycin C (25 mcg/ml) to inactivate mitotic activity. The purity of these cells are determined by FACS analysis, using anti CD1 lc⁺ antibody. The phenotype of these DCs will be determined by staining for CD80, CD86, and class II MHC, as described also in Packard et al, 2008.

Naive ovalbumin peptide 323-339 (OVA) specific, H-2K^b restricted CD4⁺ T cells from spleens of OT-II TCR transgenic mice are isolated by using anti-CD4 immunomagnetic microbeads (Miltenyi Biotec), as also described in Packard et al, 2008. The cells are cultured with the DCs at T:DC ratios of 1: 1 and 10: 1, using 2 xl0⁴ T cells, and the Ova peptide are added at 1 and 10 μg/ml. Culture supernatants are removed at 48 hours and analyzed by flow cytometry-based cytokine bead assays of culture supernatants for IFN-γ, TNFa, IL-2, IL-4 and IL-10 cytokines (Pharmingen). See Dong et al, Nat Med. 5(12): 1365- 1369, 1999. Cultures are assayed for proliferation after 64 hours by [ H]-thymidine uptake (1 μΟΛνεΙΙ), added 16 hours before harvest, following the method described in Gotsman et al., J Clin Invest. 117(10):2974-2982, 2007. Identical but separate cultures are prepared for harvesting of T cells, staining for CD4, CD25, CD69, CD62L and expression of T_reg specific markers: Foxp3, CD25^hl, CD127L and GITR, and FACS analysis. See Banham et al., Trends Immunol. 27(12):541-544, 2006.

(c) PAM3 treatment after Angll induction

AAA was induced in ApoE^_/~ mice by Angll infusion following the procedures described above. Saline or PAM3 was given i.p. 7 days after initiation of Angll infusion and the dose repeated at 7-day intervals. The development of AAAs was monitored weekly by ultrasonography as described above. After 28 days, aortas were harvested and the disease severity of each treated mouse was evaluated as described above to confirm the effectiveness of PAM3 in treating preexisting AAA.

The results obtained from this study show that while 75% of mice treated with Angll alone developed either Type II, III or IV aneurysms, 7 out of 15 mice (47%) treated with Angll followed by PAM3 demonstrated no aneurysm, 4 (27%) showed mild aneurysms (Type I), and only 4 (27%) developed Type II, III or IV aneurysms, See Figure 4. The distribution of aneurysms was significantly different between the 2 treatment groups as determined by the Likelihood Ratio χ2 test 13.9 (P = 0.008), the Goodman and Kruskal Tau test 0.373 (P = 0.024), and the Cramer's V test 0.611(P = 0.021). No significant aneurysm distribution difference was observed between the mice treated with PAM3 before Angll induction and the mice treated with PAM3 after Angll induction. EXAMPLE 2: T cell distributions in iliac draining lymph nodes in Angll- induced mice and in

PAM3-pretreated and Angll-induced mice

Two or three-color flow cytometry was performed by standard protocol to examine the distribution of T cells in spleen and draining lymph nodes in mice treated with Angll alone or in mice treated with both Angll and PAM3. Briefly, cells were prepared from spleens and draining iliac lymph nodes from control mice, mice treated with Angll alone, and mice treated with AngII+PAM3. CD4⁺, CD8⁺, and CD4⁺Foxp3⁺ cells therein were quantitated by FACS analysis.

Analysis of 3 iliac lymph nodes from 3 sets of mice demonstrated that CD4⁺ and CD8⁺ cells were increased in Angll treated mice, as compared to the control. Surprisingly, the numbers of CD4⁺ and CD8⁺ cells T cells were decreased in the draining lymph nodes of mice treated with both PAM3 and Angll. See Figure 5, Panels A and B.

Furthermore, Angll treatment demonstrated a 50% decrease in CD4⁺Foxp3⁺ T_reg cells relative to the control. Co-treatment with Angll and PAM3 resulted in a nearly 2 fold increase in CD4⁺Foxp3⁺ T_reg cells as compared with the control. See Figure 5, Panel C. No effect was seen in splenocytes from these mice. These results demonstrate that PAM3 reversed the effect of Angll on T cell distribution in ApoE^_/~ mice.

The results described above demonstrate that the amount of a particular T cell population, such as CD4⁺ T cells, CD8⁺ T cells, or CD4⁺Foxp3⁺ T_reg cells, is associated with AAA formation/severity. Thus, it is a reliable marker for diagnosing AAA occurrence and for assessing the therapeutic effectiveness of an AAA treatment. Most importantly, the CD4+Foxp3+Treg cells have been shown to exert a protective effect against inflammatory responses.

EXAMPLE 3: TLR4 signaling potentiates Angll-induced abdominal aortic aneurysm

formation

TLR4^_/~ mice (see Hoshino et al., J Immunol.162(7):3749-3752, 1999) were crossed with ApoE^_/~ mice following routine procedures and the ApoE^_/~ TLR4^~/~ double knock-out phenotype was determined by the absence of LPS stimulation of MCP-1 mRNA. AAA was induced in the ApoE^_/~ TLR4^_/~ double knock-out mice by Angll infusion as described in Example 1 above.

Surprisingly, the incidence of AAA formation and disease severity were much lower in ApoE^_/~ TLR4^~/~ double knock-out mice as compared to ApoE^_/~ mice. In one experiment, 10 out of the 13 (77%) of mice studied developed either no aneurysms, or a the mild Type I aneurysm, while 3 developed either Type II or Type III aneurysm. See Figure 6. The distribution of aneurysm severity between Angll treated ApoE-/- (n=16) and ApoE^"/ LR4^"/" (n=13) mice were significantly different as determined by the Likelihood Ratio χ test 10.1 (P = 0.039), Goodman and Kruskal Tau 0.301 (ns) (P=0.077), and Cramer's V 0.548 (ns) (P = 0.069). The differences between Angll-treated ApoE^~/ LR2^~/~ double-knockout mice (n=19) and Angll- treated ApoE^"/ LR4^"/" double -knockout mice (n=13) were also statistically significant: Likelihood Ratio 13.06 (P = 0.011), Goodman and Kruskal Tau 0.329 (P = 0.037), and Cramer' s V 0.573 (P = 0.033).

In sum, the results shown above suggest that TLR4 signaling potentiates the effects of Angll on AAA formation. In other words, reducing TLR4 signaling would be an effective approach to treating AAA.

Example 4: Effect of Angll Induction on M1/M2 Macrophage Levels in Supra Renal Aorta in

ApoE^_/~ mice

ApoE-/- mice were implanted subcutaneously with Alzet pumps calibrated to deliver either Angll at the rate of 750ng/Kg/min or saline for nine days. The abdominal aorta was then digested following the method described in Galkana et al., J. Exp. Med., 203: 1273-1282 (2006). Briefly, each aorta was individually digested in 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I-s, 60 U/ml Dnasel, and 450 U/ml collagenase type I (Sigma- Aldrich) in PBS containing 20 mM Hepes at 37 °C for one hour. Cells released from the aorta sample after digestion were filtered through a 70-μΜ strainer to obtain a single suspension. Cells were then treated with Fc-block (BD Pharmingen) prior to staining with APC-Cy7 conjugated anti-CD45.2 antibodies, phycoerythrin conjugated anti-CDl lc antibodies, APC conjugated F4/80 antibodies, (all from BD Pharmingen) and Alexa Fluor 488-conjugated CD206 antibodies (from Biolegend) for 30 minutes at 4 °C. The stained cells were analyzed on the FacsCantoll using the FloJo software. F4/80 is a macrophage specific label; CD206 is a marker for the M2 "healing" macrophage subfamily; and CD1 lc is a marker for the Ml proinflammatory classic macrophage subfamily. The numbers of the Ml and M2 macrophage cells were counted by FACS analysis and their ratio calculated.

As shown in Figure 7, the results obtained from this study indicate that the ratio of M1/M2 is significantly elevated in Angll- induced ApoE^_/~ mice as compared to the controls, indicating that the ratio of M1/M2 is associated with aortic aneurysm development. Thus, this ratio can be used as a marker in diagnosing aortic aneurysm and/or monitoring the efficacy of an aortic aneurysm therapy.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any

combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

What is claimed is:

1. A pharmaceutical composition for use in treating aortic aneurysm, the composition comprising (a) a pharmaceutically acceptable carrier, and (b) a toll-like receptor 2 (TLR2) agonist, a toll-like receptor 4 (TLR4) antagonist, or a combination thereof.

2. The pharmaceutical composition of claim 1, wherein the composition comprises a TLR2 agonist in an amount effective in treating aortic aneurysm.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the TLR2 agonist is a heat-killed bacterium, an insoluble preparation of a microorganism, a

liposaccharide, a lipoteichoic acid, a peptidoglycan, or a lipoprotein.

4. The pharmaceutical composition of any of claims 1-3, wherein the TLR2 agonist is selected from the group consisting of PAM3CSK4 (PAM3), PAM2CSK4 (PAM2), MALP-2, and FSL-1.

5. The pharmaceutical composition of any of claims 1-4, wherein the

composition comprises a TLR4 antagonist in an amount effective in treating aortic aneurysm.

6. The pharmaceutical composition of claim 5, wherein the TLR4 antagonist is a lipid A mimetic, a soluble TLR4, an anti-TLR4 antibody, an antisense oligonucleotide specific to TLR4, an interfering RNA against TLR4, or an under- acylated lipopolysaccharide.

7. The pharmaceutical composition of claim 5 or claim 6, wherein the TLR4 antagonist is selected from the group consisting of CRX-526, TAK-242, eritoran,

cynobacterial CyP, and R. sphaeroides LPS.

8. The pharmaceutical composition of any of claims 1-7, wherein the

composition comprises a TLR2 agonist, a TLR4 antagonist, or a combination thereof in an amount effective in treating AAA.

9. The pharmaceutical composition of any of claims 1-8, wherein the

composition is for use in treating AAA in a subject suffering from AAA, at risk for AAA, or suspected of having AAA.

10. The pharmaceutical composition of claim 9, wherein the subject is a male human at or above age 60.

11. A method for treating aortic aneurysm, comprising administering to a subject in need thereof an effective amount of a toll-like receptor 2 (TLR2) agonist and/or a toll-like receptor 4 (TLR4) antagonist.

12. The method of claim 11, wherein the subject is administered with an effective amount of a TLR2 agonist.

13. The method of claim 11 or claim 12, wherein the TLR2 agonist is a heat-killed bacterium, an insoluble preparation of a microorganism, a liposaccharide, a lipoteichoic acid, a peptidoglycan, or a lipoprotein.

14. The method of claim 13, wherein the TLR2 agonist is selected from the group consisting of PAM3CSK4 (PAM3), PAM2CSK4 (PAM2), MALP-2, and FSL- 1.

15. The method of any of claims 11-14, wherein the subject is administered with an effective amount of a TLR4 antagonist.

16. The method of claim 15, wherein the TLR4 antagonist is a lipid A mimetic, a soluble TLR4, an anti-TLR4 antibody, an antisense oligonucleotide specific to TLR4, an interfering RNA against TLR4, or an under- acylated lipopolysaccharide.

17. The method of claim 15 or claim 16, wherein the TLR4 antagonist is selected from the group consisting of CRX-526, TAK-242, eritoran, cynobacterial CyP, and R.

sphaeroides LPS.

18. The method of any of claims 11-17, wherein the subject is suffering from

AAA.

19. The method of any of claims 11-17, wherein the subject is at risk for AAA or is suspected of having AAA.

20. The method of claim 19, wherein the subject is a male human at or above age 60.

21. A method for diagnosing aortic aneurysm, comprising

determining the amount of at least one macrophage in a sample from a subject suspected of having aortic aneurysm; and

assessing whether the subject has aortic aneurysm based on the amount of the at least one macrophage,

wherein the amount of the at least one macrophage deviating from that in an aortic aneurysm- free sample indicates that the subject has aortic aneurysm.

22. The method of claim 21, wherein the amounts of both Ml and M2

macrophages are determined and wherein an elevated ratio of Ml to M2 in the sample as compared to that in the aortic aneurysm-free sample indicates that the subject has aortic aneurysm.

23. The method of claim 21 or 22, wherein the subject is a laboratory animal.

24. The method of any of claims 21-23, wherein the subject is a human patient.

25. A method for assessing the efficacy of an aortic aneurysm treatment in an subject having aortic aneurysm, the method comprising

obtaining samples from the subject before and after an aortic aneurysm treatment, determining the amounts of at least one immune cell in the samples, the at least one immune cell being at least a regulatory T cell (T_reg) or at least a macrophage; and

assessing whether the aortic aneurysm treatment is effective in the subject based on the amounts of the at least one immune cell,

wherein a change of the amount of the at least one immune cell after the treatment indicates that the aortic aneurysm treatment is effective.

26. The method of claim 25, wherein the amount of the T_reg cell is determined and wherein an increase in the amount of the T_reg after the treatment indicates that the aortic aneurysm treatment is effective.

27. The method of claim 26, wherein the T_reg cell is CD4 and Foxp3 positive.

28. The method of any of claims 25-27, wherein the amounts of both Ml and M2 macrophages in the samples are determined and wherein a decrease of the M1/M2 ratio after treatment indicates that the aortic aneurysm treatment is effective.

29. The method of any of claims 25-28, wherein the subject is a laboratory animal.

30. The method of any of claims 25-28, wherein the subject is a human patient.

31. A method for assessing the efficacy of an aortic aneurysm treatment in a subject, the method comprising

obtaining samples from a subject having aortic aneurysm at multiple intervals during an aortic aneurysm treatment,

determining the amounts of at least one immune cell in the samples, the at least one immune cell being at least a regulatory T cell (T_reg) or at least a macrophage, and assessing whether the aortic aneurysm treatment is effective in the subject based on changes in the amounts of the at least one immune cell.

32. The method of claim 31, wherein the amounts of the T_reg cell are determined and wherein an increase in the amount of the T_reg along the course of the treatment indicates that the aortic aneurysm treatment is effective.

33. The method of claim 32, wherein the T_reg is CD4 and Foxp3 positive.

34. The method of any of claims 31-33, wherein the amounts of both Ml and M2 macrophages in the samples are determined and wherein a decrease of the M1/M2 ratio along the course of the treatment indicates that the aortic aneurysm treatment is effective.

35. The method of any of claims 31-34, wherein the subject is a laboratory animal.

36. The method of any of claims 31-34, wherein the subject is a human patient.