**7. DCs as therapeutic targets**

Until now, it is impossible to fully inhibit the formation or progression of atherosclerotic le‐ sions in the clinic. Current therapies for atherosclerosis (e.g. statins, stent placement) focus on relieving symptoms, and consequently many patients remain at high risk for future acute coronary events. A very effective strategy in other immune-related pathologies is vaccina‐ tion, where the culprit protein or the weakened/dead version of the micro-organism is in‐ jected to the body in order to create a highly specific primary humoral immune-response [93]. New vaccines have recently been developed that deliver relevant antigens and adju‐ vants to redirect the immune system for the individual's benefit [94]. Because DCs are the most effective antigen presenting cells that initiate and regulate the immune response, they seem extremely suitable as vaccine basis. On the one hand, they can activate T cells, on the other hand, they can specifically silence unwanted immune reactions by inducing tolerance [95]. They might function as natural adjuvants for the induction of antigen-specific T-cell re‐ sponses. Approaches using DCs in atherosclerosis immunotherapy may be comparable to those already used for cancer immunotherapy [96,97,98], although a different immune re‐ sponse is required. One approach that is already intensively studied is the immunization with autologous, monocyte-derived DCs from the patient that are loaded with appropriate antigens *ex vivo* [96]. Such *ex vivo* generated and antigen-loaded DCs have nowadays been used as vaccines to improve immunity in patients with cancer [99] and chronic human im‐ munodeficiency virus (HIV) infection [100,101], providing a "proof of principle" that DC vaccines can work.

In the context of atherosclerosis, immunization of hypercholesterolemic animals with oxLDL or specific epitopes of ApoB100 has already been shown to inhibit atherosclerosis [102,103,104,105,106]. When LDL receptor-deficient (LDLr-/-) rabbits were immunized with malondialdehyde modified LDL (MDA-LDL), a reduction in the extent of atherosclerotic le‐ sions was observed in the aortic tree [102]. These observations were confirmed in LDLr-/- and apolipoprotein E deficient (ApoE-/-) mice [103,104]. Also hypercholesterolemic rabbits that were immunized with oxLDL showed reduced atherosclerotic lesions in the proximal aorta [107]. Possibly, oxLDL-pulsed DCs or DCs pulsed with immunogenic components of oxLDL could be used for vaccination as well, thereby avoiding the side effects of direct vaccination with oxLDL [108]. A series of studies have already used pulsed DCs as an immunotherapy for atherosclerosis in mice, however, results were not always consistent. Repeated injection of LDLr-/- mice with oxLDL-pulsed mature DCs resulted in attenuation of lesion develop‐ ment with a decreased amount of macrophages and increased collagen content, contributing to a more stable plaque phenotype [109]. Moreover, a similar approach was carried out us‐ ing mice expressing the full-length human ApoB100 in the liver and humanized lipoprotein profiles [110]. Those mice were repeatedly injected with mature DCs that were incubated with IL-10 and ApoB100, prior to the initiation of a Western diet. The immunosuppressive cytokine IL-10 was used to induce tolerogenic DCs [110]. This approach resulted in attenua‐ tion of atherosclerotic lesion development in the aorta, which was associated with decreased cellular immunity to ApoB100. Also, decreased Th1 and Th2 responses most likely due to enhanced regulatory T cell (Treg) expansion were observed [110]. In contrast, subcutaneous injection of DCs that were simultaneously pulsed with LPS and MDA-LDL into ApoE-/- mice at frequent intervals during lesion formation caused a significant increase in lesion size in the aortic root [111]. These differential effects may be due to different forms of antigen pre‐ sentation leading to qualitatively different immune responses. Apart from oxLDL, DCs might also be pulsed *ex vivo* by cultivating them with a total extract or suspension of athero‐ sclerotic plaque tissue, for example, from patients undergoing carotid endarterectomy [95,108] (figure 6). A major advantage of such a therapy, where a patient is vaccinated with its own DCs pulsed by its own antigens is the efficiency, because it would imitate events as they occur in plaques *in situ* in the patient.

al DNA) leading to enhanced IFN-α expression. This process amplifies inflammatory TLR-4, TNF-α, and IL-12 expression by mDCs, and correlates with plaque instability [90]. A recent study in ApoE-/- mice reported that administration of a plasmacytoid dendritic cell antigen-1 (PDCA-1) antibody to deplete pDCs protected from lesion formation [91], demonstrating that pDCs indeed exert proatherogenic functions during early lesion formation. In contrast, pDC depletion by administration of the 120G8 monoclonal antibody promoted plaque T-cell accu‐

pletion was accompanied by increased CD4⁺ T-cell proliferation, IFN-γ expression by splenic T cells, and plasma IFN-γ levels, pointing to a protective role for pDCs in atherosclerosis. Thus,

Until now, it is impossible to fully inhibit the formation or progression of atherosclerotic le‐ sions in the clinic. Current therapies for atherosclerosis (e.g. statins, stent placement) focus on relieving symptoms, and consequently many patients remain at high risk for future acute coronary events. A very effective strategy in other immune-related pathologies is vaccina‐ tion, where the culprit protein or the weakened/dead version of the micro-organism is in‐ jected to the body in order to create a highly specific primary humoral immune-response [93]. New vaccines have recently been developed that deliver relevant antigens and adju‐ vants to redirect the immune system for the individual's benefit [94]. Because DCs are the most effective antigen presenting cells that initiate and regulate the immune response, they seem extremely suitable as vaccine basis. On the one hand, they can activate T cells, on the other hand, they can specifically silence unwanted immune reactions by inducing tolerance [95]. They might function as natural adjuvants for the induction of antigen-specific T-cell re‐ sponses. Approaches using DCs in atherosclerosis immunotherapy may be comparable to those already used for cancer immunotherapy [96,97,98], although a different immune re‐ sponse is required. One approach that is already intensively studied is the immunization with autologous, monocyte-derived DCs from the patient that are loaded with appropriate antigens *ex vivo* [96]. Such *ex vivo* generated and antigen-loaded DCs have nowadays been used as vaccines to improve immunity in patients with cancer [99] and chronic human im‐ munodeficiency virus (HIV) infection [100,101], providing a "proof of principle" that DC

In the context of atherosclerosis, immunization of hypercholesterolemic animals with oxLDL or specific epitopes of ApoB100 has already been shown to inhibit atherosclerosis [102,103,104,105,106]. When LDL receptor-deficient (LDLr-/-) rabbits were immunized with malondialdehyde modified LDL (MDA-LDL), a reduction in the extent of atherosclerotic le‐ sions was observed in the aortic tree [102]. These observations were confirmed in LDLr-/- and apolipoprotein E deficient (ApoE-/-) mice [103,104]. Also hypercholesterolemic rabbits that were immunized with oxLDL showed reduced atherosclerotic lesions in the proximal aorta [107]. Possibly, oxLDL-pulsed DCs or DCs pulsed with immunogenic components of oxLDL

⁻ mice [92]. PDC de‐

mulation and exacerbated lesion development and progression in LDLr⁻/

the exact role of pDCs in atherosclerosis remains to be further unravelled.

**7. DCs as therapeutic targets**

68 Current Trends in Atherogenesis

vaccines can work.

**Figure 6.** Promising areas for further research to treat immune-mediated diseases, such as atherosclerosis. Immuniza‐ tion of patients with autologous, monocyte-derived DCs that are loaded with appropriate antigens *ex vivo*. This ap‐ proach has already been proven successful in cancer and HIV patients.

Another promising area for further research is the development of tolerogenic vaccines for immune-mediated diseases (figure 7). Both foreign and self-antigens can be targets of tolero‐ genic processes. DCs can be converted to 'tolerogenic DCs' by addition of various immuno‐ modulating agents, including IL-10, transforming growth factor-beta (TGF-β) and 1,25 dihydroxyvitamin D3 [8], or they can be generated by using small interfering RNA (siRNA) that specifically targets IL-12p35 gene [112] (figure 7). Tolerogenic DC-based immunothera‐ py has recently been tested in mice as a possible novel approach to induce immunological tolerance for prevention or treatment of atherosclerosis [110]. Hermansson et al. [110] used IL-10 to induce tolerogenic DCs. Another group showed that oral administration of calci‐ triol, the active form of vitamin D3, induced the generation of tolerogenic DCs as well as a significant increase in Foxp3+ Tregs in the lymph nodes, spleen, and atherosclerotic lesions of ApoE-/- mice, which resulted in an inhibition of atherosclerosis [113]. This was associated with increased IL-10 and decreased IL-12 mRNA expression. Furthermore, DCs from the calcitriol group showed reduced CD80 and CD86 expression and decreased proliferative ac‐ tivity of T lymphocytes, indicating that tolerogenic or maturation-resistant DCs show some similarities with immature DCs [113]. Hussain and colleagues [114] hypothesized that aspir‐ in may also induce tolerogenic DCs and CD4+ CD25+ FoxP3+ Treg cells activity/augmenta‐ tion in experimental models of autoimmune atherosclerosis. Aspirin-induced tolerogenic DCs initiated regulatory activity in responder T cells as they showed a decreased expression of costimulatory molecules and an increased expression of immunoglobulin-like transcript 3 (ILT-3), which is a co-inhibitor of T cell activation required to induce Tregs [114,115,116]. In‐ deed, the presentation of antigen complexes to T cells in the absence of costimulatory signals could lead to anergy or apoptosis of T cells, or the induction of Treg. Therefore, it might also be useful to adjust the expression of costimulatory molecules on pulsed DCs *ex vivo* prior to the vaccination [96,98,97,94,117,118].

sclerosis. Van Es et al. [119] used DCs to deplete atheroprotective Tregs by vaccinating LDLr-/- mice with DCs which were transfected with Foxp3 encoding mRNA. This approach resulted in a cytotoxic T lymphocyte (CTL) response against Foxp3 and a subsequent deple‐

crease in initial atherosclerotic lesion formation. Besides an increase in lesion size, vaccina‐ tion against Foxp3 also induced a 30% increase in cellularity of the initial lesions, which may

Another approach for therapeutic intervention against atherosclerosis might involve the di‐ rect targeting of DCs *in vivo* by manipulating the functions of different DC subsets [95]. Based on the hypothesis that cDCs act rather proatherogenic, whereas pDCs might be athe‐ roprotective (see section 4), suppression of the myeloid DC subset and activation of the lym‐ phoid subset might enable immune reactions in atherosclerosis to be regulated [95]. For future studies, it would be very useful to isolate DCs resident in plaques to be able to identi‐ fy a unique antigen(s) on their surface. That would possibly lead to new strategies where plaque DCs can be targeted to deliver biologically active substances to atherosclerotic le‐ sions. The challenge is to selectively identify regulatory molecules and novel therapies in or‐ der to inhibit DC migration and function during atherogenesis without affecting normal DC

As it is now well accepted that atherosclerosis is an immune-mediated disease, the target‐ ing of its cellular components might open possibilities for new therapeutic strategies to at‐ tenuate the progression of the disease. DCs seem to initiate and regulate immune responses in atherosclerosis and they are also involved in controlling cholesterol homeo‐ stasis by yet unknown mechanisms. It would be important to identify the pathway(s) through which CD11c+ cells may modulate the levels of plasma cholesterol. One should take into account that DCs represent a very heterogeneous population, with many subsets that have different phenotypes, functions, origin and anatomical distribution. So far, it is unclear if all DCs have equal antigen-presenting capacities, and very little is known about a preferential DC subset that is responsible for T cell-induced inflammation in the vessel wall. Moreover, there is a close relationship between DCs and macrophages, and the dis‐ tinction between both cell types is even further complicated by their plasticity. Future studies are essential to determine which DC subtypes exert pro- or anti-atherogenic ef‐ fects. It is crucial to understand the diversity in DC subsets to target DCs for immunomo‐ dulation therapies. Furthermore, functional differences between phenotypically similar mouse and human DC subtypes should also be studied. Nevertheless, DC-based vaccina‐ tion strategies have been proven successful and animal studies provide some promising data for the treatment of atherosclerosis as well. Yet, several issues, such as the most ap‐ propriate antigen(s) for loading DCs and the optimal type of DC used for vaccination re‐

indicate an increase in inflammation within the lesions [119].

function under physiological conditions.

main to be further investigated.

Tregs. Vaccination against Foxp3 aggravated atherosclerosis, it resulted in a

regulatory T cells in spleen, lymph nodes and circulation, and in an in‐

Dendritic Cells in Atherogenesis: From Immune Shapers to Therapeutic Targets

http://dx.doi.org/10.5772/52900

71

tion of Foxp3+

reduction of Foxp3+

**8. Conclusion**

**Figure 7.** Generation of tolerogenic DCs to develop tolerogenic vaccines. Tolerogenic DC-based immunotherapy has recently been successfully tested in mice as a possible novel approach to induce immunological tolerance for preven‐ tion or treatment of atherosclerosis.

A completely different strategy that might be used in therapeutic intervention implicates the use of DCs to deplete specific immune cells, such as the detrimental Th1 or Th17 cells, in atherosclerosis. The opposite approach has been shown to work in a mouse model of athero‐ sclerosis. Van Es et al. [119] used DCs to deplete atheroprotective Tregs by vaccinating LDLr-/- mice with DCs which were transfected with Foxp3 encoding mRNA. This approach resulted in a cytotoxic T lymphocyte (CTL) response against Foxp3 and a subsequent deple‐ tion of Foxp3+ Tregs. Vaccination against Foxp3 aggravated atherosclerosis, it resulted in a reduction of Foxp3+ regulatory T cells in spleen, lymph nodes and circulation, and in an in‐ crease in initial atherosclerotic lesion formation. Besides an increase in lesion size, vaccina‐ tion against Foxp3 also induced a 30% increase in cellularity of the initial lesions, which may indicate an increase in inflammation within the lesions [119].

Another approach for therapeutic intervention against atherosclerosis might involve the di‐ rect targeting of DCs *in vivo* by manipulating the functions of different DC subsets [95]. Based on the hypothesis that cDCs act rather proatherogenic, whereas pDCs might be athe‐ roprotective (see section 4), suppression of the myeloid DC subset and activation of the lym‐ phoid subset might enable immune reactions in atherosclerosis to be regulated [95]. For future studies, it would be very useful to isolate DCs resident in plaques to be able to identi‐ fy a unique antigen(s) on their surface. That would possibly lead to new strategies where plaque DCs can be targeted to deliver biologically active substances to atherosclerotic le‐ sions. The challenge is to selectively identify regulatory molecules and novel therapies in or‐ der to inhibit DC migration and function during atherogenesis without affecting normal DC function under physiological conditions.
