fimmu-11-00743 April 23, 2020 Time: 19:59 # 1 ORIGINAL RESEARCH published: 24 April 2020 doi: 10.3389/fimmu.2020.00743 Reformulating Small Molecules for Cardiovascular Disease Immune Intervention: Low-Dose Combined Vitamin D/Dexamethasone Promotes IL-10 Production and Atheroprotection in Dyslipidemic Edited by: Ursula Grohmann, Mice University of Perugia, Italy Reviewed by: Laura Ospina-Quintero†‡, Julio C. Jaramillo†, Jorge H. Tabares-Guevara and Alex Bobik, José R. Ramírez-Pineda* Baker Heart and Diabetes Institute, Australia Grupo Inmunomodulación (GIM), Instituto de Investigaciones Médicas, Facultad de Medicina, Corporación Académica para Harley Y. Tse, el Estudio de Patologías Tropicales (CAEPT), Universidad de Antioquia, Medellin, Colombia Wayne State University, United States *Correspondence: The targeting of proinflammatory pathways has a prophylactic and therapeutic potential José R. Ramírez-Pineda [email protected]; on atherosclerotic cardiovascular diseases (CVD). An alternative/complementary [email protected] strategy is the promotion of endogenous atheroprotective mechanisms that are impaired †These authors have contributed during atherosclerosis progression, such as the activity of tolerogenic dendritic cells equally to this work (tolDC) and regulatory T cells (Treg). There is a need to develop novel low cost, safe and ‡Present address: Laura Ospina-Quintero, effective tolDC/Treg-inducing formulations that are atheroprotective and that can be of Institute of Immunology, Hannover easy translation into clinical settings. We found that apolipoprotein E-deficient (ApoE−=−) Medical School, Hannover, Germany mice treated with a low-dose combined formulation of Vitamin D and Dexamethasone Specialty section: (VitD/Dexa), delivered repetitively and subcutaneously (sc) promoted interleukin-10 (IL- This article was submitted to 10) production by dendritic cells and other antigen presenting cells in the lymph nodes Immunological Tolerance draining the site of injection and the spleens. Expectedly, the treatment also increased and Regulation, C a section of the journal the numbers of IL-10-producing CD4 T cells. Concomitantly, the frequency of IFNg- Frontiers in Immunology producing CD4C and CD8C T cells in the spleen, and the IFNg response of splenocytes Received: 14 January 2020 to polyclonal stimulation ex vivo were lower after VitD/Dexa treatment, indicating a Accepted: 01 April 2020 Published: 24 April 2020 reduced proatherogenic Th1 response. Interestingly, VitD/Dexa-treated mice had smaller Citation: atherosclerotic lesions, with reduced lipid content and lower inflammatory infiltrate of Ospina-Quintero L, Jaramillo JC, macrophages and T cells in the aortic root. No hypolipidemic or antioxidant effect Tabares-Guevara JH and could be detected, suggesting that a dominantly immunomodulatory mechanism of Ramírez-Pineda JR (2020) Reformulating Small Molecules atheroprotection was engaged under the low-dose sc VitD/Dexa conditions used. for Cardiovascular Disease Immune Finally, no evidence of clinical, biochemical or immune toxicity was observed in Intervention: Low-Dose Combined −=− Vitamin D/Dexamethasone treated ApoE mice and, most importantly, C57BL/6 mice latently infected with Promotes IL-10 Production Leishmania parasites and treated with an identical VitD/Dexa dose/scheme showed and Atheroprotection in Dyslipidemic no clinical or microbiological signs of disease reactivation, suggesting the absence Mice. Front. Immunol. 11:743. doi: 10.3389/fimmu.2020.00743 of general immunosuppression. Altogether, these results indicate that a non-toxic, Frontiers in Immunology| www.frontiersin.org 1 April 2020| Volume 11| Article 743 fimmu-11-00743 April 23, 2020 Time: 19:59 # 2 Ospina-Quintero et al. Regulatory Cell-Inducing Small Molecule Formulation non-immunosuppressive, low-dose of VitD/Dexa, administered subcutaneously and repetitively, exerts atheroprotective effects in dyslipidemic mice, apparently due to the induction of an IL-10-producing network of lymphoid and myeloid immune cells. These well known, widely available, and inexpensive small molecules can be easily co- formulated into a simple and accessible agent with a potential use as a prophylactic or therapeutic immune intervention for CVD and other chronic inflammatory diseases. Keywords: atherosclerosis, immune intervention, Vitamin D3, Dexamethasone, interleukin-10, immunoregulatory cells INTRODUCTION significantly reduced the inflammatory burden and protected from secondary CVD events (8), providing a “proof of principle” Despite significant advances in the prevention and management for targeting of immune pathways, via anti-inflammatory and of atherosclerotic CVD, they are expected to remain as the immunomodulatory agents, as a therapeutic intervention against major cause of human mortality and disability worldwide (1– CVD in humans (9, 10). 3). Whereas the public health situation and economic impact Whilst direct blocking of proinflammatory atherogenic of CVD claims for novel therapies, vascular drug development pathways has been the obvious focus in the search for a has paradoxically declined in the last two decades owing to CVD immunotherapy (8), a less explored alternative form the intrinsic complexities of CVD, as compared to other life- of immunomodulation is the reinforcement/restoration of the threatening and costly chronic diseases like cancer (3). Although natural atheroprotective mechanisms that become insufficient recent advances in the understanding of atherosclerosis highlight as atherosclerosis progresses. Among the immune cell types the complexity of the pathophysiological events underlying the that promote atheroprotection, Tregs have emerged as dominant development of atheromatous plaques and subsequent vascular players (4–6, 11). Tregs not only exert classical regulatory complications, novel opportunities for therapeutic interventions functions on effector proatherogenic immune cells, but also have also been revealed (see editorial (2) and compendium influence cholesterol metabolism, plaque stability, foam cell articles therein). An overwhelming amount of information, formation and endothelial response, participate in plaque mostly derived from animal studies, has implicated the immune regression and tissue repair after ischemic events, and contribute system and the inflammatory response as key drivers/regulators to the athero/cardioprotective effects of statins, vaccination and of atherosclerotic CVD. On the one hand, macrophages and inflammation resolution inducers (4–6, 12, 13). Thus, from a T cells play a critical role in atherosclerosis progression by therapeutic perspective, it is difficult to envision a cell type, triggering and maintaining proatherogenic innate and adaptive other than Treg, that could impact atherogenic responses in a immune responses, involving proinflammatory mediators such more essential manner. Although a variety of methods have been as IL-1, IL-6, or IFNg. On the other hand, concomitant proposed to restore Treg numbers and functions, including cell atheroprotective versions of the innate and adaptive responses, therapy, biologicals or particle-based sophisticated approaches such as healing-promoting macrophages and regulatory T (Treg) (11, 12, 14, 15), their implementation for routine clinical practice cells, develop to counterbalance chronic inflammation and is hampered by high costs and/or complexity, making them promote its resolution via regulatory mediators such as IL-10 poorly realistic for widespread clinical use. Small molecules (4–6). The translational relevance of this immune hypothesis represent an alternative that might offer interesting advantages of atherogenesis remained controverted during decades, since such as lower cost, which could facilitate preclinical and clinical evidence in humans was mostly associative and circumstantial, development, and most importantly, accelerate implementation rather than causal (7). However, the CANTOS study has to clinical settings and its eventual use by the majority of patients. ended this controversy. It has demonstrated that neutralization Investigations that explore small molecule route/dose/scheme of IL-1b, a key proatherogenic/proinflammatory cytokine, of administration, delivery strategies and novel combinatory formulations could be of particular interest. Abbreviations: APC, antigen-presenting cell; ApoE−=−, apolipoprotein Many synthetic or naturally occurring small molecules E-deficient; ASA, acetylsalicylic acid; CANTOS, canakinumab anti-inflammatory have been reported as Treg inducers in vitro and in vivo, thrombosis outcomes study; CD, cluster of differentiation; cDC, conventional including immunosuppressive/anti-inflammatory drugs (such as DC; Curc, curcumin; CVD, cardiovascular diseases; Dexa, dexamethasone; dLN, draining lymph node; FACS, fluorescence-activated cell sorting; FOXP3, forkhead Dexamethasone, Dexa, Rapamycin and Acetylsalicylic Acid), box P3; GR, glucocorticoid receptor; HFD, high-fat diet; IFNg, interferon antioxidants (such as the natural polyphenols Curcumin and gamma; Ig, immunoglobulin; IL, interleukin; LDL, low-density lipoproteins; Quercetin, or the synthetic N-Acetylcysteine) and vitamins (such MDA, malondialdehyde; MDSC, myeloid derived suppressor cells; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MLN, mediastinal as Vitamin D, VitD, Vitamin A, Vitamin C, and Vitamin E) lymph node; MoDC, monocyte-derived dendritic cells; NAC, N-acetyl cysteine; (16–21). Interestingly, but not surprisingly, the Treg-inducing