Andrés A. Herrada

Spironolactone Decreases DOCA–Salt–Induced Organ Damage by Blocking the Activation of T Helper 17 and the Downregulation of Regulatory T Lymphocytes

Adaptive immune response has been implicated in inflammation and fibrosis as a result of exposure to mineralocorticoids and a high-salt diet. We hypothesized that in mineralocorticoid-salt–induced hypertension, activation of the mineralocorticoid receptor alters the T-helper 17 lymphocyte (Th17)/regulatory T-lymphocyte/interleukin-17 (IL-17) pathway, contributing to cardiac and renal damage. We studied the inflammatory response and tissue damage in rats treated with deoxycorticosterone acetate and high-salt diet (DOCA–salt), with or without mineralocorticoid receptor inhibition by spironolactone. To determine whether Th17 differentiation in DOCA–salt rats is caused by hypertension per se, DOCA–salt rats received antihypertensive therapy. In addition, to evaluate the pathogenic role of IL-17 in hypertension and tissue damage, we studied the effect of IL-17 blockade with a specific antibody (anti–IL-17). We found activation of Th17 cells and downregulation of forkhead box P3 mRNA in peripheral tissues, heart, and kidneys of DOCA–salt–treated rats. Spironolactone treatment prevented Th17 cell activation and increased numbers of forkhead box P3–positive cells relative to DOCA–salt rats. Antihypertensive therapy did not ameliorate Th17 activation in rats. Treatment of DOCA–salt rats with anti–IL-17 significantly reduced arterial hypertension as well as expression of profibrotic and proinflammatory mediators and collagen deposits in the heart and kidney. We conclude that mineralocorticoid receptor activation alters the Th17/regulatory T-lymphocyte/IL-17 pathway in mineralocorticoid-dependent hypertension as part of an inflammatory mechanism contributing to fibrosis.

Stimulation of dopamine receptor D5 expressed on dendritic cells potentiates Th17-mediated immunity.

Dendritic cells (DCs) are responsible for priming T cells and for promoting their differentiation from naive T cells into appropriate effector cells. Emerging evidence suggests that neurotransmitters can modulate T cell-mediated immunity. However, the involvement of specific neurotransmitters or receptors remains poorly understood. In this study, we analyzed the role of dopamine in the regulation of DC function. We found that DCs express dopamine receptors as well as the machinery necessary to synthesize, store, and degrade dopamine. Notably, the expression of D5R decreased upon LPS-induced DC maturation. Deficiency of D5R on the surface of DCs impaired LPS-induced IL-23 and IL-12 production and consequently attenuated the activation and proliferation of Ag-specific CD4+ T cells. To determine the relevance of D5R expressed on DCs in vivo, we studied the role of this receptor in the modulation of a CD4+ T cell-driven autoimmunity model. Importantly, D5R-deficient DCs prophylactically transferred into wild-type recipients were able to reduce the severity of experimental autoimmune encephalomyelitis. Furthermore, mice transferred with D5R-deficient DCs displayed a significant reduction in the percentage of Th17 cells infiltrating the CNS without differences in the percentage of Th1 cells compared with animals transferred with wild-type DCs. Our findings demonstrate that by contributing to CD4+ T cell activation and differentiation to Th17 phenotype, D5R expressed on DCs is able to modulate the development of an autoimmune response in vivo.

Immune complex-induced enhancement of bacterial antigen presentation requires Fcγ Receptor III expression on dendritic cells

Dendritic cells (DCs) are capable of initiating adaptive immune responses against infectious agents by presenting pathogen-derived antigens on MHC molecules to naïve T cells. Because of their key role in priming adaptive immunity, it is expected that interfering with DC function would be advantageous to the pathogen. We have previously shown that Salmonella enterica serovar Typhimurium (ST), is able to survive inside DCs and interfere with their function by avoiding activation of bacteria-specific T cells. In contrast, when ST is targeted to Fcγ receptors on the DC surface, bacteria are degraded and their antigens presented to T cells. However, the specific Fcγ receptor responsible of restoring presentation of antigens remains unknown. Here, we show that IgG-coated ST was targeted to lysosomes and degraded and its antigens presented on MHC molecules only when the low-affinity activating FcγRIII was expressed on DCs. FcγRIII-mediated enhancement of Ag presentation led to a robust activation of T cells specific for bacteria-expressed antigens. Laser confocal and electron microscopy analyses revealed that IgG-coated ST was rerouted to the lysosomal pathway through an FcγRIII-dependent mechanism. PI-3K activity was required for this process, because specific inhibitors promoted the survival of IgG-coated ST inside DCs and prevented DCs from activating bacteria-specific T cells. Our data suggest that the DC capacity to efficiently activate T cells upon capturing IgG-coated virulent bacteria is mediated by FcγRIII and requires PI-3K activity.

Modulation of nuclear factor‐κB activity can influence the susceptibility to systemic lupus erythematosus

Autoimmune diseases, such as systemic lupus erythematosus (SLE), result from deficiencies in self‐antigen tolerance processes, which require regulated dendritic cell (DC) function. In this study we evaluated the phenotype of DCs during the onset of SLE in a mouse model, in which deletion of the inhibitory receptor FcγRIIb leads to the production of anti‐nuclear antibodies and glomerulonephritis. Splenic DCs from FcγRIIb‐deficient mice suffering from SLE showed increased expression of co‐stimulatory molecules. Furthermore, diseased mice showed an altered function of the nuclear factor‐κB (NF‐κB) transcription factor, which is involved in DC maturation. Compared with healthy animals, expression of the inhibitory molecule IκB‐α was significantly decreased in mice suffering from SLE. Consistently, pharmacological inhibition of NF‐κB activity in FcγRIIb‐deficient mice led to reduced susceptibility to SLE and prevented symptoms, such as anti‐nuclear antibodies and kidney damage. Our data suggest that the occurrence of SLE is significantly influenced by alterations of NF‐κB function, which can be considered as a new therapeutic target for this disease.

Aldosterone as a modulator of immunity: implications in the organ damage

High plasmatic levels of aldosterone cause hypertension and contribute to progressive organ damage to the heart, vasculature, and kidneys. Recent studies have demonstrated a role for the immune system in these pathological processes. Aldosterone promotes an inflammatory state characterized by vascular infiltration of immune cells, reactive oxidative stress, and proinflammatory cytokine production. Further, cells of the adaptive immune system, such as T cells, seem to participate in the genesis of mineralocorticoid hormone-induced hypertension. In addition, the observation that aldosterone can promote CD4⁺ T-cell activation and Th17 polarization suggests that this hormone could contribute to the onset of autoimmunity. Here we discuss recent evidence supporting a significant involvement of the immune system, especially adaptive immunity, in the genesis of hypertension and organ damage induced by primary aldosteronism. In addition, possible new therapeutic approaches consisting of immunomodulator drugs to control exacerbated immune responses triggered by elevated aldosterone concentrations will be described.

Primary aldosteronism can alter peripheral levels of transforming growth factor β and tumor necrosis factor α

Primary aldosteronism (PA) is the most common secondary cause of hypertension that has recently been implicated in alterations of the immune system and progression of cardiovascular disease. Objective: To study the cytokines transforming growth factor β1 (TGF-β1), tumor necrosis factor α (TNF-α), and interleukin 10 (IL-10) in patients with PA and essential hypertensives (EH) and evaluate its association with the renin-angiotensin-aldosterone system. Patients and methods: We studied 26 PA and 52 EH patients as controls, adjusted by their blood pressure, body mass index, age, and gender. In both groups, PA and EH, we measured serum aldosterone (SA), plasma renin activity (PRA), and cytokines TGF-β1, TNF-α, and IL-10. In addition, 17 PA patients were treated for 6 months with spironolactone, a mineralocorticoid receptor (MR) antagonist. Results: PA patients had lower levels of TGF-β1 (17.6±4.1 vs 34.5±20.5 pg/ml, p<0.001) and TNF-α (17.0±4.4 vs 35.6±21.7 pg/ml, p<0.001) and similar IL-10 levels (99.7±18.7 vs 89.4±49.5 pg/ml, p: ns), as compared with EH controls. TGF-β1 and TNF-α levels showed a remarkable correlation with SA/PRA ratio in the total group (PA+EH). The treatment of PA patients with spironolactone increased the TGF-β1 levels (18.3±5.9 to 28.4±6.3 pg/ml, p<0.001), while TNF-α, and IL-10 remained unchanged. Conclusion: Our results showed that PA patients have lower TGF-β1 and TNF-α cytokine serum levels than EH. TGF-β1 levels were restored with spironolactone, showing a MR-dependent regulation. In this way, the chronic aldosterone excess modifies the TGF-β1 levels, which could produce an imbalance in the immune system homeostasis that may promote an early proinflammatory cardiovascular phenotype.

Haem oxygenase 1 expression is altered in monocytes from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple functional alterations affecting immune cells, such as B cells, T cells, dendritic cells (DCs) and monocytes. During SLE, the immunogenicity of monocytes and DCs is significantly up‐regulated, promoting the activation of self‐reactive T cells. Accordingly, it is important to understand the contribution of these cells to the pathogenesis of SLE and the mechanisms responsible for their altered functionality during disease. One of the key enzymes that control monocyte and DC function is haem oxygenase‐1 (HO‐1), which catalyses the degradation of the haem group into biliverdin, carbon monoxide and free iron. These products possess immunosuppressive and anti‐inflammatory capacities. The main goal of this work was to determine HO‐1 expression in monocytes and DCs from patients with SLE and healthy controls. Hence, peripheral blood mononuclear cells were obtained from 43 patients with SLE and 30 healthy controls. CD14+ monocytes and CD4+ T cells were sorted by FACS and HO‐1 expression was measured by RT‐PCR. In addition, HO‐1 protein expression was determined by FACS. HO‐1 levels in monocytes were significantly reduced in patients with SLE compared with healthy controls. These results were confirmed by flow cytometry. No differences were observed in other cell types, such as DCs or CD4+ T cells, although decreased MHC‐II levels were observed in DCs from patients with SLE. In conclusion, we found a significant decrease in HO‐1 expression, specifically in monocytes from patients with SLE, suggesting that an imbalance of monocyte function could be partly the result of a decrease in HO‐1 expression.

Harnessing DNA-induced immune responses for improving cancer vaccines.

DNA vaccines have emerged as an attractive strategy to promote protective cellular and humoral immunity against the encoded antigen. DNA vaccines are easy to generate, inexpensive to produce and purify at large-scale, highly stable and safe. In addition, plasmids used for DNA vaccines act as powerful “danger signals” by stimulating several DNA-sensing innate immune receptors that promote the induction of protective adaptive immunity. The induction of tumor-specific immune responses represents a major challenge for DNA vaccines because most of tumor-associated antigens are normal non-mutated self-antigens. As a consequence, induction of potentially self-reactive T cell responses against such poorly immunogenic antigens is controlled by mechanisms of central and peripheral tolerance as well as tumor-induced immunosuppression. Although several DNA vaccines against cancer have reached clinical testing, disappointing results have been observed. Therefore, the development of new adjuvants that strongly stimulate the induction of antitumor T cell immunity and counteract immune-suppressive regulation is an attractive approach to enhance the potency of DNA vaccines and overcome tumor-associated tolerance. Understanding the DNA-sensing signaling pathways of innate immunity that mediate the induction of T cell responses elicited by DNA vaccines represents a unique opportunity to develop novel adjuvants that enhance vaccine potency. The advance of DNA adjuvants needs to be complemented with the development of potent delivery systems, in order to step toward successful clinical application. Here, we briefly discuss recent evidence showing how to harness DNA-induced immune response to improve the potency of cancer vaccines and counteract tumor-associated tolerance.

A filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.