Belkys A. Maletto

Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice

We have previously demonstrated that subcutaneously administered ovalbumin (OVA) plus synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG‐ODN) as adjuvant stimulate cellular and humoral immunity and promote T helper cell type 1 differentiation in aged mice. The present study assessed the ability of CpG‐ODN to induce an OVA‐specific immune response after oral immunization in young (3‐month‐old) and aged (18‐month‐old) BALB/c mice. Oral OVA/CpG‐ODN immunization induces a similar OVA‐specific T cell‐proliferative response (in mucosal and systemic tissues), immunoglobulin G (IgG) in plasma, and IgA in intestinal washes in both groups of ages. The OVA‐specific humoral immune response observed in aged mice was similar to the one observed in young mice, peaking at day 7 after the last oral immunization and was present over 40 days after the last oral immunization. The pattern of cytokines released in culture supernatants in both groups of mice was similar, with specific interferon‐γ secretion in the absence of interleukin‐5 responses. These results provide evidence that orally administered OVA/CpG‐ODN induces a young‐like, specific, immune response against OVA in aged mice, showing that CpG‐ODN might be used as a mucosal adjuvant during aging.

Neutrophils Exhibit Differential Requirements for Homing Molecules in Their Lymphatic and Blood Trafficking into Draining Lymph Nodes

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Interferon-γ priming is involved in the activation of arginase by oligodeoxinucleotides containing CpG motifs in murine macrophages

Recognition of microbial products by macrophages (Mφ) stimulates an inflammatory response and plays a critical role in directing the host immune response against infection. In the present work, we showed for the first time that synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG) are able to stimulate, in the presence of interferon‐γ (IFN‐γ), both arginase and inducible nitric oxide synthase (iNOS) in murine Mφ. Unexpectedly, IFN‐γ, a cytokine believed to be an inhibitor of arginase activity, intervened in the activation of this enzyme. A significant increase in arginase activity was observed upon a short pre‐incubation (1 hr) with IFN‐γ and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG‐mediated arginase activity is dependent on IFN‐γ priming. The increase in arginase activity as a result of stimulation with CpG plus IFN‐γwas correlated with augmented expression of the arginase II isoform. The use of pharmacological specific inhibitors revealed that arginase activity was dependent on p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated protein kinase (ERK), but independent of c‐Jun N‐terminal kinase (JNK) activation. This report reveals a singular effect of the combination of CpG and IFN‐γ, one of the mayor cytokines produced in response to CpG administration in vivo.

Age-associated changes in lymphoid and antigen-presenting cell functions in mice immunized with Trypanosoma cruzi antigens.

The purpose of these studies was to analyze the role of different immune cell populations in the immune response against Trypanosoma cruzi antigens in aged mice. Mice of different ages (3 and 12 months old) were immunized i.d. with S-105 plus Bordetella pertussis as adjuvant and we compared the activities of the lymph node cells taken from 3- and 12-month-old donor animals to transfer DTH or antibody production to 3-month-old recipients. This study revealed that adherent and non-adherent immune lymph node cells of aged donor animals did not transfer response against the foreign antigen (S-105) whereas 3-month-old non-adherent lymph node cells transferred a DTH response as well as helped the specific antibody production. When total lymph node cells from 3- and 12-month-old mice were mixed, we observed an inhibition of S-105 transferred response indicating a suppressive effect of aged cells on the 3-month-old mice cells. Furthermore, we analyzed the participation of antigen-presenting cells (APC) in the immune response changes related to the previously described aged mice. Peritoneal cavities cells (PC), pulsed in vivo with S-105, obtained from 3- and 12-month-old mice were transferred to normal recipients and a DTH response to S-105 was studied. We observed that the DTH response was lower in the recipients of aged PC with respect to recipients of young PC. The results suggest that APC from aged mice are involved in controlling the cellular immune response to S-105. Age-related changes in immune T cell and APC are discussed in the context of these observations.

Diminished percentage of antigen bearing cells in the lymph nodes of immune aged rats.

We have demonstrated previously that during experimental autoimmune prostatitis (EAP), aged rats show a diminished humoral autoimmune response. In the present paper we have studied the transport of the autoantigen from the site of injection toward lymphatic organs in rats of different ages with or without EAP. We used as autoantigen prostatic components (rat accessory glands (RAG)) conjugated with fluorescein isothiocyanate (FITC). Studies of flow cytometry, fluorescent microscopy and confocal microscopy show no differences in the percentage of RAG-FITC positive cells or in the localization of the cells in the popliteal lymph nodes of not-immunized young and aged rats. On the other hand, in 18-month-old rats immunized with either RAG or Ovalbumin there were lower levels of specific IgG antibodies and fewer antigen containing cells in the draining lymph nodes than those of 3- or 12-month-old rats. In all groups fluorescent cells were MHC class II positive and some were IgM positive. Our results demonstrate that in immunized 18-month-old rats there is a diminished percentage of cells bearing the antigen in the draining lymph nodes after antigen injection in the skin, related to the levels of specific antibodies able to form antigen-antibody complexes in the periphery.

Age-related alterations in inflammatory response during experimental autoimmune prostatitis

Experimental autoimmune prostatitis (EAP) is an experimental model of autoimmune disease, developed in Wistar rats against prostatic components. The 12-and 18-month-old rats with EAP show a higher cellular autoimmune response and lower humoral autoimmune response compared to 3-month-old rats. The analysis of NO(.) and O(2)(-) production by peritoneal exudate cells (PECs) resulted in a higher NO(.) and O(2)(-) production in EAP rats at all ages, compared to control animals. PECs from 12- and 18-month-old rats produced more NO(.) and less O(2)(-) than PECs from 3-month-old rats. However, lipopolysacharide (LPS) did not stimulate PECs from aged rats for NO(.) production as much as in 3-month-old rats and thus, turning out in a lower index of LPS-stimulation of PECs from aged rats, compared to 3-month-old rats. Furthermore, the mast cells number in prostates of EAP rats, especially the number of degranulated cells, was higher than in control animals, but no significant differences were found between 3- and 12-month-old control rats. In conclusion, these results show that aging affects differentially the inflammation mediators during EAP.

Changes in the development of experimental autoimmune prostatitis (EAP) by castration in aged rats.

During Experimental Autoimmune Prostatitis (EAP), 12-month-old rats show a higher cellular autoimmune response and lower humoral autoimmune response against prostatic components than 3-month-old rats subjected to the same antigen stimulus. We analyzed if thymus recovery by orchidectomy could affect the development of EAP in 12-month-old rats. Thirty days after gonadectomy, 12-month-old rats showed an increment in the thymic mass and in the thymocytes absolute number, with percentages of the four main cell subpopulations (defined by CD4-CD8 molecules expression) similar to the 3-month-old rats. The DTH response of castrated 12-month-old with EAP were diminished in comparison with sham-castrated 12-month-old rats with EAP, resembling the values observed in 3-month-old rats with EAP. The prostates of castrated 12-month-old rats with EAP did not show inflammatory mononuclear cell infiltration, as did control 3- and 12-month-old rats with EAP. Castration seems to modulate negatively EAP in 12-month-old rats, possibly through the regeneration of thymus after testosterone deprivation.

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

CpG-ODN+IFN-γ confer pro- and anti-inflammatory properties to peritoneal macrophages in aged mice.

Aging is accompanied by a disturbance in the homeostasis of the immune system. However, research into the behavior of macrophages in aging has shown disagreements about the functional status of these cells in aged mice. In this work, we studied the influence of aging on macrophage functions by evaluating the pro- and anti-inflammatory parameters of peritoneal macrophages preserved in their natural microenvironment. Resident peritoneal macrophages from old mice, in the context of their natural milieu, were found to respond with a similar phenotype and functional pattern to macrophages from young mice. In addition, we evaluated the macrophage response to CpG-ODN, a well-known Th1 promoter. CpG-ODN+IFN-γ were able to activate not only nitric oxide to initiate the inflammatory response, but also IL-12 in resident and inflammatory peritoneal macrophages from aged mice in the context of their natural milieu, although some quantitative differences were found in IL-10 and IL-12 secretion. With this stimulus, NO secretion and arginase activation were maintained in peritoneal macrophages during aging. These results will help to elucidate potential immunization strategies with CpG-ODN in the elderly.

A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1β, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.