Expansion of myeloid-derived suppressor cells with arginase activity lasts longer in aged than in young mice after CpG-ODN plus IFA treatment.

As we age, the homeostatic function of many systems in the body, such as the immune function declines, which in turn contributes to augment susceptibility to disease. Here we describe that challenging aged mice with synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) emulsified in incomplete Freund's adjuvant (IFA), (CpG-ODN+IFA) an inflammatory stimulus, led to the expansion of CD11b+Gr1+ myeloid cells with augmented expression of CD124 and CD31. These myeloid cells lasted longer in the spleen of aged mice than in their younger counterparts after CpG-ODN+IFA treatment and were capable of suppressing T cell proliferative response by arginase induction. Myeloid cells from aged CpG-ODN+IFA-treated mice presented increased arginase-1 expression and enzyme activity. In addition, we found a different requirement of cytokines for arginase induction according to mice age. In myeloid cells from young treated mice, arginase-1 expression and activity is induced by the presence of each IL-4 or IL-6 in their extracellular medium, unlike myeloid cells from aged treated mice which need the presence of both IL-4 and IL-6 together for arginase induction and suppressor function.

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.

TLR7 triggering with polyuridylic acid promotes cross-presentation in CD8α+ conventional dendritic cells by enhancing antigen preservation and MHC class I antigen permanence on the dendritic cell surface.

ssRNA can interact with dendritic cells (DCs) through binding to TLR7, inducing secretion of proinflammatory cytokines and type I IFN. Triggering TLR7 enhances cross-priming of CD8(+) T cells, which requires cross-presentation of exogenous Ag to DCs. However, how TLR triggering can affect Ag cross-presentation is still not clear. Using OVA as an Ag model, we observed that stimulation of TLR7 in DCs by polyuridylic acid (polyU), a synthetic ssRNA analog, generates a strong specific cytotoxic response in C57BL/6 mice. PolyU stimulate CD8α(+) DCs to cross-prime naive CD8(+) T cells in a type I IFN-dependent fashion. This enhanced cross-priming is accompanied by a higher density of OVA(256-264)/H-2K(b) complexes on CD8α(+) DCs treated with polyU, as well as by upregulation of costimulatory molecules and increased secretion of proinflammatory cytokines by DCs. Cross-priming of CD8(+) T cells by DCs treated with polyU requires proteasome and Ag translocation to cytosol through the Sec61 channel in DCs. The observed enhancement in OVA cross-presentation with polyU in DCs could be mediated by a limited Ag degradation in endophagosomal compartments and a higher permanence of OVA peptide/MHC class I complexes on DCs. These observations clearly reveal that key steps of Ag processing for cross-presentation can be modulated by TLR ligands, opening new avenues for understanding their mechanisms as adjuvants of the immune response.

Arginase-dependent suppression by CpG-ODN plus IFA-induced splenic myeloid CD11b(+)Gr1(+) cells.

The ability of synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) to induce both stimulatory and counter-regulatory responses offers novel opportunities for using these molecules as immunomodulatory agents in different therapeutic strategies. Here, we investigated the potential of CpG-ODN to activate the arginase (ARG) enzyme in vivo and focused on the consequences of this activation in T-cell proliferation. Challenging mice subcutaneously with CpG-ODN emulsified in incomplete Freund's adjuvant (IFA) induced ARG and reduced T-cell proliferation associated with CD3ζ chain downregulation. Interestingly, impaired T-cell expansion correlated with elevated levels of CD11b(+)Gr1(+) myeloid cells localized near T-cell areas in the spleen. In addition, purified CD11b(+) cells obtained from the spleen of CpG-ODN+IFA-treated mice exhibited increased ARG activity and ARG I expression along with an augmented [(3)H]-L-arginine uptake. CD11b(+) myeloid cells significantly suppressed T-cell proliferation and CD3ζ chain expression induced by a polyclonal stimulus. Furthermore, these effects could be recovered by the addition of excess L-arginine or by treatment of CD11b(+) cells with a specific ARG inhibitor. This study provides a novel evidence that CpG-ODN+IFA are able to induce splenic CD11b(+) cells with ARG activity, with this population being responsible for the impaired T-cell proliferation observed after the treatment with CpG-ODN+IFA. These results underscore a key role of CpG-ODN on ARG activity in vivo and add support to the growing body of evidence in favor of a counter-regulatory role for CpG-ODN in an immune response.

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.

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

Recognition of microbial products by macrophages (Mphi) 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-gamma (IFN-gamma), both arginase and inducible nitric oxide synthase (iNOS) in murine Mphi. Unexpectedly, IFN-gamma, 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-gamma and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG-mediated arginase activity is dependent on IFN-gamma priming. The increase in arginase activity as a result of stimulation with CpG plus IFN-gamma was 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-gamma, one of the mayor cytokines produced in response to CpG administration in vivo.

Presence of neutrophil-bearing antigen in lymphoid organs of immune mice.

Neutrophils play a crucial early role during the innate response, but little is known about their possible contribution when an adaptive immune response is installed. A robust neutrophilia and a T helper 1 (Th1) immune response are present after immunization with Complete Freund Adjuvant (CFA). We show that when FITC-labeled OVA was injected into the footpad of OVA/CFA immunized mice, the main OVA-FITC+ cells recruited in draining popliteal lymph nodes (LNs) were neutrophils, with most of them arriving at the LN by means of lymphatic vessels. The development of this OVA-FITC+ neutrophil influx requires an immune response against OVA. The OVA-FITC+ neutrophils present in LNs displayed mainly intracellular TNF-alpha, and their depletion resulted in an increase in the specific IL-5 levels. These data provide new evidence about the role played by neutrophils in vivo in adaptive immunity.