María I. Crespo

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.

IFNβ Produced by TLR4-Activated Tumor Cells Is Involved in Improving the Antitumoral Immune Response

Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNβ was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNβ-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response.

Baculovirus Capsid Display Potentiates OVA Cytotoxic and Innate Immune Responses

Baculoviruses (BV) are DNA viruses that are pathogenic for insects. Although BV infect a range of mammalian cell types, they do not replicate in these cells. Indeed, the potential effects of these insect viruses on the immune responses of mammals are only just beginning to be studied. We show in this paper that a recombinant Autographa californica multiple nuclear polyhedrosis virus carrying a fragment of ovalbumin (OVA) on the VP39 capsid protein (BV-OVA) has the capacity to act as an adjuvant and vector of antigens in mice, thereby promoting specific CD4 and cytotoxic T cell responses against OVA. BV also induced in vivo maturation of dendritic cells and the production of inflammatory cytokines, thus promoting innate and adaptive immune responses. The OVA-specific response induced by BV-OVA was strong enough to reject a challenge with OVA-expressing melanoma cells (MO5 cells) and effectively prolonged survival of MO5 bearing mice. All these findings, together with the absence of pre-existing immunity to BV in humans and the lack of viral gene expression in mammalian cells, make BV a candidate for vaccination.

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.

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 Freunds 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 [3H]‐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.

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.

Association between levels of synovial anti-citrullinated peptide antibodies and neutrophil response in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by the presence of anti‐citrullinated peptide antibodies (ACPAs) and neutrophils infiltrating the synovial fluid (SF) of the affected joints. The aim of this work was to analyze whether the presence of ACPAs in SF is associated with neutrophil infiltration and with their phenotype in the inflamed joints of RA patients. We found that in the presence of ACPAs, the number of synovial neutrophils correlated with severe disease activity. The SF were divided according to synovial ACPA levels in negative‐ (<25 U/mL), low‐ (25–200 U/mL) and high level (˃200 U/mL; ACPAhigh). We observed that IL‐6, IL‐17, and IL‐8 were significantly elevated in ACPAhighSF and that IL‐8 levels correlated positively with neutrophil counts and with worse clinical manifestations. Additionally, in vitro incubation of neutrophils with ACPAhigh SF resulted in an increased ROS production and extracellular DNA release compared to neutrophils incubated with ACPA‐negative SF. These exacerbated effector functions were associated with a fraction of ICAM‐1‐positive neutrophils, which were induced by ACPAhigh SF. Likewise, in in vivo, we could also detect this subset among neutrophils present in ACPAhigh SF. In conclusion, the data presented here shed light on the role of SF‐ACPAs as inductors of a proinflammatory profile in neutrophils.