Michele M. Barsante

CCL2 and CCL5 mediate leukocyte adhesion in experimental autoimmune encephalomyelitis—an intravital microscopy study

Experimental autoimmune encephalomyelitis (EAE) models multiple sclerosis (MS) and is characterized by marked mononuclear cell influx in the brain. Several studies have demonstrated a role for chemokines during EAE. It remains to be determined whether these mediators modulate EAE primarily by mediating leukocyte influx into the CNS or by modifying lymphocyte activation and/or trafficking into lymphoid organs. After induction of EAE with MOG(35-55), leukocyte recruitment peaked on day 14 and correlated with symptom onset, TNF-alpha production and production of CCL2 and CCL5. Levels of CXCL-10 and CCL3 were not different from control animals. Using intravital microscopy, we demonstrated that leukocyte rolling and adhesion also peaked at day 14. Treatment with anti-CCL2 or anti-CCL5 antibodies just prior to the intravital microscopy prevented leukocyte adhesion, but not rolling. Our data suggest that induction of leukocyte adhesion to the brain microvasculature is an important mechanism by which CCL2 and CCL5 participate in the pathophysiology of EAE.

Schistosoma mansoni Antigens Modulate Experimental Allergic Asthma in a Murine Model: a Major Role for CD4 CD25 Foxp3 T Cells Independent of Interleukin-10

ABSTRACT In areas where schistosomiasis is endemic, a negative correlation is observed between atopy and helminth infection, associated with a low prevalence of asthma. We investigated whether Schistosoma mansoni infection or injection of parasite eggs can modulate airway allergic inflammation in mice, examining the mechanisms of such regulation. We infected BALB/c mice with 30 S. mansoni cercariae or intraperitoneally injected 2,500 schistosome eggs, and experimental asthma was induced by ovalbumin (OVA). The number of eosinophils in bronchoalveolar lavage fluid was higher in the asthmatic group than in asthmatic mice infected with S. mansoni or treated with parasite eggs. Reduced Th2 cytokine production, characterized by lower levels of interleukin-4 (IL-4), IL-5, and immunoglobulin E, was observed in both S. mansoni-treated groups compared to the asthmatic group. There was a reduction in the number of inflammatory cells in lungs of S. mansoni-infected and egg-treated mice, demonstrating that both S. mansoni infection and the egg treatment modulated the lung inflammatory response to OVA. Only allergic animals that were treated with parasite eggs had increased numbers of CD4+ CD25+ Foxp3+ T cells and increased levels of IL-10 and decreased production of CCL2, CCL3, and CCL5 in the lungs compared to the asthmatic group. Neutralization of IL-10 receptor or depletion of CD25+ T cells in vivo confirmed the critical role of CD4+ CD25+ Foxp3+ regulatory T cells in experimental asthma modulation independent of IL-10.

Phosphoinositide-3 kinases critically regulate the recruitment and survival of eosinophils in vivo : importance for the resolution of allergic inflammation

The phosphatidylinositol‐3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3Kγ‐deficient mice was inhibited at 48 h, as compared with wild‐type mice but not at earlier time‐points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3Kγ in eosinophils but not in non‐bone marrow‐derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin‐5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3Kγ for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.

Treatment with DF 2162, a non-competitive allosteric inhibitor of CXCR1/2, diminishes neutrophil influx and inflammatory hypernociception in mice.

Neutrophil migration into tissues is involved in the genesis of inflammatory pain. Here, we addressed the hypothesis that the effect of CXC chemokines on CXCR1/2 is important to induce neutrophil migration and inflammatory hypernociception.

Blockade of the chemokine receptor CXCR2 ameliorates adjuvant-induced arthritis in rats

Chemokine receptors CXCR1 and CXCR2 may mediate influx of neutrophils in models of acute and chronic inflammation. The potential benefits of oral administration of a CXCR1/2 inhibitor, DF 2162, in adjuvant‐induced polyarthritis (AIA) were investigated.

Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kγ and PI3Kδ for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kγ−/− or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kγ−/− or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kδ inhibitor, IC87114, or a specific PI3Kγ inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kγ inhibitor or in PI3Kγ−/− mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kδ in PI3Kγ−/− mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kγ−/− mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kγ−/− mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kγ. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kγ and PI3Kδ for neutrophil recruitment induced by different chemoattractants in vivo.

Peptides containing T cell epitopes, derived from Sm14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice

Sm14 and paramyosin are two major Schistosoma mansoni vaccine candidate antigens. Recently, we have identified Sm14 and paramyosin epitopes that are recognized by T cells of resistant individuals living in endemic areas for schistosomiasis. Herein, mice were immunized with these peptides separately or in association in order to evaluate their vaccine potential. Immunization of mice with Sm14 peptides alone or mixed with paramyosin peptides was able to induce 26%-36.7% or 28%-29.2% of worm burden reduction, 67% or 46% of intestinal eggs reduction and also 54%-61% or 43%-52% of liver pathology reduction, respectively. Protection was associated with a Th1 type of immune response induced by Sm14 peptide immunization. In contrast, paramyosin peptide vaccination did not engender protective immunity or liver pathology reduction and immunization was associated with a Th2 type of immune response.

Impaired host defense to Klebsiella pneumoniae infection in mice treated with the PDE4 inhibitor rolipram

The increase in levels of cAMP in leukocytes by selective inhibitors of PDE4 may result in reduction of inflammation, and may be useful in the treatment of pulmonary inflammatory disorders in humans. Here, we have assessed whether oral treatment with the prototype PDE4 inhibitor, rolipram, interfered with the antibacterial host response following pulmonary infection of mice with Klebsiella pneumoniae. K. pneumoniae infection induced a marked increase in the recruitment of neutrophils to the lungs and the production of proinflammatory cytokines and chemokines, including tumor necrosis factor‐α (TNF‐α) and keratinocyte‐derived chemokine (KC), in bronchoalveolar (BAL) fluid and lung tissue. There were also detectable amounts of interleukin‐10 (IL‐10) and significant lethality. Treatment with rolipram (3–30 mg kg−1) was associated with earlier lethality and significant inhibition of the TNF‐α production. This was associated with enhanced production of IL‐10 in lung tissue of rolipram‐treated animals. Rolipram treatment did not affect KC expression and the recruitment of neutrophils in the lung tissue. Over 70% of neutrophils that migrated into the BAL fluid following K. pneumoniae infection ingested bacteria. Treatment with rolipram inhibited the percentage of neutrophils undergoing phagocytosis of K. pneumoniae in a dose‐dependent manner. Maximal inhibition (62%) occurred at doses equal to or greater than 10 mg kg−1. Thus, treatment of mice with the PDE4 inhibitor rolipram is accompanied by earlier lethality, enhanced bacterial load and decreased capacity of the responding host to produce TNF‐α and of neutrophils to phagocytose bacteria. It will be important to investigate whether the shown ability of PDE4 inhibitors to inhibit neutrophil phagocytosis and control experimental bacterial infection will translate into an inhibition of the ability of neutrophils to deal with infectious microorganisms in the clinical setting.

Potential Role of the Chemokine Macrophage Inflammatory Protein 1α in Human and Experimental Schistosomiasis

ABSTRACT In human schistosomiasis, the concentrations of the chemokine macrophage inflammatory protein 1α (MIP-1α/CCL3) is greater in the plasma of patients with clinical hepatosplenic disease. The objective of the present study was to confirm the ability of CCL3 to detect severe disease in patients classified by ultrasonography (US) and to evaluate the potential role of CCL3 in Schistosoma mansoni-infected mice. CCL3 was measured by enzyme-linked immunosorbent assay in the plasma of S. mansoni-infected patients. CCL3-deficient mice were infected with 25 cercariae, and various inflammatory and infectious indices were evaluated. The concentration of CCL3 was higher in the plasma of S. mansoni-infected than noninfected patients. Moreover, CCL3 was greater in those with US-defined hepatosplenic than with the intestinal form of the disease. In CCL3-deficient mice, the size of the granuloma and the liver eosinophil peroxidase activity and collagen content were diminished compared to wild-type mice. In CCL3-deficient mice, the worm burden after 14 weeks of infection, but not after 9 weeks, was consistently smaller. The in vitro response of mesenteric lymph node cells to antigen stimulation was characterized by lower levels of interleukin-4 (IL-4) and IL-10. CCL3 is a marker of disease severity in infected humans, and experimental studies in mice suggest that CCL3 may be a causative factor in the development of severe schistosomiasis.

Platelet-Activating Factor Receptor Deficiency Delays Elimination of Adult Worms but Reduces Fecundity in Strongyloides venezuelensis-Infected Mice

ABSTRACT We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR−/−) and wild-type mice after a single Strongyloides venezuelensis infection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR−/− and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR−/− mice. Although PAFR−/− infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR−/− mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR−/− mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR−/− mice than in the corresponding preparations from wild-type mice. Thus, in S. venezuelensis-infected PAFR−/− mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.