TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.

MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection

The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.

CCR2 Deficiency Results in Increased Osteolysis in Experimental Periapical Lesions in Mice

Introduction: Periapical lesions are chronic inflammatory disorders of periradicular tissues caused by etiologic agents of endodontic origin. The inflammatory chemokines are thought to be involved in the latter observed osteolysis. With a murine model of experimental periapical lesion, the objective of this study was to evaluate the role of the chemokine receptor CCR2 in the lesion progression, osteoclast differentiation and activation, and expression of inflammatory osteolysis-related mediators. Methods: For lesion induction, right mandibular first molars were opened surgically with a (1)/(4) carbine bur, and 4 bacterial strains were inoculated in the exposed dental pulp; left mandibular first molars were used as controls. Animals were killed at 3, 7, 14, and 21 days after surgeries to evaluate the kinetics of lesion development. Results: CCR2 KO mice showed wider lesions than WT mice. CCR2 KO mice also expressed higher levels of the osteoclastogenic and osteolytic factors, receptor activator of nuclear factor kappa B ligand (RANKL) and cathepsin K, of the proinflammatory cytokine tumor necrosis factor alpha, and of the neutrophil migration related chemokine, KC. Conclusions: These results suggest that CCR2 is important in host protection to periapical osteolysis. (J Endod 2010;36:244-250)

Bone marrow chimeras and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Host defense at the ocular surface

Microbial infections of the cornea frequently cause painful, blinding and debilitating disease that is often difficult to treat and may require corneal transplantation. In addition, sterile corneal infiltrates that are associated with contact lens wear cause pain, visual impairment and photophobia. In this article, we review the role of Toll-Like Receptors (TLR) in bacterial keratitis and sterile corneal infiltrates, and describe the role of MD-2 regulation in LPS responsiveness by corneal epithelial cells. We conclude that both live bacteria and bacterial products activate Toll-Like Receptors in the cornea, which leads to chemokine production and neutrophil recruitment to the corneal stroma. While neutrophils are essential for bacterial killing, they also cause tissue damage that results in loss of corneal clarity. These disparate outcomes, therefore, represent a spectrum of disease severity based on this pathway, and further indicate that targeting the TLR pathway is a feasible approach to treating inflammation caused by live bacteria and microbial products. Further, as the P. aeruginosa type III secretion system (T3SS) also plays a critical role in disease pathogenesis by inducing neutrophil apoptosis and facilitating bacterial growth in the cornea, T3SS exotoxins are additional targets for therapy for P. aeruginosa keratitis.

Hypertension favors the inflammatory process in rats with experimentally induced periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Functional and Ultrastructural Analysis of Annexin A1 and Its Receptor in Extravasating Neutrophils during Acute Inflammation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo dos mecanismos envolvidos na resposta tecidual de camundongos diabéticos com doença periodontal: papel dos mastócitos

Pós-graduação em Ciência Odontólogica - FOA

Toll-like receptor 9 activation in neutrophils impairs chemotaxis and reduces sepsis outcome

Objectives: To investigate the role of toll-like receptor 9 on sepsis-induced failure of neutrophil recruitment to the site of infection. Design: Prospective experimental study. Setting: University research laboratory. Interventions: Model of polymicrobial sepsis induced by cecal ligation and puncture in wild-type and toll-like receptor 9-deficient mice. Measurements and Main Results: Toll-like receptor 9-deficient mice with cecal ligation and puncture-induced severe sepsis did not demonstrate failure of neutrophil migration and consequently had a low systemic inflammatory response and a high survival rate. Upon investigating the mechanism by which toll-like receptor 9-deficiency prevents the failure of neutrophil migration, it was found that neutrophils derived from toll-like receptor 9-deficient mice with cecal ligation and puncture induced severe sepsis expressed high levels of chemokine C-X-C motif receptor 2 (CXCR2) and had reduced induction of G-protein-coupled receptor kinase 2. Conclusions: These findings suggest that the poor outcome of severe sepsis is associated with toll-like receptor 9 activation in neutrophils, which triggers G-protein-coupled receptor kinase 2 expression and CXCR2 downregulation. These events account for the reduction of neutrophil migration to the site of infection, with consequent spreading of the infection, onset of the systemic inflammatory response, and a decrease in survival. (Crit Care Med 2012; 40:2631-2637)

Joint NOD2/RIPK2 Signaling Regulates IL-17 Axis and Contributes to the Development of Experimental Arthritis

Intracellular pattern recognition receptors such as the nucleotide-binding oligomerization domain (NOD)-like receptors family members are key for innate immune recognition of microbial infection and may play important roles in the development of inflammatory diseases, including rheumatic diseases. In this study, we evaluated the role of NOD1 and NOD2 on development of experimental arthritis. Ag-induced arthritis was generated in wild-type, NOD1(-/-)!, NOD2(-/-), or receptor-interacting serine-threonine kinase 2(-/-) (RIPK2(-/-)) immunized mice challenged intra-articularly with methylated BSA. Nociception was determined by electronic Von Frey test. Neutrophil recruitment and histopathological analysis of proteoglycan lost was evaluated in inflamed joints. Joint levels of inflammatory cytokine/chemokine were measured by ELISA. Cytokine (IL-6 and IL-23) and NOD2 expressions were determined in mice synovial tissue by RT-PCR. The NOD2(-/-) and RIPK2(-/-), but not NOD1(-/-), mice are protected from Ag-induced arthritis, which was characterized by a reduction in neutrophil recruitment, nociception, and cartilage degradation. NOD2/RIPK2 signaling impairment was associated with a reduction in proinflammatory cytokines and chemokines (TNF, IL-1 beta, and CXCL1/KC). IL-17 and IL-17 triggering cytokines (IL-6 and IL-23) were also reduced in the joint, but there is no difference in the percentage of CD4(+) IL-17(+) cells in the lymph node between arthritic wild-type and NOD2(-/-) mice. Altogether, these findings point to a pivotal role of the NOD2/RIPK2 signaling in the onset of experimental arthritis by triggering an IL-17-dependent joint immune response. Therefore, we could propose that NOD2 signaling is a target for the development of new therapies for the control of rheumatoid arthritis. The Journal of Immunology, 2012, 188: 5116-5122.

Serrumab: A human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom

In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNF alpha, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.

Intestinal Lymph-Borne Factors Induce Lung Release of Inflammatory Mediators and Expression of Adhesion Molecules After an Intestinal Ischemic Insult

Background. Intestinal ischemia and reperfusion (I/R) is a documented cause of acute lung injury (ALI) and systemic inflammation. We previously reported that obstruction of thoracic lymphatic flow during intestinal I/R blunts pulmonary neutrophil recruitment and microvascular injury and decreases the systemic levels of tumor necrosis factor. Here, we consider the existence of a gut-lung axis promoting the induction of systemic inflammation, whereby drained intestinal lymph stimulates lung expression of adhesion molecules and matrix components and generation of inflammatory mediators. Material and Methods. Upon administration of anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by 2 h of intestinal reperfusion (I/R); groups of rats were subjected to I/R with or without thoracic lymphatic duct ligation immediately before the procedure. The non-manipulated rats were used to investigate basal parameters. Results. Obstruction of thoracic lymphatic flow before intestinal I/R decreased the ability of cultured lung tissue explants to release IL-1 beta, IL-10, and VEGF. In contrast, lymphatic obstruction normalized the elevated lung expression of PECAM-1 caused by intestinal I/R. On the other hand, lung E-selectin expression was significantly reduced, whereas fibronectin expression and collagen synthesis were not affected. Lymph levels of LTB4 and TXB2 were found to be significantly increased. Conclusions. These data suggest that lymph factors drained from the intestine during ischemic trauma stimulate the lung to generate inflammatory mediators and alter the expression of adhesion molecules. Disturbances in lung homeostasis mediated by lymph might contribute to the spread of inflammatory processes, thereby accounting for the systemic inflammation induced by intestinal I/R. (C) 2012 Elsevier Inc. All rights reserved.

Gastrin-releasing peptide receptor (GRPR) mediates chemotaxis in neutrophils

Neutrophil migration to inflamed sites is crucial for both the initiation of inflammation and resolution of infection, yet these cells are involved in perpetuation of different chronic inflammatory diseases. Gastrin-releasing peptide (GRP) is a neuropeptide that acts through G protein coupled receptors (GPCRs) involved in signal transmission in both central and peripheral nervous systems. Its receptor, gastrin-releasing peptide receptor (GRPR), is expressed by various cell types, and it is overexpressed in cancer cells. RC-3095 is a selective GRPR antagonist, recently found to have antiinflammatory properties in arthritis and sepsis models. Here we demonstrate that i.p. injection of GRP attracts neutrophils in 4 h, and attraction is blocked by RC-3095. Macrophage depletion or neutralization of TNF abrogates GRP-induced neutrophil recruitment to the peritoneum. In vitro, GRP-induced neutrophil migration was dependent on PLC-beta 2, PI3K, ERK, p38 and independent of G alpha i protein, and neutrophil migration toward synovial fluid of arthritis patients was inhibited by treatment with RC-3095. We propose that GRPR is an alternative chemotactic receptor that may play a role in the pathogenesis of inflammatory disorders.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.