Daniele G. Souza

The chemokine receptors CXCR1/CXCR2 modulate antigen-induced arthritis by regulating adhesion of neutrophils to the synovial microvasculature.

OBJECTIVE The chemokine receptors CXCR1 and CXCR2 play a role in mediating neutrophil recruitment and neutrophil-dependent injury in several models of inflammation. We undertook this study to investigate the role of these receptors in mediating neutrophil adhesion, subsequent migration, and neutrophil-dependent hypernociception in a murine model of monarticular antigen-induced arthritis (AIA). METHODS AIA was induced by administration of antigen into the knee joint of previously immunized mice. Intravital microscopy studies were performed to assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated using an electronic pressure meter. Neutrophil accumulation in the tissue was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity. Levels of tumor necrosis factor alpha (TNFalpha) and the chemokines CXCL1 and CXCL2 were quantified by enzyme-linked immunosorbent assay. Histologic analysis was performed to evaluate the severity of arthritis and leukocyte infiltration. RESULTS Antigen challenge in immunized mice induced production of TNFalpha, CXCL1, and CXCL2 and also resulted in neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Treatment with reparixin or DF2162 (allosteric inhibitors of CXCR1/CXCR2) decreased neutrophil recruitment, an effect that was associated with marked inhibition of neutrophil adhesion. Drug treatment also inhibited TNFalpha production, hypernociception, and the overall severity of the disease in the tissue. CONCLUSION Blockade of CXCR1/CXCR2 receptors inhibits neutrophil recruitment by inhibiting the adhesion of neutrophils to synovial microvessels. As a consequence, there is decreased local cytokine production and reduced hypernociception, as well as ameloriation of overall disease in the tissue. These studies suggest a potential therapeutic role for the modulation of CXCR1/CXCR2 receptor signaling in the treatment of arthritis.

NLRP3 inflammasome–mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout

OBJECTIVE Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout. METHODS Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1β (IL-1β), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1β, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. RESULTS Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1β/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1β production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1β. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. CONCLUSION These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Involvement of the Chemokine RANTES (CCL5) in Resistance to Experimental Infection with Leishmania major

ABSTRACT The expression and putative role of chemokines during infection with Leishmania major in mice were investigated. CCL5 expression correlates with resistance, and blockade of CCL5 rendered mice more susceptible to infection. CCL5 is part of the cascade of events leading to efficient parasite control in L. major infection.

Cooperative role of tumour necrosis factor‐α, interleukin‐1β and neutrophils in a novel behavioural model that concomitantly demonstrates articular inflammation and hypernociception in mice

BACKGROUND AND PURPOSE Chronic joint inflammation and pain are the hallmarks of disease in patients with inflammatory arthritis, notably rheumatoid arthritis. The aim of the present study was to investigate the relative contribution of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and neutrophil influx for joint inflammation and nociception in a novel murine model of antigen‐induced arthritis (AIA).

Experimental arthritis exacerbates Aggregatibacter actinomycetemcomitans‐induced periodontitis in mice

AIM This study aimed to investigate whether chronic antigen-induced arthritis (AIA) influences infection-induced periodontitis (PD) in mice and whether PD modifies the clinical course of AIA. The contribution of anti-TNF-α therapy was also evaluated. MATERIALS AND METHODS The PD was induced in C57BL/6 mice by oral infection with Aggregatibacter actinomycetemcomitans. AIA was induced after infection. Anti-TNF-α and chlorhexidine therapies were used to investigate the role of TNF-α and oral infection on PD and AIA interaction. Maxillae, knee joints, lymph nodes and serum samples were used for histomorphometric, immunoenzymatic and/or real time-PCR analyses. RESULTS Antigen-induced arthritis exacerbated alveolar bone loss triggered by PD infection. In contrast, PD did not influence AIA in the evaluated time-points. PD exacerbation was associated with enhanced production of IFN-γ in maxillae and expression of the Th1 transcription factor tBET in submandibular lymph nodes. Increased serum levels of IL-6 and C-reactive protein were also detected. Anti-TNF-α and antiseptic therapies prevented the development and exacerbation of infectious-PD. Anti-TNF-α therapy also resulted in reduced expression of IFN-γ, TNF-α and IL-17 in maxillae. CONCLUSIONS Altogether, the current results indicate that the exacerbation of infection-induced PD by arthritis is associated with an alteration in lymphocyte polarization pattern and increased systemic immunoreactivity. This process was ameliorated by anti-TNF-α and antiseptic therapies.

MIF induces osteoclast differentiation and contributes to progression of periodontal disease in mice

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by microorganisms from the oral biofilm. Oral inoculation of mice with the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) induces marked alveolar bone loss and local production of inflammatory mediators, including Macrophage Migration Inhibitory Factor (MIF). The role of MIF for alveolar bone resorption during PD is not known. In the present study, experimental PD was induced in BALB/c wild-type mice (WT) and MIF knockout mice (MIF⁻/⁻) through oral inoculation of Aa. Despite enhanced number of bacteria, MIF⁻/⁻ mice had reduced infiltration of TRAP-positive cells and reduced alveolar bone loss. This was associated with decreased neutrophil accumulation and increased levels of IL-10 in periodontal tissues. TNF-α production was similar in both groups. In vitro, LPS from Aa enhanced osteoclastic activity in a MIF-dependent manner. In conclusion, MIF has role in controlling bacterial growth in the context of PD but contributes more significantly to the progression of bone loss during PD by directly affecting differentiation and activity of osteoclasts.

Cryptococcus gattii: In Vitro Susceptibility to Photodynamic Inactivation

Cryptococus gattii is an emergent primary human pathogen that causes meningismus, papilledema, high intracranial pressure and focal involvement of the central nervous system in immunocompetent hosts. Prolonged antifungal therapy is the conventional treatment, but it is highly toxic, selects for resistant strains, contributes to therapy failure and has a poor prognosis. Photodynamic inactivation (PDI) offers a promising possibility for the alternative treatment of cryptococcosis. The aim of this study was to test the effectiveness of toluidine blue O (TBO) and light‐emitting diode (LED) against C. gattii strains with distinct susceptibility profile to antifungal drugs (amphotericin B: 0.015–1.0 μg mL−1; itraconazole: 0.015–2 μg mL−1; fluconazole: 4–64 μg mL−1). Using 25 μm (6.76 μg mL−1) TBO and LED energy density of 54 J cm−2 these fungal isolates presented variable susceptibility to PDI. The production of reactive oxygen species (ROS)/peroxynitrite was determined, and the catalase and peroxidase activities were measured. After PDI, high amounts of ROS/peroxynitrite are produced and higher catalase and peroxidase activities could be correlated with a lower susceptibility of C. gattii isolates to PDI. These results indicate that PDI could be an alternative to C. gattii growth inhibition, even of isolates less susceptible to classical antifungal drugs, also pointing to mechanisms related to their variable susceptibility behavior.

Macrophage migration inhibitory factor drives neutrophil accumulation by facilitating IL-1β production in a murine model of acute gout.

This study evaluated the role of macrophage migration inhibitory factor in inflammation caused by monosodium urate crystals. The concentration of macrophage migration inhibitory factor was increased in synovial fluid of patients with acute gout, and there was a positive correlation between intra‐articular macrophage migration inhibitory factor and IL‐1β concentrations. In mice, the injection of monosodium urate crystals into the knee joint increased the levels of macrophage migration inhibitory factor in macrophages and in inflamed tissue. The injection of recombinant macrophage migration inhibitory factor into the joint of mice reproduced the inflammatory response observed in acute gout, including histologic changes, the recruitment of neutrophils, and increased levels of IL‐1β and CXCL1. Importantly, the accumulation of neutrophils and the amount IL‐1β in the joints were reduced in macrophage migration inhibitory factor‐deficient mice when injected with monosodium urate crystals. We observed a similar effect when we blocked macrophage migration inhibitory factor with (S,R)‐3‐(4‐hydroxyphenyl)‐4,5‐dihydro‐5‐isoxazole acetic acid or anti‐macrophage migration inhibitory factor. In addition, the blockade of IL‐1R and CXCR2 reduced recombinant macrophage migration inhibitory factor‐induced neutrophil recruitment. Mechanistically, recombinant macrophage migration inhibitory factor is important for the synthesis of il1β mRNA in vivo and in isolated macrophages. Altogether, macrophage migration inhibitory factor promotes neutrophil accumulation and is important for IL‐1β production, which are 2 crucial events contributing to the pathogenesis of acute gout.

Platelet-Activating Factor Receptor Blockade Ameliorates Aggregatibacter actinomycetemcomitans-Induced Periodontal Disease in Mice

ABSTRACT Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by oral biofilm-producing microorganisms, such as Aggregatibacter actinomycetemcomitans. The levels of the phospholipid platelet-activating factor (PAF) in the saliva, gingival crevicular fluid, and periodontal tissues are significantly increased during inflammatory conditions, such as PD, but the exact mechanism that links PAF to alveolar bone resorption is not well understood. In the current study, alveolar bone resorption was induced by experimental PD through the oral inoculation of A. actinomycetemcomitans in wild-type (WT) and PAF receptor knockout (Pafr −/−) mice. In vitro experiments using A. actinomycetemcomitans lipopolysaccharide (LPS)-stimulated RAW 264.7 cells treated with a PAF receptor antagonist (UK74505) were also performed. The expression of lyso-PAF acetyltransferase in periodontal tissues was significantly increased 3 h after A. actinomycetemcomitans LPS injection in mice. WT and Pafr −/− mice that were subjected to oral inoculation of A. actinomycetemcomitans presented neutrophil accumulation and increased levels of CXCL-1 and tumor necrosis factor alpha (TNF-α) in periodontal tissues. However, Pafr −/− mice presented less alveolar bone loss than WT mice. The in vitro blockade of the PAF receptor impaired the resorptive activity of A. actinomycetemcomitans LPS-activated osteoclasts. In conclusion, this study shows for the first time that the blockade of PAF receptor may contribute to the progression of PD triggered by A. actinomycetemcomitans by directly affecting the differentiation and activity of osteoclasts.

Multi-walled carbon nanotubes functionalized with recombinant Dengue virus 3 envelope proteins induce significant and specific immune responses in mice

BackgroundDengue is the most prevalent arthropod-borne viral disease in the world. In this article we present results on the development, characterization and immunogenic evaluation of an alternative vaccine candidate against Dengue.MethodsThe MWNT-DENV3E nanoconjugate was developed by covalent functionalization of carboxylated multi-walled carbon nanotubes (MWNT) with recombinant dengue envelope (DENV3E) proteins. The recombinant antigens were bound to the MWNT using a diimide-activated amidation process and the immunogen was characterized by TEM, AFM and Raman Spectroscopy. Furthermore, the immunogenicity of this vaccine candidate was evaluated in a murine model.ResultsImmunization with MWNT-DENV3E induced comparable IgG responses in relation to the immunization with non-conjugated proteins; however, the inoculation of the nanoconjugate into mice generated higher titers of neutralizing antibodies. Cell-mediated responses were also evaluated, and higher dengue-specific splenocyte proliferation was observed in cell cultures derived from mice immunized with MWNT-DENV3E when compared to animals immunized with the non-conjugated DENV3E.ConclusionsDespite the recent licensure of the CYD-TDV dengue vaccine in some countries, results from the vaccine’s phase III trial have cast doubts about its overall efficacy and global applicability. While questions about the effectiveness of the CYD-TDV vaccine still lingers, it is wise to keep at hand an array of vaccine candidates, including alternative non-classical approaches like the one presented here.