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Background: Cryptococcosis is a relevant invasive fungal infection that affects immunocompromised and immunocompetent individuals when caused by Cryptococcus gattii. Host innate and adaptive immune responses can be subverted by C. gattii, that blocks the differentiation of T helper (Th) 1 and Th17 cells, which are involved in the protection against cryptococcosis. Moreover, the macrophage polarization is modulated by C. gattii infection that requires a balance in the macrophage subsets to control the C. gattii infection. Toll-like receptor (TLR) 2 agonists are important immunomodulators favoring a pro-inflammatory response with potential fungicidal activity, and TLR2 agonists have been used as adjuvants in vaccines against infections caused by bacteria or viruses. Therefore, this work aimed to evaluate the immunomodulatory effect of the tripalmitoyl lipopeptide S-glycerol cysteine (Pam3CSK4 or P3C4), a TLR2 agonist, as an adjuvant in the vaccination against C. gattii infection. Methods and Results: C57BL/6 mice were immunized with 2 × 107 inactivated yeasts of C. gattii via intranasal route on day 1, 14 and 28 (Immunized group). Immunization was associated with 1µg or 10µg of adjuvant P3C4 (Immunized+P3C4-1µg or Immunized+P3C4-10 µg), followed by C. gattii infection on day 42 after the immunization protocol. Immunized+P3C4-1 µg group had reduced levels of IgG1, IgG2a and IgA and no significant difference in the IgG and IgM anti-GXM antibody titer was detected, compared to the Immunized group. High levels of IL-17 and IL-1ß in lung tissue of mice from the Immunized+P3C4-1µg group did not promote a predominance of Th17 cells, in contrast, the frequency of TLR2+ cells was increased in immunized mice that received 1 µg of P3C4. The reduction in the relative expression of T-bet and high levels of Foxp3 detected in the lungs of the Immunized+P3C4-1µg group suggest a prevalence of regulatory T cells in the tissue, which did not contribute to the control of C. gattii infection. The immunization protocol associated with 10 µg of adjuvant P3C4 induced high levels of IL-17 in the lung tissue, whereas the levels of pro-inflammatory cytokines were downregulated. To evaluate the effect of adjuvant P3C4 in the control of C. gattii infection, quantification of the fungal burden in the lungs was performed by the CFU assay, and the groups with adjuvant P3C4 showed a pulmonary C. gattii burden that was not significantly altered when compared with the immunized group. The mice that received 1 µg of adjuvant P3C4 had a lower percentage of inflammatory infiltrate in the lungs. Conclusion: The immunomodulatory effect of P3C4, associated with the immunization protocol, plays an imbalance between pro- and anti-inflammatory response in the lungs that did not favor a protection against C. gattii infection, which is related to the immune response characterized by a suppressive/regulatory profile in the pulmonary microenvironment after C. gattii infection.
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Criptococose , Cryptococcus gattii , Animais , Camundongos , Interleucina-17 , Receptor 2 Toll-Like , Camundongos Endogâmicos C57BL , Criptococose/prevenção & controle , Imunização , Vacinação , Adjuvantes Imunológicos/farmacologiaRESUMO
BACKGROUND: The macrophage lineage is characterized by plasticity due to the acquisition of distinct functional phenotypes, and two major subsets are evaluated; classical M1 activation (strong microbicidal activity) and alternative M2 activation (immunoregulatory functions). The M1 subset expresses inducible nitric oxide synthase (iNOS), which is a primary marker to identify these cells, whereas M2 macrophages are characterized by expression of Arginase-1, found in inflammatory zone 1 (Fizz1), chitinase-like molecule (Ym-1), and CD206. The micro-environmental stimuli and signals in tissues are critical in the macrophage polarization. Toll-like receptors (TLR) ligands, such as lipopolysaccharide (LPS), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and ArtinM (mannose-binding lectin) are inductors of M1 subset. The impact of TLR2 and TLR4 signals to fight against Cryptococcus gattii infection is unknown, which is a fungal pathogen that preferentially infects the lung of immunocompetent individuals. The macrophages initiate an immune response to combat the C. gattii, then we evaluated in RAW 264.7 cell the effect of TLR2 and TLR4 agonists on the macrophage polarization dynamic and the impact on the growth of C. gattii. METHODS AND RESULTS: We demonstrated that P3C4, LPS, and ArtinM induced an increase in the levels of iNOS transcripts in RAW 264.7 cells, whereas the relative expression of arginase-1, Ym-1, and Fizz1 was significantly increased in the presence of IL-4 alone. The effects of TLR2 and TLR4 agonists on repolarization from the M2 to M1 subset was evaluated, and the first stimulus was composed of IL-4 and, after 24 h of incubation, the cells were submitted to a second stimulus of P3C4, LPS, ArtinM, or Medium. These TLR agonists induced the production of TNF-α in polarized RAW 264.7 cells to the M2 subset, moreover the measurement of M1/M2 markers using qRT-PCR demonstrated that a second stimulus with LPS for 24 h induced a significant augmentation of levels of iNOS mRNA. This impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 macrophage was assayed in the presence of C. gattii, the macrophages stimulated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii, as a second stimulus, reached high levels of TNF-α even after incubation with different concentrations of C. gattii. The activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists favored the phagocytosis of C. gattii and inhibited the growth of yeast in the early period of infection. However, RAW 264.7 cells incubated with C. gattii in the presence of TLR2 and TLR4 agonists did not result a significant difference in the colony forming unit (CFU) assay in the early period of C. gattii infection, compared to negative control. CONCLUSION: Polarized RAW 264.7 cells to the M1 subset with TLR2 and TLR4 agonists did not inhibit the growth of C. gattii, whereas robust immunity was identified that could dysregulate host tolerance to this pathogen.
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The use of adjuvants in vaccine formulations is a well-established practice to improve immunogenicity and protective immunity against diseases. Previously, we have demonstrated the feasibility of intranasal vaccination with the antigen of killed Leishmania amazonensis promastigotes (LaAg) against experimental leishmaniasis. In this work, we sought to optimize the immunogenic effect and protective immunity against murine visceral leishmaniasis conferred by intranasal delivery of LaAg in combination with a synthetic TLR1/TLR2 agonist (Pam3CSK4). Intranasal vaccination with LaAg/PAM did not show toxicity or adverse effects, induced the increase of delayed-type hypersensitivity response and the production of inflammatory cytokines after parasite antigen recall. However, mice vaccinated with LaAg/PAM and challenged with Leishmania infantum presented significant reduction of parasite burden in both liver and spleen, similar to those vaccinated with LaAg. Although LaAg/PAM intranasal vaccination had induced higher frequencies of specific CD4+ and CD8+ T cells and increased levels of IgG2a antibody isotype in serum, both LaAg and LaAg/PAM groups presented similar levels of IL-4 and IFN-y and decreased production of IL-10 when compared to controls. Our results provide the first evidence of the feasibility of intranasal immunization with antigens of killed Leishmania in association with a TLR agonist, which may be explored for developing an effective and alternative strategy for vaccination against visceral leishmaniasis.
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Antígenos de Protozoários/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Lipopeptídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Citocinas/sangue , Feminino , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Lipopeptídeos/administração & dosagem , Fígado/metabolismo , Fígado/parasitologia , Linfócitos/imunologia , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Baço/metabolismo , Baço/parasitologia , VacinaçãoRESUMO
INTRODUCTION: Platelets express Toll-like receptors (TLRs) that recognise molecular components of pathogens and, in nucleated cells, elicit immune responses through nuclear factor-kappaB (NF-κB) activation. We have shown that NF-κB mediates platelet activation in response to classical agonists, suggesting that this transcription factor exerts non-genomic functions in platelets. The aim of this study was to determine whether NF-κB activation is a downstream signal involved in TLR2 and 4-mediated platelet responses. MATERIAL AND METHODS: Aggregation and ATP release were measured with a Lumi-aggregometer. Fibrinogen binding, P-selectin and CD40 ligand (CD40L) levels and platelet-neutrophil aggregates were measured by cytometry. I kappa B alpha (IκBα) degradation and p65 phosphorylation were determined by Western blot and von Willebrand factor (vWF) by ELISA. RESULTS: Platelet stimulation with Pam3CSK4 or LPS resulted in IκBα degradation and p65 phosphorylation. These responses were suppressed by TLR2 and 4 blocking and synergised by thrombin. Aggregation, fibrinogen binding and ATP and vWF release were triggered by Pam3CSK4. LPS did not induce platelet responses per se, except for vWF release, but it did potentiate thrombin-induced aggregation, fibrinogen binding and ATP secretion. Pam3CSK4, but not LPS, induced P-selectin and CD40L expression and mixed aggregate formation. All of these responses, except for CD40L expression, were inhibited in platelets treated with the NF-κB inhibitors BAY 11-7082 or Ro 106-9920. CONCLUSION: TLR2 and 4 agonists trigger platelet activation responses through NF-κB. These data show another non-genomic function of NF-κB in platelets and highlight this molecule as a potential target to prevent platelet activation in inflammatory or infectious diseases.