Daniela Carlos

Hydrogen sulfide improves neutrophil migration and survival in sepsis via K+ATP channel activation.

RATIONALE Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. OBJECTIVES To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. METHODS Sepsis was induced by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS The pretreatments of mice with H(2)S donors (NaHS or Lawessons reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80%. Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed. Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and l-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis. Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to approximately 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). CONCLUSIONS These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

α1-Acid Glycoprotein Decreases Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice

The mechanisms underlying immune deficiency in diabetes are largely unknown. In the present study, we demonstrate that diabetic mice are highly susceptible to polymicrobial sepsis due to reduction in rolling, adhesion, and migration of leukocytes to the focus of infection. In addition, after sepsis induction, CXCR2 was strongly downregulated in neutrophils from diabetic mice compared with nondiabetic mice. Furthermore, CXCR2 downregulation was associated with increased G-protein–coupled receptor kinase 2 (GRK2) expression in these cells. Different from nondiabetic mice, diabetic animals submitted to mild sepsis displayed a significant augment in α1-acid glycoprotein (AGP) hepatic mRNA expression and serum protein levels. Administration of AGP in nondiabetic mice subjected to mild sepsis inhibited the neutrophil migration to the focus of infection, as well as induced l-selectin shedding and rise in CD11b of blood neutrophils. Insulin treatment of diabetic mice reduced mortality rate, prevented the failure of neutrophil migration, impaired GRK2-mediated CXCR2 downregulation, and decreased the generation of AGP. Finally, administration of AGP abolished the effect of insulin treatment in diabetic mice. Together, these data suggest that AGP may be involved in reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice.

Dynamic changes of the Th17/Tc17 and regulatory T cell populations interfere in the experimental autoimmune diabetes pathogenesis.

A balance between proinflammatory (Th17 and Tc17) and anti-inflammatory (regulatory T cells) subsets of T cells is essential to maintain immunological tolerance and prevent the onset of several autoimmune diseases, including type 1 diabetes. However, the kinetics of these subsets and disease severity during the streptozotocin (STZ)-induced diabetes course has not been determined. Thus, susceptible C57BL/6 mice were administrated with multiple low doses of STZ and we evaluated the frequency/absolute number of these T cell subsets in the pancreatic lymph nodes (PLNs) and spleen and Th1, Th17, Treg cytokine production in the pancreatic tissue. At different time points of the disease progression (6, 11, 18 and 25 days after the last STZ administration), the histopathological alterations were also evaluated by H&E and immunohistochemistry staining. During the initial phase of diabetes development (day 6), we noted increased numbers of CD4(+) and CD8(+) T cells in spleen and PLNs. At the same time, the frequencies of Th17 and Tc17 cells in PLNs were also enhanced. In addition, the early augment of interferon gamma (IFN-γ), tumoral necrosis factor (TNF-α), IL-6 and IL-17 levels in pancreatic tissue correlated with pancreatic islet inflammation and mild β-cell damage. Notably, the absolute number of Treg cells increased in PLNs during over time when compared to control group. Interestingly, increased IL-10 levels were associated with control of the inflammatory process during the late phase of the type 1 diabetes (day 25). In agreement, mice lacking the expression of IL-17 receptor (Il17r) showed impairment in STZ-induced diabetes progression, reduced peri-insulitis and beta cells preservation when compared with wild-type mice. Our findings suggest that dynamic changes of pathogenic Th17/Tc17 and regulatory T cell subsets numbers is associated with early strong inflammation in the pancreatic islets followed by late regulatory profile during the experimental STZ-induced diabetes course.

TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection

Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection.

Gut microbiota translocation to the pancreatic lymph nodes triggers NOD2 activation and contributes to T1D onset

Streptozotocin causes T1D by inducing the translocation of intestinal bacteria into pancreatic lymph nodes and driving the development of pathogenic Th1 and Th17 cells through NOD2 receptor.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE2 in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE2 in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE2 production in response to the same stimuli. The decrease of PGE2 production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-α and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-γ. Using a variety of TLR2 ligands, we established that PGE2 release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NFκB but was not dependent on peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-IκBα formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

The IL-33/ST2 Pathway Controls Coxsackievirus B5–Induced Experimental Pancreatitis

Coxsackievirus B (CVB) is a common cause of acute and chronic infectious myocarditis and pancreatitis. Th1 cells producing IFN-γ and TNF-α are important for CVB clearance, but they are also associated with the pathogenesis of inflammatory lesions, suggesting that the modulation of Th1 and Th2 balance is likely important in controlling CVB-induced pancreatitis. We investigated the role of IL-33, which is an important recently discovered cytokine for induction of Th2-associated responses, in experimental CVB5 infection. We found that mice deficient in IL-33R, T1/ST2, significantly developed more severe pancreatitis, had greater weight loss, and contained higher viral load compared with wild-type (WT) mice when infected with CVB5. Conversely, WT mice treated with rIL-33 developed significantly lower viral titers, and pancreatitis was attenuated. Mechanistic studies demonstrated that IL-33 enhances the degranulation and production of IFN-γ and TNF-α by CD8+ T and NK cells, which is associated with viral clearance. Furthermore, IL-33 triggers the production of IL-4 from mast cells, which results in enhanced differentiation of M2 macrophages and regulatory T cells, leading to the attenuation of inflammatory pancreatitis. Adoptively transferred mast cells or M2 macrophages reversed the heightened pancreatitis in the T1/ST2−/− mice. In contrast, inhibition of regulatory T cells exacerbated the disease in WT mice. Together, our findings reveal an unrecognized IL-33/ST2 functional pathway and a key mechanism for CVB5-induced pancreatitis. These data further suggest a novel approach in treating virus-induced pancreatitis, which is a major medical condition with unmet clinical needs.

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection.

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12(-/-) and wild-type C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12(-/-) and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12(-/-) mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12(-/-) mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection.

Mast cells modulate pulmonary acute inflammation and host defense in a murine model of tuberculosis.

BACKGROUND Mast cells (MCs) participate in host resistance to several pathogens, but little is known about the role played by MCs in Mycobacterium tuberculosis infection. METHODS Compound 48/80 (C48/80)-treated mice and nontreated mice were infected intratracheally with 1 x 10(5) viable M. tuberculosis bacilli (MTB; strain H37Rv). RESULTS Infected BALB/c mice developed an acute pulmonary inflammation and had higher levels of tumor necrosis factor- alpha , interleukin-1, keratinocyte-derived chemokine, monocyte chemotactic protein-1, and macrophage inflammatory protein-2 in the lungs by day 15. In vivo degranulation of MCs by C48/80 led to a reduction in the inflammatory reaction that was associated with a marked decline in lung proinflammatory cytokine and chemokine levels. The magnitude of the cellular immune response was also partially impaired in infected mice treated with C48/80. The number of Mycobacteria bacilli recovered from the lungs of infected mice treated with C48/80 was 1 log higher than that recovered from untreated infected mice. C48/80 treatment attenuated the granulomatous inflammation in the lung parenchyma seen in untreated MTB-infected mice. CONCLUSIONS These findings suggest that MCs participate in host defense against M. tuberculosis infection through the production and secretion of cytokines and chemokines that play a role in the recruitment and activation of inflammatory cells in this experimental model.