Claudia F. Benjamim

Inhibition of Leukocyte Rolling by Nitric Oxide during Sepsis Leads to Reduced Migration of Active Microbicidal Neutrophils

ABSTRACT We developed two models of sepsis with different degrees of severity, sublethal and lethal sepsis, induced by cecal ligation and puncture. Lethal sepsis induced by cecal ligation and puncture (L-CLP) resulted in failure of neutrophil migration to the infection site and high mortality. Treatment of septic animals with aminoguanidine (AG), a nitric oxide (NO) synthase inhibitor, precluded the failure of neutrophil migration and protected the animals from death. However, cytokine-induced NO synthase (iNOS)-deficient (iNOS−/−) mice subjected to L-CLP did not present neutrophil migration failure, but 100% lethality occurred. iNOS−/− mice subjected to sublethal sepsis induced by cecal ligation and puncture (SL-CLP) also suffered high mortality despite the occurrence of neutrophil migration. This apparent paradox could be explained by the lack of microbicidal activity in neutrophils of iNOS−/− mice present at the infection site due to their inability to produce NO. Notably, SL- and L-CLP iNOS−/− mice showed high bacterial numbers in exudates. The inhibition of neutrophil migration by NO is due to inhibition of a neutrophil/endothelium adhesion mechanism, since a reduction in leukocyte rolling, adhesion, and emigration was observed in L-CLP wild-type mice. These responses were prevented by AG treatment and were not observed in the iNOS−/− L-CLP group. There was no significant change in L-selectin expression in neutrophils from L-CLP mice. Thus, it seems that the decrease in leukocyte rolling is due to a defect in the expression of adhesion molecules on endothelial surfaces mediated by iNOS-derived NO. In conclusion, the results indicate that despite the importance of NO in neutrophil microbicidal activity, its generation in severe sepsis reduces neutrophil migration by inhibiting leukocyte rolling and their firm adhesion to the endothelium, in effect impairing the migration of leukocytes and consequently their fundamental role in host cell defense mechanisms.

Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus

Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats compared with FeLV(+) symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV(+) symptomatic cats were higher than those in unstimulated neutrophils from FeLV(-) and FeLV(+) asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV(+) cats and that this feature could be considered a marker of disease progression in FeLV infection.

Role of regulatory T cells in long-term immune dysfunction associated with severe sepsis.

Objective:To investigate the role of regulatory T cells in the modulation of long-term immune dysfunction during experimental sepsis. It is well established that sepsis predisposes to development of a pronounced immunosuppression. Nevertheless, the mechanisms underlying the immune dysfunction after sepsis are still not well understood. Design:Prospective experimental study. Setting:University research laboratory. Interventions:Wild-type mice underwent cecal ligation and puncture and were treated with antibiotic during 3 days after surgery. On days 1, 7, or 15 after cecal ligation and puncture, the frequency of regulatory T cells, proliferation of CD4+ T cells and bacterial counts were evaluated. Fifteen days after cecal ligation and puncture, surviving mice underwent secondary pulmonary infection by intranasal inoculation of nonlethal dose of Legionella pneumophila. Some mice received agonistic glucocorticoid-induced tumor necrosis factor receptor antibody (DTA-1) before induction of secondary infection. Measurements and Main Results:Mice surviving cecal ligation and puncture showed a markedly increased frequency of regulatory T cells in thymus and spleen, which was associated with reduced proliferation of CD4+ T cells. Fifteen days after cecal ligation and puncture, all sepsis-surviving mice succumbed to nonlethal injection of L. pneumophila. Treatment of mice with DTA-1 antibody reduced frequency of regulatory T cells, restored CD4+ T cell proliferation, reduced the levels of bacteria in spleen, and markedly improved survival of L. pneumophila infection. Conclusion:These findings suggest that regulatory T cells play an important role in the progression and establishment of immune dysfunction observed in experimental sepsis.

Leukotriene B4 Mediates Neutrophil Migration Induced by Heme

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B4 (LTB4). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B4 receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO−/−) mice. Heme administration in vivo increased peritoneal levels of LTB4 prior to and during neutrophil recruitment. Evidence that LTB4 was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB4 was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB4 production or neutrophil influx; 4) increased levels of LTB4 were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB4. Our findings uncover a crucial role of LTB4 in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.

Oxidative stress and nitrosative stress are involved in different stages of proteolytic pulmonary emphysema

Our aim was to investigate the role of oxidative stress in elastase-induced pulmonary emphysema. C57BL/6 mice were subjected to pancreatic porcine elastase (PPE) instillation (0.05 or 0.5 U per mouse, i.t.) to induce pulmonary emphysema. Lungs were collected on days 7, 14, and 21 after PPE instillation. The control group was sham injected. Also, mice treated with 1% aminoguanidine (AMG) and inducible NO synthase (iNOS) knockout mice received 0.5 U PPE (i.t.), and lungs were analyzed 21 days after. We performed bronchoalveolar lavage, biochemical analyses of oxidative stress, and lung stereology and morphometry assays. Emphysema was observed histologically at 21 days after 0.5 U PPE treatment; tissues from these mice exhibited increased alveolar linear intercept and air-space volume density in comparison with the control group. TNF-α was elevated at 7 and 14 days after 0.5 U PPE treatment, concomitant with a reduction in the IL-10 levels at the same time points. Myeloperoxidase was elevated in all groups treated with 0.5 U PPE. Oxidative stress was observed during early stages of emphysema, with increased nitrite levels and malondialdehyde and superoxide dismutase activity at 7 days after 0.5 U PPE treatment. Glutathione peroxidase activity was increased in all groups treated with 0.5 U PPE. The emphysema was attenuated when iNOS was inhibited using 1% AMG and in iNOS knockout mice. Furthermore, proteolytic stimulation by PPE enhanced the expression of nitrotyrosine and iNOS, whereas the PPE+AMG group showed low expression of iNOS and nitrotyrosine. PPE stimulus also induced endothelial (e) NOS expression, whereas AMG reduced eNOS. Our results suggest that the oxidative and nitrosative stress pathways are triggered by nitric oxide production via iNOS expression in pulmonary emphysema.

CCR2 Receptor Is Essential to Activate Microbicidal Mechanisms to Control Toxoplasma gondii Infection in the Central Nervous System

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T. gondii infection. We orally infected CCR2(-/-) mice with five ME-49 T. gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2(-/-) mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2(-/-) mice compared with wild-type C57BL/6 mice. Additionally, CCR2(-/-) mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4(+) and MAC-1(+) and greater CD8(+) cell migration. The parasite load decreased in these organs in CCR2(-/-) mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2(-/-) mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T. gondii replication in the central nervous system.

Failure of neutrophil migration to infectious focus and cardiovascular changes on sepsis in rats : Effects of the inhibition of nitric oxide production, removal of infectious focus, and antimicrobial treatment

ABSTRACT Recently, we demonstrated that mice under lethal sepsis present failure of neutrophil migration (FNM) to infectious focus, which is mediated by nitric oxide. The aims of the present study were to investigate whether FNM is also observed in severe sepsis induced by cecal ligation and puncture in rats and the effects of the prevention of nitric oxide production and of the elimination of the infectious focus through peritoneal lavage or by antimicrobial treatment on FNM and disease outcome. Rats were submitted to several septic stimuli (low, moderate, and severe) by cecal ligation and puncture. Severe septic stimulus animals presented FNM to the peritoneal cavity that was accompanied by large numbers of bacteria in the peritoneal cavity, blood, and liver and lung tissues; high cytokines (tumor necrosis factor &agr;, interleukin [IL] 1&bgr;, IL-6, cytokine-induced neutrophil chemoattractant 1, and IL-10) concentrations in the infection site, sera, and lung tissues; marked hypotension; and high mortality rate. The exhaustive lavage of the peritoneal cavity to reduce the infectious focus did not ameliorate the disease outcome. The association of lavage procedure with aminoguanidine treatment re-established neutrophil migration, but only delayed the death of the animals. In contrast, the antimicrobial treatment of severe septic stimulus animals with sulfamethoxazole and trimethoprim significantly improved the survival rate of the severe septic stimulus but did not re-establish neutrophil migration. However, the association of aminoguanidine plus sulfamethoxazole and trimethoprim brought about a significant increase in the survival rate and re-established neutrophil migration to infectious focus; reduced the colony-forming units in the peritoneal cavity, blood, and lung tissues; and caused an improvement in the cardiovascular performance. The results showed, for the first time, that the pharmacological prevention of FNM to the infectious focus associated with the antimicrobial therapy could be a new beneficial strategy for the treatment of sepsis syndrome.

Leukotriene B4 mediates γδ T lymphocyte migration in response to diverse stimuli

Herein, we investigated the involvement of the 5‐LO‐derived lipid mediator LTB4 in γδ T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB4 triggered γδ T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli endotoxin (LPS) triggered increased levels of LTB4 in pleural cavities. The in vivo inhibition of LTB4 biosynthesis by the 5‐LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS‐induced γδ T cell accumulation into pleural cavities. Accordingly, 5‐LO KO mice failed to recruit γδ T cells into the inflammatory site after i.pl. LPS. Antagonists of the high‐affinity LTB4 receptor BLT1, CP105,696, and LY292476 also attenuated LPS‐induced γδ T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS‐simulated mice. LTB4/BLT1 also accounted for γδ T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS‐recruited γδ T cells. Isolated γδ T cells were found to undergo F‐actin cytoskeleton reorganization when incubated with LTB4 in vitro, confirming that γδ T lymphocytes can respond directly to LTB4. In addition to its direct effect on γδ T cells, LTB4 triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that γδ T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB4/BLT1.

CCL2/MCP-1 controls parasite burden, cell infiltration, and mononuclear activation during acute Trypanosoma cruzi infection

CCL2/MCP‐1 has emerged recently as a critical factor in infectious and autoimmune myocarditis. In fact, this chemokine is produced in great amounts in hearts from Trypanosoma cruzi‐infected mice and is known to enhance parasite uptake and destruction by macrophages. Herein, we studied the involvement of CCL2 in tissue inflammation and resistance to T. cruzi. Infected CCL2−/− mice developed higher parasitemias and died earlier than WT mice. Close to their death, T. cruzi‐infected CCL2−/− presented greater amounts of TNF, IFN‐γ, and IL‐10 in plasma than WTs and clinical signs of systemic inflammatory response. Amastigote nests were more frequent in hearts and livers from infected CCL2−/− tissues than in WTs, and reduced numbers of leukocytes infiltrated their tissues. Leukocytes formed diffuse but not focal infiltrates in hearts from infected CCL2−/− mice, and perivascular cuffs could still be found in their livers. Infected CCL2−/− mice had smaller percentages of activated CD11b (Mac‐1)+CD107b (Mac‐3)+ macrophages and CD8+CD69hi cells among heart and liver infiltrates than WTs (flow cytometry), indicating that CCL2 controls subset migration/activation. CCL2 accumulated among focal heart infiltrates, suggesting that this chemokine is involved in retention of mononuclear cells in particular spots. Peritoneal macrophages from CCL2−/− mice displayed decreased trypanocidal activity. Our results demonstrate that CCL2 contributes to reduce parasite growth and indicate that it does so by controlling the distribution, cellular composition, and state of activation of inflammatory infiltrates in acute T. cruzi infection.

The Chemokine CCL6 Promotes Innate Immunity via Immune Cell Activation and Recruitment

Septic syndrome is a consequence of innate immune failure. Recent studies showed that the CC chemokine CCL6 enhanced antimicrobial immunity during experimental sepsis through an unknown mechanism. The present study demonstrates that transgenic CCL6 expression abolishes mortality in a septic peritonitis model via the modulation of resident peritoneal cell activation and, more importantly, through the recruitment of IFN-producing NK cells and killer dendritic cells into the peritoneum. Thus, CCL6 attenuates the immune failure during sepsis, in part, through a protective type 1-cytokine mediated mechanism.