In the present work, we show that intracellularTrypanosoma cruziis rarely found in the livers of acutely infected mice, but inflammation is commonly observed. The presence of numerous intrahepatic amastigotes in infected gamma interferon (IFN-γ)-deficient mice corroborates the notion that the liver is protected by an efficient local immunity. The contribution of different cell populations was suggested by data showing that CD4- and CD8-deficient mice were able to restrain liver parasite growth. Therefore, we have characterized the liver-infiltrating lymphocytes and determined the sources of IFN-γ during acuteT. cruziinfection. We observed that natural killer (NK) cells increased by day 7, while T and B cells increased by day 14. Among CD3⁺cells, CD4⁺, CD8⁺, and CD4⁻CD8⁻cell populations were greatly expanded. A large fraction of CD3⁺cells were positive for PanNK, a β1 integrin expressed by NK and NK T cells. However, these lymphocytes were not classic NK T cells because they did not express NK1.1 and showed no preferential usage of Vβ8. Otherwise, liver NK T (CD3⁺NK1.1⁺) cells were not increased in acutely infected mice. The majority of PanNK⁺CD4⁺and PanNK⁺CD8⁺cells expressed T-cell receptor αβ (TCRαβ), whereas PanNK⁺CD4⁻CD8⁻cells were positive for TCRγδ. In fact, γδ T cells showed the most remarkable increase (40- to 100-fold) among liver lymphocytes. Most importantly, intracellular analysis revealed high levels of IFN-γ production at day 7 by NK cells and at day 14 by CD4⁺, CD8⁺, and CD4⁻CD8⁻TCRγδ⁺cells. We concluded that NK cells are a precocious source of IFN-γ in the livers of acutely infected mice, and, as the disease progresses, conventional CD4⁺and CD8⁺T cells and γδ T cells, but not classic NK-T cells, may provide the IFN-γ required for liver protection againstT. cruzi.

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5×10⁶ trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1⁺, CD8⁺ and CD4⁺ cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4⁺ and CD8⁺ cells were activated, increased frequencies of CD69⁺CD8⁺, CD69⁺CD4⁺ and CD25⁺CD122⁺CD4⁺ cells were observed at 24 and 48 h after challenge, and of CD25⁻CD122⁺CD4⁺ cells at 48 h. The major role of CD4⁺ cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-γ-producing CD4⁺ cells 24 h after challenge. In contrast, liver CD8⁺ cells produced little IFN-γ, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge. Chagas disease, a Latin American illness caused by the protozoan parasite Trypanosoma cruzi, has only rare spontaneous cure, and in most patients a small number of parasites persists for life in the blood and tissues, leading to chronic disorders such as cardiomyopathy. In a murine model of chronic T. cruzi infection we observed that the liver plays an important role in the clearance of blood-circulating parasites. Moreover, parasite accumulation in this organ is followed by their elimination, an effect that is not immediate but seems to depend on the recruitment of leukocytes and on the local production of IFN-γ, a cytokine known to increase the T. cruzi-killing capacity of phagocytes. Our findings contribute to the knowledge of T. cruzi-host interaction, showing the participation of a non-lymphoid organ in parasite control. In addition, they contribute to understanding the multifaceted role the liver plays in the immune response.

Studies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

Previous observations demonstrated that the delivery of recombinantSalmonella entericaserovar Dublin strains to mice via mucosal routes did not efficiently activate systemic and secreted antibody responses to either typedflagellin or genetically fused heterologous B-cell epitopes, thus reducing the usefulness of the protein as a carrier of epitopes for vaccine purposes. In this work, we investigated murine systemic and mucosal flagellin immunogenicity after oral immunization with attenuatedSalmonellastrains. The reduced anti-typedflagellin antibody responses in mice immunized via mucosal routes with three doses of flagellatedS. entericaserovar Dublin strains were not caused by oral tolerance and could not be restored by coadministration of a mucosal adjuvant. The induction of antibody responses toSalmonellaflagellins was shown to differ according to the genetic background, but not the haplotype, of the mouse lineage. Moreover, BALB/c mice orally immunized withS. entericaserovar Typhimurium strains developed anti-typeiflagellin sera and secreted antibody responses, which indicated that the serovar of theSalmonellavaccine strain also affected flagellin immunogenicity. Analyses of cytokine responses of BALB/c mice immunized with three oral doses of flagellatedS. entericaserovar Dublin vaccine strains showed that, in spite of the lack of antibody responses, elevated typedflagellin-specific CD4-cell-activation-dependent gamma interferon (IFN-γ) and interleukin-10 responses were elicited after the administration of the vaccine strains via either parenteral or mucosal routes. Similar cytokine production patterns were detected to a T-cell heterologous epitope, derived from the CFA/I fimbriae of enterotoxigenicEscherichia coli(ETEC), in mice orally immunized with aSalmonellavaccine strain expressing hybrid flagella. These results indicate that the immunogenicities ofSalmonellaflagellins can differ significantly, depending on the murine host and on the bacterial vector used, and demonstrate that the induction of CD4-cell-activation-dependent IFN-γ production represents a major immune response triggered by flagellin and in-frame fused heterologous T-cell epitopes after the oral administration of recombinantS. entericaserovar Dublin vaccine strains.

Allergic asthma is a chronic inflammatory airway disease, and has been considered a T helper-2-biased response. Studies suggest that neutrophils may be associated with exacerbation and asthma severity. We sought to evaluate the chemotactic activity and phagocytic capacity by peripheral blood neutrophils from individuals with controlled and uncontrolled allergic asthma, and compare the results with non-asthmatic controls groups. Blood neutrophils were isolated from 95 patients: 24 with controlled asthma, 24 uncontrolled asthma, 24 healthy subjects and 23 patients with IgE-mediated allergies other than asthma. The neutrophil chemotaxis, stimulated with LPS, autologous serum or homologous serum, was determined using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by NBT test. The phagocytic digestion phase and chemotaxis by neutrophils from asthmatic patients was higher than in non-asthmatic controls (p < 0.05). Autologous serum-induced neutrophil chemotaxis in patients with uncontrolled asthma was greater (p < 0.05) than in other study groups. The ingestion phase of phagocytosis showed similar values in asthmatics and non-asthmatics. We conclude that the blood neutrophil from controlled and uncontrolled asthmatic patients exhibit activation markers, particularly phagocytic digestion and chemotactic activities.