Karina R. B. Bastos

Interaction between Paracoccidioides brasiliensis and Pulmonary Dendritic Cells Induces Interleukin-10 Production and Toll-Like Receptor–2 Expression: Possible Mechanisms of Susceptibility

Dendritic cells (DCs) are the most effective antigen-presenting cells for inducing cell-mediated immune responses; it is thus important to investigate the role played by lung DCs in the pathogenesis of paracoccidioidomycosis (PCM) and their potential to initiate an immune response in mice susceptible (B10.A) and resistant (A/J) to PCM. Initially, we observed that lung DCs from susceptible mice were more phagocytic than cells from resistant mice, and we observed that phagocytosis in the presence of laminarin was inhibited only in DCs from susceptible mice. DCs from resistant mice produced a low concentration of interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)- alpha . In contrast, DCs from susceptible mice produced high concentrations of TNF- alpha and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DCs from Toll-like receptor (TLR)-2 knockout mice displayed defective production of IL-10. After 15 days of Paracoccidioides brasiliensis infection, DCs from susceptible mice produced IL-10 and expressed costimulatory molecules at a low level. We found that expression of the gene for TLR-2 is increased after infection in susceptible, but not resistant, mice. In conclusion, our data suggest that P. brasiliensis induces regulatory DCs in susceptible mice, which promotes IL-10 production and contributes to the susceptibility of mice to P. brasiliensis infection.

Contribution of NK, NK T, γδ T, and αβ T Cells to the Gamma Interferon Response Required for Liver Protection against Trypanosoma cruzi

ABSTRACT In the present work, we show that intracellular Trypanosoma cruzi is 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 acute T. cruzi infection. 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 against T. cruzi.

Pathology Affects Different Organs in Two Mouse Strains Chronically Infected by a Trypanosoma cruzi Clone: a Model for Genetic Studies of Chagas' Disease

ABSTRACT Chagas’ disease is a chronic infection caused by Trypanosoma cruzi and represents an important public health burden in Latin America. Frequently the disease evolves undetectable for decades, while in a significant fraction of the affected individuals it culminates in death by heart failure. Here, we describe a novel murine model of the chronic infection with T. cruzi using a stable clone isolated from a human patient (Sylvio X10/4). The infection in the C3H/HePAS mouse strain progresses chronically and is mainly characterized by intense cardiac inflammatory lesions that recapitulate the chronic cardiac pathology observed in the human disease. Moderate striated muscle lesions are also present in C3H/HePAS mice. Viable parasites are detected and recovered from the chronic heart lesions of C3H/HePAS mice, supporting the current notion that development of heart pathology in Chagas’ disease is related to parasite persistence in the inflamed tissue. By contrast, in infected A/J mice, chronic inflammatory lesions are targeted to the liver and the skeletal muscle, while pathology and parasites are undetectable in the heart. The phenotypic analysis of F1 (A/J × C3H/HePAS) and F2 (A/J × C3H/HePAS) mice suggests that the genetic predisposition to develop the inflammatory lesions caused by T. cruzi (Sylvio X10/4 clone) is heterogeneous because the heart and liver pathology segregate in the F2 generation. These findings raise the hypothesis that the pathology heterogeneity observed in humans with Chagas’ disease (absence and presence of cardiac or digestive chronic lesions) may be attributable to host genetic factors.

IFN-γ, But Not Nitric Oxide or Specific IgG, is Essential for the In vivo Control of Low-virulence Sylvio X10/4 Trypanosoma cruzi Parasites

Highly virulent strains of Trypanosoma cruzi are frequently used as murine models of Chagas’ disease. However, these strains do not fully represent the spectrum of parasites involved in the human infection. In this paper, we analysed parasitaemia, mortality, tissue pathology and parasite‐specific IgG serum levels in immune‐deficient mice infected with Sylvio X10/4 parasites, a T. cruzi derived from a chagasic patient that yields very low parasitaemias and in C3H/HePAS mice induces a chronic cardiopathy resembling the human disease. IFN‐γ was identified as a crucial element for parasite control as its absence determined a drastic increase in parasitaemia, tissue parasitism, leukocyte infiltrates at the heart and striated muscles and mortality. The lack of IFN‐γ or IL‐12p40, a molecule shared by IL‐12 and IL‐23, also resulted in spinal cord lesions and a progressive paralysis syndrome. Whereas IgG2a was the main Ig isotype in infected C57BL/6 mice, IL‐12p40‐KO mice produced IgG2a and IgG1 and IFN‐γ‐KO mice produced only IgG1. The IFN‐γ‐protective effect was not essentially mediated by nitric oxide (NO), inasmuch as infected iNOS‐KO mice showed no parasitaemia and low tissue damage. Mice deficient in CD4+ or CD8+ T cells showed an intermediate phenotype with increased mortality and tissue pathology but no parasitaemia. Interestingly, CD28‐KO mice were unable to produce anti‐T. cruzi IgG antibodies but presented moderate tissue pathology and managed to control the infection. Thus, differently from infections with high virulence parasites, neither IgG, NO nor CD28‐mediated signalling are essential for the non‐sterile control of Sylvio X10/4 parasites.

Role of CD28 in Polyclonal and Specific T and B Cell Responses Required for Protection against Blood Stage Malaria

The role of B7/CD28 costimulatory pathway in the polyclonal and specific lymphocyte activation induced by blood stages of Plasmodium chabaudi AS was investigated in CD28 gene knockout (CD28−/−) and C57BL/6 (CD28+/+) mice. Analysis of the spleen during the acute infection revealed a similar increase in T and B cell populations in both groups of mice. Moreover, CD28−/− mice were able to develop a polyclonal IgM response to P. chabaudi. On the contrary, the polyclonal IgG2a response was markedly reduced in the absence of CD28. Production of IFN-γ; up-regulation of CD69, CD40L, CD95 (Fas), and CD95L (Fas ligand); and induction of apoptosis were also affected by the lack of CD28. Interestingly, the ability to control the first parasitemia peak was not compromised in acutely infected CD28−/− mice, but CD28−/− mice failed to eradicate the parasites that persisted in the blood for >3 mo after infection. In addition, drug-cured CD28−/− mice were unable to generate memory T cells, develop an anamnesic IgG response, or eliminate the parasites from a secondary challenge. The incapacity of CD28−/− mice to acquire a full protective immunity to P. chabaudi correlated with an impaired production of specific IgG2a. Moreover, reinfected CD28−/− mice were protected by the adoptive transfer of serum from reinfected CD28+/+ mice containing specific IgG2a. Our results demonstrate that the polyclonal lymphocyte response is only partially affected by the absence of CD28, but this coreceptor is essential to generate specific T and B cell responses required for complete protection against P. chabaudi malaria.

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab′)2 fragments

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug‐cured mice challenged with a high inoculum of ring‐infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary‐infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont‐infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc‐dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody‐mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcγR‐mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.

Impaired Macrophage Responses May Contribute to Exacerbation of Blood-Stage Plasmodium chabaudi chabaudi Malaria in Interleukin-12-Deficient Mice

Aiming to clarify the role of endogenous interleukin-12 (IL-12) in protective immunity against blood stages of Plasmodium chabaudi chabaudi (AS), we evaluated the course of infection in IL-12p40 gene knockout (IL-12p40KO) and wild-type (WT) C57BL/6 mice, focusing (1) on the ability of T cells to develop adequate type 1 responses and (2) on the potentiality of macrophages to respond to parasites, interferon-gamma (IFN-gamma), or both. We observed that IL-12p40KO mice develop significantly higher parasitemias during the acute infection, although mice from both groups clear the parasites within a month and similarly eliminate a secondary challenge. Thus, fully protective immunity to P. c. chabaudi can be generated in the absence of IL-12. However, this cytokine may promote parasite control during the early phase of infection. The increased acute parasitemia of IL-12p40KO mice was associated with both impaired IFN-gamma and nitric oxide (NO) response by spleen cells. Because stimulation with recombinant IFN-gamma (rIFN-gamma) failed to improve the NO response in IL-12p40KO macrophages, we investigated whether these cells have an intrinsic defect. Analysis of peritoneal macrophages revealed that IL-12p40KO cells produce higher levels of transforming growth factor-beta1 (TGF-beta1) compared with WT cells and respond to infected erythrocytes or rIFN-gamma by releasing little NO. Moreover, IL-12p40KO macrophages had a severely impaired ability to internalize opsonized infected erythrocytes, suggesting that the low effector profile assumed by these cells may compromise antibody-mediated immunity. Taken together, our results support the idea that the absence of IL-12p40 not only affects IFN-gamma production but also has deep consequences in macrophage effector functions that may contribute to exacerbation of the early phase of P. c. chabaudi malaria.

Analysis of the activation profile of dendritic cells derived from the bone marrow of interleukin-12/interleukin-23-deficient mice

We have previously shown that macrophages from interleukin (IL)‐12p40 gene knockout (IL‐12/IL‐23–/–) mice have a bias towards the M2 activation profile, spontaneously secreting large quantities of transforming growth factor‐β1 (TGF‐β1) and producing low levels of nitric oxide (NO) in response to lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ). To verify whether the activation profile of dendritic cells (DCs) is also influenced by the absence of IL‐12/IL‐23, bone marrow‐derived DCs from IL‐12/IL‐23–/– and C57BL/6 mice were evaluated. At first we noticed that ≈ 50% of the C57BL/6 DCs were dead after LPS‐induced maturation, whereas the mortality of IL‐12/IL‐23–/– DCs was < 10%, a protective effect that diminished when recombinant IL‐12 (rIL‐12) was added during maturation. Similarly to macrophages, mature IL‐12/IL‐23–/– DCs (mDCs) produced higher levels of TGF‐β1 and lower levels of NO than C57BL/6 mDCs. NO release was IFN‐γ‐dependent, as evidenced by the poor response of IFN‐γ–/– and IL‐12/IL‐23–/–IFN‐γ–/– mDCs. Nevertheless, IFN‐γ deficiency was not the sole reason for the weak NO response observed in the absence of IL‐12/IL‐23. The high level of TGF‐β1 secretion by IL‐12/IL‐23–/– mDCs could explain why exogenous IFN‐γ partially restored the NO production of IFN‐γ–/– mDCs, while IL‐12/IL‐23–/– IFN‐γ–/– mDCs remained unresponsive. We also showed that CD4+ T‐cell proliferation was inhibited by C57BL/6 mDCs, but not by IL‐12/IL‐23–/– mDCs. IFN‐γ and NO appear to mediate this antiproliferative effect because this effect was not observed in the presence of mDCs from IFN‐γ–/– or IL‐12/IL‐23–/– IFN‐γ–/– mice and it was attenuated by aminoguanidine. We conclude that the presence of IL‐12/IL‐23 during LPS‐induced maturation influences the activation profile of DCs by a mechanism that is, only in part, IFN‐γ dependent.