Ises A. Abrahamsohn

Leishmania (L. ) amazonensis-induced inhibition of nitric oxide synthesis in host macrophages

Inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production was demonstrated in J774-G8 macrophages infected with Leishmania (L.) amazonensis promastigotes. The downmodulation of NO production observed in infected and LPS-stimulated J774-G8 cells correlated with a reduction in inducible nitric oxide synthase (iNOS) activity. Reduction in iNOS activity was not paralleled by decreased iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Supplementation with L-arginine or tetrahydrobiopterin did not increase NO production, suggesting that inhibition is not due to an insufficiency of substrate or co-factor. Treatment with anti-IL-10, anti-IL-4 or anti-TGF-beta neutralizing antibodies also failed to increase NO production, indicating that these cytokines are not involved in the observed parasite-induced inhibition of NO synthesis. However, treatment of the cultures with IFN-gamma resulted in a marked increase in NO production by infected LPS-stimulated cells. These results show that although L.(L.) amazonensis infection inhibits iNOS activity and NO production by J774-G8 cells, activation by IFN-gamma is capable of overriding the suppression of NO synthesis.

The role of polymorphonuclear leukocytes in the resistance to cutaneous Leishmaniasis

The massive infiltration by polymorphonuclear leukocytes (PMN) soon after skin infection with Leishmania major suggests that PMN could participate in reducing parasite load and controlling the spreading of leishmanial infection. Yet, direct evidence for the participation of PMN in host defense against L. major was lacking. We investigated L. major infection in susceptible and resistant mice treated with the monoclonal (mAb) antibody RB6-8C5 that depletes the population of mature neutrophils and eosinophils. Both BALB/c and C57BL/6 mice depleted of PMN show accelerated parasite spreading and more severe footpad swelling than similarly infected untreated mice. In addition, significant higher parasite numbers were found in the lesion draining lymph nodes from PMN-depleted C57BL/6 mice. Histopathological analysis of the paw confirmed neutrophils containing ingested parasites as the dominant cell type in the infiltrate of the first days after infection and the nearly absolute neutrophil depletion in mAb-treated mice. Our data show the importance of PMN in early control of parasite load and parasitism spreading in cutaneous leishmaniasis.

Type I IFNs Stimulate Nitric Oxide Production and Resistance to Trypanosoma cruzi Infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-αβ or anti-TNF-α Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-α (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-αβ Ab reduced NO production. IFN-γ or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-αβR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-αβ, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.

Prostaglandin and nitric oxide regulate TNF-α production during Trypanosoma cruzi infection

Abstract The mechanisms that control TNF- α production by macrophages during Trypanosoma cruzi infection are still unknown. Destruction of intracellular forms by cytokine activated macrophages is considered to be a major mechanism of parasite elimination. Although in vitro TNF- α contributes to enhanced parasite destruction by macrophages, previous work in vivo has shown that as the parasite burden increases, serum TNF- α levels decline. In this report we show that TNF- α production by peritoneal adherent cells is elevated at the initial phase of T. cruzi infection. As infection progresses TNF- α production decreases. The observed reduction is partly due to inhibition, largely exerted by endogenous PG and secondarily by NO. Inhibition of their synthesis partially restored the ability to produce high levels of TNF- α to macrophages upon stimulation by LPS. Neither endogenous IL-10 nor TGF- β seem to be involved in the negative regulation of TNF- α production.

Effects of Interleukin-4 Deprivation and Treatment on Resistance to Trypanosoma cruzi

ABSTRACT Trypanosoma cruzi (Y strain)-infected interleukin-4−/− (IL-4−/−) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-γ) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4−/− mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-γ synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-γ synthesis levels; in contrast, IL-10 neutralization increased IFN-γ production and addition of rIL-4 and/or rIL-10 diminished IFN-γ synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strain T. cruziinfection but that IL-4 can, in association with IL-10, modulate IFN-γ production and resistance.

Trypanosoma cruzi: Maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.

Resistant mice lacking interleukin-12 become susceptible to Trypanosoma cruzi infection but fail to mount a T helper type 2 response

Interleukin‐12 (IL‐12) is essential to resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon‐γ (IFN‐γ) that activates macrophages to a parasiticidal effect. Investigation of mice deprived of IL‐12 genes (IL‐12 knockout mice) has confirmed the important role of IL‐12 and IFN‐γ in controlling parasitism in T. cruzi infection. However, it has not yet been addressed whether a shift towards a T helper type 2 (Th2) pattern of cytokine response occurred in these mice that might have contributed to the aggravation of the infection caused by IL‐12 deprivation. We examined the course of T. cruzi (Y strain) infection and the regulation of cytokine responses and nitric oxide production in C57BL/6 IL‐12 p40‐knockout mice. The mutant mice were extremely susceptible to the infection as evidenced by increased parasitaemia, tissue parasitism and mortality in comparison with the control C57BL/6 mouse strain (wild‐type) that is resistant to T. cruzi. A severe depletion of parasite‐antigen‐specific IFN‐γ response, without an increase in IL‐4 or IL‐10 production, accompanied by reduced levels of nitric oxide production was observed in IL‐12 knockout mice. We found no evidence of a shift towards a Th2‐type cytokine response. In IL‐12 knockout mice, the residual IFN‐γ production is down‐regulated by IL‐10 but not by IL‐4 and nitric oxide production is stimulated by tumour necrosis factor‐α. Parasite‐specific immunoglobulin G1 antibody levels were similar in IL‐12 knockout and wild‐type mice, whereas IL‐12 knockout mice had much higher levels of immunoglobulin G2b.

Most parasite‐specific CD8+ cells in Trypanosoma cruzi‐infected chronic mice are down‐regulated for T‐cell receptor‐αβ and CD8 molecules

The present study shows that CD8+ T lymphocytes expressing low levels of T‐cell receptor (TCR)αβ, CD8 and CD3 accumulate in the spleen, blood, peritoneum and liver, but not in the lymph nodes of mice chronically infected with Trypanosoma cruzi. Analysis of spleen lymphocytes reveals that most CD8LOW TCRLOW T cells have an experienced phenotype (CD44HIGH CD62LLOW and CD45RA,B,CLOW). These cells have small size, lack activation markers such as CD69, CD25 and CD11b (Mac‐1), and do not spontaneously secrete cytokines, suggesting they are at the resting state. When stimulated in vitro with T. cruzi‐infected macrophages, TCRLOW CD8LOW T cells behave as parasite‐specific memory cells, readily responding with interferon‐γ (IFN‐γ) production. Indeed, among parasite‐activated CD8+ lymphocytes, IFN‐γ production was mostly due to TCRLOW CD8LOW cells. Upon in vitro stimulation with anti‐CD3/CD28 monoclonal antibodies, down‐regulated cells produce IFN‐γ and tumour necrosis factor‐α, but not interleukin IL‐10 or IL‐4. Our results indicate that despite parasite persistence, most T. cruzi‐specific experienced CD8+ cells are resting. Nevertheless, when encountering infected macrophages these cells differentiate to Tc1 effectors.

Immature macrophages derived from mouse bone marrow produce large amounts of IL‐12p40 after LPS stimulation

Production of IL‐12 is an important indicator of the macrophage’s ability to regulate immune responses. In this study, we investigated the IL‐12 production by macrophages in different developmental stages. To this end, macrophages were generated in vitro from precursors stimulated with M‐CSF, GM‐CSF or IL‐3. Density separation yielded populations enriched in different maturation stages. Invariably, only cells banding at the 40−50% Percoll interface produced large amounts of IL‐12p40 when stimulated with LPS, whereas only low levels of IL‐12p70 were produced. These cells represented immature macrophages, as indicated by the absence of precursor markers CD31/ER‐MP12, Ly‐6C/ER‐MP20 and ER‐MP58, and by the low level of expression of mature‐cell markers like ER‐HR3, scavenger receptor and CD11b/Mac‐1. Upon further maturation, the macrophages’ ability to produce IL‐12p40 decreased, coinciding with increased nitric oxide production upon LPS stimulation. These results show that immature macrophages produce high levels of IL‐12p40 and thus may either contribute to IL‐12p70 production or regulate it.

S-nitrosoglutathione (GSNO) is cytotoxic to intracellular amastigotes and promotes healing of topically treated Leishmania major or Leishmania braziliensis skin lesions

We read with great interest the letter by Kö ser et al., 1 responding to our article on the characterization of the embB gene in the area of Barcelona. 2 The point that Kö ser et al. 1 raise is very interesting and deserves further comment. Codon 378 has indeed been described by several authors as a phylogenetic polymorphism not related to resistance. 3,4 However, in our opinion the role of this codon remains unclear, since some studies describe the existence of ethambutol-resistant isolates with a mutation only in this codon. 5,6 Even one work 7 cited by Kö ser et al. 1 includes two isolates with a single Glu378Ala substitution and a decreased susceptibility to ethambutol. The design of our microarray was based on the existing literature , taking into account all the possible embB codons that have been implicated in ethambutol resistance. We finally included the ones that were also found in our setting. The microarray system was not designed to determine the association of each mutation with the phenotypic resistance (allelic exchange experiments would be required for this purpose), but to reflect the variety of embB substitutions prevalent in our area. Moreover, the frequency of mutations in embB378 is low (,2% in our study), so we consider that the probability of misassigning a result of embB378 mutation to phenotypic resistance rather than to an epidemiological cause is negligible and does not compromise the effectiveness of the microarray. Regarding the isolates, we included a collection of Mycobacterium tuberculosis complex (MTBC) clinical isolates (not identified to the species level). Isolate 233R has now been analysed and identified as M. tuberculosis/Mycobacterium canettii. It contains the embB378Glu variant; therefore, a homoplastic event may be present (experimental error was ruled out). Likewise, the MIRU-VNTR genotyping was performed not for epidemiological purposes (i.e. for the identification of lineages), but to establish the real frequency of embB mutations among circulating MTBC isolates in our geographical area. Finally, we stress that the main objective of this study was to highlight the relevance of mutations in embB codons apart from embB306, focusing on codon 406, which represents 20% of the embB mutations in our area. Our results show that this target (embB406) should be included in any genotypic method for rapid ethambutol resistance detection. References 1 Kö ser CU, Bryant JM, Comas I et al. Comment on: Characterization of the embB gene in Mycobacterium tuberculosis isolates …