Rita Vasconcellos

Genetic control of natural antibody repertoires: I. IgH, MHC and TCRβ loci

Global analysis of natural antibody repertoires has revealed a marked conservation of reactivity patterns within inbred mouse strains, and characteristic strain‐specific differences. We have now analyzed the genetic control of reactivity repertoires, aiming at identifying the respective selection mechanisms. Multiparametric statistics of a large number of serum antibody reactivities scored by quantitative Western blot analyses using extracts from homologous tissues and bacteria readily distinguish the reactivity patterns of C57BL/6 and BALB/c, revealing homogeneity among genetically identical individuals. Antibody repertoires in the prototype strains can also be segregated from those expressed by the respective IgH congenics, BC.8 and CB.20, demonstrating that IgH‐linked genes contribute to determining natural antibody repertoires. Conversely, strains sharing IgH haplotype also express distinct reactivity patterns, indicating that other genes participate in the selection of serum IgM repertoires. Two such non‐IgH loci were now identified. Thus, analysis of four MHC‐congenic strains demonstrated that MHC‐linked control of natural antibody repertoires is likely to operate through differential selection of T cell repertoires, since (1) mice that are congenic at the TCRβ locus, and (2) BALB/c nude mice grafted at birth with pure thymic epithelium from either C57BL/6 or BALB/c also differ in their natural antibody repertoires.

Significant association between the skewed natural antibody repertoire of Xid mice and resistance to Trypanosoma cruzi infection.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self‐antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre‐ and post‐infected Xid mice. In particular, relatively increased natural (pre‐existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivies to self and non‐self proteins. These results suggest that self‐reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self‐aggressive components responsible for pathology.

Interleukin-27 early impacts Leishmania infantum infection in mice and correlates with active visceral disease in humans

The complexity of Leishmania–host interactions, one of the main leishmaniasis issues, is yet to be fully understood. We detected elevated IL-27 plasma levels in European patients with active visceral disease caused by Leishmania infantum, which returned to basal levels after successful treatment, suggesting this cytokine as a probable infection mediator. We further addressed this hypothesis recurring to two classical susceptible visceral leishmaniasis mouse models. BALB/c, but not C57BL/6 mice, showed increased IL-27 systemic levels after infection, which was associated with an upregulation of IL-27p28 expression by dendritic cells and higher parasite burdens. Neutralization of IL-27 in acutely infected BALB/c led to decreased parasite burdens and a transient increase in IFN-γ+ splenic T cells, while administration of IL-27 to C57BL/6 promoted a local anti-inflammatory cytokine response at the site of infection and increased parasite loads. Overall, we show that, as in humans, BALB/c IL-27 systemic levels are infection dependently upregulated and may favor parasite installation by controlling inflammation.

Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-γ production

Mdx mouse, the animal model of Duchenne muscular dystrophy, develops an X‐linked recessive inflammatory myopathy with an apparent sustained capacity for muscle regeneration. We analysed whether changes in the skeletal muscle during myonecrosis and regeneration would correlate with functional alterations in peripheral lymphoid tissues. Here we show that during the height of myonecrosis, mdx mice display marked atrophy of peripheral lymph nodes and extensive muscle inflammation. In contrast, enlargement of draining lymph nodes with accumulation of CD4+ CD44+, CD4+ CD25+, CD8+ CD44+ T lymphocytes and type‐2 B cells was consistently observed during amelioration of the muscle lesion. In addition, regeneration of the muscular tissue was accompanied by concomitant increase of immunoglobulin‐secreting cells in regional lymph nodes and bone marrow. Double immunolabelling analysis revealed intense B cell proliferation and formation of germinal centre in the follicles of dystrophic regional lymph nodes. Furthermore, lymph node cells produced large amounts of IFN‐γ but not IL‐4, IL‐6 or IL‐10 after in vitro mitogen stimulation with Concanavalin A. As these alterations occurred mainly during the recovery period, we suggested that local activation of the immune system could be an influence which mitigates the myonecrosis of muscular tissue in the mdx dystrophic mouse.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1beta (3.8X) and TNF-alpha (6.0X). IFN-gamma was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Apoptotic lymphocytes treated with IgG from Trypanosoma cruzi infection increase TNF-α secretion and reduce parasite replication in macrophages

Phagocytic removal of apoptotic lymphocytes exacerbates replication of Trypanosoma cruzi in macrophages. We investigated the presence of Ab against apoptotic lymphocytes in T. cruzi infection and the role of these Ab in parasite replication. Both control and chagasic serum contained IgG Ab that opsonized apoptotic lymphocytes. Treatment of apoptotic lymphocytes with purified IgG from chagasic, but not control serum, reduced T. cruzi replication in macrophages. The protective effect of chagasic IgG depended on Fcγ receptors, as demonstrated by the requirement for the intact Fc portion of IgG, and the effect could be abrogated by treating macrophages with an anti‐CD16/CD32 Fab fragment. Chagasic IgG displayed increased reactivity against a subset of apoptotic cell Ag, as measured by flow cytometry and immunoblot analyses. Apoptotic lymphocytes treated with chagasic IgG, but not control IgG, increased production of TNF‐α, while decreasing production of TGF‐β1 by infected macrophages. Increased control of parasite replication required TNF‐α production. Previous immunization with apoptotic cells or injection of apoptotic cells opsonized with chagasic IgG reduced parasitemia in infected mice. These results indicate that Ab raised against apoptotic cells could play a protective role in control of T. cruzi replication by macrophages.

Antibody Repertoires Identify β-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.