Trypanosoma cruzi Experimental Infection Impacts on the Thymic Regulatory T Cell Compartment.

The dynamics of regulatory T cells in the course of Trypanosoma cruzi infection is still debated. We previously demonstrated that acute murine T. cruzi infection results in an impaired peripheral CD4+Foxp3+ T cell differentiation due to the acquisition of an abnormal Th1-like phenotype and altered functional features, negatively impacting on the course of infection. Moreover, T. cruzi infection induces an intense thymic atrophy. As known, the thymus is the primary lymphoid organ in which thymic-derived regulatory T cells, known as tTregs, differentiate. Considering the lack of available data about the effect of T. cruzi infection upon tTregs, we examined tTreg dynamics during the course of disease. We confirmed that T. cruzi infection induces a marked loss of tTreg cell number associated to cell precursor exhaustion, partially avoided by glucocorticoid ablation- and IL-2 survival factor depletion. At the same time, tTregs accumulate within the CD4 single-positive compartment, exhibiting an increased Ki-67/Annexin V ratio compared to controls. Moreover, tTregs enhance after the infection the expression of signature markers (CD25, CD62L and GITR) and they also display alterations in the expression of migration-associated molecules (α chains of VLAs and chemokine receptors) such as functional fibronectin-driven migratory disturbance. Taken together, we provide data demonstrating profound alterations in tTreg compartment during acute murine T. cruzi infection, denoting that their homeostasis is significantly affected. The evident loss of tTreg cell number may compromise the composition of tTreg peripheral pool, and such sustained alteration over time may be partially related to the immune dysregulation observed in the chronic phase of the disease.

Immunoendocrine dysbalance during uncontrolled T. cruzi infection is associated with the acquisition of a Th-1-like phenotype by Foxp3(+) T cells.

We previously showed that Trypanosomacruzi infection in C57BL/6 mice results in a lethal infection linked to unbalanced pro- and anti-inflammatory mediators production. Here, we examined the dynamics of CD4(+)Foxp3(+) regulatory T (Treg) cells within this inflammatory and highly Th1-polarized environment. Treg cells showed a reduced proliferation rate and their frequency is progressively reduced along infection compared to effector T (Teff) cells. Also, a higher fraction of Treg cells showed a naïve phenotype, meanwhile Teff cells were mostly of the effector memory type. T. cruzi infection was associated with the production of pro- and anti-inflammatory cytokines, notably IL-27p28, and with the induction of T-bet and IFN-γ expression in Treg cells. Furthermore, endogenous glucocorticoids released in response to T. cruzi-driven immune activation were crucial to sustain the Treg/Teff cell balance. Notably, IL-2 plus dexamethasone combined treatment before infection was associated with increased Treg cell proliferation and expression of GATA-3, IL-4 and IL-10, and increased mice survival time. Overall, our results indicate that therapies aimed at specifically boosting Treg cells, which during T. cruzi infection are overwhelmed by the effector immune response, represent new opportunities for the treatment of Chagas disease, which is actually only based on parasite-targeted chemotherapy.

Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

Reciprocal influences between leptin and glucocorticoids during acute Trypanosoma cruzi infection.

Leptin and glucocorticoids (GCs) are involved in metabolic functions, thymic homeostasis and immune activity through complex interactions. We recently showed that C57BL/6 mice infected with Trypanosoma cruzi revealed a fatal disease associated with a dysregulated immune-endocrine response characterized by weight loss, deleterious synthesis of pro-inflammatory cytokines and GCs-driven thymus atrophy. Extending this study, we now explored the relationship between leptin and GCs, in terms of infection outcome, thymic and metabolic changes. T. cruzi-infected mice showed a food intake reduction, together with hypoglycemia and lipolysis-related changes. Infected animals also displayed a reduction in systemic and adipose tissue levels of leptin, paralleled by a down-regulation of their receptor (ObR) in the hypothalamus. Studies in infected mice subjected to adrenalectomy (Adx) showed a worsened course of infection accompanied by even more diminished systemic and intrathymic leptin levels, for which GCs are necessary not only to decrease inflammation but also to sustain leptin secretion. Adx also protected from thymic atrophy, independently of the reduced leptin contents. Leptin administration to infected mice aggravated inflammation, lowered parasite burden and attenuated GCs release, but did not normalize thymic atrophy or metabolic parameters. Acute T. cruzi infection in C57BL/6 mice coexists with a dysregulation of leptin/hypothalamic ObR circuitry dissociated from body weight and food intake control. Endogenous GCs production attempted to reestablish systemic leptin concentrations, but failed to improve leptin-protective activities at the thymic level, suggesting that the leptin/GCs intrathymic relationship is also altered during this infection.

Tumor necrosis factor-α regulates glucocorticoid synthesis in the adrenal glands of Trypanosoma cruzi acutely-infected mice. the role of TNF-R1.

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1(-/-)) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1(-/-) mice undergoing acute infection (Tc-WT and Tc-TNF-R1(-/-) groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1(-/-) compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1(-/-) mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1(-/-) mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1(-/-) mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.