T follicular cytotoxic (Tfc) cells are a specialized subset of CD8+ T lymphocytes that combine helper and cytotoxic functions. While their phenotypic and functional properties have been described in several infectious and inflammatory settings, little is known whether Tfc cells exhibit distinct characteristics across secondary lymphoid organs. Using acute Trypanosoma cruzi infection as a model of systemic immune activation, we performed a comparative phenotypic, transcriptomic, metabolic, and functional characterization of Tfc cells arising in the spleen and inguinal lymph nodes. Tfc cells emerged transiently in both organs with similar kinetics and antigen specificity yet segregated primarily according to tissue of analysis at the transcriptional level. Splenic Tfc cells exhibited enhanced glycolytic and mTORC1-associated signatures, increased mitochondrial mass, and stronger cytotoxic and B cell helper functions, promoting immunoglobulin production and plasmablast death. In contrast, Tfc cells from lymph node preferentially displayed memory-associated features, including higher CD127 and CD122 expression, increased IL-2 production, and preferential persistence during chronic infection. Together, these findings demonstrate that Tfc cells are not a homogeneous population but can adopt organ-associated transcriptional, metabolic, and functional states during T. cruzi infection.

Vulvovaginal candidiasis (VVC) is a mucosal infection predominantly caused by Candida albicans, affecting over three-quarters of immunocompetent women worldwide. While the female genital tract mucosa is the primary defense against the fungus, the specific immune mechanisms involved in this host-pathogen interaction remain largely unknown. In this study, we explored the relevance of type-I interferons (IFNs-I) pathway using both in vitro and in vivo models of VVC. Our quantitative proteomic analysis revealed that C. albicans induces the activation of the IFNs-I pathway in human epithelial cells (ECs) of the female genital tract shortly after exposure to the fungus. Additionally, we identified β-glucans as a crucial fungal component involved in triggering this pathway. Using a VVC model in IFN-α/β receptor-deficient (Ifnar1-/-) mice, we demonstrated that IFNs-I regulate the fungal burden, C. albicans epithelial invasion, polymorphonuclear neutrophils (PMNs) recruitment, inflammatory tissue response, local cytokine balance, and the composition of T cell subsets in the draining lymph nodes. These findings underscore the pivotal role of the IFNs-I pathway in ECs-mediated responses against C. albicans, especially in the early stages of VVC development, offering insights into potential therapeutic targets for this condition.

Anaplastic thyroid cancer (ATC) is a clinically aggressive form of undifferentiated thyroid cancer with limited treatment options. Tumor-associated macrophages (TAMs) constitute over 50% of ATC-infiltrating cells, and their presence is associated with a poor prognosis. We have previously shown that paracrine signals released by ATC cells induced pro-tumor M2-like polarization of human monocytes. However, which soluble factors derived from ATC cells drive monocyte activation, are largely unknown. In this study we investigated the participation of transforming growth factor β1 (TGFβ1) on the phenotype of macrophage activation induced by ATC cell-derived conditioned media (CM). THP-1 cells exposed to CM derived from ATC cells and recombinant human TGFβ1 induced M2-like macrophage polarization, showing high CD163 and Dectin1 expression. Moreover, we showed that TGFβ1 induced the messenger RNA (mRNA) and protein expression of the transcription factors SNAIL and SLUG. Accordingly, increased TGFβ1 secretion from ATC cells was confirmed by enzyme-linked immunosorbent assay (ELISA). Addition of SB431542, a TGFβ receptor inhibitor, significantly decreased the Dectin1, CD163, SNAIL and SLUG expression stimulated by ATC cell-derived CM. We validated the clinical significance of the expression of TGFβ ligands, their receptors, as well as SNAIL and SLUG in human ATC by analyzing public microarray datasets. We found that the expression of the main TGFβ ligands, TGFβ1 and TGFβ3, along with their receptors, TGFR1 and TGFR2, as well as SLUG, was significantly higher in human ATC tissue samples than in normal thyroid tissues. Our findings indicate that ATC cell-secreted TGFβ1 may play a key role in M2-like macrophage polarization of human monocytes and in the up-regulation of SNAIL and SLUG transcription factors. Thus, ours results uncovered a novel mechanism involved in the activation of TAMs by soluble factors released by ATC cells, which suggest potential therapeutic targets for ATC.

The thymus plays a crucial role in T cell differentiation, a complex process influenced by various factors such as antigens, the microenvironment and thymic architecture. The way the thymus resolves infections is critical, as chronic persistence of microbes or inflammatory mediators can obstruct the differentiation.Here, we illustrate that following inflammatory T helper 1 infectious processes like those caused by C. albicans or T. cruzi, single positive thymocytes adopt a mature phenotype. Further investigations focused on T. cruzi infection, reveal a substantial existence of CD44+ cells in both the cortical and medullary areas of the thymus at the onset of infection. This disturbance coincides with heightened interferon gamma (IFNγ) production by thymocytes and an increased cytotoxic capacity against T. cruzi-infected macrophages. Additionally, we observe a reduced exportation capacity in T. cruzi-infected mice. Some alterations can be reversed in IFNγ knockout mice (KO). Notably, the majority of these effects can be replicated by systemic expression of interleukin (IL)-12+IL-18, underlining the predominantly inflammatory rather than pathogen-specific nature of these phenomena.Understanding the mechanisms through which systemic inflammation disrupts normal T cell development, as well as subsequent T cell exportation to secondary lymphoid organs (SLO) is pivotal for comprehending susceptibility to diseases in different pathological scenarios.

Aim: Benznidazole (BNZ), a class-II drug, is the primary treatment for Chagas disease, but its low aqueous solubility presents challenges in formulation and efficacy. Nanosuspensions (NS) could potentially address these issues. Methods: BNZ-NS were prepared using a simple, organic solvents-free nano-milling approach. Physicochemical characterizations were conducted on both NS and lyophilized solid-state BNZ-nanocrystals (NC). Results: BNZ-NS exhibited particle size <500 nm, an acceptable polydispersity index (0.23), high Z-potential, and physical stability for at least 90 days. BNZ-NC showed tenfold higher solubility than pure BNZ. Dissolution assays revealed rapid BNZ-NS dissolution. BNZ-NC demonstrated biocompatibility on an eukaryotic cell and enhanced BNZ efficacy against trypomastigotes of Trypanosoma cruzi. Conclusion: BNZ-NS offers a promising alternative, overcoming limitations associated with BNZ for optimized pharmacotherapy.

BackgroundCOVID-19 severity has been linked to an increased production of inflammatory mediators called “cytokine storm”. Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the “cytokine storm”, offering added value to the field.MethodsWe investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected.ResultsComparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave.ConclusionsOur data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3+ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4+ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB+ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8+ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8hi and CD8lo T cells within the CD8+ population in HD are similar, a significant increase was observed in CD8lo T cell frequency in COVID-19 patients. CD8lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8hi T cells. Interestingly, the frequency of CD8lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8hi/CD8lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8hi/CD8lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

This work focusses on the chemical diversification of an Ambrosia tenuifolia extract and its bioguided fractionation, aiming to unveil the chemical entity responsible for the trypanocidal activity. Besides, a revision of the phytochemical study of this species, based on previous reports of the antiparasitic psilostachyins A and C as main compounds, was conducted. To improve the biological properties of a plant extract through a simple chemical reaction, the oxidative diversification of the dichloromethane extract of this plant species was carried out. A bioguided fractionation of a chemically modified extract was performed by evaluating the inhibitory activity against Trypanosoma cruzi trypomastigotes. This experiment led to the isolation of one of the most active compounds. In general terms, epoxidized metabolites were obtained as a result of the oxidation of the major metabolite of the species. The trypanocidal activity of some tested metabolites overperformed the reference drug, benznidazole, displaying no cytotoxicity at trypanocidal concentrations. Key structure-activity relationships were obtained for designing previously undescribed antiparasitic sesquiterpene lactones.

Anaplastic thyroid cancer (ATC) is a highly aggressive type of thyroid cancer (TC). Currently, no effective target treatments are available that can improve overall survival, with ATC representing a major clinical challenge because of its remarkable lethality. Tumor-associated macrophages (TAMs) are the most evident cells in ATCs, and their high density is correlated with a poor prognosis. However, the mechanisms of how TAMs promote ATC progression remain poorly characterized. Here, we demonstrated that the treatment of human monocytes (THP-1 cells) with ATC cell-derived conditioned media (CM) promoted macrophage polarization, showing high levels of M2 markers. Furthermore, we found that STAT3 was activated, and this was correlated with an increased expression and secretion of the inflammatory cytokine interleukin-6. Remarkably, the M2-like macrophages obtained revealed tumor-promoting activity. A cytokine array analysis demonstrated that M2-like macrophage-derived CM contained high levels of TIM3, which is an important immune regulatory molecule. Consistently, TIM3 expression was up-regulated in THP-1 cells cultured with ATC cell-derived CM. Moreover, TIM3 blockade significantly reversed the polarization of THP-1 cells induced by ATC cell-secreted soluble factors. We validated the clinical significance of the TIM3 in human TC by analyzing public datasets and found that the expression of TIM3 and its ligand galectin 9 was significantly higher in human TC tissue samples than in normal thyroid tissues. Taken together, our findings identified a new mechanism by which TIM3 induces tumor-promoting M2-like macrophage polarization in TC. Furthermore, TIM3 interference might be a potential tool for treatment of patients with ATC.

Chagas disease caused by Trypanosoma cruzi parasite is an endemic infection in America. It is well known that T. cruzi causes a strong immunosuppression during the acute phase of infection. However, it is not clear whether T. cruzi infection is related to metabolic alterations in CD4 T cells that prevent downstream effector function. Here, we evaluated the CD4 T cell metabolic and mitochondrial profiles from non-infected (NI), acute phase (AP) and chronic phase (CP) T. cruzi infected mice. CD4 T cells from all groups showed increased glucose uptake after stimulation. Moreover, the bioenergetic analysis revealed a rise in glycolysis and a higher oxidative metabolism in CD4 T cells from the AP. These cells showed increased proton leak and uncoupling protein 3 (UCP3) expression that correlated with mitochondrial ROS (mROS) accumulation, mitochondrial membrane potential (MMP) depolarization and expression of PD-1. In addition, CD4 T cells with mitochondrial alteration displayed an activated phenotype, and were less functional and more prone to apoptosis. In contrast, mitochondrial alterations were not observed during in vivo activation of CD4 T cells in a model of OVA-immunization. The Mn-superoxide dismutase (SOD2) expression, which is involved in mROS detoxification, was increased during the AP and CP of infection. Remarkably, the apoptosis observed in CD4 T cells with MMP depolarization was prevented by incubation with N-acetyl cysteine (NAC). Thus, our results showed that infection triggered an exacerbated metabolism together with mROS production in CD4 T cells from the AP of infection. However, antioxidant availability may not be sufficient to avoid mitochondrial alterations rendering these cells more susceptible to apoptosis. Our investigation is the first to demonstrate an association between a disturbed metabolism and an impaired CD4 T cell response during T. cruzi infection.

Current understanding of key cellular pathways, which are activated by the interaction between T. cruzi and host immunity, is crucial for controlling T. cruzi infection and also for limiting the development of the immunopathological symptoms of Chagas´ disease. Here, we focus on recent advances in the knowledge of modulation of innate receptors such as TLRs and NLRs, especially NLRP3, by T. cruzi in different cells of the immune system. On the other hand, the modulation of macrophage activation may be instrumental in allowing parasite persistence and long-term host survival. In this sense, we discuss the importance of the metabolism of two amino acids: L-arginine and tryptophan, and evaluate the role of iNOS, arginase and IDO enzymes in the regulation of innate and adaptive immune response during this infection; and, finally, we also discuss how T. cruzi exploits the AhR, mTOR and Wnt signaling pathways to promote their intracellular replication in macrophages, thus evading the host's immune response.

Thyroid cancer is the most common endocrine malignancy. Anaplastic thyroid cancer is one of the most aggressive thyroid tumors. It is known that activation of oncogenes and/or inactivation of tumor suppressor genes in tumor cells promotes tumorigenesis. The microenvironment of the tumor also plays a key role on cancer development and progression in a variety of tumors. However, the mechanisms by which tumor-stroma crosstalk in thyroid cancer remains poorly characterized. In this study we aimed to understand how interactions between fibroblasts and anaplastic thyroid cancer cells contribute to thyroid carcinogenesis. We first characterized the phenotypic changes of human fibroblasts in vitro through co-cultures by using transwells as well as by using anaplastic thyroid cancer cells-derived conditioned media. We found that fibroblasts acquired an activated phenotype or also known as cancer-associated fibroblast phenotype after being in contact with soluble factors secreted from anaplastic thyroid cancer cells, compared to the fibroblasts in mono-cultures. All the changes were partly mediated through Src/Akt activation. Treatment with the antioxidant N-acetyl-cysteine reversed in part the metabolic phenotype of activated fibroblasts. Remarkably, conditioned media obtained from these activated fibroblasts promoted cell proliferation and invasion of follicular thyroid cancer cell line, FTC-133 cells. Thus, a reciprocal and dynamic interaction exists between tumor and stromal cells, which results in the promotion of thyroid tumorigenesis. The present studies have advanced the understanding of the molecular basis of tumor-stroma communications, enabling identification and targeting of tumor-supportive mechanisms for novel treatment modalities.

Parastrephia lucida (Compositae), Tessaria absinthioides (Compositae), and Ephedra multiflora (Ephedraceae), three plant species from the Argentinean Puna (3600 m.a.s.l.) were selected for their anti-inflammatory and antioxidant properties to prepare mixtures to evaluate their use as nutraceuticals. Seven binary and ternary herbal mixtures made of ethanol 20% extracts of the selected plant species were prepared (Mixtures A to G), and they were analyzed for their effect on proinflammatory enzymes and their antioxidant activity in two cellular systems and in cell free systems. Toxicity tests were also carried out, and they were analyzed by high-performance liquid chromatography with a diode-array detector (HPLC-DAD) to quantify chemical markers. Mix A (equal parts of the three selected plant species) showed an important inhibitory capacity of different proinflammatory enzymes. Its potency on COX-2 was also higher than that of ibuprofen. Mix A and Mix G (P. lucida and T. absinthioides 1:1) showed a high antioxidant capacity in cellular and in cell-free systems. Toxicity assays further demonstrated their safety. This work shows the potential use of herbal mixtures made of medicinal plant species from the Argentinean Puna as nutraceutical or dietary supplements with antioxidant and anti-inflammatory activities. PRACTICAL APPLICATION: P. lucida, T. absinthioides, and E. multiflora are three plant species that are commonly used by Argentinean Puna inhabitants with medicinal purposes. Their proven safety, their antioxidant activity as well as their capacity to inhibit different proinflammatory enzymes make them attractive candidates to be used in combination as part of a dietary supplement aimed to prevent or palliate gastrointestinal and systemic inflammatory diseases. The use of native plant species as an alternative to more common and commercial plant species would have a positive impact on local communities' economies.

Foxp3+ Regulatory T cells (Tregs) are pivotal for the maintenance of tolerance. Alterations in their number and/or function have been proposed to occur in the autoimmune-prone non-obese diabetic (NOD) mouse. Comparing the frequencies and absolute numbers of CD4+Foxp3+CD25+ Tregs among 4 to 6-week old NOD, B6, and BALB/c mice, we observed differences in counts and Foxp3 expression in Tregs from secondary lymphoid organs, but not in the thymus. Upon TCR and IL-2 stimulation, NOD Tregs showed lower responses than Tregs from B6 and BALB/c mice. Indeed, NOD Tregs responded with less proliferation and with smaller increments in the expression of CD25, LAP-1, CD39, PD-1, PD-L1, and LAG-3, when in vitro cultured for 3 days with anti-CD3/CD28 in the absence or presence of IL-2, Tregs from NOD mice showed to be highly dependent on IL-2 to maintain Foxp3 expression. Moreover, NOD Tregs become producers of IL-17 and INF-gamma more easily than Tregs from the other strains. In addition, NOD Tregs showed lower responsiveness to IL-2, with significantly reduced levels of pSTAT5, even at high IL-2 doses, with respect to B6 and BALB/c Tregs. Interestingly, NOD Tregs exhibit differences in the expression of SOCS3, GRAIL, and OTUB1 when compared with Tregs from B6 and BALB/c mice. Both, at steady state conditions and also after activation, Tregs from NOD mice showed increased levels of OTUB1 and low levels of GRAIL. In addition, NOD Tregs had differences in the expression of ubiquitin related molecules that play a role in the maintenance of Foxp3 cellular pools. Indeed, significantly higher STUB1/USP7 ratios were detected in NOD Tregs, both at basal conditions and after stimulation, compared to in B6 and BALB/c Tregs. Moreover, the addition of a proteasome inhibitor to cell cultures, conferred NOD Tregs the ability to retain Foxp3 expression. Herein, we provide evidence indicating a differential expression of SOCS3, GRAIL, and STUB1/USP7 in Tregs from NOD mice, factors known to be involved in IL-2R signaling and to affect Foxp3 stability. These findings add to the current knowledge of the immunobiology of Tregs and may be related to the known insufficiency of Tregs from NOD mice to maintain self-tolerance.

The causative agent of Chagas’ disease, Trypanosoma cruzi, affects approximately 10 million people living mainly in Latin America, with macrophages being one of the first cellular actors confronting the invasion during T. cruzi infection and their function depending on their proper activation and polarization into distinct M1 and M2 subtypes. Macrophage polarization is thought to be regulated not only by cytokines and growth factors but also by environmental signals. The metabolic checkpoint kinase mammalian target of rapamycin (mTOR)-mediated sensing of environmental and metabolic cues influences macrophage polarization in a complex and as of yet incompletely understood manner. Here, we studied the role of the mTOR pathway in macrophages during T. cruzi infection. We demonstrated that the parasite activated mTOR, which was beneficial for its replication since inhibition of mTOR in macrophages by different inhibitors decreased parasite replication. Moreover, in rapamycin pretreated and infected macrophages, we observed a decreased arginase activity and expression, reduced IL-10 and increased interleukin-12 production, compared to control infected macrophages treated with DMSO. Surprisingly, we also found a reduced iNOS activity and expression in these macrophages. Therefore, we investigated possible alternative mechanisms involved in controlling parasite replication in rapamycin pretreated and infected macrophages. Although, cytoplasmic ROS and the enzyme indoleamine 2, 3-dioxygenase (IDO) were not involved, we observed a significant increase in IL-6, TNF-α, and IL-1β production. Taking into account that IL-1β is produced by activation of the cytoplasmic receptor NLRP3, which is one of the main components of the inflammasome, we evaluated NLRP3 expression during mTOR inhibition and T. cruzi infection. We observed that rapamycin-pretreated and infected macrophages showed a significant increase in NLRP3 expression and produced higher levels of mitochondrial ROS (mtROS) compared with control cells. Moreover, inhibition of mtROS production partially reversed the effect of rapamycin on parasite replication, with there being a significant increase in parasite load in rapamycin pretreated and infected macrophages from NLRP3 KO mice compared to wild-type control cells. Our findings strongly suggest that mTOR inhibition during T. cruzi infection induces NLRP3 inflammasome activation and mtROS production, resulting in an inflammatory-like macrophage profile that controls T. cruzi replication.

During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), β-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/β-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/β-catenin pathway and then activating the Wnt/Ca+2 pathway. Wnt signaling pathway activation was critical to sustain the parasite's replication in BMM; since the treatments with specific inhibitors of β-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts' interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.

Background Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens and is characterized by decreased IL-2 synthesis. In addition, the acquisition of the anergic phenotype is correlated with upregulation of “gene related to anergy in lymphocytes” (GRAIL) protein in CD4 T cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during T. cruzi infection. Methodology/Principal Findings Balb/c mice were infected intraperitoneally with 500 blood-derived trypomastigotes of Tulahuen strain, and spleen cells from control non-infected or infected animals were obtained. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenic T cells was measured by real-time PCR, flow cytometry and Western blot. We found increased GRAIL expression at the early stages of infection, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN-γ secretion in response to plate-bound antibodies. In addition, we showed that the expression of GRAIL E3-ubiquitin ligase in CD4 T cells during the acute phase of infection was complemented by a high expression of inhibitory receptors such as PD-1 and CTLA-4. We demonstrated that GRAIL expression during infection was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post infection led to a reduction in GRAIL expression. Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation. Conclusions We hypothesize that GRAIL expression induced by the parasite may be maintained by the increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. Consequently, the GRAIL regulator Otubain-1 was not expressed and GRAIL maintained the brake on T cell proliferation. Our findings reveal a novel association between increased GRAIL expression and impaired CD4 T cell proliferation during Trypanosoma cruzi infection.

Macrophage plasticity is critical for controlling inflammation including those produced by helminth infections, where alternatively activated macrophages (AAM) are accumulated in tissues. AAM expressing the co-inhibitory molecule programmed death ligand 2 (PD-L2), which is capable of binding programmed death 1 (PD-1) expressed on activated T cells, have been demonstrated in different parasitic infections. However, the role of PD-L2 during F. hepatica infection has not yet been explored. We observed that F. hepatica infection or a F. hepatica total extract (TE) injection increased the expression of PD-L2 on peritoneal macrophages. In addition, the absence of PD-L2 expression correlated with an increase in susceptibility to F. hepatica infection, as evidenced by the shorter survival and increased liver damage observed in PD-L2 deficient (KO) mice. We assessed the contribution of the PD-L2 pathway to Th2 polarization during this infection, and found that the absence of PD-L2 caused a diminished Th2 type cytokine production by TE stimulated splenocytes from PD-L2 KO infected compared with WT mice. Besides, splenocytes and intrahepatic leukocytes from infected PD-L2 KO mice showed higher levels of IFN-γ than those from WT mice. Arginase expression and activity and IL-10 production were reduced in macrophages from PD-L2 KO mice compared to those from WT mice, revealing a strong correlation between PD-L2 expression and AAM polarization. Taken together, our data indicate that PD-L2 expression in macrophages is critical for AAM induction and the maintenance of an optimal balance between the Th1- and Th2-type immune responses to assure host survival during F. hepatica infection.

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.