Systemic inflammatory Th1 cytokines during Trypanosoma cruzi infection disrupt the typical anatomical cell distribution and phenotypic/functional characteristics of various cell subsets within the thymus.

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 Candida albicans or Trypanosoma 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.

COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity.

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.

Different cytokine and chemokine profiles in hospitalized patients with COVID-19 during the first and second outbreaks from Argentina show no association with clinical comorbidities.

Background: COVID-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. Methods: We 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. Results: Comparison 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. Conclusions: Our 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.

Psilostachyins as trypanocidal compounds: Bioguided fractionation of Ambrosia tenuifolia chemically modified extract.

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.

Secreted Factors by Anaplastic Thyroid Cancer Cells Induce Tumor-Promoting M2-like Macrophage Polarization through a TIM3-Dependent Mechanism.

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.

Signaling pathways that regulate Trypanosoma cruzi infection and immune response.

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.

Author Correction: Interplay of fibroblasts with anaplastic tumor cells promotes follicular thyroid cancer progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

T Regulatory Cells From Non-obese Diabetic Mice Show Low Responsiveness to IL-2 Stimulation and Exhibit Differential Expression of Anergy-Related and Ubiquitination Factors.

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.

Argentinean Puna Plants with In Vitro Antioxidant and Anti-Inflammatory Activities as a Potential Nutraceutical.

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.

Interplay of fibroblasts with anaplastic tumor cells promotes follicular thyroid cancer progression.

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.

Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages.

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), ß-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/ß-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/ß-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 ß-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.

Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection.

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-1ß production. Taking into account that IL-1ß 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.

GRAIL and Otubain-1 are Related to T Cell Hyporesponsiveness during Trypanosoma cruzi Infection.

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.

PD-L2 negatively regulates Th1-mediated immunopathology during Fasciola hepatica 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.

The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages.

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

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.

Programmed death ligand 2 regulates arginase induction and modifies Trypanosoma cruzi survival in macrophages during murine experimental infection.

The programmed death ligands 1 (PD-L1) and 2 (PD-L2) that bind to programmed death 1 (PD-1) have been involved in peripheral tolerance and in the immune escape mechanisms during chronic viral infections and cancer. However, there are no reports about the role of these molecules during Trypanosoma cruzi infection. We have studied the role of PD-L1 and PD-L2 in T. cruzi infection and their importance in arginase/inducible nitric oxide synthase (iNOS) balance in the immunomodulatory properties of macrophages (Mφ). In this work, we have demonstrated that expression of the PD-1/PD-L pathway is modified during T. cruzi infection on Mφs obtained from peritoneal cavity. The Mφs from T. cruzi-infected mice suppressed T-cell proliferation and this was restored when anti-PD-1 and anti-PD-L1 antibodies were added. Nevertheless, anti-PD-L2 antibody treatment did not re-establish T-cell proliferation. PD-L2 blockade on peritoneal cells from infected mice showed an increase in arginase expression and activity and a decrease in iNOS expression and in nitric oxide (NO) production. Additionally, interleukin-10 production increased whereas interferon-γ production was reduced. As a result, this microenvironment enhanced parasite proliferation. In contrast, PD-1 and PD-L1 blockage increased iNOS expression and NO production on peritoneal Mφs from T. cruzi-infected mice. Besides, PD-L2 knockout infected mice showed an increased in parasitaemia as well as in arginase activity, and a reduction in NO production. Taken together, our results demonstrate that PD-L2 is involved in the arginase/iNOS balance during T. cruzi infection having a protective role in the immune response against the parasite.

Arginase in parasitic infections: macrophage activation, immunosuppression, and intracellular signals.

A type 1 cytokine-dependent proinflammatory response inducing classically activated macrophages (CaMvarphis) is crucial for parasite control during protozoan infections but can also contribute to the development of immunopathological disease symptoms. Type 2 cytokines such as IL-4 and IL-13 antagonize CaMvarphis inducing alternatively activated macrophages (AaMvarphis) that upregulate arginase-1 expression. During several infections, induction of arginase-1-macrophages was showed to have a detrimental role by limiting CaMvarphi-dependent parasite clearance and promoting parasite proliferation. Additionally, the role of arginase-1 in T cell suppression has been explored recently. Arginase-1 can also be induced by IL-10 and transforming growth factor-beta (TGF-beta) or even directly by parasites or parasite components. Therefore, generation of alternative activation states of macrophages could limit collateral tissue damage because of excessive type 1 inflammation. However, they affect disease outcome by promoting parasite survival and proliferation. Thus, modulation of macrophage activation may be instrumental in allowing parasite persistence and long-term host survival.

Cruzipain and SP600125 induce p38 activation, alter NO/arginase balance and favor the survival of Trypanosoma cruzi in macrophages.

Cruzipain (Cz), an antigen of Trypanosoma cruzi, mediates the activation of arginase involving p38 MAPK. In this work, it was studied whether the phosphorylation of MAPKs into macrophages (Mvarphi) could be induced by Cz and/or by the parasite. We found that Cz induced activation of p38, while the parasite produced phosphorylation of JNK and p44/p42. MAPK phosphorylation changed and JNK activation was blocked when Mvarphi were pre-incubated with Cz, before coming into contact with T. cruzi. We investigated the role of JNK inhibitor SP600125 on T. cruzi infection, since it also induces p38 phosphorylation. Thus, J774 cells were pre-treated with SP600125 and then infected with T. cruzi. This set of cells showed a decrease in nitric oxide (NO) production and an increase in arginase I expression. Another group of J774 cells was pre-treated with SP600125 and incubated with Cz before being infected with T. cruzi. This second group showed a greater reduction in NO production. These results can be correlated with the parasitic growth since the ex vivo treatment with SP600125 on adherent spleen cells (ASC) of BALB/c infected mice also increased the parasitic growth. Therefore, Cz and SP600125 favor the T. cruzi survival in Mvarphi by changing the iNOS/arginase balance.

Macrófagos e inducción de arginasa como mecanismo de evasión de parásitos / Macrophages and arginase induction as a mechanism for parasite escape

Aunque existen varios mecanismos inmunológicos para eliminar a los patógenos intracelulares, éstos han elaborado una variedad de estrategias para escapar de la respuesta del sistema inmune y asegurarse su supervivencia y replicación en el huésped. Algunos parásitos modulan la producción de numerosas moléculas tóxicas sintetizadas por el sistema inmune. Varios parásitos son altamente sensibles al óxido nítrico (ON) y sus derivados. El ON es producido en macrófagos (MΦ) luego de la estimulación con productos microbianos o con citoquinas. En el pasado, los MΦ se identificaban como células puramente inflamatorias (MΦ activados en forma clásica), capaces de secretar mediadores inflamatorios, actuar como células presentadoras de antígenos y matar patógenos intracelulares. Sin embargo, los MΦ activados representan un grupo más heterogéneo de células con distintos marcadores biológicos que pueden llevar a cabo diferentes funciones inmunológicas. Los MΦ activados alternativamente, fallan en producir ON en virtud de la inducción de la enzima arginasa y consecuentemente tienen disminuida su capacidad para matar patógenos intracelulares. Se ha comunicado la inducción de arginasa por parte de varios parásitos, por lo tanto este mecanismo podría favorecer su supervivencia en el huésped. En un modelo de infección con Trypanosoma cruzi, en nuestro grupo estudiamos la participación de arginasa y de las señales intracelulares involucradas en su inducción, durante la replicación de este parásito en los MΦ. La información obtenida a partir de nuestros trabajos permitiría comprender algunos mecanismos por los cuales distintas células del sistema inmune pueden ser programadas para favorecer el establecimiento de infecciones parasitarias crónicas.

Although there are several immunological mechanisms to eliminate the intracellular pathogens, they have elaborated a variety of strategies to escape of the immune response and to make possible their survival and replication in the host. Some parasites modulate the production of several toxic molecules synthesized by the immune system. Several parasites are highly sensitive to nitric oxide (ON) and their derivatives. ON is produced in macrophages (MΦ) after stimulation with microbial products or cytokines. In the past, M Φ were defined as inflammatory cells (classically activated MΦ), able to produce inflammatory mediators, to act like antigens presenting cells and to kill intracellular pathogens. Nevertheless, activated MΦ involve a more heterogeneous group of cells with different biological markers that can carry out different immunological functions. Alternatively activated MΦ fail to produce ON due to the arginase induction and consequently they have diminished their capacity to kill intracellular pathogens. It has been reported the induction of arginase by different parasites; therefore this mechanism could favor their survival in the host. In our group, we studied the participation of arginase in a model of Trypanosoma cruzi infection and the intracellular signals involved in the replication of this parasite in MΦ. The data obtained from our works would allow the understanding of some mechanisms by which cells can be programmed to favor the establishment of chronic parasitic infections.