EDITORIAL article Front. Public Health, 07 June 2022Sec. Infectious Diseases – Surveillance, Prevention and Treatment https://doi.org/10.3389/fpubh.2022.913507

Chagas disease (CD) is caused by the parasitic protozoan T. cruzi. The progression of CD in ~30% of patients results in Chagasic Cardiomyopathy (CCM). Currently, it is known that the inflammatory system plays a significant role in the CCM. Interferon-gamma (IFN-γ) is the major cytokine involved in parasitemia control but has also been linked to CCM. The L-type calcium current (ICa,L) is crucial in the excitation/contraction coupling in cardiomyocytes. Thus, we compared ICa,L and the mechanical properties of cardiomyocytes isolated from infected wild type (WT) and IFN-γ(−/−) mice in the first stage of T. cruzi infection. Using the patch clamp technique, we demonstrated that the infection attenuated ICa,L in isolated cardiomyocytes from the right and left ventricles of WT mice at 15 days post-infection (dpi), which was not observed in the IFN-γ(−/−) cardiomyocytes. However, ICa,L was attenuated between 26 and 30 dpi in both experimental groups. Interestingly, the same profile was observed in the context of the mechanical properties of isolated cardiomyocytes from both experimental groups. Simultaneously, we tracked the mortality and MCP-1, TNF-α, IL-12, IL-6, and IL-10 serum levels in the infected groups. Importantly, the IFN-γ(−/−) and WT mice presented similar parasitemia and serum inflammatory markers at 10 dpi, indicating that the modifications in the cardiomyocyte functions observed at 15 dpi were directly associated with IFN-γ(−/−) deficiency. Thus, we showed that IFN-γ plays a crucial role in the electromechanical remodeling of cardiomyocytes during experimental T. cruzi infection in mice.

The capacity of tumor cells to shift dynamically between different states could be responsible for chemoresistance and has been commonly linked to the acquisition of stem cell properties. Here, we have evaluated the phenotype switching associated with drug resistance in breast cancer cell lines and cell lineage obtained from Brazilian patients. We have highlighted the role of the cancer stem cell marker CD24 in the dynamics of cell plasticity and the acquirement of drug resistance. We showed that the translocation of CD24 from cytosol to cell membrane is a triggering event for the phenotype change of breast tumor cells exposed to drug stress. Here, we provide evidence that the phenotype switching is due to the presence of a cytosolic pool of CD24. Importantly, the cellular localization of CD24 was correlated with the changes in the dynamics of p38 MAPK activation. A strong and continuous phosphorylation of the p38 MAPK led to the overexpression of Bcl-2 after treatment in persistent cells presenting high density of CD24 on cell membrane. This phenotype enabled the cells to enter in slow-down of cell cycle, after which several weeks later, the dormant cells proliferated again. Importantly, the use of a p38 activity inhibitor sensitized cells to drug treatment and avoided chemoresistance.

BackgroundThe adaptation capacity of tumor cells to shift dynamically between different states could be responsible for chemoresistance and has been commonly linked to the acquisition of stem cell properties. Here, we have provided new elements concerning the role of the Cancer Stem Cell marker CD24 in the dynamics of tumor cell plasticity and the acquirement of drug resistance in breast cancer.MethodologyBreast cancer cell lines and cell lineage obtained from Brazilian patients were treated with cytotoxic and non-cytotoxic drugs and the phenotype switching associated with drug resistance was evaluated. Different markers were quantified like the expression of CD24 through microscopy and FACS, the regulation of pro- and anti-apoptotic proteins Bax and Bcl-2, epigenetic changes and MAPK activation using western blotting in wild type and CD24 silencing cells. ResultsThe translocation of CD24 from cytosol to cell membrane was a triggering event for the phenotype change of tumor cells exposed to drug stress. Importantly, this phenomenon was observed in all the cells of the different population that was rendered possible thanks to the presence of an intracellular pool of CD24 in each given breast tumor cell. In these CD24+ cells, a strong and continuous phosphorylation of the p38 MAPK leading to drug resistance through the tri-methylation of H3K9 and overexpression of the anti-apoptotic protein Bcl-2 was observed. Importantly, the drug-resistant CD24+ cells entered into slow-down of cell cycle and restarted to proliferate after several weeks. The reawakened cells presented constitutive p38 activation, higher drug resistance and higher migration capacity. Notably, the use of a p38 activity inhibitor sensitized cells to drug treatment and avoided the appearance of chemoresistance.Conclusions/significance In this study, we have associated the cellular localization of CD24 with the changes in the dynamics of p38MAPK activation in breast tumor cells under drug stress. The tandem CD24/p38 plays a crucial role in the acquisition of drug resistance and new cell identity. The use of p38 inhibitor was able to disrupt this partnership and consequently to avoid chemotherapy-induced cell state transition. These results suggest that targeting p38 in breast cancer can overcome adaptive resistance to doxorubicin treatment.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, has become a global health problem due to the massive migration flow from Latin America to other parts of the globe, including Europe [1,2].The disease is the most common infective cause of cardiomyopathy and it is observed in 11-19% of patients in Europe [3].The time course of the disease is complex and 20-30% of patients with 10-30 years after infection will develop heart failure and/or arrhythmias.Despite its importance, there is scarce information about the molecular physiopathology of the heart in CD.It is known that inducible nitric oxide synthase (iNOS) plays an important role in the macrophage-driven control of parasitic infection, through the formation of peroxynitrite [4][5][6].However, nitric oxide (NO) is an important modulator of cell machinery and its unbalanced production aggravates electrical disturbances during experimental CD [7][8][9][10].We hypothesized that activation of iNOS plays an important role on the electrical remodeling of cardiomyocytes in the acute phase of experimental CD.Thus, we evaluated if iNOS takes part in electrical remodeling of isolated cardiomyocyte in the time course of the acute phase of experimental CD.Eight-week-old male/female C57BL/6 and iNOS -/-mice were infected intraperitoneally with 50 bloodstream trypomastigote form of colombian strain of T. cruzi.Mouse Inflammation Kit from BDTM was used to detect serum cytokine levels.The animals were kindly provided by Professor Dr. Ricardo T. Gazzinelli.Left ventricular cardiomyocytes were obtained using an enzymatic dissociation method with collagenase type II, as previously described [11][12][13].Only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied.Isolated cardiomyocytes from non-infected and infected mice were studied at 15 and 30 days post-infection (dpi).Patch-clamp recordings were obtained using an EPC-9.2 patchclamp amplifier.To measure action potentials (AP) and outward K + currents (I K ) the pipette solution had (in mM): 130 K-aspartate, 20 KCl, 10 HEPES, 2 MgCl 2 , 5 NaCl, 5 EGTA, pH set to 7.2 with KOH.We used Tyrode's as bath solution (in mM) 140 NaCl, 5.4 KCl, 1 MgCl 2 , 1.8 CaCl 2 , 10 HEPES, 10 glucose (pH set at 7.4).To record total outward I K cardiac myocytes were bathed with recording solution containing 100 μM of Cd 2+ to block L-type Ca 2+ currents.Total I K was measured by depolarization steps from -40 to 50 mV (3 s duration) from a holding potential of -80 mV every 15 s.For measurements of L type Ca 2+ current (I CaL ), recording pipettes were filled with internal solution containing (in mM): 120 CsCl, 20 TEACl, 5 NaCl, 10 HEPES, 5 EGTA, pH set to 7.2 with CsOH.I CaL was measured using 1.8 mM of Ca 2+ in extracellular solution.Membrane potential was first stepped from a holding potential of -80 mV to -40 mV for 50 ms and then stepped to different membrane voltages from -40 to 50 mV (300 ms duration).Junction potential was not corrected.The complete patch-clamp methodology is described in [12].Data are expressed as mean ± standard error (SE) and the number of samples is shown as n.Statistical significance of parametric data between multiple groups was determined by one-way or two-way ANOVA, followed by Tukey's post-test.For patch-clamp experiments cardiomyocytes were obtained from at least three animals for each measurement.Survival rate was analyzed using Kaplan-Meier plot.Comparisons were considered to be statistically significant when p < 0.05.During the acute phase of experimental CD excessive NO production contributes to the impairment of electrical properties in cardiomyocytes [12].Thus, we hypothesized that the deletion of iNOS, which is highly activated through IFN-γ activation pathway during the course of the disease, would attenuate the electrical remodeling of cardiomyocytes.Parasitemia were similar for both groups at 15 dpi.However, it was higher in iNOS -/-mice at 30 dpi (Fig. 1A).Higher parasitism was associated with increased, yet not significant, mortality rate in knockout (KO) mice (Fig. 1B) between 20 and 40 dpi, however,

Abstract BackgroundThe adaptation capacity of tumor cells to shift dynamically between different states could be responsible for chemoresistance and has been commonly linked to the acquisition of stem cell properties. Here, we have provided new elements concerning the role of the Cancer Stem Cell marker CD24 in the dynamics of tumor cell plasticity and the acquirement of drug resistance in breast cancer.MethodologyBreast cancer cell lines and cell lineage obtained from Brazilian patients were treated with cytotoxic and non-cytotoxic drugs and the phenotype switching associated with drug resistance was evaluated. Different markers were quantified like the expression of CD24 through microscopy and FACS, the regulation of pro- and anti-apoptotic proteins Bax and Bcl-2, epigenetic changes and MAPK activation using western blotting in wild type and CD24 silencing cells. ResultsThe translocation of CD24 from cytosol to cell membrane was a triggering event for the phenotype change of tumor cells exposed to drug stress. Importantly, this phenomenon was observed in all the cells of the different population that was rendered possible thanks to the presence of an intracellular pool of CD24 in each given breast tumor cell. In these CD24+ cells, a strong and continuous phosphorylation of the p38 MAPK leading to drug resistance through the tri-methylation of H3K9 and overexpression of the anti-apoptotic protein Bcl-2 was observed. Importantly, the drug-resistant CD24+ cells entered into slow-down of cell cycle and restarted to proliferate after several weeks. The reawakened cells presented constitutive p38 activation, higher drug resistance and higher migration capacity. Notably, the use of a p38 activity inhibitor sensitized cells to drug treatment and avoided the appearance of chemoresistance.Conclusions/significance In this study, we have associated the cellular localization of CD24 with the changes in the dynamics of p38MAPK activation in breast tumor cells under drug stress. The tandem CD24/p38 plays a crucial role in the acquisition of drug resistance and new cell identity. The use of p38 inhibitor was able to disrupt this partnership and consequently to avoid chemotherapy-induced cell state transition. These results suggest that targeting p38 in breast cancer can overcome adaptive resistance to doxorubicin treatment.

Toll-like receptors (TLR)s are central in immune response by recognizing pathogen-associated molecular patterns (PAMP)s. If they are essential to eliminate pathogens in earlier stages of infection, they also might play a role in homeostasis and tissue repair. TLR versatility parallels the plasticity of monocytes, which represent an heterogeneous population of immune cells. They are rapidly recruited to sites of infection and involved in clearance of pathogens and in tissue healing. This review underlines how TLRs have proved to be an interesting tool to study the properties of monocytes and why different therapeutic strategies exploring monocyte plasticity may be relevant in the context of chronic inflammatory disorders.

Chagas disease is caused by the trypanosomatid Trypanosoma cruzi, which chronically causes heart problems in up to 30% of infected patients. Chagas disease was initially restricted to Latin America. However, due to migratory events, this disease may become a serious worldwide health problem. During Chagas disease, many patients die of cardiac arrhythmia despite the apparent benefits of anti-arrhythmic therapy (e.g., amiodarone). Here, we assimilate the cardiac form of Chagas disease to an inflammatory cardiac disease. Evidence from the literature, mostly provided using experimental models, supports this view and argues in favor of new strategies for treating cardiac arrhythmias in Chagas disease by modulating cytokine production and/or action. But the complex nature of myocardial inflammation underlies the need to better understand the molecular mechanisms of the inflammatory response during Chagas disease. Here, particular attention has been paid to tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGF-β) although other cytokines may be involved in the chagasic cardiomyopathy.

Tumor cells capture the signaling pathways used by normal tissue to promote their own survival and dissemination and among them, the NF-κB and MAPK pathways (ERK, JNK and p38). MAPK activation has ambiguous effects on tumor cell fate depending on cell type, cancer stage and the engaged MAPK isoforms. A synthetic peptide named LyeTx II, derived from the venom of the Brazilian spider Lycosa erythrognatha, was capable of increasing MDA-MB-231 aggressive breast cancer cell proliferation as indicated by MTT and BrdU (5-bromo-2'-deoxyuridine) incorporation assay and cell migration. A correlation has been established between the accelerated proliferation and migration observed in the presence of LyeTx II and the upregulation of p38 MAPK phosphorylation. The use of the selective inhibitor of p38α/β (SB203580) abrogated the peptide effect in MDA-MB-231 cells. Besides, an augment of the canonical NF-κB pathway activation considered as crucial in cancer progression was noted after cell incubation with LyeTx II. Importantly, activation of p38 and NF-κB pathways was dependent on TAK1 activity. Together, these data suggest that TAK1-p38 pathway may represent an interesting target for treatment of aggressive breast cancers.

The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer.

This study continues to explore the plasticity of Toll-like receptor 2 (TLR2) previously described in immune response during Trypanosoma cruzi infection. Here, we have shown that Ly6ChiTLR2hi monocytes were involved in TNF-α and IL-12 production, whereas Ly6CloTLR2hi monocytes were mainly committed to IL-10 and TNF-α production during T. cruzi infection independently of TLR agonist used (i.e. TLR2 or TLR9 agonists). Another difference between the monocyte populations is that the adapter Mal (encoded by TIRAP) has appeared crucial for the cytokine production by Ly6Clo but not by Ly6Chi monocytes. The protein Mal was necessary to induce cytokine synthesis by Ly6Clo monocytes after triggering TLR2 or TLR9. Finally, our data have suggested that TLR2, TLR9, and Mal/TIRAP controlled differentially the emergence of the different TLR2hi monocyte populations in the spleen. In summary, this study highlights the central role of the TLR2/Mal tandem in the distinct activity among the monocyte subsets during T. cruzi infection. Such findings provide a basis for understanding the challenge posed by the use of TLR2 agonist in immunotherapy.

During oral infection, mucosal immunity assumes a predominant role. Here, we addressed the role of mast cells (MCs), which are mainly located in mucosa during oral infection with Toxoplasma gondii, using MC‐deficient (W/Wv) mice. We show that in the absence of MCs the resistance of W/Wv mice to oral infection was considerably reduced. W/Wv mice uniformly succumbed within 15 days of infection after administration of cysts of the ME49 strain of T. gondii. The rapid lethality of T. gondii in W/Wv mice correlated with a delayed Th1‐cell response, since IFN‐γ and IL‐12 levels peaked in the later phase of the infection. In vitro, BM‐derived MCs were able to recognize parasite lysate in a MyD88‐dependent way, reaffirming the role of this TLR adapter in immune responses to T. gondii. The importance of MCs in vivo was confirmed when W/Wv mice reconstituted with BM‐derived MCs from control mice retrieved an early strong Th1‐cell response and specially a significant IL‐12 production. In conclusion, MCs play an important role for the development of a protective immune response during oral infection with T. gondii.

BACKGROUND:Chagas' disease is one of the leading causes of heart failure in Latin American countries. Despite its great social impact, there is no direct evidence in the literature explaining the development of heart failure in Chagas' disease. Therefore, the main objective of the study was to investigate the development of the Chagas' disease towards its chronic phase and correlate with modifications in the cellular electrophysiological characteristics of the infected heart.METHODS AND RESULTS:Using a murine model of Chagas' disease, we confirmed and extended previous findings of altered electrocardiogram and echocardiogram in this cardiomyopathy. The observed changes in the electrocardiogram were correlated with the prolonged action potential and reduced transient outward potassium current density. Reduced heart function was associated with remodeling of intracellular calcium handling, altered extracellular matrix content, and to a set of proteins involved in the control of cellular contractility in ventricular myocytes. Furthermore, disruption of calcium homeostasis was partially due to activation of the PI3Kinase/nitric oxide signaling pathway. Finally, we propose a causal link between the inflammatory mediators and heart remodeling during chagasic cardiomyopathy.CONCLUSION:Altogether our results demonstrate that heart failure in Chagas' disease may occur due to electrical and mechanical remodeling of cardiac myocytes, and suggest that AKT/PI3K/NO axis could be an important pharmacological target to improve the disease outcome.

Pathogens express ligands for several TLRs that may play a role in the induction or control of the inflammatory response during infection. Concerning Trypanosoma cruzi, the agent of Chagas disease, we have previously characterized glycosylphosphatidylinositol (GPI) anchored mucin-like glycoproteins (tGPI-mucin) and unmethylated CpG DNA sequences as TLR2 and TLR9 agonists, respectively. Here we sought to determine how these TLRs may modulate the inflammatory response in the following cell populations: F4/80(+)CD11b(+) (macrophages), F4/80(low)CD11b(+) (monocytes) and MHCII(+)CD11c(high) (dendritic cells). For this purpose, TLR2(-/-) and TLR9(-/-) mice were infected with Y strain of T. cruzi and different immunological parameters were evaluated. According to our previous data, a crucial role of TLR9 was evidenced in the establishment of Th1 response, whereas TLR2 appeared to act as immunoregulator in the early stage of infection. More precisely, we demonstrated here that TLR2 was mainly used by F4/80(+)CD11b(+) cells for the production of TNF-α. In the absence of TLR2, an increased production of IL-12/IL-23p40 and IFN-γ was noted suggesting that TLR2 negatively controls the Th1 response. In contrast, TLR9 was committed to IL-12/IL-23p40 production by MHCII(+)CD11c(high) cells that constitute the main source of IL-12/IL-23p40 during infection. Importantly, a down-regulation of TLR9 response was observed in F4/80(+)CD11b(+) and F4/80(low)CD11b(+) populations that correlated with the decreased TLR9 expression level in these cells. Interestingly, these cells recovered their capacity to respond to TLR9 agonist when MHCII(+)CD11c(high) cells were impeded from producing IL-12/IL-23p40, thereby indicating possible cross-talk between these populations. The differential use of TLR2 and TLR9 by the immune cells during the acute phase of the infection explains why TLR9- but not TLR2-deficient mice are susceptible to T. cruzi infection.

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.