HERV-W upregulation expression in bipolar disorder and schizophrenia: unraveling potential links to systemic immune/inflammation status.

BACKGROUND: Bipolar disorder (BD) and schizophrenia (SZ) are the two main mental disorders with unknown etiology that significantly impact individuals' quality of life. The potential pro-inflammatory role in their pathogenesis is postulated and Human Endogenous Retrovirus W (HERV-W) is an emerging candidate to modulate this pathogenic finding. HERVs, ancient retroviruses in the human genome, may play roles in inflammation and disease pathogenesis. Despite HERVs' involvement in autoimmune diseases, their influence on mental disorders remains underexplored. Therefore, the aim of this study was to assess the level of HERV-W-env expression and the systemic inflammatory profile through the concentration of IL-2, IL-4, IL-6, IL-10, TNF-α and INF-γ cytokines in BD and SZ patients. RESULTS: All participants showed HERV-W-env expression, but its expression was higher in mental disorder patients (p < 0.01) than in control. When separated, SZ individuals exhibited higher HERV-W expression than the control group (p < 0.01). Higher serum levels of TNF-α and IL-10 were found in BD (p = 0.0001 and p = 0.001, respectively) and SZ (p = 0.01) and p = 0.01, respectively) than in the control group, while SZ showed decreased levels IFN-γ and IL-2 as compared to controls (p = 0.05) and BD patients (p = 0.05), respectively. Higher TNF-α/IL-4 and TNF-α/IL-10 ratios, and lower IFN-γ/IL-10 were observed in BD and SZ patients than controls. Significant negative correlation between HERV-W-env expression and IL-10 (r=-0.47 p < 0.05), as well as positive correlations between HERV-W-env expression and TNF-α/IL-10 or IFN-γ/IL-10 ratios (r = 0.48 p < 0.05 and r = 0.46 p < 0.05, respectively) were found in BD patients. CONCLUSION: These findings suggest not only a potential link between HERV-W-env expression both in BD and SZ, but also a possible involvement of systemic inflammatory status in BD patients.

Epidemiological profile and genetic resistance of Neisseria gonorrhoeae infection in women in a poor region of São Paulo, Brazil.

BACKGROUND AND AIM: Gonorrhea is a bacterial infection in the urogenital tract, transmitted by sexual or perinatal contact, caused by Neisseria gonorrhoeae, a gram-negative diplococcus. The present study evaluates the frequency of N. gonorrhoeae in women treated at Hospital Wladimir Arruda in poor area of São Paulo and also verifies the presence of genetic resistance against three antimicrobials of different classes: Tetracycline, Azithromycin and Ciprofloxacin. METHODS: This is an observational and descriptive study with a quantitative approach. Samples were collected at Hospital Escola Wladimir Arruda. The volunteers are women from 16 to 65 years of age. Sociodemographic, gynecological, sexual and health data are collected through a questionnaire, their symptoms/clinical manifestation were requested by the medical records, and then the participant is referred for collection of samples of cervical vaginal smear. The samples were screened for N. gonorrhoeae (dcmH gene) and tested for resistance genes to Tetracycline, Azithromycin and Ciprofloxacin through PCR. RESULTS: In the total of 127 samples analyzed by Real-Time PCR, 23 were positive and correspond to a general prevalence of a gonococcal infection in the studied population of 17% (CI:95%), and the participants were married (43.4%), had active sexual life (56.5%) and did not use any type of condom during sexual intercourse (52.1%). The resistance to the tetM ribosomal gene was found in 14 samples, prevalence of 60% (CI= 95%). CONCLUSIONS: We have described a concerning frequency of N. gonorrhoeae infection in females attended in an outcare patient. Also, most of the strains detected presented resistance to one or more antimicrobials.

Lipophosphoglycan From Dermotropic New World Leishmania Upregulates Interleukin-32 and Proinflammatory Cytokines Through TLR4 and NOD2 Receptors.

Interleukin-32 (IL-32) is produced during Leishmania infection, but the components of the parasite that induce its production are unknown. An important multivirulence factor of Leishmania spp. protozoa is the lipophosphoglycan (LPG), which plays a crucial role in the host-parasite interaction. Here, the ability of LPGs from two dermotropic Leishmania species to induce IL-32 production was evaluated in human peripheral blood mononuclear cells (PBMCs). Additionally, the potential receptors involved in this activation were assessed. PBMCs from healthy individuals were stimulated with LPGs from L. amazonensis (La) or L. braziliensis (Lb), live promastigotes of La or Lb and E. coli lipopolysaccharide (LPS, TLR4 agonist) as control. Blockers of TLR4 (Bartonella quintana LPS or monoclonal antibody) and Ponatinib (RIPK2 inhibitor, NOD2 pathway) were used to evaluate the receptors. ELISA was performed for IL-32 expression and cytokine (IL-1ß and IL-6) production in cell lysates and in supernatants, respectively. Expression of TLR4 (2 h, 24 h) was assessed by flow cytometry. IL-32γ mRNA transcript was analyzed by qPCR. It was observed that LPG from Leishmania, like whole parasites, induced the production of IL-32, IL-1ß and IL-6. Both LPGs induced the expression of IL32γ mRNA. The production of IL-32 was earlier detected (6 h) and positively associated with the production of IL-1ß and IL-6. The induction of cytokines (IL-32, IL-1ß and IL-6) was dependent on TLR4 and NOD2. The TLR4 was internalized after interaction with LPG. Therefore, our data suggest that LPGs from La and Lb are components of Leishmania able to upregulate IL-32 and other pro-inflammatory cytokines in a TLR4- and NOD2-dependent manner. In addition, LPG-induced IL-32 seems to be necessary for IL-1ß and IL-6 production. To identify the parasite factors and host receptors involved in IL-32 induction is crucial to reveal potential targets for novel strategies to control leishmaniasis.

Low frequency of SARS-CoV2 infection in daycare centers during the reopening of school activities in the Southeast's poor area of Brazil

ABSTRACT SARS-Cov2 has already infected over 482 million people and caused more than 6.1 million deaths. The beginning of the pandemic has led the health authorities of several countries to adopt non-pharmacological preventive measures such as daycare closures. The reopening took place when the country had the highest rates of infection and mortality (mainly due to the gamma variant (P.1) outbreak) and the beginning of the vaccination program. Therefore, we aimed to investigate the prevalence of SARS-CoV2 in daycare after educational activities resumed. The study was conducted in seven childcare facilities. Swab samples from the nasopharynx were collected from children and staff members. The viral RNA was obtained through PureLink RNA extraction kit purification and SARS-CoV2 presence was detected using the All plex SARS-CoV2 kit. The study population included 201 participants, including daycare workers and children. The average age of the workers and children is 40 and 3 years old, respectively. Among the children, 47.5% are female and among the workers, 91.4%. One (0.5%) test came out positive for the presence of SARS-CoV-2, which was from a sample of an asymptomatic childcare worker, and no secondary infections were detected. Considering that the return to daycare activities occurred during a period with a high number of deaths and a lack of vaccines throughout the country, the small number of cases indicates the effectiveness of the several preventive measures used by daycare centers in preventing SARS-CoV2 transmission.

PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection.

Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1ß both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1ß production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1ß is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1ß production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1ß secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1ß production. Leishmania-dependent suppression of IL-1ß secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1ß production.

Iron prevents the development of experimental cerebral malaria by attenuating CXCR3-mediated T cell chemotaxis.

Cerebral malaria is a severe neurological complication of Plasmodium falciparum infection. Previous studies have suggested that iron overload can suppress the generation of a cytotoxic immune response; however, the effect of iron on experimental cerebral malaria (ECM) is yet unknown. Here we determined that the incidence of ECM was markedly reduced in mice treated with iron dextran. Protection was concomitant with a significant decrease in the sequestration of CD4+ and CD8+ T cells within the brain. CD4+ T cells demonstrated markedly decreased CXCR3 expression and had reduced IFNγ-responsiveness, as indicated by mitigated expression of IFNγR2 and T-bet. Additional analysis of the splenic cell populations indicated that parenteral iron supplementation was also associated with a decrease in NK cells and increase in regulatory T cells. Altogether, these results suggest that iron is able to inhibit ECM pathology by attenuating the capacity of T cells to migrate to the brain.

Absence of metalloprotease GP63 alters the protein content of Leishmania exosomes.

Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm vesicles that are freed by many eukaryotic cells. To better understand the GP63-dependent immune modulation of the macrophage by Leishmania parasites and their exosomes, we compared the immunomodulatory properties of Leishmania major (WT) and L. major gp63-/- (KO) as well as their exosomes in vitro and in vivo. Importantly, we observed that Leishmania exosomes can modulate macrophage PTPs and TFs in a GP63-dependent manner. In addition, our qRT-PCR analyses showed that WT parasites were able to downregulate multiple genes involved in the immune response, especially cytokines and pattern recognition receptors. KO parasites showed a strongly reduced modulatory capacity compared to WT parasites. Furthermore, comparison of WT versus KO exosomes also showed divergences in alteration of gene expression, especially of chemokine receptors. In parallel, studying the in vivo inflammatory recruitment using a murine air pouch model, we found that exosomes have stronger proinflammatory properties than parasites and preferentially induce the recruitment of neutrophils. Finally, comparative proteomics of WT and KO exosomes surprisingly revealed major differences in their protein content, suggesting a role for GP63 in Leishmania exosomal protein sorting. Collectively our data clearly establish the crucial role of GP63 in dampening the innate inflammatory response during early Leishmania infection, and also provides new insights in regard to the role and biology of exosomes in Leishmania host-parasite interactions.

TAK1 contributes to the enhanced responsiveness of LTB(4)-treated neutrophils to Toll-like receptor ligands.

Pattern-recognition receptors such as Toll-like receptors (TLRs) are essential sensors implicated in the early and efficient innate immune response against pathogens. We have previously demonstrated that leukotriene B(4)(LTB(4)) has the capacity to enhance leukocyte responses to TLR9 ligands and to control viral infection. In this report, we provide evidence that LTB(4) treatment of human neutrophils leads to a potentiation in proinflammatory cytokine secretion induced by various myeloid differentiation factor 88-dependent TLR agonists. LTB(4) failed to enhance TLR mRNA levels as well as expression of TLR2 and TLR4 receptors, suggesting that LTB(4) acts through intracellular mechanism(s) to potentiate neutrophil responses to TLR ligands. We found that while IRAK can be activated by LTB(4), this process is dispensable to LTB(4) to potentiate neutrophil responses to TLR ligands since pretreatment of neutrophils with IRAK1/4 inhibitor did not affect its potentiating effects. However, our data clearly show that LTB(4) treatment of neutrophils led to the phosphorylation of downstream signaling molecules, TAK1 and p38, a process found essential to observe an increased secretion of cytokines by neutrophils activated with TLR ligands. Pretreatment of neutrophils with TAK1 or p38 kinase inhibitors strongly repressed the effect of LTB(4) on cytokine synthesis by neutrophils stimulated with LTA, LPS or CpG. The same pattern was observed in agonist-treated human embryonic kidney 293 cells transfected with TAK1-targeting siRNA where secretion of IL-8 was significantly reduced to basal levels. These results indicate that TAK1 and p38 kinases appear to be central in the 'priming effect' of LTB(4) on neutrophils to enhance response to TLR ligands.

Leishmania repression of host translation through mTOR cleavage is required for parasite survival and infection.

The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.

Protein tyrosine phosphatases are regulated by mononuclear iron dicitrate.

The involvement of macrophages (Mvarphis) as host, accessory, and effector cells in the development of infectious diseases, together with their central role in iron homeostasis, place these immune cells as key players in the interface between iron and infection. Having previously shown that the functional expression of NRAMP-1 results in increased protein phosphorylation mediated in part by an iron-dependent inhibition of Mvarphi protein-tyrosine phosphatase (PTP) activity, we sought to study the mechanism(s) underlying this specific event. Herein we have identified the mononuclear dicitrate iron complex [Fe(cit)(2)H(4-x)]((1+x)-) as the species responsible for the specific inhibition of Mvarphi PTP activity. By using biochemical and computational approaches, we show that [Fe(cit)(2)](5-) targets the catalytic pocket of the PTP SHP-1, competitively inhibiting its interaction with an incoming phosphosubstrate. In vitro and in vivo inhibition of PTP activity by iron-citrate results in protein hyperphosphorylation and enhanced MAPK signaling in response to LPS stimulation. We propose that iron-citrate-mediated PTP inhibition represents a novel and biologically relevant regulatory mechanism of signal transduction.

Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif.

Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKalpha/beta) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen.

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57Bl/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies.

Cross-regulation of iNOS and COX-2 by its products in murine macrophages under stress conditions.

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPS-induced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43 degrees C, 30 min) and stimulated with LPS (1 microg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE(2) by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE(2) and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NO-synthesis inhibitor) and of iNOS by nimesulide (PGs-synthesis inhibitor). Such cross-regulation was not observed in cells at 37 degrees C. These treatments significantly reduced MTT levels in cells at 37 degrees C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.

PAF is involved in the Mycoplasma arthritidis superantigen-triggering pathway for iNOS and COX-2 expression in murine peritoneal cells.

We investigated the capacity of Mycoplasma arthritidis mitogen (MAM) to induce (a) expression of the inducible enzymes cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS), (b) production of prostaglandin E2 (PGE2) and nitric oxide (NO), and (c) involvement of platelet-activating factor (PAF) in the MAM-induced activation pathway. Resident peritoneal cells from C3H/HePas mice were incubated with MAM in the presence or absence of a PAF-antagonist (WEB2170) or COX-2 inhibitors (nimesulide or NS398). Enzyme expression was evaluated by immunoblotting, PGE2 by EIA, and NO by Griess reaction. Following MAM-stimulation of peritoneal cells, expression of COX-2 was detected at 3 h (peak levels at 12 h) and of iNOS at 6 h (peak levels at 20 h). PGE2 increased till 20 h, decreasing thereafter, whereas NO increased with time. WEB2170 (5 x 10(-5) M) treatment caused 44% inhibition of NO output and reduced iNOS expression (48% at the peak of expression). Concomitant treatment with WEB2170 and nimesulide (10(-5) M) reversed these inhibitory effects. WEB2170 reduced COX-2 expression (43% at the peak of expression) and prevented the decline in PGE2 levels after 20 h. These results suggest the involvement of PAF in the signaling pathway triggered by MAM that leads to expression of iNOS and COX-2, and show that PAF regulates the production of NO, possibly by controlling levels of PGE2.

Mycoplasma arthritidis superantigen (MAM)-induced macrophage nitric oxide release is MHC class II restricted, interferongamma dependent, and toll-like receptor 4 independent.

Mycoplasma arthritidis causes arthritis in rodents that resembles human rheumatoid arthritis. It produces a superantigen (MAM) that stimulates production of cytokines by making a bridge between lymphocyte T-cell receptor with the appropriate Vbeta chain, and H-2 1-Ealpha MHC class II molecules. Here we studied MAM-induced nitric oxide (NO) production in mouse peritoneal macrophages and found that it was: (1) time and concentration dependent, (2) possibly derived from inducible NOS synthase since it was reduced significantly by amino guanidine pretreatment, (3) restricted to H-2(K) (C3H/HePas and C3H/HeJ) and H-2(d) strains (BALB/c), (4) independent of TLR4 signaling since the coisogenic strains C3H/HePas and C3H/HeJ (TLR4 deficient) produced similar levels of NO following MAM stimulation, (5) potentiated by lipopolysaccharide, and (6) dependent on the presence of nonadherent peritoneal cells. Neutralization of interferon-gamma (IFNgamma in the peritoneal cell cultures with monoclonal antibodies abolished MAM-induced NO production. Addition of rIFNgamma to the adherent cells substituted the nonadherent cells for MAM-induced NO production. A macrophage cell line, J774A.1 (H-2(d)), also produced NO upon MAM stimulation but only when BALB/c spleen lymphocytes were added. Thus, in murine macrophages, MAM induces NO production that is dependent on signaling through MHC class II molecules and IFNgamma but independent of TLR4 expression.