Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice.

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.

Influence of silvopastoral systems on gastrointestinal nematode infection and immune response of Nellore heifers under tropical conditions.

Among the strategies for integrating crops, livestock, and forestry, silvopastoral systems must be highlighted due to their inherent microclimatic conditions, mainly in tropical countries such as Brazil, where cattle are frequently subjected to unfavorable thermal conditions. However, according to some studies, shading can potentially worsen herds´ parasitism due to better microclimatic condition for the parasites. This study aimed to assess fecal egg count in Nellore heifers reared in two silvopastoral arrangements (pasture with single or triple tree rows), in a crop-livestock system, and open pasture. In the silvopastoral treatment composed of triple rows, lesser parasite burden means were found, with a peak infection in February/March and another in October. Regarding the effect of seasons over the year, there was an environmental influence on the egg counts, with higher averages during the late rainy season and the beginning of the dry season. An immunological investigation of animals from each group showed that cattle kept on the silvopastoral arrangements with either single or triple rows have significantly higher lymphocyte proliferation when stimulated with specific antigens than those kept on open pastures. Based on our results, it can be concluded that both silvopastoral systems were not considered as a risk factor for nematode egg counts in Nellore heifers. Indeed, the shadiest system promoted milder parasitism and higher immunological lymphocyte responses in animals.

IL-17RA receptor signaling contributes to lung inflammation and parasite burden during Toxocara canis infection in mice.

IL-17 is a cytokine produced by innate and acquired immunity cells that have an action against fungi and bacteria. However, its action in helminth infections is unclear, including in Toxocara canis infection. Toxocariasis is a neglected zoonosis representing a significant public health problem with an estimated seroprevalence of 19% worldwide. In the present study, we describe the immunopathological action of IL-17RA in acute T. canis infection. C57BL/6j (WT) and IL-17RA receptor knockout (IL-17RA-/-) mice were infected with 1000 T. canis eggs. Mice were evaluated 3 days post-infection for parasite load and white blood cell count. Lung tissue was harvested for histopathology and cytokine expression. In addition, we performed multiparametric flow cytometry in the BAL and peripheral blood, evaluating phenotypic and functional changes in myeloid and lymphoid populations. We showed that IL-17RA is essential to control larvae load in the lung; however, IL-17RA contributed to pulmonary inflammation, inducing inflammatory nodular aggregates formation and presented higher pulmonary IL-6 levels. The absence of IL-17RA was associated with a higher frequency of neutrophils as a source of IL-4 in BAL, while in the presence of IL-17RA, mice display a higher frequency of alveolar macrophages expressing the same cytokine. Taken together, this study indicates that neutrophils may be an important source of IL-4 in the lungs during T. canis infection. Furthermore, IL-17/IL-17RA axis is important to control parasite load, however, its presence triggers lung inflammation that can lead to tissue damage.

Nucleolar organizer region proteins enhancement in nucleoplasm's of renal tubular cells is an indication of kidney impairment in Leishmania-infected dogs.

Hyperactivation of tubular cells contributes for the progression of kidney lesions. The exacerbated expression of immunological proteins and ribosomal DNA (rDNA) transcriptional activity are observed in tubular cells. This intensified expression results in more prominent hypertrophic changes and is often accompanied by increased expression of factors involved in different phases of ribosomal biosynthesis, such as the nucleolar organizer regions (NOR). The aim of this study was to evaluate whether there is an association between NOR proteins, renal impairment, and clinical status in Leishmania-infected dogs (CanL). Forty-five dogs with CanL and six uninfected controls were assessed in this study. PCR was performed to detect parasites' nucleic acids in kidney. Histopathological analyses were performed in kidney fragments, and NOR was detected by Ag stain (AgNOR). Leishmania-infected dogs showed more intense inflammation and collagen deposition compared with uninfected controls. Biochemical alterations were observed only in Leishmania-infected dogs. AgNORs per cell were significantly higher in clinically affected dogs and higher histopathological lesion score was observed in Leishmania-infected dogs. Positive correlations between number of NORs per cell in medullary region and histopathological lesion score were observed. Furthermore, AgNOR expression, intensity of renal lesions, and clinical sigs was associated in Leishmania-infected dogs. We propose that the detection of AgNOR proteins could be used to better estimate the kidney tubular damage at the time of examination in Leishmania-infected dogs as a marker to estimate renal impairment in dogs with CanL.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Resinous adhesive systems differentially affect the expression of cytokines by human monocytes stimulated or not with Streptococcus mutans in vitro.

OBJECTIVES: The polymerization of adhesive systems is incomplete and the residual monomers that have been released have a cytotoxic capacity. Some teeth develop into pulp necrosis after composite resin restorations. Considering frequent pulpal inflammation in response to cariogenic bacteria, substances released from the patches could affect the cells of the inflammatory infiltrate and interfere with the mechanisms of defense against microorganisms and protection of pulpal tissue. The aim of this study was to evaluate the effect of substances released by different resinous adhesive systems on cell viability and cytokine expression by human monocytes stimulated in vitro with Streptococcus mutans. DESIGN: Peripheral blood mononuclear cells from 10 healthy subjects were stimulated with S. mutans and then incubated with supernatants obtained from the Single Bond Universal (SBU) or Clearfil SE Bond (CSEB) adhesive systems for eight hours. Staining with Annexin V and 7AAD for analysis of apoptosis were performed and detection of monocytes expressing cytokines IL-1α, IL-6, IL-8, IL-10, IL-12 and TNF-α were performed by flow cytometry. RESULTS: No treatment significantly affected apoptosis in monocytes. SBU supernatant increased the frequency of monocytes expressing IL-8 and decreased the monocytes expressing IL-10. Considering S. mutans-stimulated cells, while SBU increased the frequency of IL-8+ monocytes, CSEB reduced the frequency of IL-6 and TNF-α positive monocytes. CONCLUSIONS: Products released from SBU seem to induce proinflammatory effects on monocytes while those from CSEB show an anti-inflammatory outcome. These effects may interfere in the control of cytokine-mediated immunoinflammatory pulp reactions, both in the presence and absence of stimulation by cariogenic bacteria.

Effect of porous tantalum on the biological response of human peripheral mononuclear cells exposed to Porphyromonas gingivalis.

AIM: To evaluate biological behavior of human peripheral mononuclear cells (PBMC) in contact with porous tantalum (PT) and Porphyromonas gingivalis (Pg). METHODS: Pg was incubated for 8 hours. The groups formed were: PBMC (control), PBMC + PT, PBMC + Pg and PBMC + PT + Pg. Cell viability was evaluated using MTT assay. The morphology and adhesion of PBMC to PT was evaluated using scanning electron microscopy. Expression of interleukin (IL)-10, transforming growth factor (TGF)-ß, matrix metallopeptidase (MMP)-9 and receptor activator of nuclear factor-κΒ ligand (RANKL) was determined by enzyme-linked immunosorbent assay. RESULTS: MTT assay revealed that PT did not interfere in the mitochondrial activity of PBMC (P > .05). Scanning electron microscopy showed the adherence of PBMC to PT. IL-10 levels in PBMC + PT were similar to PBMC and lower than PBMC + Pg. TGF-ß levels in PBMC + PT were higher than PBMC and PBMC + Pg. MMP-9 levels in PBMC + PT were similar to PBMC and lower than PBMC + Pg and PBMC + PT + Pg. RANKL levels in PBMC + PT were lower than in PBMC. CONCLUSION: PT did not affect PBMC viability, allowed cell adhesion, reduced expression of RANKL and enhanced TGF-ß in comparison with the control group.

Leishmania infantum induces expression of the negative regulatory checkpoint, CTLA-4, by human naïve CD8+ T cells.

AIMS: CD8+ T cells are important in mediating protective responses to intracellular pathogens. However, an uncontrolled response may lead to pathology. The role of CD8+ T cells in different clinical manifestations of human leishmaniasis is controversial and poorly understood. We aim to study the response of CD8+ T cells to the first exposure to different strains of Leishmania, seeking to correlate these findings with clinical manifestations of disease. METHODS AND RESULTS: We have evaluated the expression of granzyme A, inflammatory and anti-inflammatory cytokines, as well as CTLA-4 by human naïve CD8+ T cells exposed to Leishmania braziliensis and two different strains of Leishmania infantum in vitro. We observed that while exposure to L braziliensis induced an inflammatory profile, as measured by the expression of granzyme A, IFN-gamma and IL-17, as well as a higher IFN/IL-10 ratio, exposure to L infantum led to a regulatory profile, as measured by lower IFN/IL-10 ratio and higher expression of CTLA-4. CONCLUSION: These results may help explain why patients with the visceral clinical form present a weaker cellular response and, consequently, a worse outcome of the disease. The use of CTLA-4 checkpoint inhibitors may emerge as a potential immunotherapy to ameliorate the immune response in visceral leishmaniasis patients.

Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis.

OBJECTIVES: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. METHODOLOGY: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1ß, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. RESULTS: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1ß and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1ß and TNF-α, whereas UDMA reduced IL-1ß release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. CONCLUSIONS: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1ß and TNF-α secretion by PBMC.

Corrigendum: Activation of Human CD11b+ B1 B-Cells by Trypanosoma cruzi-Derived Proteins Is Associated With Protective Immune Response in Human Chagas Disease.

[This corrects the article DOI: 10.3389/fimmu.2018.03015.].

Activation of Human CD11b+ B1 B-Cells by Trypanosoma cruzi-Derived Proteins Is Associated With Protective Immune Response in Human Chagas Disease.

B-cells mediate humoral adaptive immune response via the production of antibodies and cytokines, and by inducing T-cell activation. These functions can be attributed to distinct B-cell subpopulations. Infection with Trypanosoma cruzi, the causative agent of Chagas disease, induces a polyclonal B-cell activation and lytic antibody production, critical for controlling parasitemia. Individuals within the chronic phase of Chagas disease may remain in an asymptomatic form (indeterminate), or develop severe cardiomyopathy (cardiac form) that can lead to death. Currently, there is no effective vaccine to prevent Chagas disease, and no treatment to halt the development of the cardiomyopathy once it is installed. The pathology associated with cardiac Chagas disease is a result of an inflammatory reaction. Thus, discovering characteristics of the host's immune response that favor the maintenance of favorable heart function may unveil important immunotherapeutic targets. Given the importance of B cells in antibody production and parasite control, we investigated T. cruzi-derived antigenic fractions responsible for B-cell activation and whether frequencies and functional characteristics of B-cell subpopulations are associated with different clinical outcomes of human Chagas disease. We stimulated cells from indeterminate (I) and cardiac (C) Chagas patients, as well as non-infected individuals (NI), with T. cruzi-derived protein- (PRO), glycolipid- (GCL) and lipid (LIP)-enriched fractions and determined functional characteristics of B-cell subpopulations. Our results showed that the frequency of B-cells was similar amongst groups. PRO, but not GCL nor LIP, led to an increased frequency of B1 B-cells in I, but not C nor NI. Although stimulation with PRO induced higher TNF expression by B1 B-cells from C and I, as compared to NI, it induced expression of IL-10 in cells from I, but not C. Stimulation with PRO induced an increased frequency of the CD11b+ B1 B-cell subpopulation, which was associated with better cardiac function. Chagas patients displayed increased IgM production, and activation of gamma-delta T-cells, which have been associated with B1 B-cell function. Our data showed that PRO activates CD11b+ B1 B-cells, and that this activation is associated with a beneficial clinical status. These findings may have implications in designing new strategies focusing on B-cell activation to prevent Chagas disease cardiomyopathy.

Effects of Bio-Oss® and Cerasorb® dental M on the expression of bone-remodeling mediators in human monocytes.

In contribution to diverse techniques of bone reconstruction involving biomaterials in contemporary dentistry, this study aimed to evaluate the effect of the bone-grafting materials Bio-Oss® and Cerasorb® Dental M on the expression of cytokines associated with bone remodeling by human monocytes in vitro. Bio-Oss® and Cerasorb® Dental M were incubated in separate culture media, and their supernatants were added to mononuclear cells of human peripheral blood, some of which had been stimulated with Porphyromonas gingivalis. The frequency of total monocytes and CD14+ monocytes producing cytokines interleukin 6 (IL-6), IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α) were determined by flow cytometry. One-way analysis of variance with repeated measures, followed by Tukey's post hoc test, revealed that stimulation with P. gingivalis increased the expression of IL-6 and IL-8 and reduced the expression of TNF-α compared to effects demonstrated in the control group (p < 0.05). Adding biomaterial supernatants did not significantly affect the expression of any cytokine evaluated, however, either in the absence or in the presence of bacterial stimulation. Our data suggest that Bio-Oss® and Cerasorb® Dental M neither stimulate cytokine production in human monocytes nor interfere with mechanisms of cell communication mediated by cytokines evaluated during stimulation with P. gingivalis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2066-2073, 2017.

Infection of Human Monocytes with Leishmania infantum Strains Induces a Downmodulated Response when Compared with Infection with Leishmania braziliensis.

Human infection with different species of Leishmania leads to distinct clinical manifestations, ranging from relatively mild cutaneous (Leishmania braziliensis) to severe visceral (Leishmania infantum) forms of leishmaniasis. Here, we asked whether in vitro infection of human monocytes by Leishmania strains responsible for distinct clinical manifestations leads to early changes in immunological characteristics and ability of the host cells to control Leishmania. We evaluated the expression of toll-like receptors and MHC class II molecules, cytokines, and Leishmania control by human monocytes following short-term infection with L. braziliensis (M2904), a reference strain of L. infantum (BH46), and a wild strain of L. infantum (wild). The induction of TLR2, TLR9, and HLA-DR were all lower in L. infantum when compared with L. braziliensis-infected cells. Moreover, L. infantum-infected monocytes (both strains) produced lower TNF-alpha and a lower TNF-alpha/IL-10 ratio, resulting in a weaker inflammatory profile and a 100-fold less effective control of Leishmania than cells infected with L. braziliensis. Our results show that L. infantum strains fail to induce a strong inflammatory response, less activation, and less control of Leishmania from human monocytes, when compared with that induced by L. braziliensis infection. This functional profile may help explain the distinct clinical course observed in patients infected with the different Leishmania species.

Blocking of CD1d Decreases Trypanosoma cruzi-Induced Activation of CD4-CD8- T Cells and Modulates the Inflammatory Response in Patients With Chagas Heart Disease.

The control of inflammatory responses to prevent the deadly cardiac pathology in human Chagas disease is a desirable and currently unattained goal. Double-negative (DN) T cells are important sources of inflammatory and antiinflammatory cytokines in patients with Chagas heart disease and those with the indeterminate clinical form of Chagas disease, respectively. Given the importance of DN T cells in immunoregulatory processes and their potential as targets for controlling inflammation-induced pathology, we studied the involvement of CD1 molecules in the activation and functional profile of Trypanosoma cruzi-specific DN T cells. We observed that parasite stimulation significantly increased the expression of CD1a, CD1b, CD1c, and CD1d by CD14(+) cells from patients with Chagas disease. Importantly, among the analyzed molecules, only CD1d expression showed an association with the activation of DN T cells, as well as with worse ventricular function in patients with Chagas disease. Blocking of CD1d-mediated antigen presentation led to a clear reduction of DN T-cell activation and a decrease in the expression of interferon γ (IFN-γ) by DN T cells. Thus, our results showed that antigen presentation via CD1d is associated with activation of DN T cells in Chagas disease and that CD1d blocking leads to downregulation of IFN-γ by DN T cells from patients with Chagas heart disease, which may be a potential target for preventing progression of inflammation-mediated dilated cardiomyopathy.

Trypanosoma cruzi-induced activation of functionally distinct αß and γδ CD4- CD8- T cells in individuals with polar forms of Chagas' disease.

CD4(-) CD8(-) (double-negative [DN]) T cells have recently been shown to display important immunological functions in human diseases. They express γδ or αß T-cell receptors that recognize lipid/glycolipid antigens presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently demonstrated that while αß DN T cells serve primarily to express inflammatory cytokines, γδ DN T cells express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi experimental infection. In this work, we sought to investigate whether αß or γδ DN T cells display distinct immunoregulatory potentials in patients with polar forms of human Chagas' disease. Our data showed that in vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe cardiac clinical forms of the disease. However, while αß DN T cells primarily produce inflammatory cytokines in both forms of the disease, γδ DN T cells display a marked, significant increase in antigen-specific IL-10 expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-producing γδ DN T cells were correlated with improved clinical measures of cardiac function in the patients, suggesting a protective role for these cells in Chagas' disease. Taken together, these data show distinct functional characteristics for αß and γδ DN T cells associated with distinct morbidity rates and clinical forms in human Chagas' disease.