PD-L1 may mediate T-cell exhaustion in a case of early diffuse Leishmaniasis caused by Leishmania (L.) amazonensis

Introduction: Diffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the “anergic” pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry. Case presentation: A 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation. Conclusion: The dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient

Balance of IL-10 and Interferon-γ plasma levels in human visceral leishmaniasis: Implications in the pathogenesis

BACKGROUND: Leishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis. METHODS: To explore immunoregulatory mechanisms, 20 visceral leishmaniasis (VL) patients were evaluated during active disease and at different periods up to one year after treatment determining their plasma cytokine levels, clinical parameters (palpable spleen and liver) and antibody levels. RESULTS: Elevated plasma levels of IFN-γ and of IL-12 p40 were observed during active disease, significantly decreasing after treatment whereas in vitro Leishmania antigen-stimulated IFN-γ production by PBMC exhibited an inverse pattern being low during disease and increasing steadily thereafter. Absence of IFN-γ activity is a hallmark of VL. The main candidate for blunting IFN-γ activity is IL-10, a cytokine highly elevated in plasma with sharp decrease after treatment. Activity of IL-10 is inferred by high levels of anti-Leishmania specific IgG1 and IgG3. TGF-β had elevated total, but not of active, levels lessening the likelihood of being the IFN-γ counterpart. Spleen or liver size presented a steady decrease but return to normal values at only 120 days after treatment. Anti-Leishmania IgG (total and subclasses) levels and DTH or Leishmania-stimulated lymphocyte proliferation conversion to positive also present a slow decrease after treatment. IL-6 plasma levels were elevated in only a few patients. CONCLUSION: Taken together our results suggest that IFN-γ and IL-10 are the molecules most likely involved in determining fate of disease. After treatment, there is a long delay before the immune profile returns to normal what precludes using plasma cytokine levels as criteria of cure as simpler clinical evaluations, as a palpable spleen or liver, can be used

Leishmania infantum and Leishmania braziliensis: differences and similarities to evade the innate immune system

Visceral Leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs) act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human dendritic cells biology to better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood’s healthy volunteers donors and infected with L. infantum or L. braziliensis for 24 hours. We observed similar rates of infection (around 40%) as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of dendritic cells after 24 hours. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model

PD-L1 May Mediate T-Cell Exhaustion in a Case of Early Diffuse Leishmaniasis Caused by Leishmania (L.) amazonensis

IntroductionDiffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the “anergic” pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry.Case presentationA 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation.ConclusionThe dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient

Lutzomyia longipalpis Salivary Gland Homogenate Impairs Cytokine Production and Costimulatory Molecule Expression on Human Monocytes and Dendritic Cells

In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Mφs), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Mφs, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Mφs, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SGH-specific antibodies abolished the effects of SGH on cytokine production by LPS-stimulated monocytes

New Role of P. brasiliensis α-Glucan: Differentiation of Non-conventional Dendritic Cells

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-12-10T17:06:41Z No. of bitstreams: 1 Souza,A.C.O. New Role.pdf: 3104697 bytes, checksum: 823c2a6d2f52c4147d1c02de861826fb (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-12-10T17:15:42Z (GMT) No. of bitstreams: 1 Souza,A.C.O. New Role.pdf: 3104697 bytes, checksum: 823c2a6d2f52c4147d1c02de861826fb (MD5)Made available in DSpace on 2019-12-10T17:15:42Z (GMT). No. of bitstreams: 1 Souza,A.C.O. New Role.pdf: 3104697 bytes, checksum: 823c2a6d2f52c4147d1c02de861826fb (MD5) Previous issue date: 2019Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), Conselho Nacional de Pesquisa (CNPq), and Decanato de Pesquisa e Pós-Graduação da Universidade de Brasília (DPP/UnB) for financial support, and CAPES for graduate students’ grants.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Universidade de Brasília. Departamento de Biologia Celular. Brasília, DF, Brasil.The cell wall has a critical role in the host immune response to fungal pathogens. In this study, we investigated the influence of two cell wall fractions of the dimorphic fungi Paracoccidioides brasiliensis (Pb) in the in vitro generation of monocyte-derived dendritic cells (MoDCs). Monocytes were purified from the peripheral blood of healthy donors and cultivated for 7 days in medium supplemented with IL-4 and GM-CSF in the presence of Pb cell wall fractions: the alkali-insoluble F1, constituted by β-1,3-glucans, chitin and proteins, and the alkali-soluble F2, mainly constituted by α-glucan. MoDCs phenotypes were evaluated regarding cell surface expression of CD1a, DC-SIGN, HLA-DR, CD80, and CD83 and production of cytokines. The α-glucan-rich cell wall fraction downregulated the differentiation of CD1a+ MoDCs, a dendritic cell subset that stimulate Th1 responses. The presence of both cell fractions inhibited DC-SIGN and HLA-DR expression, while the expression of maturation markers was differentially induced in CD1a- MoDCs. Differentiation upon F1 and F2 stimulation induced mixed profile of inflammatory cytokines. Altogether, these data demonstrate that Pb cell wall fractions differentially induce a dysregulation in DCs differentiation. Moreover, our results suggest that cell wall α-glucan promote the differentiation of CD1a- DCs, potentially favoring Th2 polarization and contributing to pathogen persistence

Analysis of immunoproteasome mRNA levels during <i>T. cruzi</i> infection.

<p>Semi-quantitative RT-PCR analysis of β1i, β2i and β5i expression were done using total RNA from HeLa cells treated with IFN-γ and/or infected with <i>T. cruzi</i>. (A) The PCR products were analyzed by electrophoresis in 1.2% agarose gels stained with ethidium bromide. The reactions were carried as duplex-PCR, using <i>GAPDH</i> as internal control (arrows). (B) mRNA levels were determined by densitometry and plotted using the expression of <i>GAPDH</i> as normalizer. Each value represents the mean ± standard deviation of three individual experiments. The abundance of α1, β1i, β2i, β5i and PA28β mRNAs were also determined by real time RT-qPCR. The relative expression of the transcripts was calculated by normalization with <i>GAPDH</i> and <i>HPRT1</i> housekeeping genes using the 2^−ΔΔCt method. (C) The mRNA levels were plotted relatively to “IFN-γ” experimental condition (HeLa treated 24 h with IFN-γ). Each value represents the mean ± standard deviation of three independent experiments.</p

Analysis of mRNA levels and protein composition of HeLa constitutive proteasome during <i>T. cruzi</i> infection.

<p>Semi-quantitative RT-PCR analysis of α1, α6, β1, β2 and β5 expression were done using total RNA from HeLa cells treated with IFN-γ and/or infected with <i>T. cruzi</i>. (A) The PCR products were analyzed by electrophoresis in 1.2% agarose gels stained with ethidium bromide. The reactions were carried as duplex-PCR, using <i>GAPDH</i> as internal control (arrows). (B) mRNA levels were determined by densitometry and plotted using the expression of <i>GAPDH</i> as normalizer. Each value represents the mean ± standard deviation of three individual experiments. (C) Two-dimensional gels of immunoprecipitated proteasomes from HeLa uninfected and infected with <i>T. cruzi</i>. HeLa cells in standard culture conditions were exposed or not to <i>T. cruzi</i> and cultured for 24 h. In the twenty-first hour of culture, cells were metabolically labeled with [³⁵S]-methionine for three hours. Cell lysates (100 µg) were immunoprecipitated with anti-human proteasome antibodies and analyzed by two-dimensional electrophoresis. Panel D show the protein levels of proteasome α and β subunits quantified by densitometry. Each value represents the mean ± mean deviation of two independent experiments.</p

Analysis of immunoproteasome protein expression during <i>T. cruzi</i> infection.

<p>(A) Lysates (25–50 µg) of HeLa cells treated with IFN-γ and/or infected with <i>T. cruzi</i> were analyzed by western blot using anti-immunoproteasome subunits antibodies as indicated. (B) Protein levels were determined by densitometry and plotted using the expression of α6 subunit as experimental normalizer. Infection was confirmed using anti-tubulin antibody. (C) Western blot analysis of PA28β expression during different times of infection. HeLa cells were pre-treated with IFN-γ for 24 h and then infected with <i>T. cruzi</i> for 24, 48 and 72 h. (D) Protein levels were plotted using the expression of α1 as experimental normalizer. All values in this figure represent mean ± standard deviation of three individual experiments.</p