Role of Aryl Hydrocarbon Receptor (AhR) in the Regulation of Immunity and Immunopathology During Trypanosoma cruzi Infection

Resistance to Trypanosoma cruzi infection is dependent on a rapid induction of Th1-type and CD8+ T cell responses that should be promptly balanced to prevent immunopathology. T. cruzi-infected B6 mice are able to control parasite replication but show a limited expansion of Foxp3+regulatory T (Treg) cells that results in the accumulation of effector immune cells and the development of acute liver pathology. AhR is a ligand-activated transcription factor that promotes Treg cell development and suppression of pro-inflammatory cytokine production in dendritic cells, altering the course of adaptive immune response and the development of immunopathology. Here, we used different AhR-dependent activation strategies aiming to improve the Treg response, and B6 congenic mice carrying a mutant AhR variant with low affinity for its ligands (AhRd) to evaluate the role of AhR activation by natural ligands during experimental T. cruzi infection. The outcome of TCDD or 3-HK plus ITE treatments indicated that strong or weak AhR activation before or during T. cruzi infection was effective to regulate inflammation improving the Treg cell response and regularizing the ratio between CD4+ CD25- to Treg cells. However, AhR activation shifted the host-parasite balance to the parasite replication. Weak AhR activation resulted in Treg promotion while strong activation differentially modulated the susceptibility and resistance of cell death in activated T and Treg cells and the increase in TGF-β-producing Treg cells. Of note, T. cruzi-infected AhRd mice showed low levels of Treg cells associated with strong Th1-type response, low parasite burden and absence of liver pathology. These mice developed a Treg- and Tr1-independent mechanism of Th1 constriction showing increased levels of systemic IL-10 and IL-10-secreting CD4+ splenocytes. In addition, AhR activation induced by exogenous ligands had negative effects on the development of memory CD8+ T cell subsets while the lack/very weak activation in AhRd mice showed opposite results, suggesting that AhR ligation restricts the differentiation of memory CD8+T cell subsets. We propose a model in which a threshold of AhR activation exists and may explain how activation or inhibition of AhR-derived signals by infection/inflammation-induced ligands, therapeutic interventions or exposure to pollutants can modulate infections/diseases outcomes or vaccination efficacy

Dynamics of soluble immune mediators in COVID-19 patients from an Argentinean cohort with moderate and severe symptoms

The cytokine storm, a form of systemic inflammatory response syndrome, is one of the most dreadful complications that can occur during COVID-19. The severity of infection is associated at different levels of these immune mediators and many molecules are considered marker of COVID mortality. Because of its central role in the pathogenesis of SARS-CoV-2 infection, the cytokine storm have become a therapeutic target in the treatment of COVID-19 patients.In this work, we aimed at studying the concentration of different pro- and anti-inflammatory cytokines in a cohort of COVID-19 patients from Córdoba (Argentine). The immunological reaction triggered by infection with SARS-CoV-2 mobilizes numerous cytokines, mainly of proinflammatory character. Changes in their levels are associated with the presence of the disease and with a more severe prognosis. Although our data have similarities with those in international reports, the complete profiling of different parameters (cytokine/chemokines, risk factors, epidemiological and clinical characteristics) in the local cases add value by identifying particularities that may be relevant for the management and prognosis during SARS-CoV2 infection in Argentine.Fil: Almada, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Angiolin, Sofia C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Dho, Nicolás. Universidad Nacional de Córdoba; ArgentinaFil: Dutto, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gazzon, Yamila. Universidad Nacional de Córdoba; ArgentinaFil: Manzone, Clarisa. Universidad Nacional de Córdoba; ArgentinaFil: Marin, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ponce, Nicolás Eric. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Iribarren, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Cerban, Fabio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Morón, Gabriel. Universidad Nacional de Córdoba; ArgentinaFil: Amezcua Vesely, Carolina. Universidad Nacional de Córdoba; ArgentinaFil: Ana, Yamile. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Chiapello, Laura Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fozzatt, Laura. Universidad Nacional de Córdoba; ArgentinaFil: Icely, Paula Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Maccioni, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Mena, Cristian Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Montes, Carolina Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Motrán, Cristina. Universidad Nacional de Córdoba; ArgentinaFil: Rodríguez Galán, Cecilia. Universidad Nacional de Córdoba; ArgentinaFil: Stempin, Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Viano, María Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bertone, M.. Hospital Privado Universitario de Córdoba; ArgentinaFil: Abiega, Claudio Daniel. Hospital Privado Universitario de Córdoba; ArgentinaFil: Escudero, Daiana Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Privado Universitario de Córdoba; ArgentinaFil: Kahn, Adrian Mario. Hospital Privado Universitario de Córdoba; ArgentinaFil: Caeiro, Juan Pablo. Hospital Privado Universitario de Córdoba; ArgentinaFil: Arroyo, Daniela Soledad. Hospital Privado Universitario de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maletto, Belkys Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Acosta Rodriguez, Eva Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gruppi, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Sotomayor, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaLXVI reunión anual de la sociedad argentina de investigación clínica (saic), LXIX reunión anual de la sociedad argentina de inmunología (sai), LIII reunión anual de la asociación argentina de farmacología experimental (aafe), XI reunión anual de la asociación argentina de nanomedicinas (nanomed-ar)Buenos AiresArgentinaSociedad Argentina de Inmunologí

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

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against <i>Fasciola hepatica</i> Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.Facultad de Ciencias Veterinaria

Helminth Antigens Enable CpG-Activated Dendritic Cells to Inhibit the Symptoms of Collagen-induced Arthritis through Foxp3+ Regulatory T Cells

<div><p>Dendritic cells (DC) have the potential to control the outcome of autoimmunity by modulating the immune response. In this study, we tested the ability of <em>Fasciola hepatica</em> total extract (TE) to induce tolerogenic properties in CpG-ODN (CpG) maturated DC, to then evaluate the therapeutic potential of these cells to diminish the inflammatory response in collagen induced arthritis (CIA). DBA/1J mice were injected with TE plus CpG treated DC (T/C-DC) pulsed with bovine collagen II (CII) between two immunizations with CII and clinical scores CIA were determined. The levels of CII-specific IgG2 and IgG1 in sera, the histological analyses in the joints, the cytokine profile in the draining lymph node (DLN) cells and in the joints, and the number, and functionality of CD4+CD25+Foxp3+ T cells (Treg) were evaluated. Vaccination of mice with CII pulsed T/C-DC diminished the severity and incidence of CIA symptoms and the production of the inflammatory cytokine, while induced the production of anti-inflammatory cytokines. The therapeutic effect was mediated by Treg cells, since the adoptive transfer of CD4+CD25+ T cells, inhibited the inflammatory symptoms in CIA. The <em>in vitro</em> blockage of TGF-β in cultures of DLN cells plus CII pulsed T/C-DC inhibited the expansion of Treg cells. Vaccination with CII pulsed T/C-DC seems to be a very efficient approach to diminish exacerbated immune response in CIA, by inducing the development of Treg cells, and it is therefore an interesting candidate for a cell-based therapy for rheumatoid arthritis (RA).</p> </div

Depletion of CD25+ cells abrogated the ability of CII pulsed T/C-DC to inhibit the symptoms of CIA in mice.

<p>A) Mice were injected i.p. with four injections of 500 µg/mouse/day of anti-CD25 Ab (clone PC61) or the same amount of isotype control Ab (clone GL113), as shown in the scheme. B) Depletion of CD4+CD25+ T cells was checked in DLN by FACS with anti-CD4 and anti-CD25 Abs (clone 7D4). A representative staining pattern is shown. C) Mice were injected with PBS; CII pulsed T/C-DC, CII pulsed T/C-DC plus the treatment with anti-CD25 Ab or CII pulsed T/C-DC plus the treatment with anti-isotype control Ab. The arthritis score and incidence were assessed by clinical scoring during 15 days of onset. Values in arthritis score are the mean ± SEM of 7–10 mice per group and the incidence shows data compiled from 3 independent experiments, *<i>P</i><0.05 for T/C-DC versus T/C-DC-PC61 on days 3, 7 and 15 of onset.</p

Decreased severity of CIA following adoptive transfer of CD4+CD25+ T cells induced by the CII pulsed T/C-DC treatment.

<p>A) Scheme representing the experimental protocol followed to induce and transfer CD4+CD25+ T cells, which is described in material and methods. B) Mice were injected with PBS, CII pulsed T/C-DC (5–8×10⁵/mouse), CD4+CD25-, CD4+CD25+ T cells, (3×10⁵/mouse), and the arthritis severity and incidence were assessed by clinical scoring during 15 days of onset. Values in arthritis score are the mean ± SEM of 5 mice per group, *<i>P</i><0.05 for CD4+CD25+ versus PBS on days 6, 9, 12 and 15. C) Histopathological examination of representative joints of PBS, CD4+CD25−, CD4+CD25+ T cells or CII pulsed T/C-DC recipient mice on day 15 of onset, original magnifications 10× (small squares) and 40× (big squares). The pictures show: inflammatory cell infiltrates (i), bone erosion (b), cartilage erosion (c), joint space (js). D) Graphs representing the measurement of the mean length of the articular cartilage and inflammation area from 5 points in each sample. Values are the mean ± SD of 5 mice per group. **<i>P</i><0.005 versus PBS on day 15 of onset.</p

Enrichment of CD4+CD25+Foxp3+ in DLN cells by CII pulsed T/C-DC treatment.

<p>A) Cells from DLN of differentially-treated DC recipient mice were collected on day 7 of onset and stained with fluorescent antibodies for CD4, CD25 and Foxp3. Plots show the percentage of CD25+Foxp3+ cells gated on CD4+ cells. B) Cells from DLN of CII immunized mice cultured with T/C-DC plus CII for 5 days in the presence of neutralizing antibodies against TGF-β or 1-MT. Cells were gated on CD4+ cells, and bars represent the percentage of CD25+Foxp3+ cells. #<i>P</i><0.05 versus DLN+DC, *<i>P</i><0.05 versus DLN+T/C-DC. C) DLN cells from CII immunized mice were cultured with CII pulsed differentially treated DC in the presence of anti TGF-β and IL-10 production was analyzed by ELISA. **<i>P</i><0.005 versus DLN + T/C-DC αTGF-β. Data are representative of two independent experiments.</p

Cytokine production, phenotype and IDO expression by differentially-maturated DC.

<p>A) The DC were generated from DBA/1J mice and cultured for 18 h with LPS, Zymosan or CpG in presence or absence of TE. IL-10 and TNF were detected by ELISA. *<i>P</i><0.05, **<i>P</i><0.005 B) DC were cultured for 18 h with medium, TE, CpG and T/C. Supernatants were tested for IL-12p70, TNF, IL-6, IL-10, IL-23, TGF-β by ELISA. *<i>P</i><0.05, **<i>P</i><0.005 in IL-12p70, TNF, IL-6 and IL-23 versus CpG group, and *<i>P</i><0.05, **<i>P</i><0.005 in IL-10 and TGF-β versus the rest of the groups. C) DC were stimulated as described above and the expression of MHCII and costimulatory molecules (thick lines) was performed by FACS gating the cells on the basis of CD11c⁺ cells. Thin lines show isotype control and values represent the mean fluorescent intensity (MFI). D) Western blot analysis for IDO detection in whole lysates from DC treated as decribed above, using anti-β-actin Ab as the loading control. Data obtained were analyzed by scanning densitometry and were normalized according to the ratio IDO/β-actin expression and these values were compared to medium treated-DC (left panels). L-kynurenine concentration (IDO activity), was determined by a colorimetric assay in lysates or supernatants (right panels) respectively from DC treated as described above. Data shown are representative of two (D) or three (A, B and C) independent experiments. Bars in A, B and D are the means ± SD of triplicate wells per group.</p