Hemozoin is a key factor in the induction of malaria-associated immunosuppression

Infection-associated immunoincompetence during malaria might result from macrophage dysfunction. In the present study, we investigated the role of macrophages as target for immunosuppression during infection, using the murine Plasmodium c. chabaudi model. Special attention has been paid to the analysis of processing/presentation of protein antigens and presentation of peptides, using cocultures of peritoneal exudate cells (PECs) from infected mice and antigen-specific T-cell hybridomas. The results obtained indicate a defective processing of protein antigens that becomes maximal at acute parasitemias. In addition, macrophages from acutely infected mice suppress the interleukin-2 production by the antigen-activated T-cell hybridomas. This effect was independent of prostaglandin and nitric oxide production by the macrophage. The possible role of parasite components in the impaired accessory cell function of PECs was investigated and hemozoin, the end-product of the hemoglobin catabolism by intraerythrocytic malaria parasites, was found to induce similar infection-associated deficiencies in vitro. Moreover, hemozoin, was shown to mimic the immunosuppressive effects induced in PECs during in-vivo infections with P. chabaudi. In conclusion, we propose that hemozoin is a key factor in the malaria-associated immunosuppression, affecting both the antigen processing and immunomodulatory functions of macrophages

Impact of in vitro HIV infection on human thymic regulatory T cell differentiation

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment

Impact of Erythropoietin Production by Erythroblastic Island Macrophages on Homeostatic Murine Erythropoiesis

Erythropoietin (EPO) is an essential hormone for erythropoiesis, protecting differentiating erythroblasts against apoptosis. EPO has been largely studied in stress or pathological conditions but its regulatory role in steady state erythropoiesis has been less documented. Herein, we report production of EPO by bone marrow-derived macrophages (BMDM) in vitro, and its further enhancement in BMDM conditioned with media from apoptotic cells. Confocal microscopy confirmed EPO production in erythroblastic island (EBI)-associated macrophages, and analysis of mice depleted of EBI macrophages by clodronate liposomes revealed drops in EPO levels in bone marrow (BM) cell lysates, and decreased percentages of EPO-responsive erythroblasts in the BM. We hypothesize that EBI macrophages are an in-situ source of EPO and sustain basal erythropoiesis in part through its secretion. To study this hypothesis, mice were injected with clodronate liposomes and were supplied with exogenous EPO (1–10 IU/mouse) to evaluate potential rescue of the deficiency in erythroid cells. Our results show that at doses of 5 and 10 IU, EPO significantly rescues BM steady state erythropoiesis in mice deficient of macrophages. We propose existence of a mechanism by which EBI macrophages secrete EPO in response to apoptotic erythroblasts, which is in turn controlled by the numbers of erythroid precursors generated

Preconditioning with Hemin Decreases <em>Plasmodium chabaudi adami</em> Parasitemia and Inhibits Erythropoiesis in BALB/c Mice

<div><p>Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during <em>Plasmodium chabaudi adami</em> infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and T_h1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following <em>P.c.adami</em> infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology.</p> </div

<i>Plasmodium chabaudi adami</i> parasites preferentially infect RBCs that have not been conditioned by HE.

<p>At peak parasitemia (day 7 post-infection), whole blood was fixed in 1% paraformaldehyde overnight, washed, stained and analyse with confocal microscopy. The parasites were labeled with DAPI (blue), and distinction between saline/HE-treated RBCs (strep⁺) and RBCs generated after treatments (strep^-) was made by APC-conjugated streptavidin staining (red): reticulocytes were stained with FITC-labeled anti-CD71 antibody (green). DIC and fluorescence images were captured with Nikon A1 confocal microscope (plan Apo VC 60x, NA 1.4, λs oil immersion), and analysed with NIS-Elements Viewer 4.20 imaging software. Parasitemia within strep⁺ and strep^- RBCs (A; infected strep^+/- RBCs / total strep^+/- RBCs) and proportions of strep⁺ and strep^- RBCs charge with multiple ring (B; multiple infected strep^+/- RBCs / infected strep^+/- RBCs) were evaluated on >150 ring-iRBCs per mouse. DAPI, anti-CD71-FITC, streptavidin-APC fluorescence, DIC and merged channels are shown for a control and a HE-treated mouse (C). Data are the means ± SEM of one experiment (n = 4). All data from Ctrl and HE-treated mice were compared with an unpaired Student <i>t</i> test, **p<0.01, (Fig 5B; Ctrl strep^- vs HE strep^-, p = 0.0593).</p

Red Blood Cells Preconditioned with Hemin Are Less Permissive to <i>Plasmodium</i> Invasion <i>In Vivo</i> and <i>In Vitro</i>

<div><p>Malaria is a parasitic disease that causes severe hemolytic anemia in <i>Plasmodium</i>-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of <i>Plasmodium</i> immunity <i>in vitro</i> and <i>in vivo</i>, mice preconditioned with hemin develop lower parasitemia when challenged with <i>Plasmodium chabaudi adami</i> blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and <i>P</i>. <i>c</i>. <i>adami</i> infection. This strategy allowed discriminating hemin-treated from d<i>e novo</i> generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased <i>P</i>. <i>c</i>. <i>adami</i> red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in <i>in vitro P</i>. <i>falciparum</i> cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during <i>in vivo</i> infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.</p></div

<i>Plasmodium chabaudi adami</i> schizogony and invasion stage.

<p>Six days post-infection, thin blood smears of iRBCs were prepared at the time of schizogony and stained with DAPI (blue). DIC and fluorescence images were captured with Nikon A1 confocal microscope (objective plan Apo VC 60x, NA 1.4, λs oil immersion), and analysed with NIS-Elements Viewer 4.20 imaging software. The proportion of merozoites (nuclei) per mature schizont (containing ≥4 merozoites) was determined in >150 iRBCs per mouse (A). DAPI-stained blood smears were also analysed at the time of merozoite invasion, and the proportion of extra-erythrocytic merozoites was measured in ≥600 merozoites (number of extra-erythrocytic merozoites/total number of merozoites) (B). DIC, DAPI fluorescence and merged images at the time of merozoite invasion are shown for a control and HE-treated mouse (C). The results represent the mean ± SEM of one experiment (Ctrl; n = 4, HE; n = 3) and were compared with an unpaired Student <i>t</i> test, *p<0.05.</p

Markers of oxidative stress and ageing in RBCs from HE-preconditioned mice infected with <i>Plasmodium chabaudi adami</i>.

<p>BALB/c mice received intravenous injection of sulfo-NHS-LC-biotin (1 mg/100μl/mouse) to label RBCs in circulation, one day prior to a first HE treatment. Saline (control) or HE (10 mg/kg/day) were administered by the intraperitoneal (ip) route for 3 days and followed by ip injection of 5x10⁵<i>P</i>. <i>c</i>. adami iRBCs, on the 4^th day. Tail-tip blood smears stained with Giemsa were performed daily to follow the parasitemia (A), determine the magnitude of peak parasitemia (B) and estimate cumulative parasitemia (calculated as the sum of daily parasitemia) (C). Three hours after the 3^rd and last saline/HE injection, the mean volume of RBCs was estimated from FSC values (D), and gmeanFI relative to Annexin-V binding (E), DCFDA labeling (F) and CD47 expression (G) were estimated by flow cytometry on 40 000 RBCs positive for streptavidin. These parameters were also analysed 7 days after <i>P</i>. <i>c</i>. <i>adami</i> infection (H-K). Data in A-C are the means ± SEM of three independent experiments (n = 11), and data in D-K are the means ± SEM of one experiment (Ctrl; n = 4, HE; n = 3). An unpaired Student <i>t</i> test was performed to compare the control group to the HE-treated one, *p<0.05, **p<0.01, *** p<0.001.</p

<i>Plasmodium chabaudi adami</i> selectivity for red blood cells.

<p>At day 6 and 7 post- <i>P</i>. <i>c</i>. <i>adami</i> infection, thin blood smears of parasite ring stages iRBCs were fixed and stained with DAPI (blue). DIC and fluorescence images were captured with Nikon A1 confocal microscope (plan Apo VC 60x, NA 1.4, λs oil immersion), and analysed with NIS-Elements Viewer 4.20 imaging software. Single and multiple-iRBCs were counted in >150 ring-iRBCs per mouse (A, C). Parasite SI was calculated for each day by dividing the observed value of multiple iRBCs by the value expected from a Poisson distribution (B, D). DIC, DAPI fluorescence, and merged channels are shown at day 6 post-infection during ring stage for a control and a HE-treated mouse (E). The results represent the means ± SEM of one experiment (Ctrl; n = 4, HE; n = 3) at day 6 post-infection and two experiments (Ctrl; n = 8, HE; n = 7) at day 7 post-infection. Data were compared with an unpaired Student <i>t</i> test, **p<0.01, ***p<0.001.</p