1. Volume 19, Issue 2, Pages 194-203 (February 2016)
Anti-Self Phosphatidylserine Antibodies Recognize Uninfected Erythrocytes Promoting Malarial Anemia 
Cristina Fernandez-Arias, Juan Rivera-Correa, Julio Gallego-Delgado, Rachel Rudlaff, Clemente Fernandez, Camille Roussel, Anton Götz, Sandra Gonzalez, Akshaya Mohanty, Sanjib Mohanty, Samuel Wassmer, Pierre Buffet, Papa Alioune Ndour, Ana Rodriguez 
Cell Host & Microbe 
Volume 19, Issue 2, Pages (February 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 19, 194-203DOI: (10.1016/j.chom.2016.01.009)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Anti-Self Antibodies Recognize Uninfected RBCs during Malaria
(A) Average of total (black line) and infected (gray line) RBCs per volume of blood in P. yoelii infected mice (n = 5).
(B) Antibodies (IgG) against RBC lysates (black line) and MSP 1 (gray line) were detected in the sera of P. yoelii-infected mice (n = 10) by ELISA.
(C–E) RBCs from control uninfected mice (C) or from P. yoelii-infected mice (day 14 postinfection) separated into uninfected (D) or infected (E) were incubated with affinity-purified anti-RBC antibodies (red line), isotype-matched irrelevant antibodies (blue line), or buffer alone (gray line), followed by secondary fluorescent antibodies. As control, we used RBCs from uninfected mice (filled gray plot) incubated with secondary antibodies. Insets show the average of mean fluorescence intensity for each condition from three independent mice. One representative experiment of three is shown. Average SD is shown in each panel. ∗p < See also Figure S1.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2
Anti-Self Antibodies Anti-RBCs Mediate Phagocytosis of Uninfected RBC and Contribute to Anemia in Malaria
(A) RBCs from control uninfected mice (control), uninfected RBCs (uRBC), and infected RBCs (iRBC) from P. yoelii-infected mice (day 14 postinfection) were labeled with DDAO and preincubated with buffer alone (control), isotype-matched irrelevant antibodies (IgG), or affinity-purified anti-RBC antibodies (anti-RBC). Phagocytosis was measured after incubation with splenocytes from uninfected control mice. DDAO fluorescence of F4/80+CD11b+ cells is shown. Lower panels show the average of mean fluorescence intensity for each condition from three independent mice. One representative experiment of two is shown.
(B) P. yoelii-infected or control groups of mice (n = 5) were injected with one dose of purified anti-RBC antibodies or isotype-matched irrelevant immunoglobulins 1 day after infection. Number of RBCs per volume (cells × 106/μl; gray bars), hemoglobin concentration (g/dl; black bars), and hematocrit (percentage; white bars) was measured at day 7 postinfection. All three parameters were found to be significantly lower in P. yoelii-infected mice injected with anti-RBC antibodies when compared to infected mice injected with isotype control antibodies (p < 0.01). Average of parasitemia of mice groups at day 7 postinfection is shown in the right panel. One representative experiment of three is shown.
(C) P. yoelii-infected groups of mice (n = 5) were injected with one dose of purified anti-RBC antibodies (black) or isotype-matched irrelevant antibodies (gray) on day 11 postinfection. Number of RBCs per volume (cells × 106/μl) was measured at the indicated times and expressed as the ratio to the value at day 11. One representative experiment of two is shown.
(D) Parasitemia of mice groups described in (c). Average ± SD is shown in each panel. ∗p < 0.05; ∗∗p < See also Figure S2.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 3
Anti-Self Antibodies Recognize PS on Uninfected RBCs from P. yoelii-Infected Mice and Mediate Their Phagocytosis
(A) Surface expression of PS in RBCs in P. yoelii-infected (black line) or uninfected (gray line) groups of mice (n = 5). Data are expressed as percentage of RBCs positive for annexin V staining. One representative experiment of three is shown. Insets show uninfected (DAPI negative, top) and infected (DAPI positive, bottom) annexin V-stained RBCs from infected mice.
(B and C) Antibodies against PS, IgM (B) and IgG (C) were detected in the sera of P. yoelii-infected mice (n = 3 for each time point) by ELISA.
(D) The percentage of antibody secreting cells (ASCs) for each antigen from the total ASCs in spleen was calculated by ELISPOT in plates coated with control (BSA), RBC lysates, iRBC lysates, or PS, in control uninfected (gray bars) and infected (black bars) mice. One representative experiment of three is shown.
(E) Uninfected RBCs from groups of mice (n = 6) control (gray bars) or P. yoelii-infected (day 14 postinfection; black bars) were incubated independently with affinity purified anti-PS antibodies from other infected mice (anti-PS) or isotype-matched irrelevant antibodies (IgG) followed by secondary fluorescent antibodies. RBCs were also preincubated with annexin V followed or not by anti-PS antibodies incubation. One representative experiment of two is shown.
(F) Uninfected RBCs from control (gray bars) or P. yoelii-infected mice (n = 3 mice; day 14 postinfection; black bars) were labeled with DDAO and preincubated with isotype-matched irrelevant antibodies (IgG), anti-RBC (anti-RBC) or anti-PS (anti-PS) antibodies. RBCs were also preincubated with annexin V followed or not by anti-PS or anti-RBC antibodies as indicated. Phagocytosis was measured after incubation with splenocytes from control uninfected mice, as percentage of DDAO-positive F4/80+CD11b+ cells. Average ± SD is shown in each panel. ∗p < 0.05, ∗∗p < One representative experiment of two is shown. See also Figures S3 and S4.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4
Uninfected RBCs Exposing PS during Infection Are Reticulocytes that Express High Levels of the “Do-Not-Eat-Me Signal” CD47
Expression of different markers were analyzed in RBCs of P. yoelii-infected mice (n = 3).
(A) Average of percentage RBCs showing high levels of: surface PS (white squares), surface CD47 (white circles), thiazole orange labeling (reticulocytes, black circles). Parasitemia (dashed line) and RBC density as percentage of day 0 (dash-dot line).
(B and D) Analysis of surface expression of PS (annexin V), CD47, and thiazole orange (RNA) labeling (at day 15 postinfection) in RBCs from control uninfected mice (left panels) and uninfected RBCs from infected mice (right panels). Gating strategy in Figure S5.
(C and E) Quantification of RBCs double positive for surface PS and CD47 (C) or surface PS and thiazole orange (E). Average ± SD is shown in each panel. One representative experiment of two is shown. See also Figure S5.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 5
Anti-Self PS Antibodies Correlate with Late Postmalarial Anemia in Humans
(A and B) Hemoglobin (Hb) and anti-PS antibody levels in the day of treatment (day 0) for 23 falciparum patients in Rourkela, India (A), and 35 patients in France (B). Lines show no correlation between variables (R2 > 0.03).
(C and D) Hemoglobin (black) and anti-PS antibody (white) levels in patients with late (n = 8) (C) or early (n = 12) (D) postmalarial anemia. Significant differences for each point with its day 0 value are indicated (∗p < 0.05, ∗∗p < 0.01).
(E) Anemia progression (hemoglobin on day 21/hemoglobin on day 3) and anti-PS antibody levels three weeks after treatment for falciparum patients in France (n = 11). Line shows strong inverse correlation between variables (R2 = 0.44). Average ± SD is shown in each panel. ∗p < 0.05; ∗∗p < 0.01.
Cell Host & Microbe  , DOI: ( /j.chom )
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8. Figure 6 Anti-Self PS Antibodies Contribute to Anemia in Mouse Models
(A) P. yoelii-infected groups of mice (n = 5) were injected with one dose of purified anti-PS antibodies (gray line) or isotype-matched irrelevant antibodies (black line) on day 11 postinfection. Number of RBCs per volume was measured over time and expressed as the average of the ratio of variation for each mice (value on each day versus value at the day of injection (day 11).
(B) Average of parasitemia of mice groups described in (A).
(C) P. yoelii-infected groups of mice (n = 5) were injected with annexin V (gray line) or vehicle alone (black line) at days 6 and 10 postinfection. Number of RBCs per volume was measured in the indicated days and expressed as the average of the ratio for each mice of the value on the day of injection (day 11) versus the value on each day.
(D) Average of parasitemia of mice groups described in (C). Average ± SD is shown in each panel. ∗p < 0.05; ∗∗p < One representative experiment of two is shown.
Cell Host & Microbe  , DOI: ( /j.chom )
Copyright © 2016 Elsevier Inc. Terms and Conditions