Increased Expression of Complement Regulators CD55 and CD59 on Peripheral Blood Cells in Patients with EAHEC O104:H4 Infection

<div><p>Background</p><p>An outbreak of Shiga Toxin 2 (Stx-2) producing enterohemorrhagic and enteroaggregative <i>E.coli</i> (EAHEC) O104H4 infection in May 2011 caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). The monoclonal anti-C5 antibody Eculizumab (ECU) has been used experimentally in EAHEC patients with HUS but treatment efficacy is uncertain. ECU can effectively prevent hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) caused by a lack of complement-regulating CD55 and CD59 on blood cells. We hypothesized a low expression of CD55 and CD59, as seen in PNH, might correlate with HUS development in EAHEC patients.</p><p>Methods</p><p>76 EAHEC patients (34 only gastrointestinal symptoms [GI], 23: HUS, 19: HUS and neurological symptoms [HUS/N]) and 12 healthy controls (HC) were tested for the expression of CD55 and CD59 on erythrocytes and leukocytes retrospectively. Additionally, the effect of Stx-2 on CD55 and CD59 expression on erythrocytes and leukocytes was studied <i>ex vivo</i>.</p><p>Results</p><p>CD55 expression on erythrocytes was similar in all patient groups and HC while CD59 showed a significantly higher expression in HUS and HUS/N patients compared to HC and the GI group. CD55 and CD59 expression on leukocytes and their subsets was significantly higher in all patient groups compared to HC regardless of treatment type. However, CD59 expression on erythrocytes was significantly higher in HUS and HUS/N patients treated combined with plasma separation (PS) and ECU compared to HC. Adding Stx-2 <i>ex vivo</i> had no effect on CD55 and CD59 expression on leukocytes from HC or patients.</p><p>Conclusion</p><p>HUS evolved independently from CD55 and CD59 expression on peripheral blood cells in EAHEC O104:H4 infected patients. Our data do not support a role for CD55 and CD59 in HUS development during EAHEC O104:H4 infection and point to a different mechanism within the complement system for HUS development in EAHEC patients.</p></div

Blood parameters for hemoglobin, thrombocytes, urea and creatinine of all patients included in the study.

a<p>The data are shown as means ± standard deviations.</p>b<p>HC = healthy controls, GI = gastrointestinal symptoms, HUS = hemolytic uremic syndrome, HUS/N = HUS and neurological symptoms reference levels: hemoglobin: 14–17.5 g/dL, thrombocytes: 150–400 10⁹/L, urea: 8–26 mg/dL, creatinine: 0.6–1.3 mg/dL.</p

CD55 and CD59 expression are increased on erythrocytes and leukocytes of former EAHEC-infected patients.

<p>Erythrocytes and leukocytes were incubated with CD45-, CD55- and CD59-specific antibodies and analyzed via flow cytometry. The CD45 marker was used to exclude or include the leukocyte population whereas the leukocyte subsets were distinguished via FSC/SSC-plot. Patients were grouped according to their clinical course into 3 groups with either severe gastrointestinal symptoms (GI, n = 34), HUS without neurological symptoms (HUS, n = 23) and HUS with neurological symptoms only (HUS/N, n = 19). Healthy controls were also screened (HC, n = 12). A Erythrocytes, B Leukocytes, C Granulocytes, D Monocytes, E Lymphocytes. All values are given as mean fluorescence intensity (MFI) ± S.D. ANOVA, following symbols are used to pinpoint significant differences: vs. HC *, vs. GI #. One symbol equals 0.05, two symbols 0.01, three symbols 0.001.</p

Pearson-Bravais correlation tests between CD55 and CD59 expression and hemoglobin, thrombocytes, urea and creatinine levels of all patients included in the study.

a<p>HC = healthy controls, GI = gastrointestinal symptoms, HUS = hemolytic uremic syndrome, HUS/N = HUS and neurological symptoms.</p

Stx-2 does not reduce the expression of CD55 and CD59 on erythrocytes and leukocytes.

<p>After incubating erythrocytes and leukocytes in whole blood with 0, 0.1 and 10/ml Shiga toxin 2 for 24 h the expression levels for CD55 and CD59 were analyzed via flow cytometry. Patients were grouped according to their clinical course into 3 groups with GI (n = 4), HUS (n = 4) and HUS/N (n = 4). Healthy controls were also screened (HC, n = 4) A CD55 expression, B CD59 expression on erythrocytes, C CD55 expression, D CD59 expression on leukocytes. All values are given as mean fluorescence intensity (MFI) ± S.D.</p

Plasma separation and ECU do not affect CD55 and CD59 expression on erythrocytes and leukocytes.

<p>Erythrocytes and leukocytes were incubated with CD45-, CD55- and CD59-specific antibodies and analyzed via flow cytometry. The CD45 marker was used to exclude or include the leukocyte population whereas the leukocyte subsets were distinguished via FSC/SSC-plot. HUS patients were grouped according to their therapy into 4 groups with HUS patients who received plasma separation (n = 9), HUS patients who received plasma separation and ECU (n = 14), HUS/N patients who received plasma separation (n = 3) and HUS/N patients who received plasma separation and ECU (n = 16). Healthy controls were also screened (HC, n = 12). A Erythrocytes, B Leukocytes. All values are given as mean fluorescence intensity (MFI) ± S.D. ANOVA, following symbols are used to pinpoint significant differences: vs. HC *. One symbols equals 0.05, two symbols 0.01, three symbols 0.001.</p

Characteristics of all subjects included in the study.

a<p>The data are shown as means ± standard deviations.</p>b<p>The groups HC and GI were neither treated with plasma seperation or eculizumab.</p>c<p>HC = healthy controls, GI = gastrointestinal symptoms, HUS = hemolytic uremic syndrome, HUS/N = HUS and neurological symptoms.</p

Baseline characteristics.

<p>Baseline characteristics.</p

Distribution of ALT values according to Anti-HDV IgM categories in the HIDIT-2 cohort.

<p>ALT values were significantly associated with the different anti-HDV IgM groups based on univariate ANOVA (p = 0.04) and univariate T- test in the HIDIT-2 cohort.</p

Distribution of ALT values according to Anti-HDV IgM categories in the MHH cohort (only significant p-values).

<p>The association of anti-HDV IgM and ALT was confirmed in the MHH cohort with significance over all groups of 0.03.</p