Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis-relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated β-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β-catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach

Chronic HIV infection enhances the responsiveness of antigen presenting cells to commensal Lactobacillus.

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation

Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal <i>Lactobacillus</i>

<div><p>Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to <i>Lactobacillus plantarum</i> WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.</p></div

Increased expression of pattern recognition receptors on APCs from HIV-infected patients.

<p>(<b>A</b>) Heatmap and (<b>B</b>) fold changes of PRR gene expression in isolated CD11c+ APCs from HIV-infected patients were compared to HIV-negative controls (HIV− n = 4, HIV+ n = 4) using DNA microarray analysis. P values determined by unpaired t test (*P<0.05, **P<0.01, ***P<0.001). (<b>C</b>) Representative flow cytometry histograms of TLR2 expression of APCs from HIV-negative controls (black) and HIV+ patients (gray). (<b>D</b>) Median fluorescence intensity of TLR2 expression of APCs from HIV-negative controls (n = 20) and HIV-infected patients (n = 46). (<b>E</b>) Representative flow cytometry histograms of CD36 expression of APCs from HIV-negative controls (black) and HIV+ patients (gray). (<b>F</b>) Median fluorescence intensity of CD36 expression of APCs from HIV-negative controls (n = 20) and HIV-infected patients (n = 46). (<b>G</b>) Frequencies of APCs from HIV-infected patients (n = 7) producing IL-6, IL-12/IL23p40, and TNFα in response to <i>L. plantarum</i> WCFS1 with or without TLR2 blocking antibody. (<b>H</b>) Concentrations of IL-6, IL-12/IL-23p40, and TNFα following stimulation with <i>L. plantarum</i> WCFS1 with or without TLR2 blocking antibody as determined by ELISA (HIV+ n = 3–6). Each dot represents an individual subject. In the HIV+ group, open circles represent therapy-naïve patients, closed circles represent patients on HAART. Bars indicate median value. Bar graphs represent mean +/− SEM. P values determined using Mann Whitney U test or paired t test.</p

Enhanced APC inflammatory response to commensal lactobacilli signals predominantly through p38-MAPK.

<p>(<b>A</b>) Representative flow cytometry plot and frequencies of APCs from HIV-infected patients producing proinflammatory cytokines IL-6, IL-12/IL-23p40, and TNFα in response to (<b>B</b>) <i>L. plantarum</i> WCFS1 (n = 16), (<b>C</b>) <i>L. gasseri</i> 1SL4 (n = 12), and (<b>D</b>) <i>L. casei</i> BL23 (n = 11) with or without BIRB796 pretreatment. (<b>E</b>) Relative expression of MKP-1 in unstimulated and bacterial stimulated APCs as determined by real-time PCR (HIV− n = 3–9, HIV+ n = 5–16). Bar graphs represent mean +/− SEM. P values determined using paired t or Mann Whitney U test (*P<0.05, **P<0.01, ***P<0.001).</p

Patient cohorts and characteristics.

<p>Age, T cell counts, viral loads, years diagnosed and years on HAART are expressed as mean values with the range indicated in parentheses. CD4+ T cell counts and plasma viral load are not applicable (N/A) for HIV-negative controls. Plasma viral load was under 50 HIV RNA copies/mL plasma in HIV-infected, HAART-treated group. (PI = protease inhibitor, NNRTI = non-nucleoside reverse transcriptase inhibitor, combination = PI+NNRTI). P values determined using ANOVA followed by Bonferroni’s Multiple Comparisons test (**P<0.01, ****P<0.001 compared to HIV-negative controls.</p>†<p>P<0.001 compared to HIV, therapy naïve.).</p

Distribution of antigen presenting cells in peripheral blood during chronic HIV infection.

<p>(<b>A</b>) Multicolor flow cytometric gating strategy for the identification of APC populations in peripheral blood. APCs (CD3-CD19-CD56-HLADR+CD11c+CD123-), monocytes (CD3-CD19-CD56-HLADR+CD123-CD11c+CD14+), and mDCs (CD3-CD19-CD56-HLADR+CD123-CD11c+CD14+) were detected following the exclusion of dead cells and CD3+, CD19+ and CD56+ cells. The HLA-DR-positive cells were gated for CD11c and CD123. Data were analyzed using FlowJo. (<b>B</b>) Percentages of circulating APCs (HIV− n = 37, HIV+ n = 94). (<b>C</b>) Monocyte percentage of the peripheral blood APC population. (<b>D</b>) Myeloid dendritic cell population of the peripheral blood APC population. (<b>E</b>) Plasma sCD14 concentrations as determined by ELISA (HIV− n = 25; HIV+ n = 66). Each dot represents an individual patient. In the HIV+ group, open circles represent therapy-naïve patients and closed circles represent patients on HAART. Bars indicate median value. P values determined using Mann Whitney U test. P values as indicated.</p

Enhanced inflammatory response by APCs from HIV-infected patients to commensal lactobacilli.

<p>(<b>A</b>) Representative flow cytometry plots of APCs producing proinflammatory cytokines in response to <i>L. plantarum</i> WCFS1. (<b>B</b>) Frequencies of APCs from HIV-negative controls (n = 29) and HIV-infected patients (n = 62) producing IL-6, IL-12/IL-23p40, and TNFα in response to <i>L. plantarum</i> WCFS1 determined by multicolor flow cytometry. (<b>C</b>) Concentrations of IL-6, IL-12/IL-23p40, and TNFα following stimulation with <i>L. plantarum</i> WCFS1 as determined by ELISA (HIV− n = 4; HIV+ n = 10). Frequencies of APCs from HIV-negative controls (n = 14) and HIV-infected patients (n = 23) producing IL-6, IL-12/IL-23p40, and TNFα in response to (<b>D</b>) <i>L. gasseri</i> 1SL4 and (<b>E</b>) <i>L. casei</i> BL23 as measured by multicolor flow cytometry. Each dot represents an individual subject. In the HIV+ group, open circles represent therapy-naïve patients, closed circles represent patients on HAART. Bars indicate median value. Bar graphs represent mean +/− SEM. P values determined using Mann Whitney U test, P values as indicated.</p