PONE-D-16-46185_R2 cumulative data

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Reduced internalization of TNF-ɑ/TNFR1 down-regulates caspase dependent phagocytosis induced cell death (PICD) in neonatal monocytes

<div><p>Phagocytosis-induced cell death (PICD) is diminished in cord blood monocytes (CBMO) as compared to cells from adults (PBMO) due to differences in the CD95-pathway. This may support a prolonged pro-inflammatory response with sequels of sustained inflammation as seen in neonatal sepsis. Here we hypothesized that TNF-α mediated induction of apoptosis is impaired in CBMO due to differences in the TNFR1-dependent internalization. Monocytes were infected with <i>Escherichia coli-</i>GFP (<i>E</i>. <i>coli-</i>GFP). Monocyte phenotype, phagocytic activity, induction of apoptosis, and TNF-α/TNF-receptor (TNFR) -expression were analysed. In the course of infection TNF-α-secretion of CBMO was reduced to 40% as compared to PBMO (p<0.05). Neutralization of TNF-α by an αTNF-α antibody reduced apoptotic PICD in PBMO four-fold (p < 0.05 vs. infection with <i>E</i>. <i>coli</i>). PICD in CBMO was reduced 5-fold compared to PBMO and showed less responsiveness to αTNF-α antibody. CBMO expressed less pro-apoptotic TNFR1, which, after administration of TNF-α or infection with <i>E</i>. <i>coli</i> was internalized to a lesser extent. With similar phagocytic capacity, reduced TNFR1 internalization in CBMO was accompanied by lower activation of caspase-8 (p < 0.05 vs. PBMO). Stronger caspase-8 activation in PBMO caused more activation of effector caspase-3 and apoptosis (all p < 0.05 vs. PBMO). Our results demonstrate that TNFR1 internalization is critical in mediating PICD in monocytes after infection with <i>E</i>.<i>coli</i> and is reduced in CBMO.</p></div

Internalization of TNFR1 induces caspase-8 and -3 cleavage after <i>E</i>.<i>coli</i> infection.

<p>Assessment of TNFR1 internalization in monocytes 2 hrs p.i. (A; n = 11, left panel) and proteolytic cleavage of caspases-8 in monocytes with internalized TNFR1 (A; n = 3,panel with dotted bars;*p < 0.05, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, one- and two-way ANOVA). Monocytes which exhibited surface retained TNFR1 (A, panel to the right, hatched bars) were also tested for cleaved caspase-8 (second right panel, both, *p < 0.05, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, one- and two-way ANOVA).The percentage of monocytes expressing cleaved caspase-8 is given (B). Pre-treatment with either zVAD or αTNF-α antibody as described (n = 10, *p < 0.05, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, two-way ANOVA, brunched clamped bars, one-way ANOVA). The cleavage of RIP was monitored by calculating the quotient of RIP/RIPc after quantification of RIP and RIPc signals. If RIPc outweighs RIP the quotient is less than 1 which is the value found in non-treated probes (C; n = 3; *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test, blunt-ended bars, two-way ANOVA).</p

TNFR1 dependent internalization of TNF-α causes apoptosis.

<p>Assessment of internalized TNFR1 in PBMO and CBMO after indicated intervals (A, n = 8; *p < 0.05, ***p < 0.005, clamped bars, student`s t-test; ***p < 0.005, blunt-ended bars, two-way ANOVA). Detection of TNF-α on the plasma-membrane of monocytes (B, n = 3; p < 0.05; clamped bars, student`s t-test; blunt-ended bars, two-way ANOVA). Apoptosis induced by addition of TNF-α was measured in monocytes exhibiting internalized TNFR1 (C, n = 7, *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, two-way ANOVA).</p

TNFR1 expression after infection is functional in induction of monocytic apoptosis.

<p>TNFR1 expression on the plasma-membrane of PBMO and CBMO before and after infection depicted as mean expression of TNFR1 on all monocytes (A, n = 6; *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, two-way ANOVA). To groups indicated, the metalloprotease inhibitor GM6001 was added. TNF-α secretion is diminished by GM6001 (B, n = 3; *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test). Detection of apoptotic PBMO and CBMO after infection and/or addition of GM6001 and TNF-α (C, n = 8, *p < 0.05, **p < 0.01, clamped bars, student`s t-test *within bars, p < 0.005, two-way ANOVA).</p