Verminderter phagozytose-induzierter Zelltod neonataler Monozyten im In-vitro-Sepsismodell mit Candida albicans

BACKGROUND: Besides bacteria candida albicans is a frequently found pathogen in neonatal sepsis. Phagocytosis of bacteria induces apoptosis in monocytes (phagocytosis-induced cell death, PICD). Although adult (peripheral blood monocytes, PBMO) and neonatal monocytes (cord blood monocytes, CBMO) phagocyte the same amount of e. coli and streptococcus species, adult monocytes (PBMO) show less apoptosis (PICD). HYPOTHESIS: Phagocytosis of candida albicans induces PICD in monocytes, as well. In comparison with bacterial infections CBMO show likewise less apoptosis (PICD). PBMO and CBMO differ in TLR2- and TLR4-modulation, which are important to pathogen recognition. METHODS: Monocytes were isolated, phenotyped and infected by GFP-expressing candida albicans. Kinetics of phagocytosis and apoptosis were determined by measurement of hypodiploide DNA (Nicoletti). Expression of TLR2, TLR4, CD86, CD14 were detected and quantified by antibody staining. Positive controls were performed (TLR2: 5µg/ml Pam3Cys; TLR4: 10ng/ml LPS).FINDINGS: PBMO and CBMO showed identical phagocytosis (70,46 ± 5,03% and 66,27 ± 7,88%). Two hours post infection CBMO showed a significant lower PICD compared to PBMO (13,91 ± 3,53% vs. 6,5 ± 1,71%; p < 0,05). Post infection both populations showed an identical up-modulation of CD86. TLR2 was significantly higher expressed on PBMO after stimulation with c. albicans (18,42 ± 8.45% vs. 7,71 ± 3,16%; p < 0,005). Before stimulation the expression on PBMO and CBMO had been the same (1,13 ± 0,28 % and 0,93 ± 0,35 %). Positive controls with Pam3Cys showed identical up-modulation on CBMO and PBMO (37,59 ± 18,72% bzw. 34,15 ± 11,86%). TLR4 was down modulated the same way on PBMO and CBMO after stimulation with c. albicans (46,68 ± 17,63% bzw. 57,26 ± 15,45%).CONCLUSION: CBMO showed the same rate of phagocytosis but a reduced PICD compared to PBMO. Also CBMOs TLR2-expression is reduced. Non-apoptotic monocytes may be involved in perpetuation of neonatal sepsis and could contribute to organ failure

Data Sets DOI10.1371journal.pone.0166648

Excel Table containing data given in the figures of the manuscript

Data from: Reduced PICD in monocytes mounts altered neonate immune response to Candida albicans

Background: Invasive fungal infections with Candida albicans (C. albicans) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of C.albicans initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO). Hypothesis: Phagocytosis of C. albicans causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses. Methods: The ability to phagocytose C. albicans, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor). Results: Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and C. albicans induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-alpha which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO). Conclusion: Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO

Reduced PICD in Monocytes Mounts Altered Neonate Immune Response to Candida albicans

<div><p>Background</p><p>Invasive fungal infections with <i>Candida albicans</i> (<i>C</i>. <i>albicans</i>) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of <i>C</i>.<i>albicans</i> initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO).</p><p>Hypothesis</p><p>Phagocytosis of <i>C</i>. <i>albicans</i> causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses.</p><p>Methods</p><p>The ability to phagocytose <i>C</i>. <i>albicans</i>, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor).</p><p>Results</p><p>Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and <i>C</i>. <i>albicans</i> induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-α, which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO).</p><p>Conclusion</p><p>Our data suggest direct involvement of TLR2-signalling in <i>C</i>. <i>albicans</i>-induced PICD in monocytes and an alteration of this pathway in CBMO.</p></div

Phagocytic properties of neonatal and adult monocytes.

<p>Comparative flow cytometric analysis of the PI and PC of gated CD14⁺ monocytes in CBMO and PBMO following infection with GFP⁺ <i>C</i>. <i>albicans</i> for 2h at the indicated MOI (n = 3; student`s t-test, * p < 0.05).</p

<i>C</i>. <i>albicans</i> infection-induced caspase-8.

<p>Mean fluorescence intensity (MFI) of the intracellular staining of cleaved caspase-8 in CD14⁺ PBMO (A) or CBMO (B) following infection with <i>C</i>. <i>albicans</i> at a MOI of 1:5. Pre-treatment with TLR2 bAb or MyD88i peptide (n = 5; student`s-t-test marked by solid (PBMO groups) or crotched (CBMO groups) lines, *p<0.05, **p<0.01, ***p<0.005; 1-way_ANOVA marked by blunt ended lines, *p<0.05, **p<0.01, ***p<0.005; 2-way-ANOVA marked by asterisks in chart bars, **p<0.01).</p

TLR2 and TLR4 surface expression by neonatal or adult monocytes following infection with <i>C</i>. <i>albicans</i> or TLR2 stimulation.

<p>CD14⁺ CBMO and PBMO were infected with <i>C</i>. <i>albicans</i> at a MOI of 5 for 2h (A, C), or treated with indicated TLR2 agonists (B, D). The surface expression of TLR2 (A, B) and TLR4 (C, D) were assessed by flow cytometric analysis (n = 8; 1-way-ANOVA test marked by crotched solid (between PBMO groups) and dotted (between CBMO groups) lines, *p<0.05; **p<0.01, ***p<0,005; 2-way ANOVA test marked by blunt ended lines and asterisks in chart bars testing groups in C and D; **p<0.01).</p

<i>C</i>. <i>albicans</i>-induced monocyte apoptosis.

<p><i>C</i>. <i>albicans</i>-induced monocyte apoptosis.</p

<i>C</i>. <i>albicans</i> induced TNF and IL-6 expression in neonatal and adult monocytes.

<p>CBMO and PBMO were infected with <i>C</i>. <i>albicans</i> at a MOI of 5 for 4h, or were treated with the indicated TLR agonists. The role of TLR2 for intracellular TNF-α expression was determined by administration of a TLR2 bAb (A, n = 5; 1-way ANOVA solid/dashed crotched lines, **p<0.01; 2-way-ANOVA blunt-ended lines and stars within bars, *p<0.05;**p<0.01; ***p<0.005). IL-6 secretion 4 h p.i. with or without TLR2 bAb administration as indicated (B, n = 3; student`s t-test solid/dashed crotched lines, **p<0.01; 2-way-ANOVA blunt-ended lines,*p<0.05; **p<0.01).</p