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-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). Conclusion Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO.


Introduction
Although C. albicans is part of the normal microbiota, it may emerge as opportunistic pathogen in the event of innate or acquired immunodeficiency. C. albicans and other Candida species are also common pathogens in preterm infants. Very low birthweight (VLBW) and extremely low birthweight (ELBW) infants are extremely vulnerable to invasive Candida infection (ICI) with adverse neurodevelopmental outcome and high mortality. The spectrum of clinical disease comprises mucocutaneous infections, blood stream infection and invasive infections of various internal organs [1,2].
Preterm infants exhibit a particular sensitivity to fungal infections. These opportunistic pathogens take advantage of the immature neonatal immune system and the need for prolonged antibiotic treatment that facilitates fungal growth, replication and dissemination [3]. C. albicans accounts for 9.8% of early onset sepsis (EOS) in newborns and additionally significantly contributes to neonatal late-onset sepsis (LOS), affecting approximately 7% of VLBW infants before hospital discharge and, varying from country to country up to 20% of ELBW [4]. Candida thereby represents the fourth most common species of LOS. There is growing evidence, that the epidemiology of Candida-infection still may be underestimated [3].
Insight in the pathogenesis of Candida infection of the neonate is required to understand the associated morbidity and mortality and develop new preventive and therapeutic strategies. C. albicans grows in different morphological forms (yeast, pseudohyphae, and hyphae) dependent on the tissue milieu. C. albicans ability to grow as hyphae was shown to contribute enhanced pathogenicity and virulence [3].
At least three groups of innate immune receptors are involved in the recognition of C. albicans, namely toll-like receptors (TLRs), C-type lectin receptors (CLR) and nucleotide-bindingdomain-leucine-rich-repeat-receptors (NLRs) [5]. Among these receptors, TLR2 is one of the key receptors that play a critical role in recognition of pathogens and activation of host innate immune signalling. Although the signalling mechanisms following infection are not fully understood [6,7] [8], they significantly contribute to host immune responses during candidiasis [9]. The individual host response is most likely due to differences in the innate immune recognition and the release of pro-inflammatory cytokines and chemokines [10,11]. The downstream signalling of TLR 2 is modulated by the formation of heterodimers with TLR1 and TLR6 [12]. Stimulation of TLR2 leads to the recruitment of the adaptor molecule MYD88 [13] and the IL-1 receptor-associated kinases IRAK-1 and -4 activating a cytosolic signalling cascade that culminates in the stimulation of mitogen activated protein (MAP) kinases and nuclear factor-κB (NF-κB). Both transcription factors induce the expression of cytokines with pro-and anti-inflammatory activity. During infection, TLRs act as double edged sword triggering antimicrobial host responses but also apoptotic pathways [14].
We previously identified an involvement of the CD95/CD95L and TNF-α-pathway in the phagocytosis-induced cell death of monocytes, following infection with E. coli [15,16]. Unexpectedly, we noted a significantly reduced PICD in neonatal monocytes despite a similar phagocytic capacity. This reduced PICD was due to defects in the CD95-/CD95L-pathway [15]. Two experimental challenges had to be considered performing in-vitro C. albicans infection. Infection at 37 C˚leads to overgrowing yeasts. Co-cultivation of leukocytes and C. albicans at RT initiates hyphae growth which rules out any FACS analysis. Therefore, experiments were restricted to 4 hours p.i. at 37 C˚followed by fixation.
Microscopical analysis revealed that both, CD14-positive PBMO and CBMO were capable to incorporate the 4-10 μm sized yeast blastospores within the selected time interval, independent of the temperature of cultivation.

Mononuclear cell cultures
Peripheral blood and cord blood mononuclear cells (PBMO and CBMO) were isolated by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare, Little Chalfont, UK) as described previously [19]. Washed cells were resuspended in RPMI-1640 (Invitrogen, Carlsbad, CA). For analysis of post-phagocytic reactions, cells were counted in a Neubauer hemocytometer, placed at 1x10 6 cells/ml in flat bottom 24 well cell culture plates (Costar, Bodenheim, Germany) containing 10% heat-inactivated fetal calf serum (FCS) and 1% Penicillin/Streptomycin (both Invitrogen) and incubated at 37˚C.

Staining procedures and flow cytometry
Because of infectiousness of C. albicans the samples were fixed with 2% paraformaldehyde (1:1) for 30 min and washed with PBS + 3% FCS prior to FACS analysis. Monocytes were gated by forward (FSC), side scatter (SSC), and CD14 expression. Lymphocytes could be identified by their ability to show a characteristic CD3/CD4/CD8-positive population in FACS analysis.
TLR2-and TLR4-antibodies were added for 20 min at RT in 1:500 dilution prior to fixation. After fixation for 1 hour at RT, cells were washed twice in PBS + 3% FCS prior to FACS analysis (S1C Fig). For the detection of the cleaved forms of caspase-3 and -8, fixed leucocyte samples were permeabilized with 0.1% (v/v) Triton-X-100 in PBS for 5 min, washed twice in PBS/ FCS (1% v/v) and incubated with the appropriate antibodies, diluted 1:100 for 30 min at RT. Again, samples were washed twice in PBS/FCS and incubated with the secondary antibodies, diluted 1:1000 for 5 min at RT, followed by subsequent washing. As a control, samples were incubated with secondary antibodies only or incubated with non-specific isotype control (S1D Fig). For intracellular staining, utilizing anti-TNF-α and anti-phosphorylated-P38 antibodies, cells were permeabilized by PERM/WASH buffer supplied by Becton Dickinson Biosciences (Franklin Lakes, New Jersey, USA) for 20 min at RT. The antibodies named above or corresponding isotype controls were diluted 1:200 in PERM/WASH buffer and incubated for 1 h at 4˚C. After subsequent washings, cells were analyzed via FACS. The TUNEL activity staining was performed according to the manufacturer's recommendations and as previously described [16]. To avoid false-positive signals due to interference with staining of yeast genome, we corrected our gating strategy (S1A and S1B Fig). A daily calibrated FACS-Canto II flow cytometer (Becton Dickinson, MountainView, CA) was used to perform phenotypic analysis.

Stimulation and blocking of TLR and MYD88
In indicated experiments, the culture medium was supplemented with TLR agonists: Pam3Cys (1 μg/ml; TLR2/TLR1 agonist), LPS (10 ng/ml, TLR4 agonist), FSL-1 (1μg/ml, TLR2/TLR6 agonist), and Zymosan (5 μg/ml, TLR2/TLR6, Dectin-1). In general, stimulation intervals were identical to infection intervals and lasted 2 hours. In an ex-ante experiment, the agonist Pam3Cys was given for shorter and longer intervals to determine the onset of intracellular signalling (S3 Fig).The TLR2 blocking procedure followed the protocol of the manufacturer (InVivoGen, Toulouse, France). In brief, to 2x10 6 PBMC and CBMC the antibody (PAb-hTLR2) was added 15 min prior to infection or addition of agonists in a final concentration of 5μg/ml. In control assays an equivalent concentration of non-specific antibody (Rat PAb Control) was added. Mononuclear cells were cultivated under standard conditions. The MYD88i blocking peptide and the corresponding control peptide, consisting of the antennapedia region only, were administered two hours before infection and stimulation in a final concentration of 20μM. In precursory phagocytic assays we checked that incubation with either blocking antibody (TLR2 bAb) and MYD88i did not interfere with phagocytosis.

Phagocytosis assay
Phagocytosis assays were performed by adding GFP-expressing yeasts (SCADH1G4A) in different concentrations to 1x10 6 cells. Infection was performed at a multiplicity of infection (MOI) of 1:1 to 1:10 which was achieved by dilution with 0.9% NaCl. The phagocytosis index (CD14+GFP+ monocytes: CD14+ monocytes) and the phagocytic capacity (PC, mean GFP (MFI)) were analyzed.

Hypodiploid nuclei
DNA fragmentation was assessed according to Nicoletti [20] and previously described [16]. In brief, mononuclear cells were stained with CD14 antibody for 15 minutes at RT to identify monocytes. A fixation with paraformaldehyde (2% v/v in PBS) for 30 min at RT was performed. Afterwards, cells were permeabilized by incubation in PBS-T (PBS, Triton X-100 0,1% w/v) for 20 minutes at RT, washed twice in PBS, resuspended in PBS-PI (PBS, 70μg/ml PI and 13 units RNAse) and incubated for 10 minutes at RT before analysis by flow cytometry.

Cytokine detection
The IL-6 Elecsys1 IL-6 (Interleukin-6) assay (Roche Diagnostics GmbH; D-68305 Mannheim, Germany) were used according to the manufacturer's recommendations for analysis of IL-6 levels in the cell supernatant fluid. The TNF-α ELISA was used according to the manufacturer's recommendations. The read-out was executed in a spectra max 340PC ELISA reader (molecular devices, Sunnyvale, CA, USA) with a sensitivity from 4 -500pg/ml.

Statistical analysis
Results are expressed as mean +/-standard deviation. Error bars represent standard deviations. Statistical analysis was performed using a student's t-test and 1-or 2-way ANOVAs, respectively. Results of 2-way-Anova calculation were validated with a Bonferroni post test. Values of p < 0.05 were considered significant. Analyses were done with statistical software (GraphPad Software Statistical Package, La Jolla, CA 92037 USA).

Phagocytic properties of PBMO and CBMO are similar after infection with C. albicans
We assessed the dose dependent uptake of C. albicans by FACS analysis and quantified the phagocytosis index (PI) and phagocytic capacity (PC) of monocytes, as previously described for bacterial infection assays [21] (Fig 1A and 1B). PBMO and CBMO revealed no significant differences in PI after co-incubation with increasing numbers of fungi with a MOI varying between 1 and 10 ( Fig 1A). Analysis of the PC revealed that increasing numbers of C. albicans saturated the phagocytic process at a MOI of 2.5 for PBMO and at a MOI of 5 for CBMO ( Fig  1B). At a MOI of 5, the PC was equal in PBMO and CBMO.

C. albicans infection regulates surface expressions of TLR2 and TLR4
We assessed the basal cell surface expression of TLR2 and TLR4 and followed it after C. albicans infection and incubation in the presence of TLR2 and TLR4 agonists (Fig 2). TLR2 and TLR4 expression was modulated differently in PBMO and CBMO by C. albicans and TLR-agonists. Basal expression of TLR2 was low in CBMO and PBMO with slightly more TLR2 expression on CBMO as compared to PBMO (Fig 2A). C. albicans infection raised the number of TLR2-expressing PBMO significantly stronger as compared to CBMO (Fig 2A). Stimulation with the TLR2 agonists Pam3Cys or Zymosan resulted in almost identical TLR2 up-regulation on PBMO and CBMO.
The TLR4 ligand LPS also induced a slight up-regulation of TLR2 but to a weaker extent as compared to Pam3Cys and Zymosan ( Fig 2B). FSL-1, a TLR2/TLR6 agonist, induced TLR2 up-regulation comparable to what was observed after LPS stimulation (Fig 2B). The results indicate that C. albicans and the TLR2/TLR1 agonist Pam3Cys, but not the TLR4 and the TLR2/TLR6 agonists LPS and FSL-1 efficiently up-regulate TLR2 expression.
Compared to TLR2, surface expression of TLR4 on PBMO was significantly higher as compared to CBMO (Fig 2C). Infection with C. albicans down-regulated TLR4 on PBMO, but had no significant effect on CBMO (Fig 2C). LPS up-regulated TLR4 on CBMO but had no effect on the high basal expression of TLR4 on PBMO (Fig 2D). The TLR2 agonist Pam3Cys did not change the TLR4 expression pattern (Fig 2D). These results suggest that C. albicans infection influences TLR2 expression more profoundly than TLR4 expression on PBMO. The expression of TLR2 and TLR4 (MFI) were found similar before and after stimulation in PBMO and CBMO (S2 Fig). CD14, which serves as a co-receptor for TLR4 and TLR2 [22], was stronger down-regulated on PBMO as compared to CBMO after C.albicans infection decreasing within 2 h p.i. (196 ± 52.5 MFI to 121.3 ± 28.9 MFI; p < 0.05). CD14 expression in non-infected CBMO was

Induction of P38 MAP Kinase (MAKP) is TLR2 dependent and differs in CBMO and PBMO
To monitor cellular signal transduction downstream of TLR2 and TLR4, we assessed the phosphorylation of the P38 MAPK (Fig 3). Within an infection interval of 2 h, the P38-phosphorylation in PBMO increased 2-fold stronger than in CBMO (Fig 3A, p < 0.05). Comparable results were obtained after stimulation with the TLR2/TLR1 agonist Pam3Cys (Fig 3A).
The potential to phosphorylate P38 MAPK was not due to a critical reduced in the expression of the adaptor protein MYD88. MYD88 was found to be abundantly expressed in both PBMO and CBMO before and after C. albicans infection (Fig 3B).
Blocking of TLR2 with an inhibitory Ab (Fig 3C, PBMO; Fig 3D, CBMO) reduced the C. albicans-and Pam3Cys-induced P38 phosphorylation by 50% in PBMO (Fig 3C, first and second column). In contrast, the C. albicans-induced P38-phosphorylation in CBMO was lower than in PBMO (p < 0.05) and was not abolished in the presence of the TLR2 blocking Ab ( Fig  3D). This was not due to an impaired signalling via TLR2/TLR1, since Pam3Cys induced P38 phosphorylation could readily be abolished by the TLR2 bAb ( Fig 3C). The TLR2 bAb did not inhibit LPS induced P38 phosphorylation in PBMO and CBMO, confirming the specificity of the blocking antibody.
Inhibition of MYD88 resulted in a decreased P38 phosphorylation after C. albicans infection or Pam3Cys stimulation of PBMO (Fig 3C). In CBMO, MYD88 blockage decreased the P38 phosphorylation after Pam3Cys stimulation, but not after C. albicans infection (Fig 3D).
In summary, these results suggest that C. albicans-induced P38 phosphorylation in PBMO occurs predominantly via TLR2/TLR1 and MYD88. Interestingly, Pam3Cys administration in CBMO was accompanied with a lower concentration of phosphorylated P38 compared to PBMO (Fig 3), although Pam3Cys up-regulates TLR2 equally in CBMO and PBMO (Fig 2).

TNF-α expression and IL-6 secretion following infection with C. albicans
Phosphorylation of the P38 MAPK promotes downstream-signalling via JNK and NFκB, resulting in enhanced expression of cytokines such as TNF-α [14]. We, therefore, analyzed the intracellular TNF-α 4 h p.i. with C. albicans or following stimulation with TLR2 and TLR4 agonists ( Fig 4A).
The requirement of TLR2 was confirmed by administration of a TLR2 bAb, which significantly reduced the C. albicans-as well as Pam3Cys-induced TNF-α production in PBMO. In CBMO, the addition of TLR2 bAb did not decrease the TNF-α production. The specificity of the TLR2 bAb was demonstrated by the absence of a detectable effect after administration of LPS (Fig 4A, 4 th columns). For C. albicans infected PBMO, the TLR2-dependency of TNF-α secretion could also be demonstrated by ELISA analysis (135.3.5 ± 23.4 pg/ml to 90.6 ± 20.7 pg/ml; p < 0.05 vs. TLR2 bAb treated).
Assessment of IL-6 secretion 4 h p.i. showed an almost 10 times stronger production of IL-6 in CBMO compared to PBMO (Fig 4B). Furthermore, the secreted IL-6 was diminished after application of a TLR2 bAb, whereas TLR2 signalling was not obligatory for IL-6 secretion in PBMO.
C. albicans infection activates caspase-8 and caspase-3 via TLR2 to drive apoptosis in PBMO but not CBMO Activation of caspase-8 by the TLR1-/TLR2-/MYD88-signalling may induce apoptosis [23]. We, therefore, tested whether caspase-8 and caspase-3 were cleaved to generate the active forms after C. albicans infection in PBMO and CBMO 2 h after infection (Figs 5 and 6). In PBMO cleaved caspase-8 products were found more prominently as compared to CBMO (Fig  5A and 5B; p < 0.05). The increase in caspase-8 cleavage was dependent on TLR2 and could be abrogated by pre-incubation with the TLR2 bAb. The TLR2/TLR1 agonist Pam3Cys initiated only a weak TLR2-dependent caspase-8 cleavage.
MYD88 was not engaged in the TLR2-induced caspase-8 activation, since MYD88 inhibition had no effect on caspase-8 cleavage (Fig 5). In PBMO but not CBMO, cleavage of effector caspase-3 was initiated by C. albicans infection (Fig 6A and 6B). Again, the involvement of TLR2 in caspase-3 activation was shown by administration of the TLR2 bAb ( Fig 6A). Addition of MYD88 inhibitory peptide abrogated activation of caspase-3 (Fig 6B)

C. albicans infection initiates apoptosis more dominantly in PBMO than in CBMO
Induction of apoptosis in C. albicans infected monocytes was assessed by measuring processing of caspase-3 (Fig 6A and 6B) determining the hypodiploid DNA content (Fig 6C and 6D) and detecting DNA strand breaks by TUNEL staining (Table 1). PICD was increased 5-fold following C. albicans infection in PBMO (Fig 6C), but only 2-fold in CBMO (Fig 6A; p < 0.05). Apoptosis in PBMO and CBMO was reduced to the levels of non-infected controls by administration of the TLR2 bAb, demonstrating the critical role of TLR2 (Fig 6C). Also, inhibition of MYD88 reduced monocyte apoptosis (Fig 6D), demonstrating the requirement of MYD88 signalling in TLR2-induced apoptosis.

Discussion
The present work describes marked differences in the activation of P38 MAPK phosphorylation, TNF-α production, caspase-8 and -3 processing, and phagocytosis induced cell death (PICD) in response to C. albicans infection between CBMO and PBMO despite similar phagocytosis capacity. The critical involvement of TLR2 in the recognition of C. albicans by PBMO suggests that reduced innate immune sensitivity might be responsible for the enhanced susceptibility of preterm neonates to fungal infection.
The phagocytosis of non-opsonized C. albicans by polymorphonuclear cells was previously demonstrated to be low in both, adult and neonatal neutrophils (10-12%), leading to the conclusion, that dysfunctional phagocytosis may not contribute to the increased susceptibility of preterm neonates to infection with C. albicans [24]. This is consistent with reports on similar phagocytic properties of PBMO and CBMO in respect to various bacterial species with pathogenic potential in the neonate (E. coli, Group B streptococci) [25].
During the first few weeks after birth, the TLR2 and TLR4 receptor densities on blood monocytes of human preterm newborns increases rapidly to reach levels that are similar to those of full-term newborns and adults [26,27]. This is in line with our results on the Age-Dependent Monocyte Response to Candida albicans Infection expression of TLR2 and TLR4 by non-stimulated PBMO and CBMO (Fig 2A and 2C). Clinical investigations [28] and animal studies [29,30] have demonstrated reduced TLR2 and TLR4 expression and diminished stimulus-induced cytokine secretion at birth both in preterm infants and term newborns. Our results demonstrating reduced TLR2 expression in CBMO are well in accordance with these studies. Though present work shows altered innate immune response against C. albicans in healthy full-term neonatal compared to adults. It seems that innate immunity is differentially regulated among neonatal subsets, depending on gestational age and birth weight, involving pattern recognition receptor-and TLR4-signalling [31,32].
doi:10.1371/journal.pone.0166648.g004 In accordance with previous studies [33] we observed a reduced MAPK P38 phosphorylation in CBMO after administration of a TLR2 ligand or C. albicans. In the published literature, the secretion of several pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8 and TNF-α downstream of P38 MAPK activation was reported after stimulation of PBMO and CBMO. The results, however, have remained controversial. Some studies found the production of moderately less TNF-α, but similar or even higher IL-1 and IL-6 levels in CBMO as compared to adult monocytes [35], as it was shown here (Fig 4B). Other studies reported on a generally lower cytokine production [33].
Our results blend into these findings since infection with C. albicans resulted in an increased TNF-α production in PBMO (Fig 4). Stimulation of TLR2/TLR1 by Pam3Cys also induced significant TNF-α production in PBMO and CBMO (Fig 4) although TLR2 was found to be stronger up-regulated in PBMO (Fig 2B). Moreover, neutralizing TLR2 did not inhibit Pam3Cys initiated TNF-α production in CBMO. Additionally, MYD88 inhibition abrogated P38 phosphorylation completely after C.albicans infection but only partially after TLR2/TLR1 stimulation by Pam3Cys (Fig 3B and 3C), suggesting that P38 phosphorylation and TNF-α production in PBMO requires the engagement of co-receptors. Recent publications reported on a functional cross-talk between TLR2 and Dectin-1 [7,36], which may account for the mismatching effects of TLR2 neutralizing antibodies and MYD88 inhibition. In CBMO, P38 phosphorylation can be blocked by a MYD88 inhibitor and a neutralizing antibody. Hence there is evidence for another P38-independent activation pathway that explains the intracellular TNF-α levels in CBMO after C.albicans infection and TLR2/TLR1 stimulation (Fig 4).
Although, the present study cannot thoroughly rule out a contribution of TLR4, which is down-regulated after C. albicans infection (Fig 2B), we suggest a minor role of TLR4 signalling. We could show that administration of LPS led to an ineffectual activation of P38 in comparison to the TLR2 agonists (Fig 2A). The TNF-α production, however, did not differ between TLR2/TLR1 stimulation and C. albicans infection (Fig 4), delimiting the role of TLR4. Several studies on foetal cells [26] and on cells obtained during the early postnatal period [27] indicate that the level of TLR4 expression does not correlate with TNF-α production. Albeit publications describe functional TLR2/TLR4 heterodimers which could initiate cytokine production, the designated TLR2/TLR4 heterodimer is formed after brain damage and exposure to haemoglobin [37].
TLR2 was one of the first TLR family members to be described as a death inducing receptor following overexpression in various cell types such as macrophages and neutrophils [14]. Proapoptotic signalling involves MYD88-dependent recruitment of FADD and caspase-8 leading to cleavage of caspase-3 and executing the apoptotic program [23,38]. The pro-apoptotic and the cytokine-inducing signal transduction pathway downstream of TLR2 converge at the level of the P38 MAPK, which activates caspase-7 and -3. This may involve activation of the apoptosis-signal-regulating-kinase-1 (ASK1) leading to sustained P38 MAPK phosphorylation and activation of caspase-3 [14]. In previous studies, the involvement of TLR2 in apoptosis induction was established using blocking anti-TLR2 antibodies or comparison between TLR2 sufficient and deficient cells. More recently, the participation of TLR2 also in virus-triggered apoptosis was demonstrated [39]. We observed a pronounced TLR2-dependent activation of caspase-8 and caspase-3 in PBMO resulting in a C. albicans-mediated monocyte apoptosis (Figs 5 and 6). Caspase-8 activation could be reduced by TLR2 neutralizing antibodies but were resistant to MYD88 inhibition, suggesting an indirect contribution of TLR2. We suggest a trans-activation of caspase-8 which is caused by TLR2/Dectin-1 triggered TNF-α production (see above). Ligation of TNFα to TNFR1, in turn, activates caspase-8, which is supported by the finding that TNF-α inhibitory antibodies reduce caspase-8 activation (unpublished results). The TLR2 ligand Pam3Cys induced caspase-8 equally in CBMO and PBMO, but exposure to C. albicans induced caspase-8 stronger in PBMO, pointing again to a contribution of a TLR-2 co-receptor.
The question that remains to be clarified is whether trans-activated caspase-8 contributes to caspase-3 activation and apoptosis. Here, we observed effector caspase-3 activation after C. albicans infection (Fig 6A), which could be blocked by TLR2 neutralizing antibodies and MYD88 inhibition, demonstrating a direct requirement of TLR2. Further studies should thus identify signal transducing factors linking P38 phosphorylation and activation of the intrinsic apoptotic machinery.
The reduced PICD of CBMO following C. albicans infection (Fig 5C) is consistent with our previous report on the response of cord blood monocytes to bacterial pathogens [15,21]. Although a similar phagocytic index was detected in PBMO and CBMO following exposure to E. coli and GBS a reduced level of PICD was noted in CBMO. The results further point towards a crucial role of CD95L [15,21] and TNF-α in these processes [16]. Together, this indicates age-dependent differences in the lifespan of macrophages following exposure to both bacterial and fungal pathogens although different receptor pathways appear to be involved. Since monocytes represent a major source of pro-inflammatory cytokines such as TNF-α or IL-6 during infection, this may explain the prolonged pro-inflammatory response and sustained inflammation in organs such as brain, lung and eye in neonates suffering from bacterial or fungal infection.