extfieldsofstudy: Biology Medicine

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MicroRNAs miR-26a, miR-26b, and miR-29b accelerate osteogenic differentiation of unrestricted somatic stem cells from human cord blood

Background MicroRNAs are a population of short non-coding RNAs with widespread negative regulatory impact on mRNA translation. Unrestricted somatic stem cells (USSC) are a rare population in human cord blood that can be induced into cells representative of all three germinal layers. Here we analyzed the functional impact of miRNAs on the osteogenic differentiation in USSC. Results Gene expression profiling identified 20 microRNAs that were consistently upregulated during osteogenic differentiation of two different USSC cell lines (SA5/73 and SA8/25). Bioinformatic target gene prediction indicated that among these microRNAs, miR-10a, -22, -26a, -26b, and -29b recognize transcripts that encode a set of proteins inhibiting osteogenesis. We subsequently verified osteo-inhibitory CDK6, CTNNBIP1, HDAC4, and TOB1 and osteo-promoting SMAD1 as targets of these microRNAs. In Western blot analyses demonstrated that endogenous levels of CDK6 and HDAC4 were downregulated during osteogenic differentiation of USSC and reduced following ectopic expression of miR-26a/b and miR-29b. In contrast, endogenous expression of SMAD1, targeted by miR-26a/b, was unaltered during osteogenic differentiation of USSC or following ectopic expression of miR-26a/b. Functional overexpression analyses using microRNA mimics revealed that miR-26a/b, as well as miR-29b strongly accelerated osteogenic differentiation of USSC as assessed by Alizarin-Red staining and calcium-release assays. Conclusions miR-26a/b and miR-29b are upregulated during osteogenic differentiation of USSC and share target genes inhibiting osteogenesis. Furthermore, these microRNAs accelerate osteogenic differentiation, likely mediated by osteo-inhibitory proteins such as CDK6 and HDAC4.

Unrestricted somatic stem cells (USSC) are a rare CD45-negative population in human cord blood [33]. These cells can be discriminated from CB-MSC and BM-MSC by their HOX expression pattern which resembles that of H9 embryonic stem cells [34]. Adherently-growing in vitro USSC can be induced to cells representative of all three germinal layers on a clonal level [35] and have been successfully reprogrammed to a pluripotent ES-like state [36]. Undergoing miRNA-supported cell cycle arrest, USSC can be differentiated into cells of neural lineage with miRNAs acting as network-like regulators [37][38][39]. USSC also differentiate into functional hepatic-like cells [40,41] as well as along osteogenic and chondrogenic lineages [33]. Upon induction with dexamethasone, ascorbic acid, and ß-glycerol phosphate (DAG), USSC differentiate into osteoblasts as evidenced by calcium phosphate deposition, bone-specific ALP-activity, increase in Ca 2+ -release, and expression of the osteogenic marker proteins osteocalcin, osteopontin, bone sialo-protein, and collagen type I [33]. Bony reconstitution was observed following implantation of USSC into nude rat femurs [33]. Beside their differentiation potential, USSC also fulfil regenerative functions in acute spinal cord trauma [42].
Here we analyzed the impact of miRNAs on osteogenic differentiation of USSC. We identified a set of miRNAs upregulated upon induction of osteogenesis, co-ordinately regulating a distinct set of genes known to inhibit osteogenesis. Among these inhibitors, CDK6, CTNNBIP1, HDAC4, TGFB3, and TOB1 were experimentally identified as targets of miR-26a, miR-26b, and miR-29b. These miRNAs were functionally identified as accelerators of osteogenic differentiation of USSC.

Differential miRNA expression during osteogenic differentiation of USSC
To assess the impact of miRNAs on osteogenic differentiation of USSC we studied two USSC lines (USSC SA5/73 and USSC SA8/25) that were induced to osteogenic differentiation using DAG as described [33]. As strong calcification of USSC during osteogenic differentiation impacts RNA isolation, we restricted our analyses to day 7 of differentiation. miRNA expression profiles of native and day 7 osteo-differentiated USSC were analyzed using the RT-PCR-based TaqMan Assay (Pool A) covering 377 miRNAs [43]. In SA5/73, 220 miRNAs were expressed and 124 miRNAs were upregulated by a factor ≧ 2 in differentiated cells. In SA8/25, 225 miRNAs were expressed and 196 miRNAs were upregulated during osteogenic differentiation. Interestingly, only 30 miRNAs were commonly upregulated in both USSC lines. In follow-up analyses we focused on 20 of these microRNAs (Figure 1), which were not only upregulated by a factor ≧ 2 but also present at high expression levels (< Ct 26) in differentiated USSC. We omitted those upregulated miRNAs that were weakly expressed in differentiated USSC due to their expected minor biological impact. Among the most prominently expressed miRNAs were miR-10a, miR-152, miR-22, miR-26a/b, miR-29b, miR-30b/c, miR-345, and miR-532-5p. Complete miRNA expression data from USSC SA5/73 and SA8/25 osteogenic differentiation experiments are presented in Additional file 1.

Bioinformatic target gene predictions
To investigate the biological impact of our selected set of 20 miRNAs, we computationally identified miRNA targets using the DIANA miRGen target gene prediction software [44], which combines the prediction results of several web-based algorithms (TargetScan, PicTar-4, PicTar-5, mi-Randa, DIANA microT). This approach resulted in an extensive list of putative targets (>10 4 genes), many of which were recognized by more than one of our 20 miRNAs. As our study is focused on miRNAs that are upregulated during osteogenic differentiation, we reasoned that their biological impact should be posttranscriptional downregulation of proteins inhibiting osteogenic differentiation. We thus filtered our list of putative targets using the Gene Ontology (GO) terms function implemented in the DAVID database, applying search terms like "bone" and "osteo". Table 1 presents an overview of 21 proteins inhibiting osteogenic differentiation that are putatively recognized by our set of 20 miRNAs. Consistent with the view that miRNAs regulate networks [38], Table 1 demonstrates target gene redundancy: certain proteins were predicted to be targeted by more than one miRNA and other miRNAs (e.g. miR-29b) putatively regulate up to 11 proteins (Table 1).

Target gene expression analysis and impact of regulating microRNAs
As the availability of USSC lines SA5/73 and SA8/25 became limited in the course of our study, we focused on the newer USSC lines 86b and 77 for target gene expression analyses. Using qPCR, we analyzed transcript expression of validated targets CDK6, HDAC4, CTNNBIP1, SMAD1, and TOB1 during osteogenic differentiation of USSC line 86b at time points day 0 (native cells), and days 7 and 12 post DAG induction. Figure 4 demonstrates that CDK6, HDAC4, SMAD1, and CTNNBIP1 were all steadily downregulated at days 7 and 12 compared to day 0. HDAC4 was downregulated at day 7, followed by a slight increase in expression at day 12, albeit weaker than at day 0. We were unable to identify TOB1 transcripts in USSC. These results indicate inverse transcriptional regulation of miRNAs and target-mRNAs inhibiting osteogenic differentiation of USSC.
As miRNAs downregulate their targets on a posttranscriptional level, we also analyzed HDAC4, CDK6, and SMAD1 protein expression during osteogenic differentiation (up to day 12) of USSC 86b and in response to ectopic expression of miR-26a, miR-26b, and miR-29b in native USSC86b.
As seen in Figure 5A, HDAC4 was weakly expressed in native USSC 86b and downregulated by a factor 5 in day 9 osteo-differentiated USSC 86b, thereby matching our qPCR results ( Figure 4). The HDAC4 protein level was also reduced following transfection with miR-29b ( Figure 5B).
CDK6 behaved differently on the protein level; this protein was initially upregulated at day 7 of osteogenic differentiation in USSC 86b, and slightly downregulated at day Bioinformatic target predictions for the subset of 20 miRNAs upregulated in osteogenic differentiation of USSC on osteo-inhibitory proteins: miR-26a, -26b, and -29b were experimentally assessed for impact on proteins denoted in bold. The second column ("Total") illustrates the number of miRNAs from the analyzed set that targets an individual protein. Predictions with an asterix were already validated in the literature.
12 although still more abundant than in native cells ( Figure 5C). To further examine this unexpected result, we tested an additional USSC line (USSC 77), which yielded the same dynamic CDK6 expression pattern ( Figure 5C). Upon independent transfection with (i) an equimolar mixture of miR-26a and miR-26b mimics (both miRNAs share their seed sequence and differ in only two nucleotides) and (ii) with miR-29b mimics, CDK6 protein abundance was reduced compared to native and negative control cells 48h after transfection ( Figure 5D). As with HDAC4, our results confirm that miR-26a, miR-26b, and miR-29b target CDK6. Interestingly, SMAD1 expression remained unchanged at day 9 post DAG induction compared to native USSC 86b ( Figure 5E) and was not affected by transfection with miR-26a and miR-26b mimics ( Figure 5F). We were unable to detect the weakly transcribed CTNNBIP1 with any of several established antibodies (data not shown). (B) fragments of the 10235nt 3 0 -UTR of CDK6 (CDK6-1 and CDK6-2, shown as blue bars with indicated putative miRNA binding sites) for miRNAs miR-22, -26a, -26b, -29b, and -137 in HEK293T-Cells. To assess the influence of endogenous miRNAs, empty firefly/renilla dual reporter vector pmirGLO and pmirGLO/CDK6-3 0 -UTRs were each transfected into HEK293T-cells. Normalized firefly-activities were compared to those of pairwise co-transfections of these vectors with the miRNA mimic of interest (including an unspecific siRNA negative control) to test for unspecific effects of the given miRNA-mimic on firefly/renilla per se and for validation of the particular target prediction. Dark grey columns show normalized firefly activities from pmirGLO (co)-transfections, light grey columns those from pmirGLO/CDK6-3 0 -UTRs (co)-transfections. Percent reductions of firefly activities of pmirGLO/CDK6-3 0 -UTRs compared to pmirGLO are given as well as their statistical significancies (at least two biological replicates with four technical replicates each, Student's t-test, unpaired, ** p ≤ 0.01, ***: p ≤ 0.001). Summarizing results from both CDK6-3 0 -UTR fragments, significant regulatory miRNA effects were seen for miR-22, miR-26a, miR-26b, and miR-29b, whereas miR-137 had no significant effect.

Functional impact of miR-26a/b and miR-29b on osteogenic differentiation of USSC
Our experimental target validations indicate that miR-26a, miR-26b, and miR-29b likely have the strongest impact on osteogenic differentiation of USSC by reducing osteo-inhibitory CDK6 and HDAC4 proteins. We thus tested whether overexpression of miR-26a/b and miR-29b using miRNA mimics influences DAG-induced osteogenic differentiation. USSC SA5/73 and USSC 86b were each transfected with (i) a small unspecific negative control RNA, (ii) an equimolar batch of miR-26a and miR-26b, (iii) miR-29b, and (iv) an equimolar batch of miR-26a, miR-26b, and miR-29b mimics (SA5/73 only), each followed by DAG induction. Osteogenic differentiation was assessed by alizarin-red staining and calcium release at day 7 (both USSC lines). Figures 6A and 6B, both USSC lines started to differentiate at day 7 post DAG induction. Transfection of negative control RNA did not substantially influence alizarin-red staining in both USSC SA5/73 ( Figure 6A) and USSC 86b ( Figure 6B). In contrast, miR-26a/b-mimic transfected cells of both USSC lines showed significantly increased staining (Figures 6A and 6B). Transfection with miR-29b-mimic also resulted in accelerated osteogenic differentiation of both lines (Figures 6A and 6B). Transfection of USSC SA5/73 with miR-26a/miR-26b/miR-29b mimics further increased differentiation ( Figure 6A).

Discussion
USSC are primary cells from human cord blood and are available in limited amounts from different patient sources. In general, USSC cell lines, such as SA5/73, SA8/25, 86b, and 77, are capable of comparable osteogenic differentiation as assessed by alizarin-red staining (data not shown). During osteogenic differentiation, only 30 miRNAs were commonly upregulated in two USSC lines (SA5/73 and SA8/25), although 124 and 196 miRNAs were respectively upregulated in each cell line. However, as expected for target-redundant miRNA networks, evaluation of commonly upregulated miRNAs revealed multiple bioinformatically osteo-inhibitory target genes. miRNA expression profiling followed by target validation indicated that miR-26a, miR-26b, and miR-29b had the highest impact on osteogenic differentiation in our USSC lines. In the mouse osteoblast model, miR-29b is a positive regulator of osteogenic differentiation, able to increase differentiation on ectopic expression, with HDAC4, TGFB3, ACVR2A, CTNNBIP1 and DUSP2 as validated targets [32]. Here we show a comparable function for miR-29b in osteogenic differentiation of human somatic stem cells confirming human CTNNBIP1 and HDAC4 as miR-29b targets in our HEK293T-cell based validation assay. CTNNBIP1 was also regulated by miR-10a and CDK6 [45] was targeted by miR-22, miR-26a, miR-26b and miR-29b. miR-26a modulates late osteogenic differentiation of hADSC through SMAD1 targeting [31] and we showed In HEK293T-cells we tested the effects of a total of six miRNA-mimics (miR-10a, -22, -26a, 26b, 29b, and -137) on the 3 0 -UTRs of 8 predicted proteins (CDK6, CTNNBIP1, DUSP2, HDAC4, SMAD1, SMAD6, TGFB3, and TOB1) related to osteogenic differentiation and/or function. Percent reductions of normalized firefly-activities from pmirGLO/3 0 -UTR/miRNA-mimic co-transfections compared to pmirGLO/miRNAmimic co-transfections are given in the colored squares. Red squares: lacking or non-significant miRNA effects, orange: weak but significant (p ≤0,05) miRNA effects, light green: moderate microRNA effects, dark green: strong microRNA effects. Among 22 individual miRNA-3 0 -UTR interactions analyzed, 10 were counted as positive (light and dark green squares). *: predictions already validated in [32], **: predictions already validated in [31]. These were analyzed again here for reasons of comparability to our other validations. Detailed data from all experimental validations are listed in Additional file 2.
that both, miR-26a and miR-26b regulate SMAD1, this protein is known as a positive mediator of osteogenic differentiation [59]. We also identified the osteo-inhibitory BMP/SMAD regulator TOB1 [51] as a target of miR-26a and miR-26b. Substantial homology between these two miRNAs (identical seed sequences) is reflected in their similar behavior in our target validation assays. To clarify the contradictory roles of osteo-promoting SMAD1 and osteo-inhibitory CDK6 and TOB1 as parallel targets of miR-26a and miR-26b, we directly analyzed target protein abundance by quantitative Western blotting (i) during osteogenic differentiation of USSC and (ii) in response to transfection of USSC with miRNA mimics.
Despite targeting of the SMAD1 3 0 -UTR by miR-26a and miR-26b in our luciferase assay (Figure 3), SMAD1 protein abundance remained unaltered upon transfection with miR-26a/b mimics ( Figure 5F). This finding indicates either a long intracellular half-life for SMAD1 in USSC, unaltered by miRNA transfection in the experimental timeframe, or secondary regulatory mechanisms such as increased transcription that keep SMAD1 levels constant. Accordingly, SMAD1 protein levels also remained constant at day 9 in osteo-differentiated USSC 86b ( Figure 5E) as expected for an osteo-promoting protein. However, our qPCR data indicate that SMAD1 transcription decreased steadily from day 0 to day 7 and to day 12 of osteogenic differentiation (Figure 4), further supporting a long halflife for SMAD1 protein. Due to strong calcification of USSC at day 12, we were unable to measure SMAD1 protein and it remains unclear whether this protein has a long half-life or is regulated by other unidentified mechanisms.
In contrast to osteo-promoting SMAD1, osteoinhibitory CDK6 protein expression was indeed reduced 48h post transfection with miR-26a, miR-26b, and miR-29b mimics. This finding indicates a comparatively strong regulatory influence of miR-26a/b and miR-29b on CDK6. However, despite steady transcript downregulation, CDK6 protein expression peaked at day 7 of osteogenic differentiation, before decreasing. As miRNAs act postranscriptionally [4,60], the observed decrease of CDK6 abundance at day 12 was likely due to continued upregulation of miRNAs targeting osteo-inhibitory factors beyond day 7, after achieving sufficient levels to alter CDK6 protein abundance.
The HDAC4 gene product was reduced on both transcript ( Figure 4) and protein level ( Figure 5B) during osteogenic differentiation. Similarly, the HDAC4 protein level was reduced upon transfection with miR-29b mimic, consistent with our target validation assays. It should be noted that we were unable to detect either TOB1 transcripts or TOB1 and CTNNBIP1 gene products in USSC (data not shown).
Using alizarin-red staining and calcium release assays, we clearly demonstrated that miR-26a/b and miR-29b mimic transfections specifically accelerate osteogenic differentiation in both USSC lines tested. This result is consistent with the observation that miR-29b contributes to osteogenic differentiation of mouse osteoblasts [32]. It should be noted that attempts to functionally analyze miR-10a and miR-22 failed due to a nearly complete loss of transfected USSC from the culture plates (data not shown). Since miR-26a/b and miR-29b regulate osteo-inhibitory and osteo-promoting factors in parallel, the osteo-inhibitory effects of CDK6 and HDAC4 likely outweigh the osteopromoting effects of SMAD1; this finding is further supported by the unaltered abundance of SMAD1 in miR-26a/b transfected USSC.
The strongest effect on osteogenic differentiation was observed by transfecting an equimolar mixture of miR-26a, miR-26b, and miR-29b mimics. It is likely that miR-26a/b and miR-29b influence a common set of target genes with each miRNA making additional contributions through targeting exclusive genes e.g. HDAC4 and CTNNBIP1, which are regulated by miR-29b but not by miR-26a/b.
Recently miR-135b was reported to be 100-fold upregulated in USSC following DAG-induction [61]. In contrast, Li and coworkers reported that miR-135b (and also miR-133a/b) were downregulated during mouse osteoblast differentiation after 16 hours [30]. In USSC, miR-133a and miR-133b as well as miR-135b are only weakly expressed even in native cells and virtually unchanged during osteogenic differentiation (see Additional file 1).
A recent study of miRNA expression signatures asssociated with osteogenic commitment of USSC showed upregulation of similar miRNAs (miR-26b, miR-30b, let-7a, let-7f and miR-181a) as determined in our analysis [62]. However the authors used USSC from a different source, employed different miRNA expression analysis methodology (hybridisation array), and focused on other miRNAs, precluding direct comparison.
Taken together, our results further establish the importance of miRNAs in differentiation processes of USSC. We clearly demonstrated the combined functional impact of miR-26a/b and miR-29b, which had individually been identified as modulators of osteogenic differentiation in hADSC [31] and mouse osteoblasts [32]. Target gene similarities and differences between these miRNAs imply that these miRNAs act in a synergistic manner to improve and accelerate osteogenic differentiation of USSC.

Conclusions
In summary, we detected a subset of miRNAs, notably miR-26a, miR-26b and miR-29b, which is consistently upregulated during osteogenic differentiation of USSC. We experimentally identified specific osteo-inhibitory proteins as regulatory targets for these miRNAs in reporter gene analyses and in direct measurements of target protein abundance. Functional analyses demonstrated that miR-26a, miR-26b and miR-29b positively modulate osteogenic differentiation of USSC, most likely by downregulating osteo-inhibitory target proteins. Together with our previous studies on neuronal lineage differentiation [38], [39] these findings further support the notion that differentiation of the unique somatic stem cell type USSC follows established biochemical pathways wherein miRNAs are important regulatory molecules.

Generation and osteogenic lineage differentiation of USSC
USSC lines were isolated from human cord blood and characterized in a designated unit in our institute under GMP conditions as described in detail in [33] and [63]  and were supplied to us for this study. Informed consent was obtained from each participant.
USSC lines SA5/73 and SA8/25 were induced to osteogenic differentiation on addition of DAG (dexamethasone, ascorbic acid, ß-glycerolphosphate). Cells were incubated for 7 days and osteogenic differentiation was assessed using Alizarin-Red staining as described [33]. Calcium release was measured following incubation of cells in 6M HCl for 24h at 37°C; calcium content in the supernatant was analyzed using the Calcium Colorimetric Assay (BioVision) according to the manufacturer's instructions followed by normalization to the protein content of the sample.

miRNA expression analysis
Small and large RNA fractions were isolated from native and differentiated USSC lines SA5/73 and SA8/25 using the Ambion mirVana miRNA Isolation kit (Applied Biosystems, Darmstadt, FRG) according to the manufacturer's instructions; adherent USSC were directly lysed without cell trypsinization. Total RNA for small RNA sequencing was prepared using TRIzol reagent (Invitrogen, FRG). miRNA expression analyses were performed on small RNA fractions using the TaqMan miRNA Megaplex array (pool A, Applied Biosystems, Darmstadt, FRG) [43] according to the manufacturer's instructions. Briefly, 10ng small RNA fraction was reverse transcribed and preamplified for 12 PCR cycles, with subsequent TaqMan-probe based array-amplification for 40 additional PCR cycles. Raw Ct-values were normalized to U6 RNA data and ddCt as well as 2 -(ddCt) data were calculated.

Experimental verification of bioinformatic target gene predictions
PCR-products of full length or fragments of 3 0 -UTRs as well as double-stranded oligonucleotides covering the predicted miRNA binding sites on the target mRNA of interest were cloned at the 3 0 -end of firefly luciferase ORF in dual reporter (firefly and renilla luciferases) vector pmirGLO (GenBank accession FJ376737, Promega Mannheim, FRG) using restriction enzyme pairs SacI/ XbaI or SalI/XhoI. PCR primers, sense and antisense oligonucleotides, and 3 0 -UTR-and fragment-lengths are listed in Additional file 3. To normalize for effects of endogenous miRNAs on a given 3 0 -UTR, 100ng pmirGLO and pmirGLO/3 0 -UTR were transfected into 5×10 4 HEK293T cells using 0.5 μl Lipofectamine 2000 (Invitrogen, Karlsruhe, FRG). Pairwise cotransfections of 100ng empty pmirGLO with the 2.5 pmol miRNA mimic (Dharmacon, Bonn, FRG) of interest and pmirGLO/3 0 -UTR with the miRNA mimic of interest were performed to determine the influence of the given miRNA on the 3 0 -UTR. Firefly and renilla activities were measured 24h after transfection using Beetlejuice and Renillajuice reagents (PJK, Kleinblittersdorf, FRG). All transfection experiments were performed in at least two independent biological experiments with quadruple transfections each and statistical significances were calculated by a student's t-test, unpaired.

Real-time PCR analysis
Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNAs were reverse transcribed using M-MLV reverse transcriptase (Promega, Mannheim, FRG) and real-time PCRs were performed using the Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, St. Leon-Rot, FRG). Primers for CDK6 [64], CTNNBIP1 [65], SMAD1 [31] and HDAC4 (this work) used in RT-reactions are listed in Additional file 3. Results were analyzed using Step One Software v2.1 and statistical significances were calculated by a student's t-test, unpaired.
For functional analyses, USSC were cultivated on 24-well plates and transfected with 10pmol miRNA-mimic/well using Dharmafect1 reagent according to the manufacturer's instructions. Transfected cells were induced to osteogenic differentiation 24 h post transfection as described above. Differentiation was analyzed at day 7 by alizarin-red stainig and calcium released as described above.

Ethics statement
Work with USSC was approved by the Ethics Committee of the Medical Faculty, University Düsseldorf, study numbers 3436 and 2975.

Additional files
Additional file 1: Comprehensive miRNA expression data from native (d0) USSC SA5/73 and SA8/25 and osteo-differentiated (day 7) SA5/73 and SA8/25. This Excel file contains three separate sheets, "USSC SA573", "USSC SA825", and "Deep Sequencing". In the first two, raw Ct, U6-RNA normalized dCt, ddCt, and 2 -ddCt values (corresponding to fold changes of miRNA expression in day 7 osteo vs. native cells, in grey columns) are given for each miRNA analyzed. The sheet "deep sequencing" indicates results from deep-sequencing derived expression analysis of miR-26a and miR-26b in native USSC SA5/73, SA8/25, SA8/77, and SA4/101 and osteo-differentiated USSC SA8/77 and SA4/101. Total sequence reads for the miRNA indicated as well as frequency of specific sequence reads among all sequence reads are given. Additional file 3: Primers and oligonucleotides. This Excel file summarizes primers and sense-and antisense-oligonucleotides used for 3 0 -UTR cloning and qRT-PCR analysis. Sheet "3 0 UTR cloning": Primers for generation of PCR products from 3 0 -UTRs, corresponding NCBI accession numbers, and sense-and antisense-oligonucleotides used for experimental target validation of predicted target proteins. Restriction sites used for cloning are given in bold as well as sticky and recessed ends synthesized into oligonucleotides. "Ⓟ" denotes the phosphate group added to the 5 0 -end of oligonucleotides. Mutations in oligonucleotide sequences corresponding to the seed of microRNAs are given in red. Sheet "qRT-PCR": Forward and reverse primers used for RTreaction.


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Mathematical Modelling of Polyamine Metabolism in Bloodstream-Form Trypanosoma brucei: An Application to Drug Target Identification

We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.

Part 1. Modelling the ODC-catalyzed reaction reversibly
One of the refinements we made during the model building procedure was to represent the ODC-catalyzed reaction with the reversible rate law, as defined in Equation 1 in the section of Materials and Methods in the main text (also given below).
In this final model (Model V5 in Supplementary Table S2), we used the best set of parameter estimates obtained from solving Model V4, based on which we analytically derived the newly-added parametersthe equilibrium constant K ODC eq and the half-saturation constant K ODC mP ut . Due to the lack of information on these two parameters, we assumed the half-saturation constant K ODC mP ut to hold the same value as the known parameter K ODC mOrn and we analytically derived the equilibrium constant K ODC eq against the experimental observations of AdoMetDC RNAi induction and prozyme knockout.
We illustrate in Figure ST1 the effect of different values of parameter K ODC eq on the dynamic behaviour of Put in the cases of AdoMetDC RNAi induction and prozyme knockdown. This figure shows that our choice of K ODC eq (which was assigned the value of 5) best reflects the dynamics and exact concentration values of Put under the perturbed conditions and when compared with simulation results from modelling ODC irreversibly, Put dynamics could reach steady state gradually over time. When AdoMetDC or prozyme were inhibited, direct downstream metabolites of the ODC-catalyzed reaction -Spd and T SH tot , were not different from the model simulations where ODC was described with the irreversible rate law, in terms of both the basal condition and dynamic behaviours (see Figure ST2), which is also the case under all other perturbed conditions considered in the manuscript. The similar effect on model behaviours exerted by varying K ODC eq was also seen on parameter K ODC mP ut (varied between 0.1 to 100 fold of the assumed value, 350 µM). We also found that there is no obvious difference in model behaviour when the term representing production inhibition by Put is present or not, which is most likely due to the weak inhibitory effect exerted by Put.

Part 2. The postulated regulatory correlation of enzyme SpdS on ODC
We postulated in the main text that the regulation of enzyme SpdS on ODC is helpful for avoiding Put accumulation.
The regulation of SpdS on ODC is reflected by the term e (−λ SpdS · t) as defined in Equation ST1. In the model where the regulatory effect is not considered, parameter λ SpdS is set to zero and the maximum velocity of ODC becomes time-invariant, whereas the model having this effect enabled allows the maximum velocity of ODC to vary with respect to changes in SpdS activity (in this case a value of 0.0016 is assigned to parameter λ SpdS as used in the main text).
We examined this postulation by comparing Put dynamics (over a simulated time span of 6 days) with ODC modelled reversibly against the situation where ODC is modelled irreversibly, which is further compared in the presence and absence of the postulated regulation of SpdS on ODC. Figure ST3 shows that, in the absence of the postulated regulation, the reversible ODC kinetics helped bring down Put level in response to SpdS knockdown compared with the result from the irreversible kinetics; however, the resulting concentration of Put still increased nearly 20 fold of the control level. These comparison results imply that the postulated regulation of SpdS on ODC is still necessary in preventing Put accumulation regardless and the conclusion remains the same with different values of the equilibrium constant.


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Hyperresponsiveness of mice deficient in plasma-secreted sphingomyelinase reveals its pivotal role in early phase of host response

Plasma secretion of acid sphingomyelinase is a hallmark of cellular stress response resulting in the formation of membrane embedded ceramide-enriched lipid rafts and the reorganization of receptor complexes. Consistently, decompartmentalization of ceramide formation from inert sphingomyelin has been associated with signaling events and regulation of the cellular phenotype. Herein, we addressed the question of whether the secretion of acid sphingomyelinase is involved in host response during sepsis. We found an exaggerated clinical course in mice genetically deficient in acid sphingomyelinase characterized by an increased bacterial burden, an increased phagocytotic activity, and a more pronounced cytokine storm. Moreover, on a functional level, leukocyte-endothelial interaction was found diminished in sphingomyelinase-deficient animals corresponding to a distinct leukocytes’ phenotype with respect to rolling and sticking as well as expression of cellular surface proteins. We conclude that hydrolysis of membrane-embedded sphingomyelin, triggered by circulating sphingomyelinase, plays a pivotal role in the first line of defense against invading microorganisms. This function might be essential during the early phase of infection leading to an adaptive response of remote cells and tissues.

Survival analysis
For survival analysis, aSMase WT and KO animals (n = у 15 per genotype) were monitored every 3 h after peritonitis induction over 72 h. During this experiment, animals received volume resuscitation (balanced saline solution) of 25 µl/g BW twice per day subcutaneously over the period of the survival study. Logrank statistics were used for data analysis.

Bacterial burden in blood and organs
Blood and organs were collected from deeply anesthetized WT and KO mice (n = у 4 per genotype per time point) 6 h after PCI. Sodium citrate was used as an anticoagulant. After blood was harvested, whole liver and lungs were collected in a sterile manner. To avoid bacterial contamination, collected organs were briefl y washed in 70% ethanol. The organs were later homogenized in sterile 0.9% saline solution. Enriched brain-heart infusion (2 ml) was added to each sample, and dilutions up to fi ve orders of magnitude were prepared. These dilutions were plated on Schädler's and blood agars and incubated under aerobic and anaerobic conditions for 48 h. The number of bacterial colonies was counted. Data were evaluated with consideration of blood volume or normalized to wet weight of organs.

Blood analysis
Whole blood was collected by heart puncture from shamtreated and septic animals (n = у 15 per genotype per time point) 6 h after peritonitis induction. Leukocyte and platelet counts were determined for aSMase WT and KO animals using the automated veterinary hematology analyzer Poch-100iv-Diff (Sysmex, Leipzig, Germany).
Leukocyte subpopulations were also analyzed in whole blood collected from both genotypes. Blood smears were prepared and stained using Giemsa. The slides were then processed for analysis using the automated analyzer Cellavision DM 96 (Sysmex, Leipzig, Germany).
Serum was collected from aSMase WT and KO animals at three time points (n = у 8 per genotype per time point) for analysis of laboratory markers of organ dysfunction using the clinical chemistry analyzer Fuji Dri-Chem 3500i (Sysmex, Leipzig, Germany) according to the manufacturer's instructions.

Measurement of phagocytotic activity
Phagocytotic activity of granulocytes and monocytes was determined by fl ow cytometry after incubating whole blood samples with FITC-labeled and opsonized Escherichia coli (Phagotest; Becton Dickinson, Franklin Lakes, NJ). Briefl y, 6 h after PCI, blood was collected from sham and septic WT and KO animals (n = у 4 per genotype per time point) using Li-heparin as an anticoagulant. Diluted blood was incubated with labeled E. coli (1.7 × 10 9 /ml) for 20 min at 37°C followed by washing, lysis, and staining. The samples were measured by fl ow cytometry according to the manufacturer's instructions (software: CellQuest Pro, version 5.1.1). After "gating" FITC-positive monocytes and granulocytes, groups were compared with each other.
The secreted acid sphingomyelinase (aSMase) is the only isoform associated with extracellular hydrolysis of sphingomyelin and has been found to be secreted by macrophages, human skin fi broblasts, and human vascular endothelial cells ( 13,14 ). Apical secretion by endothelial cells was stimulated by a variety of proinfl ammatory mediators and cytokines, such as tumor necrosis factor (TNF)-␣ , interleukin (IL)-6, IFN-␤ , or IL-1 ␤ , but also by membrane constituents of gram-negative bacteria ( 14 ). It was previously shown that patients with chronic or acute systemic infl ammation or infection such as sepsis exhibited an enhanced sphingolytic activity in comparison to controls ( 15,16 ). During progression of the disease, a further increase in the activity of secreted aSMase parallel to severity of illness predicts a fatal outcome ( 15 ). Furthermore, in mononuclear cells of septic patients, the concentration of ceramide was increased and correlated positively with plasma TNF-␣ levels and was higher among patients who developed dysfunction of remote organs ( 17,18 ). A 2-to 3-fold rise in plasma sphingolytic activity was also observed in animal models after application of endotoxin or proinfl ammatory cytokines ( 15,19 ). As the inert membrane constituent, sphingomyelin is preferentially distributed in the outer leafl et of cellular membranes. After infl ammation-triggered secretion, aSMase gains unlimited access to its substrate and produces huge amounts of ceramide, affecting membrane organization ( 20,21 ). In an instantaneous manner, ceramide induced the release of reactive oxygen species in endothelial cells, decreasing endothelium-dependent vasorelaxation ( 22,23 ). These observations support the concept that secreted aSMase is a key player in cytokine secretion during host response. This sequence of cellular response to infl ammatory stress also put forward the hypothesis that an unfavorable outcome of sepsis-associated remote organ failure might be linked to regulating the secretion and activation of aSMase.

Animals
Transgenic mice with a defi ciency in aSMase function (knockout [KO]) (aged 8-10 weeks) and their wild-type (WT) littermates (aged 8-12 weeks) were used in this study ( 24 ). Animals were randomly selected for each experiment, were maintained under artifi cial day-night conditions at room temperature, and received a standard diet and water ad libitum. All experiments were performed in accordance with the German legislation on protection of animals and with permission of the regional animal welfare committee. Mice were anesthetized with a combination of ketamine and xylazin injected intraperitoneally before any surgical procedure. During the surgical procedures, body temperature was maintained at 37°C.

Sepsis model
Sepsis was induced using the peritoneal contamination and infection (PCI) model as previously described ( 25 ). In the present study, peritonitis was induced by the injection of a 1:4 diluted Chromatographic separations were obtained using a ZORBAX Eclipse XDB-C18 as described previously (Agilent Technologies, Waldbronn, Germany) ( 28 ). The injection volume per sample was 10 µl. Best results were obtained with an isocratic elution (acetonitrile/2-propanol 3:2 with 1% formic acid) at a fl ow rate of 1 ml/min for 15 min. For mass spectrometric measurements, we used the following ion source conditions and gas settings for positive LC-MS/MS: sheath gas temperature: 400°C, sheath gas fl ow: 9 l/min, nebulizer pressure = 30 psig, drying gas temperature = 350°C, drying gas fl ow = 8 l/min, capillary voltage = 2,000 V, fragmentor voltage = 355 V, nozzle voltage = 2,000 V. All ceramides gave the same fragment ion of m/z 264.27 at different retention times depending on their chain length. Quantifi cation was performed using Mass Hunter software (Agilent). Calibration curves of reference ceramide values were performed from 1 to 100 pmol and were constructed by linear fi tting using the least squares linear regression calculation. The resulting slope of the calibration curve was used for calculating the concentration of the respective analyte in the unknowns.

Measurement of aSmase activity in plasma
Citrated plasma was collected from aSMase WT and KO animals (n = у 6 per genotype per time point) 6 h after the insult. aSMase activity was determined by the hydrolysis of fl uorescently labeled sphingomyelin (NBD-SM; Molecular Probes, Eugene, OR) as a substrate, chromatographic product separation, and image analysis as described previously ( 15,29 ). Plasma samples were diluted 1:10 with the incubation buffer (sodium acetate, pH 5.0) before analysis. The fi nal composition of the reaction mixture was 12 l plasma, and the extraction was carried out using the Speed-Vac concentrator Plus (Eppendorf, Hamburg, Germany).

Determination of reactive oxygen species formation in circulating granulocytes
Whole blood was collected from aSMase WT and KO animals at baseline and 6 h after sepsis induction (n = 6 per genotype per time point). Granulocytes were labeled with a PE-labeled Ly-6G antibody (eBioscience Inc., San Diego, CA). For the detection of reactive oxygen species (ROS), we used the FagoFlowEx kit according to the manufacturer's instructions (EXBIO, Vestec, Czech Republic). Briefl y, blood samples were incubated with E. coli and dihydrorhodamine-123. After red blood cell lysis, the resuspended leukocytes were analyzed for detection of ROS formation using fl ow cytometry according to the manufacturer's instructions (software: CellQuest Pro, version 5.1.1). The fl uorescence signal was expressed as MFI. We measured MFI for sham samples and those incubated with E. coli. Results are represented as a stimulation index, which was calculated by normalization of the E. coli mix against the control.

Gene expression analysis and qPCR
Whole blood and tissue samples (liver and lung) were collected from aSMase KO mice and their WT littermates 6 and 24 h after sepsis (n = 2 per genotype per time point). Organs were shock frozen for later isolation of total RNA. Samples were analyzed by a pangenomic microarray mouseWG-6 v1.1 expression bead chips using an iScan platform (Illumina, San Diego, CA) measuring the variation of expression rate of >42.000 transcripts. Subsequent to biostatistical analysis, validation of expression data of representative genes was performed by quantitative real-time PCR. Data were normalized to unchanged reference transcripts ( Actb , Gapdh , Hprt ), and relative changes were plotted against sham-treated WT littermates.

Statistics
Data were obtained from randomly selected animals for each time point. Analyses were generally performed in an independent

Granulocyte-migration studies
Livers were collected from WT and KO animals (n = у 4 per genotype per time point) at three time points. Transmigration of leukocytes into liver tissue was evaluated by specifi c staining of granulocytes in liver sections. These sections (3 µm) were deparaffi nized with decreasing ethanol series. The LEUCOGNOST ® NASDCL kit (Merck Millipore, Darmstadt, Germany) was used for staining transmigrated granulocytes following the user's manual. At 400× magnifi cation, stained cells were counted in at least 20 visual fi elds by two independent and experienced scientists blinded to treatment or genotype.

Intravital microscopy
In vivo imaging of the liver was performed with an epifl uorescence microscope as described in detail previously ( 26 ) to evaluate leukocyte endothelium interactions 6 h after insult in sham-treated and in septic aSMase WT and KO animals (n = 5 per genotype per time point). Leukocytes were labeled in vivo with carboxyfl uorescein diacetate succinimidyl ester (1 mg/kg BW) administered via a central line 10 min before surgical instrumentation. At least fi ve regions of interest per mouse of either postsinusoidal venules (40× objective lens) or sinusoids (10× objective lens) were recorded over 30 s and 5 s, respectively (GFP fi lterset, 470-495 nm excitation and 525-550 nm emission band pass fi lters). Leukocytes were considered as "rollers" when they rolled across the endothelium or stuck to the endothelium for a few seconds and then detached. Leukocytes were considered as "stickers" when they remained stuck to the endothelium throughout the observation period. The total number of leukocytes in the postsinusoidal venules was calculated per mm 2 endothelial surface (length of observed vessel segment × diameter × ). Rollers and stickers in postsinusoidal venules were calculated per 100 counted leukocytes. Stickers in sinusoids as well as transmigrated leukocytes were calculated per mm 2 liver surface ( 27 ).

Protein expression on leukocytes
Expression of surface proteins CD49d, CD62L, and CD11b was analyzed using fl ow cytometry according to the manufacturer's instructions (software: CellQuest Pro, version 5.1.1). Briefl y, whole blood was collected from aSMase WT and KO animals (n = 9 per genotype per time point). Samples of whole blood were separately incubated with FITC-labeled CD49d, CD62L, and CD11b antibodies. The geo-mean of fl uorescence intensity (MFI) of all gated leukocytes was documented.

Determination of ceramide content in circulating leukocytes
Whole blood was collected from aSMase WT and KO animals at baseline and 6 h after sepsis induction (n = у 12 per genotype per time point). The ceramide pattern was fi xed by the addition of an unspecifi c, broad-spectrum inhibitor of hydrolases (diisopropylfl uorophosphate) during the lysis of red blood cells. Washed and pelleted leukocytes were pooled (to obtain 1-2 million cells per sample; six samples per group) and resuspended in methanol. After addition of 20 pmol C17-ceramide, chloroform, methanol. and distilled water to the cells followed by intense vortexing and centrifugation, the lower organic phase was obtained for further analysis. Lipids were then resolved in methanol after evaporation of the organic phase via the SpeedVac SC201 ARC vacuum system (Thermo Fisher Scientifi c GmbH, Dreieich, Germany).
Analysis was performed using a quadrupole/time-of-fl ight mass spectrometer (Agilent, Waldbronn, Germany) connected to a rapid-resolution liquid chromatograph. High-purity nitrogen for the mass spectrometer was produced by a nitrogen generator (Parker Balston, Maidstone, UK). compared with WT animals. CFUs of anaerobes did not signifi cantly increase in the KO animals 6 h after sepsis induction ( Fig. 2 ). As a negative control, vehicle-treated control mice (sham) exhibited no CFU in blood or in liver and lung.
The levels of proinfl ammatory mediators refl ect important markers of disease progression and are linked to the activation status of the innate immune system. After PCI, cytokine levels increased over time (baseline, 6 h, and 24 h) independent of the genotype ( Fig. 3 ). In WT and KO animals, 6 h after sepsis induction, cytokine levels of IL-6 and MCP signifi cantly increased in both genotypes. However, we measured a signifi cantly higher increase in the levels of the prototypical anti-and proinfl ammatory cytokines TNF-␣ and IL-10 in KO animals 6 h after sepsis induction compared with WT animals. At 24 h, a further increase in cytokine levels of TNF-␣ and MCP could be observed in KO animals compared with WT littermates without reaching signifi cance.
To evaluate the distinct cytokine profi les as a cause or consequence of sphingomyelinase activity and ceramide formation, we further investigated the infl ammatory response in a systems biology approach using a tissue-specifi c transcriptomal analysis. Across all tissues (circulating leukocytes, liver, and lung), more than 26,000 transcripts were found expressed. After normalization and hierarchical clustering analysis, only seven annotated transcripts exhibited a different expression compared with untreated animals of each genotype (e.g., Gpnmb , Hexa , CD83 ). In aSMase KO animals with peritonitis, transcripts were found differentially regulated when compared with WT littermates at both time points (n = 258 at 6 h; n = 315 at 24 h) ( Fig. 4A ). The transcripts for Tnfa , Nfkbia , Casp4 , Ccl4 , Ccl7 , Mapkapk2 , Csf3 , Stat3 , and Il1rn were found to be differentially regulated (supplementary Table I). These transcripts included those crucially involved in infl ammation, immune regulation, and infl ammatory response against invading pathogens. qPCR confi rmed a sharp increase of selected transcripts relevant to regulation of infl ammatory response, such as cytokines and cytokine-regulated transcripts (e.g., Tnf , Tnfaip3 ) in KO animals ( Fig. 4B ).
The data obtained from the quantifi cation of bacterial burden, cytokine profi ling, and transcriptome analysis urged us to further determine the total count of monocytes and their ability to phagocytize circulating pathogens. Although WT and KO groups revealed a signifi cant decrease in monocyte counts 6 h after the septic insult ( Fig. 5A ), phagocytotic activity signifi cantly increased in both genotypes. However, in KO animals, there was a signifi cantly higher increase in phagocytotic activity compared with WT littermates ( Fig. 5B ). In contrast, no changes in absolute counts or phagocytotic activity of granulocytes were detected (data not shown).
Characterizing the distinct phenotypes upon sepsis induction, we established parameters of host response, such as complete blood and platelet counts. In whole blood, leukocyte and platelet counts were signifi cantly reduced in both WT and KO groups 6 h after sepsis induction ( Fig.  5C, D ). Only minor changes in leukocyte subpopulations setting with respect to the measure. Thus, each animal refl ects a discrete set of data. For ethical reasons, when possible and appropriate different tissues, organs, and whole blood samples from one animal were used for several measurements (i.e., samples for determination of leukocyte counts, leukocyte subpopulations, and platelet counts in whole blood were obtained from the same animals). The same applied for the analyses of bacterial burden from blood and organs and granulocyte migration studies. All data were examined for normal distribution, and appropriate tests were applied. Statistical differences between groups were analyzed using ANOVA followed by Student-Newman-Keuls post hoc test or ANOVA (Kruskal-Wallis test) followed by pairwise multiple comparison procedures (Dunn's method) or Mann-Whitney rank-sum test. Data are given as boxplots, which show the median and the fi rst and third quartiles and whiskers (10th and 90th percentiles). P values below 0.05 were considered statistically signifi cant.

RESULTS
Discriminating values of ceramide content in circulating leukocytes highlighted the differences between the two genotypes. Despite the loss of sphingomyelinase, cellbound ceramide was found signifi cantly increased at baseline in KO animals compared with the WT littermates. Refl ecting the biological signifi cance of ceramide generation, levels of ceramide in leukocytes signifi cantly increased only in WT animals 6 h after the septic insult ( Fig. 1 ).
For characterization of the bacterial load, solid organs and blood samples collected from WT and KO mice were investigated for their content of aerobic and anaerobic bacteria. The two genotypes exhibited differences in bacterial load subsequent to sepsis. In KO animals, the number of colony-forming units (CFUs) of aerobes and anaerobes in liver as well as the aerobes in blood were signifi cantly higher compared with WT animals. Blood anaerobes were similarly high in both WT and KO groups. Surprisingly, the number of CFU in lungs of KO was lower the change in leukocyte counts in both genotypes and the increase in the number of rollers only in the WT animals ( Fig. 6C ). Similarly, the number of stickers in liver sinusoids signifi cantly increased in the WT animals from 38 to 66 sticking leukocytes per mm 2 liver surface 6 h after the septic insult. No signifi cant changes were observed in the KO group ( Fig. 6D, E ).
Analysis of leukocyte-endothelium interaction suggests that leukocytes are transmigrating from the circulation into the tissue. To confi rm this process, we estimated the number of granulocytes transmigrated into liver parenchyma by cell-specifi c immunohistochemical staining. Both genotypes revealed a signifi cant increase in the number of transmigrated granulocytes 6 h after the septic insult. However, these values signifi cantly dropped in both genotypes 24 h after the septic insult ( Fig. 7A ).
To explain the differences in leukocyte rolling and sticking between the genotypes, we characterized the activation status of the cells. For this purpose, we monitored the expression of surface proteins relevant for rolling and sticking were observed in the WT group ( Fig. 5E ). However, KO animals revealed signifi cant changes in the leukocyte subpopulations with a drop in lymphocyte and monocyte counts and a subsequent increase in the number of neutrophils ( Fig. 5F ).
Based on the differences in leukocyte subpopulation profi les between the genotypes, we were interested in the fate of leukocytes during sepsis. For this purpose, leukocyte-endothelial interaction in postsinusoidal venules and sinusoids were analyzed by intravital microscopy. The number of leukocytes signifi cantly decreased in both groups 6 h after the septic insult, which is in line with the quantifi cation of leukocytes in whole blood ( Fig. 6A ). However, there was a difference in the leukocyte-endothelial interaction between the genotypes highlighted by differences in rolling and attachment. The number of rollers (per 100 counted leukocytes) increased signifi cantly 6 h after PCI from 6.26 to 11.45% in WT animals. However, there was no increase in the number of rollers in the KO group ( Fig. 6B ). The representative image demonstrates Measurement of kidney function markers BUN and creatinine revealed differences between the two genotypes in sham-treated and septic animals. After the septic insult, BUN levels increased in WT and KO groups, reaching signifi cance only in the KO animals. The levels of BUN continued to signifi cantly increase at 18 h in both genotypes and more so in KO animals, reaching signifi cance. Levels of creatinine were signifi cantly higher in KO animals at the baseline and 6 h time points compared with WT littermates. These levels signifi cantly increased in both genotypes 18 h after the septic insult. This increase was more pronounced in the KO animals, reaching signifi cance ( Fig. 8B ).
Our model of abdominal cavity infection was found to be effective with respect to triggering severe infection and host response ( 25 ). With the absence of aSMase in the KO animals and the signifi cant increase in the activity in WT littermates after sepsis ( Fig. 9A ), we expected to observe a higher survival rate in KO animals compared with the WT animals due to the limited ceramide generation. Surprisingly, aSMase WT and KO animals showed a similar pattern in mortality with an onset of mortality at 21 h and an end-survival of approximately 20% ( Fig. 9B ).

DISCUSSION
Here we characterize the role of aSMase in the early phase of host response to life-threatening infection. For this purpose, we used the model of PCI to obtain a severe polymicrobial sepsis that closely refl ects the clinical situation as observed in around 50% of patients admitted to the functions. Six hours after sepsis induction, the expression of CD49d was signifi cantly down-regulated in KO animals. There was no change in the expression of CD49d in WT animals over time ( Fig. 7B ). A slight numerical increase in the expression of CD62L was only observed in WT animals 6 h after the septic insult without reaching signifi cance ( Fig. 7C ). The expression of CD11b was signifi cantly upregulated only in WT animals 6 h after sepsis ( Fig. 7D ).
When determining the release of ROS from circulating leukocytes as a measure of the activated phenotype, we found a signifi cant 2-fold increase 6 h after sepsis induction in both genotypes compared with baseline (shamtreated animals). No signifi cant differences were monitored with respect to aSMase defi ciency ( Fig. 7E ).
The development of organ dysfunction is a major consequence of transmigration of activated leukocytes into the tissue. Therefore, we measured established markers of hepatic and renal dysfunction or injury during the course of our sepsis model. At baseline, there were no differences between the genotypes in liver markers of cholestasis and hepato-cellular injury (aspartate transaminase [AST], alanine transaminase [ALT], gamma glutamyl-transferase [GGT], and total bilirubin [T-Bil]). Six hours after the septic insult, there were changes in the markers of hepatocellular injury where AST and ALT were signifi cantly increased only in KO animals but dropped 18 h after the septic insult. There was a signifi cant increase in levels of T-Bil in WT and KO animals in the late phase. The levels of T-Bil were slightly more increased in KO animals compared with WT animals without reaching significance. No changes in the levels of GGT were observed in WT and KO animals 6 and 18 h after sepsis ( Fig. 8A ).  well as a cytokine storm ( 27,(31)(32)(33). In the present study, the high bacterial burden in liver and blood and the rise in the levels of cytokines as well as parameters of organ dysfunction 6 h after the septic insult confi rm an infectious focus and the development of severe sepsis. With the development of sepsis, secreted aSMase activity is increased and breaks down inert cell membrane-embedded sphingomyelin into highly active ceramide, which accumulates, forming lipid rafts. It has been recently speculated ICU due to sepsis, severe sepsis, and septic shock ( 30 ). This method involves the collection of stools from nonvegetarian donors followed by extensive microbiological characterization, preparation, and storage. This model refl ects a reproducible and standardized, nontrauma method that results in a diffuse peritonitis similar to the clinical situation in cases such as perforated viscus due to diverticulitis. An acute onset of the disease is followed by systemic infl ammation highlighted by an elevated bacterial burden as content in circulating leukocytes. Due to variation in the number of white blood cells analyzed, we normalized the ceramide values to protein content. In KO animals, levels of ceramide were signifi cantly higher compared with WT littermates but did not increase after sepsis induction. These ceramide levels at baseline (as well as after sepsis induction), resulting from unknown compensatory mechanisms, are enough to maintain a normal function at a healthy state but not under septic conditions. The missing increase of ceramide in the KO animals after sepsis outlines the pivotal role of this enzyme in the rapid and transient formation of ceramide. Here we describe that the increase in the activity of aSMase ( 12 ) is refl ected by a biological correlate. This increase might safeguard an appropriate cellular stress response but might also be, in an overwhelming fashion, a trigger of tissue damage ( 35 ). Without overinterpretation of our present data with the WT model, we could speculate that the increase in activity could be the course or the consequence with respect to tissue damage and the development of organ failure. Although the increase in sphingomyelinase activity might originate from (active) release by stimulated (endothelial) cells ( 20 ) or passively from the lysosomes of damaged tissues, the biological activity in circulation per se might be interpreted as a hallmark of systemic stress response. There are several reports addressing the abundance of lysosomal proteins, such as chitinase or granulocytic elastase, during sepsis ( 36,37 ). We also determined that the enzyme might be relevant for triggering cellular stress response in remote cells and tissues. During sepsis, the lack of lysosomal activity in transgenic mice might hinder an essential signaling pathway of cellular stress response, leading to macrophage activation and infl ammation due to sphingomyelin accumulation ( 38 ). To perform best practice procedures and to avoid a potential bias caused by substrate accumulation, we enrolled in our experimental setting animals aged 8-10 weeks ( 24 ) where this phenotype is not yet developed. On the other hand, aSMasedefi cient animals with cystic fi brosis appear to be more resistant to Pseudomonas infections with a decreased pulmonary infl ammation. However, the absence of lysosomal aSMase could play a role, together with the more prominent course of infection, in the development of a more pronounced organ dysfunction observed in the present study (panel 8) as compared with WT littermates. Thus, the absence of lysosomal activity could possibly obviate benefi ts obtained through the decreased levels of secreted aSMase ( 8 ).
We expected a better outcome with the KO model when compared with WT littermates. The results instead highlighted a crucial role of aSMase in the early phase of host response to sepsis. The complete loss of function model resulted in a hyperresponsive state due to signifi cantly higher bacterial burden mirrored by a more potent infl ammatory response, thereby resulting in a similar outcome as compared with WT littermates through different trajectories.
The importance of aSMase during the early phase of sepsis was highlighted by the analysis of bacterial burden, that in lipid storage diseases such as aSMase defi ciency, immune cells may not obviate an appropriate infl ammatory response ( 34 ).
To clarify a potential benefi cial or maladaptive role of aSMase in the host response to sepsis, we compared data obtained from animals with a complete loss of function model to their WT littermates. The difference between the genotypes was elucidated in the measurement of ceramide multinucleated macrophages, which also registered an increased production of chemokines ( 38 ).
As key humoral mediators, we investigated the cytokine pattern in plasma. We noticed an earlier anti-infl ammatory response in KO animals. Therefore, impairment of aSMase activation exhibited distinct effects on cytokine release. Moreover, kinetic and peak concentrations were in part different from the WT at the two observation time points in our study. This is an interesting observation because the pharmacological inhibition of aSMase has been characterized with a decreased transcription and secretion of TNF-␣ directly acting on relevant peripheral cell types in a polymicrobial sepsis model ( 42,43 ). Also, in an in vitro model of cellular stress response triggered by prototypic electrophiles as triggers of cellular damage, aSMase activation resulted in an increase in the expression of TNF-␣ , amplifying generation of IL-6. Both knock-down and pharmacological inhibition abrogated cytokine response under these conditions ( 44 ), whereas the overexpression of aSMase, resulting in ceramide generation, induced TNF-␣ sensitivity measured by IL-6 secretion ( 44 ). which suggested a pivotal role of the enzyme in eliminating invading bacteria. Six hours after sepsis induction, bacterial burden of aerobes and anaerobes in the liver was signifi cantly higher in KO animals compared with WT littermates. KO animals also registered a signifi cantly higher CFU of aerobes in blood compared with WT animals. This suggests an inability of aSMase KO animals to effi ciently eliminate microorganisms. Ceramide produced by activated aSMase plays a crucial role in innate immune response with respect to the elimination of microorganisms ( 39 ). Ching et al. (40) suggested that the absence of the enzyme left murine animals more susceptible to Sindbis virus-induced fatal encephalomyelitis. Unlike WT animals, aSMase KO mice were unable to eliminate P. aeruginosa from the lung upon acute pulmonary infection and died from sepsis a few days later ( 41 ). Without reaching significance, we registered a lower CFU in lungs of KO animals compared with WT littermates. This might be explained by the altered lung pathology in aSMase KO animals characterized by a signifi cant increase in cell counts in the pulmonary spaces consisting mainly of enlarged and The hyper-responsive state in the KO animals revealed an overall higher bacterial burden in the liver compared with the lung. This more pronounced infl ammatory response of the liver along with its pivotal role in host response in combination with higher organ mass, the exorbitant bacterial burden, and the cytokines levels in blood as well as the distinct role and response pattern of each organ during host response ( 46 ) enlighten the trajectory of the expression patterns in the different organs.
A panel of the relevant transcripts for immune regulation and bacterial elimination were differentially expressed in the lungs of aSMase-defi cient animals compared with the WT littermates. Lysozyme and the astrocyte-elevated gene 1 (Aeg1, syn. Mtdh) were only found up-regulated in the lung tissue of aSMase-defi cient animals (cluster 3). Aeg1 is known as a TLR-4 specifi c regulator of NF-Bmediated cytokine synthesis ( 47 ). On the other hand, lysozyme (EC 3.2.1.17) is one of the most important secreted proteins hydrolyzing certain mucopolysaccharides of bacterial cell walls ( 48 ). Also functioning as primary regulators of ROS generation and essential components of the microbicidal oxidase system of phagocytes, both ␣ and ␤ subunits of the membrane-bound cytochrome b oxidase were found down-regulated in KO animals. These observations support the hypothesis that, beyond the altered lung physiology in KO animals, a genotype-specifi c regulation of transcripts involved in recognition and elimination of bacteria might have contributed to a better clearance of bacteria in the lungs.
In circulating leukocytes, expression of transcripts encoding for cyto-and chemokines as well as their receptors was found up-regulated in both genotypes during the course of sepsis, corresponding to the results of cytokine measurements in the blood.
We compared the Tnfa transcript expression in an ex vivo LPS-triggered stimulation of expression and release of the cytokine ( 45 ) with systemic TNF levels and organ-specifi c expression in our model of polymicrobial infection. We found an unaffected expression in the lung homogenates 6 h after peritonitis, similar to unchanged expression of Tnfa in peritoneal macrophages ( 45 ). On the other hand, in the present study, we registered an up-regulated expression of Tnfa in whole tissue homogenates of the liver A: Laboratory markers of liver dysfunction. * P < 0.05 versus sham; # P < 0.05 between genotypes; § P < 0.05 versus 6 h sepsis. AST, aspartate transaminase; ALT, alanine transaminase; GGT, gamma glutamyl-transferase; T-Bil, total bilirubin. B: Laboratory markers of kidney dysfunction. * P < 0.05 versus sham; # P < 0.05 between genotypes; § P < 0.05 versus 6 h sepsis. BUN, blood urea nitrogen; Crea, creatinine. their intracellular elimination ( 53 ). These results support our concept of the crucial role of aSMase in the early response to infection.
Although both genotypes registered a drop in total leukocyte counts 6 h after sepsis, we observed a different subpopulation profi le in KO animals compared with WT animals. At baseline, both genotypes had similar subpopulation profi les. However, KO animals revealed a drop in lymphocytes and monocytes, mirrored by an increase in neutrophils 6 h after sepsis induction. With the development of sepsis, bacteria migrate into the tissue, inducing an infl ammatory response. This includes the recruitment of neutrophilic granulocytes, as fi rst line of defense, to the focus of infection and infl ammation to combat invading microorganisms ( 54,55 ). In the present study, KO animals have an overwhelming infection due to their inability to effi ciently eliminate the microorganisms. This situation could explain the increased production of neutrophils in KO animals after sepsis as a combat strategy to overcome the overwhelming infection.
Another interesting fi nding was the differentiated leukocyte expression in KO animals compared with their WT littermates. Indeed, 6 h after sepsis induction, the expression of CD49d was signifi cantly down-regulated in KO animals, whereas CD11b was signifi cantly up-regulated only in WT animals. The missing increase in CD11b expression in KO animals might be due to their inability to produce ceramide and a subsequent lack of lipid raft formation necessary for CD11b recruitment ( 56 ).
This described phenotype might be one cause of a different course of the disease in the KO animals. The different leukocyte expression profi le of CD49d and CD11b resulted in a different leukocyte-endothelium interaction between the two genotypes. CD49d, CD62L, and CD11b expression are essential for the rolling and sticking phenomenon leading to leukocyte-endothelium interaction ( 57,58 ). In the present study, WT animals exhibited a signifi cant increase in leukocyte rolling and sticking in the liver sinusoids and postsinusoidal venules 6 h after sepsis induction attributed to the up-regulation of expression markers on leukocytes. The down-regulation or missing increase in expression of these markers in the KO animals consequently resulted in an unchanged leukocyte-endothelium interaction between sham-treated and septic animals. Sepsis causes important changes in the liver, with alterations in hepatic macro-and microcirculation as well as leukocytes-endothelium interaction ( 59 ). The latter leads to neutrophil sequestration with subsequent local release of proinfl ammatory mediators and therefore plays a role in the development of liver dysfunction, which is a crucial event in the development of sepsis ( 27 ). It is unknown whether the phenotype of sequestered aSMase-defi cient leukocytes have a rather benefi cial or detrimental role with respect to development of organ dysfunction.
The proinfl ammatory cytokine TNF-␣ was found to be crucially involved in the generation of ROS (specifically H 2 O 2 ) in a ceramide-dependent manner ( 60 ). The impotence in rapid ceramide generation in leukocytes of aSMase KO animals might be adjusted by the 2-fold Sepsis induced up-regulation of cathepsin G ( Ctsg ) was found diminished in aSMase-defi cient mice. Ctsg is a serine protease participating in the killing and digestion of engulfed bacteria and regulates phagocytosis, degranulation, and ROS release ( 49,50 ). The down-regulation of Ctsg in septic aSMase KO animals could therefore explain the inability of leukocytes to effectively eliminate phagocytised bacteria, which resulted in an overwhelming infection.
The dynamic, insult-specifi c variations of platelet and leukocyte counts were acknowledged as critical hints with respect to disease progression. Similar to the course in patients with sepsis, a signifi cant drop in leukocyte and platelet counts was observed in both genotypes 6 h after sepsis induction. With the progression of systemic infl ammation and infection, leukopenia and thrombocytopenia, which may function as indicators of disseminated intravascular coagulation, occur ( 50,51 ). Further studies are needed to elucidate the proposed mechanism and to understand the role of aSMase in platelet activation and thrombocytopenia during sepsis.
The observation of leukopenia in WT and KO animals 6 h after sepsis induction raised the question of the fate of leukocytes during sepsis. This drop in cell count was confi rmed with intravital microscopy of the liver where both genotypes exhibited a decrease in leukocytes in circulation after sepsis, which suggested the transmigration of the cells into the remote tissues. Indeed, 6 h after the septic insult, extravasation of leukocytes was refl ected by a signifi cant increase in transmigrated granulocytes into liver tissue in WT and KO animals. In patients with sepsis, the liver plays a principle role as a source of infl ammatory mediators and acts as a remote organ for the effects of circulating infl ammatory mediators released from other tissues. Transmigration of neutrophilic granulocytes can result from microcirculatory changes and leukocyte-endothelium interaction ( 52 ). The number and the activation status of transmigrated granulocytes are held responsible for the induction and retention of hepato-cellular dysfunction. Further analysis of the phenotype of transmigrated granulocytes could provide insight into the expression profi le and the activation status of these leukocytes.
Speculating a distinct phenotype triggered by the enhanced cytokine storm and bacterial burden, we were able to detect this phenotype with a more elevated phagocytotic activity, a different leukocyte subpopulation profi le, and a differentiated leukocyte expression pattern in KO animals compared with WT littermates. Phagocytotic activity increased signifi cantly in both genotypes 6 h after the septic insult. However, this activity was more signifi cantly increased in KO animals, refl ected by a more elevated bacterial burden and higher levels of MCP in the blood. Although a more signifi cant increase in phagocytotic activity was registered in KO mice, these animals still registered higher CFU values in blood and organs. This might suggest the inability of granulocytes to control intracellular microorganisms due to impairment in effector mechanisms. The lack of aSMase is not essential for the uptake of bacteria by the phagocytes but could play a crucial role in invading microorganisms but could have detrimental effect during the late phase of sepsis. In line with this conclusion, stratifi cation of patients pretreated with pharmacological inhibitors for other underlying conditions (i.e., major depression) might be of great value. In fact, a chronological inhibition of aSMase has been presenting a more favorable outcome in chronic infectious diseases such as cystic fi brosis with striking differences to the KO model ( 67 ). These results and the data from the present study encourage further research using a pharmacological-inhibition model as well as clinical studies with inhibitors of aSMase in patients with well-defi ned sepsis.
increase in circulating TNF levels to yield a similar ROS release in the two genotypes as observed here during sepsis. Thereby, with respect to the cytokine pattern, the earlier and more pronounced response might refl ect a hyper-responsive status to overcome the inadequacy in ceramide-dependent pathways of host response.
The development of multiple organ injury is a major complication of sepsis and septic shock, increasing the lethality of sepsis to 70-80% ( 61,62 ). To investigate the course of sepsis in remote organs, which were primarily affected during the continuum of the disease, different laboratory markers of organ dysfunction were measured. Dysfunction of the liver is considered a crucial complication in the course of human sepsis, is often limiting for prognosis ( 63 ), and was refl ected here by a pronounced increase in markers of hepatocellular injury (AST and ALT) in KO animals in the early phase and in the marker of cholestasis (T-Bil) in the late phase of sepsis. Markers of hepatocellular injury dropped in the later phase of sepsis in KO animals, with an increase in the marker of cholestasis. In WT animals, markers of cholestasis and hepatocellular injury continued to rise in the later phase. A paradoxical up-regulation of a key determinant of transactivation of hepatic stellate cells was associated with the genetic ablation of aSMase, which was not restricted to the liver ( 64 ).
Similarly, acute renal failure is a common complication of critical illness. Levels of creatinine drastically increased (more so in KO animals) in the later phase of the disease. However, at baseline creatinine levels in KO animals were signifi cantly higher compared with their WT littermates. This suggests that our aSMase KO animals are prone to kidney preinjury ( 65,66 ).
The role of aSMase in diseases such as diabetes, cystic fi brosis, and chronic heart failure has been highly characterized in recent years ( 8,16,67 ). In the present study, we elucidated a crucial role of aSMase secretion through measurement of the stress-induced enzyme activity matched to accumulation of its product in circulating leukocytes in the early phase of sepsis. In a complete loss-of-function model, the pronounced amplitude of principle infl ammation mediators such as cytokines and bacterial burden supported the concept that this enzyme is crucial in the fi rst line defense against invading microorganisms. Loss of function resulted in a hyperresponsive state due to an overwhelming spread of microorganisms and the inability to effectively eliminate invading bacteria, leading to the generalization of host response.
The end result is highlighted in the survival analysis. Opposite to expectation, the complete loss-of-function model did not lead to a better survival. Instead, the overwhelming infection and infl ammatory response led to a high mortality in KO similar to WT animals, which overall highlights the pivotal role of aSMase in sepsis.
In patients with sepsis, the increase of aSMase might function as an adaptive response mechanism. In our translational model we provided further insight into this process. It is evident that the conservation of this enzyme is crucial during the early phase through elimination of