Mobilized Peripheral Blood versus Cord Blood: Insight into the Distinct Role of Proinflammatory Cytokines on Survival, Clonogenic Ability, and Migration of CD34+ Cells

Inflammation may play a role in cancer. However, the contribution of cytokine-mediated crosstalk between normal hemopoietic stem/progenitor cells (HSPCs) and their (inflammatory) microenvironment is largely elusive. Here we compared survival, phenotype, and function of neonatal (umbilical cord blood (CB)) and adult (normal G-CSF-mobilized peripheral blood (mPB)) CD34+ cells after in vitro exposure to combined crucial inflammatory factors such as interleukin- (IL-) 1β, IL-6, tumor necrosis factor- (TNF-) α, or tissue inhibitor of metalloproteinases-1 (TIMP-1). To mimic bone marrow (BM) niche, coculture experiments with normal BM stromal cells (BMSCs) were also performed. We found that combined inflammatory cytokines increased only the in vitro survival of CB-derived CD34+ cells by reducing apoptosis. Conversely, selected combinations of inflammatory cytokines (IL-1β + TNF-α, IL-6 + TNF-α, and IL-1β + TNF-α + TIMP-1) mainly enhanced the in vitro CXCR4-driven migration of mPB-derived CD34+ cells. TNF-α, alone or in combination, upregulated CD44 and CD13 expression in both sources. Finally, BMSCs alone increased survival/migration of CB- and mPB-derived CD34+ cells at the same extent of the combined inflammatory cytokines; importantly, their copresence did not show additive/synergistic effect. Taken together, these data indicate that combined proinflammatory stimuli promote distinct in vitro functional activation of neonatal or adult normal HSPCs.


Introduction
Hemopoietic stem/progenitor cell (HSPC) activation and retention are modulated by the bone marrow (BM) niche where they are located. In response to inflammation and/or BM injury, long-term quiescent hemopoietic stem cells (HSCs) are efficiently recruited into the cell cycle progression returning back to quiescence after reestablishment of homeostasis [1,2]. Inflammation is a fundamental response that protects tissues from damage and preserves internal homeostasis. However, chronic inflammation may hinder functionality of different tissues and has been suggested to cover a key role in cancer [3].
Proinflammatory cytokines are emerging as key regulators of steady-state and infection-driven hemopoiesis. Recent findings contributed to highlight how HSPC fate could be dictated by inflammatory factors in the BM microenvironment as HSPCs may actively respond to danger signals and proinflammatory cytokines [4,5]. However, excessive chronic signalling can have negative effects on HSPC regulation and function [6]. Moreover, abnormalities in the inflammatory signalling pathways have been discovered in both preleukemic and leukemic diseases [7]. BM mesenchymal stromal cells (BMSCs) are one of the most important components of the BM microenvironment. They respond to various microenvironment stimuli by changing their secretory capacity and displaying immune-suppressive activity through direct or indirect production of prostaglandin E-2, indoleamine 2,3-dioxygenase, interleukin-(IL-) 10 [8][9][10], and soluble receptors for IL-1 and tumor necrosis factor-α (TNF-α) [11]. However, a crosstalk between HSPCs and the stromal cells may also create a proinflammatory environment that promotes malignant transformation and disease progression [12]. In such process, several factors and pathways have been implicated but it is not clear how inflammation could affect or transform HSPCs. Understanding the direct cellular target(s) of proinflammatory cytokines is a critical step to better clarifying how HSCs/HSPCs are regulated in the BM niche.
Granulocyte colony-stimulating factor-(G-CSF-) mobilized peripheral blood (mPB) and umbilical cord blood (CB) are two of the current sources of HSPCs for transplantation in hematological malignancies [13]; however, insights into the effects mediated by inflammation on neonatal and adult HSPCs are still elusive. In the last years, several phenotypic and functional differences between CB and mPBderived HSPCs have been described [14][15][16][17][18][19]. However, so far, studies analyzing the adaptations of HSPCs from these two sources to inflammatory cytokines were focused on a limited number of cytokines which were individually tested [20][21][22][23][24].
To mirror the in vivo inflammatory microenvironment, here we investigated the role of combined crucial proinflammatory cytokines (IL-1β, TNF-α, IL-6, and tissue inhibitor of metalloproteases (TIMP-1)) on the in vitro functional behavior of CB-or mPB-derived CD34 + cells in the presence or absence of BMSCs.

Materials and Methods
2.1. Sample Collection. CB samples (n = 14) from normal fullterm deliveries were provided by the Cord Blood Bank of the University Hospital of Bologna after written informed consent. mPB samples (n = 14) were obtained from hemopoietic stem cell transplantation donors. This study was approved by the medical Ethical Committee of the University Hospital of Bologna and was conducted in accordance with the Declaration of Helsinki.

Cell Isolation.
Mononuclear cells (MNCs) were separated from CB and mPB samples (maximum after 1 day from harvesting) by stratification on Lympholyte-H 1.077 g/cm 3 gradient (Gibco-Invitrogen, Milan, Italy), followed by red blood cell lysis for 15 min at 4°C. MNCs were then processed on magnetic columns for CD34 + cell isolation (mean purity 94 ± 4%) (CD34 Isolation kit; Miltenyi Biotec, Bologna, Italy), as previously described [25], and treated with our combination of cytokines on the same day. In selected cases, CD34 + cells from CB or mPB were cryopreserved in liquid nitrogen and then thawed before testing with the combined inflammatory cytokines. Of note, to minimize the influence of freezing/thawing, only thawed CD34 + cells with a survival rate > 80% were used and the thawed CB/mPB cells were studied in the same experiment.
2.3. Phenotype of Circulating CD34 + Cells. The phenotype of circulating CD34 + cells was evaluated in CB and mPB samples by conventional flow cytometry, as previously described [20]. Antibodies used to characterize the CD34 + cells are listed in Supplementary Table 1. A minimum of 1 × 10 4 CD34 + cells were acquired by a BD Accuri C6 flow cytometer (Becton Dickinson, Milan, Italy). Analysis was performed excluding cellular debris in a SSC/FSC dot plot. The percentage of positive cells was calculated subtracting the value of the appropriate isotype controls. The absolute number of positive cells/μL was calculated as follows: percentage of positive cells × white blood cell count/100.
2.6. Migration Assay. Migration of CB/mPB-purified CD34 + cells was assayed in transwell chambers (diameter 6.5 mm, pore size 8 μm; Costar, Corning), as previously described [25]. In order to highlight the effects of the selected inflammatory cytokines, suboptimal CXCL12 gradient (120 ng/ mL) was employed. Briefly, 50 μL of RPMI 1640 plus 10% FBS containing 0.5 × 10 5 cells was added to the upper chamber and 150 μL of medium with or without CXCL12 ± IL-6 (10 ng/mL), IL-1β (1 ng/mL), TNF-α (10 ng/mL), and TIMP-1 (100 ng/mL) (alone or in combination) was added to the bottom chamber. After overnight incubation at 37°C in 5% humidified CO 2 atmosphere, inserts (upper chambers) were removed and cells transmigrated into the lower chamber were recovered and counted by the Trypan Blue exclusion test. The amount of migrated cells was expressed as a percentage of the input, applying the following formula: (number of migrated cells recovered from the lower compartment/total number of cells loaded in the upper compartment) × 100. In addition, migrated cells were assayed in methylcellulose-based medium for their ability to form hematopoietic colonies (as above described).

BMSCs Coculture Assay.
In selected experiments, CBand mPB-derived CD34 + cells were cocultured either without stromal support or directly seeded on a confluent layer of BMSCs in 96-well plates for 24 hours before use. CD34 + cells were then harvested and used to perform clonogenic and migration assay as above described; in addition, the selected cytokines were added to the bottom chamber. After overnight incubation at 37°C in 5% humidified CO 2 atmosphere, cells transmigrated into the lower chamber were recovered and counted, as previously described.
2.9. Statistical Analysis. Data are presented as mean ± SEM of at least three independent determinations. Statistical differences between groups were determined by a two-tailed Student t-test and one-or two-way ANOVA, as appropriate. All analyses were performed using GraphPad Prism software (version 6.0; La Jolla, CA, USA); p ≤ 0 05 was considered to indicate statistical significance.

Selected Combinations of Proinflammatory Cytokines
Promote the In Vitro Survival of CB-Derived CD34 + Cells. To test the role of proinflammatory factors on HSPCs, we firstly evaluated the in vitro survival of CB-and mPBderived CD34 + cells in the presence of IL-6, IL-1β, TNF-α, and TIMP-1, at concentrations previously shown by us to be effective in dose-response experiments [20].
These data suggest that CD34 + cells from CB are more actively responsive to inflammatory cues than their mPB counterparts; however, multiple combinations are required to promote their survival.
Taken together, these results demonstrate that selected combinations of inflammatory cytokines, along with the promotion of the survival of CB-derived CD34 + cells, stimulate the expression of CD13, which is an early and late myeloid marker.

Proinflammatory Cytokines Poorly Stimulate the
Clonogenic Output of CD34 + Cells from CB and mPB. To confirm the capacity of selected proinflammatory cytokines to drive the HSPCs toward a myeloid lineage, we performed clonogenic assay.
We therefore evaluated the CFU-C growth in the presence of proinflammatory factors in a two-by-two combination. As shown in Figure 2(a) and Supplementary Table 3, CFU-C growth from CB or mPB-derived CD34 + cells was not significantly affected by incubation with the proinflammatory cytokines as compared with untreated cells. When we used multiple combinations of cytokines ( Figure 2(b)), we found an increased clonogenic output in the presence of IL-1β + TNF-α + TIMP-1 for CBderived CD34 + cells and in the presence of IL-6 + IL-1β + TIMP-1 for the mPB counterparts (p ≤ 0 05, resp.). Comparing the two sources, IL-1β + TNF-α + TIMP-1 led to an increase in the clonogenic output of CD34 + cells (p ≤ 0 05).
These findings demonstrate that inflammatory cytokines slightly stimulate the hemopoietic functions of CB-and mPB-derived HSPCs.

Selected Combinations of Inflammatory Cytokines Mainly
Enhance the In Vitro Migration of mPB-Derived CD34 + Cells. It has been reported that CXCL12 is chemotactic for CD34 + cells and that the migratory behavior of CD34 + cells depends on their source of origin [28].
We firstly evaluated CXCR4 expression in CB-and mPB-CD34 + cells. As shown in Figure 3(a), the absolute number of CD34 + cells coexpressing CXCR4 was significantly increased in mPB as compared with the CB counterparts (p ≤ 0 01). Accordingly, we observed a slight increase in the geometric mean value of CXCR4 in mPB CD34 + cells as compared to CB-derived CD34 + cells (Figures 3(b) and 3(c)).
Subsequently, we compared spontaneous versus CXCL12driven migration and no significant differences were found in the migration rate when CXCL12 was added in culture (Figures 3(d) and 3(e)). This was probably due to the fact that, to highlight the effects of the inflammatory cytokines, low CXCL12 dose (120 ng/mL) was used.
These results demonstrate the capacity of selected combinations of inflammatory cytokines to increase the CXCR4-driven migration of CD34 + cells from mPB and CB. This effect was more prominent when mPB cells were assayed.
3.4. CD34 + Cells from mPB Show Increased Clonogenic Ability after In Vitro Migration toward Selected Combinations of Proinflammmatory Cytokines. Cell migration could be considered a selection of cells with different function and properties; for this reason, we tested the clonogenic potential of migrated CB and mPB CD34 + cells (Figure 2 and Supplementary Table 4).
Overall, here we demonstrate that selected combinations of proinflammatory cytokines promoted the CXCR4-driven migration of mPB-derived CD34 + cells with higher clonogenic ability and granulomonocytic potential.

The Copresence of BMSCs and Combined Inflammatory Cytokines Does Not Show Additive/Synergistic Effect in
Terms of Hemopoietic Supportive Role. To mimic the in vivo niche and to investigate the role of normal BMSCs in the inflammation-driven functional behavior of normal HSPCs, we cocultured CB-or mPB-derived CD34 + cells with BMSCs from HD in the presence or absence of combined proinflammatory cytokines.
As shown in Figure 5(a), the survival of CD34 + cells, either from CB or mPB, was significantly promoted by BMSCs (p ≤ 0 01 and p ≤ 0 05, resp.). Interestingly, cocultures with BMSCs decreased the percentage of apoptotic CB CD34 + cells compared with the monoculture counterparts, with 33.5 ± 8.8% of apoptosis in cocultures versus 76.71 ± 8.9% in monocultures (p ≤ 0 05, data not shown). Similar results were obtained when cocultures of mPB CD34 + cells were performed, with 48.63 ± 2.1% of apoptosis in cocultures versus 78.92 ± 6.05% in monocultures (p ≤ 0 05) (data not shown). However, in the presence of BMSCs (Figures 5(b) and 5(c)), the viability of cocultured CD34 + cells from CB or mPB was not significantly modified by the combined inflammatory factors as compared with the untreated counterparts. Comparing the two sources, only IL-6 + TIMP-1 (p ≤ 0 01) significantly increased the number of viable cocultured CB-derived CD34 + cells.
Altogether, these findings demonstrate that (1) the survival of normal CD34 + cells is highly promoted by normal BMSCs through a strong protection from apoptosis, (2) BMSCs alone or the combined proinflammatory cytokines stimulate the survival of normal HSPCs at the same extent, and (3) the copresence of BMSCs and the combined inflammatory cytokines does not show additive/synergistic effect in terms of hemopoietic supportive role.

Proinflammatory Cytokines Do Not Modify the In Vitro
Migration of CD34 + Cells from CB and mPB toward BMSCs. The BMSCs produce CXCL12 as mediator of migratory response of different cell types. CXCL12 is constitutively expressed by murine and human BM stromal cells [29]. To explore the effects of inflammation on the CXCR4-driven migratory ability of CD34 + cells in the presence or absence of BMSCs, we set up a migratory assay towards CXCL12 alone and BMSCs alone or in combination with various inflammatory cytokines. Of note, in order to mimic the in vivo pattern, along with inflammatory cytokines, a suboptimal concentration of CXCL12 (120 ng/mL) was also added. As shown in Figure 6(a), we compared the spontaneous migration of CD34 + cells from CB and mPB with the migration towards BMSCs seeded on the bottom of the transwell system, as chemoattractant. We found that the migration of CD34 + cells from both sources was promoted by BMSCs, being significant (p ≤ 0 05) with CB only. However, the migration rate of CB or mPB CD34 + cells was not increased by the presence of CXCL12 + BMSCs as compared with that of CXCL12 alone or BMSCs alone (Figures 6(b) and 6(c)). When we added various combinations of proinflammatory cytokines in the presence of CXCL12 and BMSCs, once again we did not find any significant difference in the migration rate of CD34 + cells between treated and untreated cells or between the two sources (Figures 6(b) and 6(c)).
These experiments demonstrate that the BMSCs exert a potent chemoattractive effect on normal HSPCs; moreover, in the presence of BMSCs, these combined proinflammatory factors are unable to significantly modify the CXCR-4-driven migratory behavior of HSPCs from both sources.

Discussion
Several cytokine-based strategies enhancing hemopoiesis, homing, and subsequent engraftment of CB/mPB-derived HSCs have been previously described [30]. However, critical steps are involved in these processes and further insights are necessary to better understand HSPCs homing and engraftment [31]. Along with a role as activators of immune cell function, a growing evidence now demonstrates that proinflammatory cytokines strongly affect the size and lineage distribution of the blood cells via reprogramming of HSC/HSPC and the supporting BM niche [5,32]. Along with the cytokine storm, the network created by dangerassociated molecular patterns (PAMPs/DAMPs) and alarmins could deviate HSCs fate, directly or indirectly via stromal cells [33]. Based on these evidences and due to the lack of informative data, it is of utmost importance to clarify the impact of proinflammatory cytokines on the biology of the normal HSPC and its BM microenvironment. A better understanding of the mechanisms driven by the inflammatory milieu in HSPCs may lead to better transplantation outcomes and knowledge of hematological defects or malignancies.
Here we tested various combinations of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and TIMP-1 in order to investigate their functional role on the in vitro behavior of young (CB-derived) and adult (mPB-derived) CD34 + cells. To mirror the in vivo condition, tested cytokines were two to two or multiple combined. We selected these inflammatory cytokines for the following features: (i) IL-6 is a pleiotropic proinflammatory cytokine that acts on many cell types including hemopoietic cells. It has been implicated as a critical activator of myelopoiesis in response to pathogen infection and chronic inflammation [34]; (ii) IL-1β is a potent inflammatory cytokine that mediates leukocytosis and thrombocytosis under inflammatory conditions by inducing various cytokines (i.e., granulocyte colony-stimulating factor and IL-6) [7]. Moreover, Pietras et al. [35] recently demonstrated that while IL-1β is dispensable for steady-state hemopoiesis, acute exposure to IL-1β accelerates HSC proliferation and instructs HSC priming for a myeloid fate. Lastly, it is involved in the pathogenesis of solid tumors and hematological malignancies [36]; (iii) TNF-α negatively regulates the expansion and self-renewal of HSPCs [37]. However, other evidence suggests that TNF signalling may enhance HSC function [38,39]; (iiii) TIMP-1, through receptor (CD63) binding, has a role in multiple biological processes, including inflammation and immune regulation. We recently demonstrated that it displays cytokine-like features in the normal and leukemic HSPC compartment [25,26]. In addition, experimental evidence demonstrated that combined proinflammatory cytokines such as IL-6 and TNF-α are critical for both inflammation and cancer by activating STAT3 and the NF-κB complex [40]. Furthermore, other inflammatory cytokines and pathways (such as IL-6) are induced by IL-1β and are involved in malignancy [36]. Thus, the combined action of these cytokines could constitute a central signalling pathway that promotes inflammation and tumor growth.
Comparing the two sources (neonatal versus adult) of HSPCs, here we demonstrated that the following: (1) Various combinations of inflammatory cytokines mainly enhance the in vitro survival of CB-derived CD34 + cells.
(2) As compared to CB, mPB-derived CD34 + cells are susceptible to selected combinations of inflammatory factors in terms of proliferation and hemopoietic function.
(3) TNF-α, alone or in combination, promotes the expression of the CD13 myeloid marker and CD44, an adhesion/proliferation marker of normal and leukemic cells.
(4) Along with an increased number of circulating CXCR4 + CD34 + cells, selected combinations of inflammatory cytokines mainly enhance the in vitro migration of mPB-derived CD34 + cells.
(5) BMSCs alone or combined inflammatory cytokines promote survival/migration of HSPCs from both sources at the same extent; moreover, their copresence does not show additive/synergistic effect in terms of hemopoietic supportive role.
Of note, despite that the use of frozen/thawed CD34 + cells in some cases might have influenced the variability of phenotype/clonogenic ability, our results clearly demonstrate that the selected network of proinflammatory factors has the potential to activate either neonatal or adult normal hemopoiesis and acts as regulator of HSPCs. Moreover, these results are consistent with previously described promoting effects of the inflammatory microenvironment on hemopoiesis [4,7,12,]. Taking into account that the functional consequences of inflammation-related molecules depend on the duration of exposure (acute versus chronic), our results may provide a starting point to investigate whether the inflammatory cues contribute to creating a favorable milieu for the development of hematological malignancies through hemopoietic activation.
Several groups evaluated the expression of hematopoietic markers, identifying various subpopulations of CD34 + cells in CB or mPB samples [41,42]. Interestingly, we found that, when CB-or mPB-derived CD34 + cells were treated with combined proinflammatory cytokines including TNF-α, CD13 expression was highly promoted. The CD13/aminopeptidase N is expressed on various cell types, including myeloid hematopoietic cells, and regulates biological phenomena such as differentiation, proliferation, apoptosis, motility, and tumor cell invasion. Importantly, CD13 degrades the chemokine CXCL11 and modulates CXCL12-induced migration [43,44]. It has also been reported that high levels of human CD13 correlate with leukemic cell resistance to apoptosis [45]. Moreover, CD13 is differentially expressed in discrete states of differentiation of neoplastic myeloid cells [46]. Along with CD13, here we found modulation of the homingassociated cell adhesion molecule CD44. Recently, CD44, as a receptor for hyaluronan, emerges as mediator of cell-cell and cell-matrix interactions and as pivotal trigger in cancer stem cell communication with their microenvironment [47,48]. Here we identified combined proinflammatory cytokines including TNF-α, which are able to upregulate CD44 expression on the surface of normal CD34 + cells. Although TNF-αdriven modulation of CD44 expression was already reported in several cancers [49], this is the first time that a strong link has been found between combined inflammatory cytokines and CD44 expression on HSPCs. Therefore, our results clearly indicate that the inflammation-driven CD13 and CD44 upregulation on neonatal or adult CD34 + cells has the potential of modulating key functional pathways (i.e., survival/differentiation) of the normal hemopoietic progenitor cells. Interestingly, these pathways may also play a role in myeloproliferation and leukemogenesis.
Several studies have shown that HSC can be expanded in cytokine-driven culture and by MSC feed layers [50,51]. Consistently, our data clearly showed that normal BMSCs enhance survival and migration of CB-and mPB-derived CD34 + cells. Due to their capacity to modulate oxidative stress, it is likely that BMSCs are capable of inhibiting apoptosis; moreover, producing CXCL12 [52], they enhance cell migration. Interestingly, even though it is likely that different mechanisms are involved, the BMSC-driven promoting effect of the CB-derived CD34 + cell survival is similar to that induced by the combined inflammatory cytokines. A similar trend was observed in mPB. However, for the first time, we investigated the copresence of BMSCs and various combinations of selected proinflammatory cytokines. Surprisingly, the copresence of inflammatory stimuli with BMSCs did not significantly modify the survival-migration rate of normal HSPCs as compared with that observed after stimulation with BMSCs alone or combined inflammatory cytokines alone. Of note, the BMSCs are capable to sustain the survival of mPB-derived CD34 + cells in the presence of IL-1β + TIMP-1. These findings demonstrate that in our culture system an acute inflammatory stimulus does not impair the hemopoietic-supportive role of BMSCs. Interestingly, based on murine models, it has been previously demonstrated that MSCs can modulate inflammation by secreting soluble receptors for IL-1 and TNF, which bind to IL-1 and TNF-α and neutralize the activity of the cytokines [53][54][55]. We can therefore hypothesize that in our cocultures the promotion of normal HSPCs survival/migration is mainly due to BMSCs which show regulatory properties of the hemopoietic function of HSPCs because they are capable of balancing the proinflammatory signal-driven hemopoietic activation. These results suggest that exploiting or modulating the thin balance between pro-and anti-inflammatory pathways may be a clinically relevant approach in hematological malignancies.

Conclusion
The goal of this study is the demonstration that an inflammatory microenvironment promotes distinct in vitro functional activation of neonatal and adult HSPCs and that an acute inflammatory stress does not impair the hemopoietic promoting effect of BMSCs. Moreover, this study may represent a starting point for future studies aiming at addressing the role of inflammation and the balance with antiinflammatory signals in the functional behavior of normal HSPCs and their transformation to a leukemic phenotype.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
An earlier version of this work has been presented at XIV Congress of the Italian Society of Experimental Hematology, Rimini, Italy, 2016.

Supplementary Materials
Supplementary Figure 1: survival of CD34 + cells derived from CB or mPB in the presence of 517 proinflammatory cytokines alone. Supplementary Figure 2: phenotype of CD34 + cells derived from CB or mPB in the presence of proinflammatory cytokines alone. Supplementary Figure 3: colony composition in the presence or absence of 521 combined inflammatory cytokines. Supplementary Figure 4: colony composition of CB-and mPB-derived CD34 + cells after migration toward CXCL12 alone and CXCL12 plus various combinations of inflammatory factors. Supplementary Table 1: antibodies used to characterize the CD34 + cells. Supplementary Table 2: absolute numbers of gMFI for CD13 and CD44 expression in CD34 + cells from CB or mPB after incubation for 24 hours in the presence/absence of 528 inflammatory stimuli. Supplementary Table 3: absolute numbers of CFU-C, CFU-GM, and BFU-E after incubation of CD34 + cells from CB or mPB in methylcellulose-based medium for 14 days in the presence/absence of inflammatory stimuli. Supplementary Table 4: absolute numbers of CFU-C, GM-CFU, and BFU-E in CD34 + derived from CB or mPB counted after migration towards inflammatory stimuli and seeded in methylcellulose-based medium for 14 days. (Supplementary  Materials)