Transcriptomic analysis identifies lactoferrin-induced quiescent circuits in neonatal macrophages

Introduction Upon birth, a hitherto naïve immune system is confronted with a plethora of microbial antigens due to intestinal bacterial colonization. To prevent excessive inflammation and disruption of the epithelial barrier, physiological mechanisms must promote immune-anergy within the neonatal gut. As high concentrations of human lactoferrin (hLF), a transferrin glycoprotein shown to modulate macrophage function, are frequently encountered in colostrum, its direct interaction with intestinal macrophages may satisfy this physiological need. Thus, the primary objective of this study was to investigate transcriptional changes induced by human lactoferrin in neonatal monocyte-derived macrophages. Methods Cord blood-derived monocytes were differentiated with M-CSF in presence or absence of 500 µg/mL hLF for 7 days and afterwards stimulated with 1 ng/mL LPS or left untreated. RNA was then isolated and subjected to microarray analysis. Results Differentiation of cord blood-derived monocytes in presence of hLF induced a distinct transcriptional program defined by cell cycle arrest in the G2/M phase, induction of IL-4/IL-13-like signaling, altered extracellular matrix interaction, and enhanced propensity for cell-cell interaction. Moreover, near-complete abrogation of transcriptional changes induced by TLR4 engagement with LPS was observed in hLF-treated samples. Discussion The global transition towards an M2-like homeostatic phenotype and the acquisition of quiescence elegantly demonstrate the ontogenetical relevance of hLF in attenuating pro-inflammatory signaling within the developing neonatal intestine. The marked anergy towards proinflammatory stimuli such as LPS further underlines the glycoprotein’s potential therapeutic relevance.


Introduction
Human lactoferrin (hLF) is an 80 kDa, 703 amino acid-long protein belonging to the conserved family of transferrin glycoproteins (1).Structurally, it consists of two globular lobes connected by a three-turn helix, capable of binding ferric iron (Fe 3+ ) with higher affinity than serum transferrin (2,3).Displaying a low dissociation constant at acidic pH, lactoferrin is thought to act in concert with plasma-transferrin by reducing iron availability in the microenvironment of inflamed tissue.This activity aids the inhibition of bacterial RNA-and DNA-synthesis, metabolism, enzymatic function, and ultimately microbial growth, virulence and pathogenicity (4).HLF's functional properties are also exploited by human immune cells, as the molecule is highly abundant in secondary and tertiary granules of granulocytes.Vastly mobilized during the initial stages of host-pathogen interaction, 10 6 granulocytes may release up to 15 µg of hLF (5).Therefore, elevated local concentrations of hLF are present in inflamed tissue and shape the microenvironment at the site of immunological action (6).
In addition to its bacteriostatic and tissue-protective function, hLF also elicits direct bactericidal effects (7), and interacts with the cellular fraction of the immune system (8).The latter is especially important to the function of human monocytes.These cells enter the site of infection and differentiate into macrophages in an hLF-rich microenvironment.
As their role largely depends on polarization into M1-or M2phenotypes, it is tempting to speculate that direct interactions of hLF with monocyte lactoferrin receptor (MC-LfR) and low-density lipoprotein receptor-related proteins (LRPs) shape distinct cellular responses (9,10).
Adding an additional layer of complexity, both local and systemic lactoferrin concentrations follow a distinct trajectory over time.In term-born infants, lactoferrin reaches systemic levels between 200 and 800 µg/L within the first two weeks of life (11).Considering its high concentration in colostrum (up to 7 g/L), hLF seems pivotal for the development of a favorable neonatal gut-microbial composition and maintenance of intestinal barrier integrity in newborns (12).
Although macrophages have been shown to be crucially involved in maintaining intestinal barrier function (13) and hLF appears to prevent the development of intestinal hyperinflammation in preterm infants (14) by inducing a distinct anti-inflammatory/anergic state (15), a deeper understanding of the transcriptional networks that regulate the activity of neonatal macrophages upon birth is currently missing.Therefore, we aimed to identify the transcriptional profile of hLF-treated neonatal monocyte-derived macrophages and the mechanisms that underlie its immunomodulatory properties.We were able to show that hLF drastically alters the cell cycle progression of neonatal macrophages, leading to the development of a distinct transcriptional program that drives polarization towards an M2-tissue homeostatic phenotype unresponsive to pro-inflammatory stimuli.

Study population
The umbilical cord blood of term-born infants (n = 4, 38-40 weeks of gestation) was collected aseptically into heparin tubes after cesarean section.Infants with congenital malformations and congenital infections and infants whose mothers suffered from autoimmune disease, or inborn or acquired immune deficiencies were not considered eligible for study inclusion.All procedures were approved by the ethics committee of the Medical University of Vienna (ref. 1923/2012) and informed consent was obtained from the mothers.

RNA-extraction and microarray processing
Total RNA was isolated from 1.5 x 10 6 differentiated macrophages using the RNeasy Plus Micro Kit (Qiagen, Venlo, the Netherlands) according to the manufacturer's instructions.RNA was then quantified using a NanoDrop 8000 device (Thermo Scientific, Waltham, MA, USA) and RNA integrity number (RIN) was determined employing the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA).To prepare samples for microarray analysis, fragmented and biotin-labeled sense-stranded cDNA was prepared from 100 ng of total RNA (20 ng/µl) using the GeneChip Whole Transcript (WT) PLUS Reagent Kit (Affymetrix, Santa Clara, CA, USA).cDNA was then hybridized to the GeneChip Human Gene 2.1 ST Array Plate (Affymetrix, Santa Clara, CA, USA) and automatically processed and analyzed using the GeneTitan instrument (Affymetrix, Santa Clara, CA, USA).

Gene set enrichment analysis
For gene set enrichment analysis (GSEA), genes with similar expression values to respective differentially expressed genes (DEG) were selected from the dataset and used as a background for each experimental condition (16).Reactome Pathway Enrichment Analysis was then carried out using ReactomePA (24), biological theme comparison performed with clusterProfiler (25), and results depicted utilizing the enrichplot (26) package.Chord diagrams were generated with GoPlot (22) by manually computing respective adjacency matrices between DEG and enriched pathways.Results were filtered for biologically meaningful interactions to reduce noise, by only selecting genes with a logFC higher than 2.5 or lower than -2.5 for visualization.

Gene regulator enrichment analysis
For Gene Regulator Enrichment Analysis, normalized expression values were used as input for the RegEnrich algorithm (27).Thus, using a predefined set of human transcription regulators, a regulator-target network inference based on weighted gene co-expression network analysis was conducted.Subsequently, enrichment analysis was performed using Fisher's exact test and the RegEnrich score, a metric corresponding to the biological importance of respective regulators, was modeled.Data were visualized in a heatmap by extracting the normalized expression values of the top-25 regulators for each respective condition.Additionally, RegEnrich scores of the top-20 regulatory molecules were depicted as a bar chart.Using the previously computed adjacency matrices of enriched pathways and differentially expressed genes, a second matrix that numerically estimates the effect each transcriptional regulator exerts on a cellular function was created.This was achieved by assigning a point to a regulator for each shared gene with a respective pathway and extracting the five regulators with the highest scores.Thus, functional relations between transcription factors and enriched pathways were modeled and visualized in a Sankey plot using the networkD3 package (21).Furthermore, to depict connectivity between transcriptional regulation and biological functionality, a protein-protein interaction network (PPI) was generated using the STRING database (28) (Version 11.5) for proteins shown to be involved in the regulation of enriched pathways upon hLF-treatment and all DEG found in the dataset.The retrieved network was then filtered for entries with a minimum required interaction score of 0.900 (highest confidence), exported, and processed in Cytoscape (29) (Version 3.10.0).

Cytokine expression and membrane protein abundance
To analyze differences in cytokine expression between conditions, DEG were filtered for cytokines annotated in the ImmPort Cytokine Registry (30).Similarly, using InnateDB's (31) annotation of proteins relevant to innate immunity and the Human Protein Atlas' (32) annotation of subcellular protein locations, DEG relevant to the innate surfaceome of macrophages were extracted.Additionally, a more targeted analysis of immunologically relevant genes was conducted.Differences between target means were calculated using ANOVA and Tukey's post-hoc test employed to retrieve adjusted p-values for pairwise comparisons.

Cell-cell interaction model
With the purpose of modelling the propensity of hLF-treated macrophages to interact with other immune cells via direct physical-or indirect soluble-interactions, all DEG of membrane proteins and cytokines were extracted from the dataset using annotations stored in the ImmPort Cytokine Registry (30) and the Human Protein Atlas (32).Moreover, experimentally curated physical protein-protein interactions annotated in InnateDB (31), as well as cytokine-receptor interactions denoted in the KEGG pathway database (33), were retrieved.Subsequently, two adjacency matrices incorporating normalized expression data of selected immune cells stored in the RNA Monaco immune cell gene data (34) available from The Human Protein Atlas (32) and our experimental results, were modeled.Because the product of two interacting proteins within adjacency matrices of standardized expression sets yields a constant arbitrary value, the sum of respective products per interacting cell-pair produces a metric for interaction likelihood between two cells.Thus, differences in interaction probability between treated macrophages and all immune cells present in the RNA Monaco immune cell gene data (34) were determined and depicted in a radarchart using the R package fmsb (35).Additionally, mean expression of cytokines and membrane proteins with the highest difference in interaction scores between uMf +LPS, hlfMf +LPS were retrieved, ordered according to their number of interactions with other cells ("interaction score"), and displayed as a bar chart.

Statistical analyses
For differential gene expression analysis, we employed the empirical Bayes variance moderation method to compute moderated t-statistics.If not indicated otherwise within this section, we selected genes with a false discovery rate (FDR) < 0.05 and log2-fold changes below -0.58 and above 0.58, respectively, for subsequent analyses.Pathway enrichment analysis was performed on input genes with an FDR < 0.1 in line with the statistical settings recommended in the maEndToEnd Affymetrix differential gene expression workflow (16).For biological theme comparison, DEG with an FDR < 0.05 and log2fold changes below -0.58 or above 0.58 were used as input.Transcription regulator network target inference with RegEnrich (27) was performed by constructing an undirected, unsigned coexpression network on differentially expressed genes employing a minimum scale-free topology fitting index (R 2 ) of 0.85 and retaining the top 5% of edges in the full network.Regulator enrichment was subsequently calculated for genes with a differential analysis p-value < 0.05, using a cutoff of 0.05 for adjusted enrichment p-value and 0.2 for enrichment q-value.

HLF induces a distinct transcriptional pattern capable of repressing LPS-induced effects
Highly abundant in breast milk, human lactoferrin exerts a diverse set of immunological and developmental functions.Next to direct bactericidal effects, hLF might also act as an immunological primer of macrophages, a cell subset capable of propagating inflammatory damage in the evolving human gut.Thus, we established an in vitro culture system to differentiate neonatal monocytes into macrophages in the presence or absence of hLF.Additionally, we used LPS-treatment to mimic pro-inflammatory stimuli encountered in the intestine upon bacterial colonialization (Figure 1A).Thus, following our previous work on hLF-mediated attenuation of pro-inflammatory macrophage polarization, we analyzed transcriptomic changes elicited by hLF to decipher biological patterns that underlie its manifold of immunological functions (15).Transcriptomic signatures of macrophages differentiated with M-CSF in the absence (uMf) or presence (hlfMf) of hLF, either stimulated with LPS (uMf+LPS, hlfMf+LPS) or left untreated (uMf, hlfMf), were investigated using RNA microarray technology.Differential gene expression analysis revealed 406 differentially expressed genes (DEG) between hlfMf and uMf.Moreover, stimulation of uMf with LPS induced an increase in transcriptional activity (971 DEGs) that vastly differed from that observed in hlfMf+LPS (823 DEGs).This was of particular interest, as the comparison between hlfMf and hlfMf+LPS revealed no DEG (Figure 1B).To completely exclude the existence of DEG between hlfMf and hlfMf+LPS we re-analyzed the comparison without employing logFC cut-offs.Even after this adaptation, no DEG were found between the two conditions (Supplementary Figure 1).Correspondingly, hierarchical clustering of DEG deriving from all conditions revealed a distinct transcriptomic profile of hLF-treated cells when compared to uMf and uMf+LPS (Figure 1C).This finding is also in line with the high expressional overlap between stimulated and unstimulated hlfMf observable in the Venn diagram (Figure 1D) and the clear separation between uMf, uMf+LPS and hLF-treated cells shown by principal component analysis (Figure 1E).
Taken together, the differentiation of neonatal monocytes in the presence of hLF induced a distinct transcriptomic profile and severely impaired the responsiveness of derived macrophages to LPS stimulation.

Human lactoferrin modulates cell cycle progression and induces M2 polarization
To understand the cellular responses hLF evokes in neonatal monocyte-derived macrophages, a gene set enrichment analysis (GSEA) was performed using the Reactome database of curated cellular pathways (Figure 2A).Biological theme comparison revealed cell-cycle specific effects of hLF, as displayed by marked downregulation of cell cycle progression, DNA replication, and deposition of CENP-A nucleosomes needed for maintaining centromere identity.Moreover, hLF-treated cells presented with an abrogation of LPS-induced effects on cytokine signaling, including engagement in interferon-alpha/beta and interferon-gamma pathways, as exemplified by diametrically opposed enrichment results, whilst concomitantly maintaining cell-cycle specific properties (Figure 2A).To gain a deeper understanding of the drivers of hLF-function and its immunosuppressive properties, chord diagrams displaying the most disparate expression profiles for respective Reactome pathways were generated (Figures 2B, C).In this context, genes of several histones, proteins (NDC80, CENPF, SKA1, TPX2), cyclins (CCNA2, CCNB1, CCNB2), and cyclin-dependent kinases (CDK1) were downregulated in hlfMf when compared to uMf -all of which relevant to chromosome segregation, kinetochore function, or cell cycle regulation.In addition, hLF seemed to promote interactions with extracellular matrix proteins via upregulation of transmembrane receptors ITGB3 and ITGA2 as well as FN1 and SCD4.It also triggered downstream effects comparable with IL-4-and IL-13-induced signaling by increasing the expression of JAK3, FOS, SOCS3, ALOX5, CCL22, and PIM1 (Figure 2B).Additionally, the upregulation of anti-inflammatory signaling molecules FOS, EGR1, and JUN, as well as increased expression of decoy receptor IL1R2 and anti-inflammatory cytokine IL-10 seem to stand in stark contrast to an LPS-induced proinflammatory phenotype (Figure 2C).In line with this finding, hLF also potently blocked LPSinduced upregulation of cytokines and chemokines CXCL8, CXCL10, CCL5, EBI3, and CCL20 as well as interferon-stimulated genes IFITM1, ISG15, IFIT1, and OAS2.
Overall, functional analyses revealed that hLF-induces a 'quiescent' state in macrophages due to modification of cell cycle regulation, an important mechanism recently described to drive macrophages into an M2-like phenotype (36).

HLF induces distinct gene regulatory networks that drive neonatal macrophages into quiescence
In order to understand the gene regulatory programs that underlie hLF-induced quiescence, we performed a gene regulator enrichment analysis using RegEnrich (27).This algorithm identifies genetic regulators based on differential gene expression analysis and regulator-target network inference, and provides a metric for their regulatory importance.Hierarchical clustering of the top-25 regulators per condition revealed prominent changes in the mean expression of gene regulatory molecules in hLF-treated samples, irrespective of LPS stimulation (Figure 3A).These gene regulatory changes were driven by the downregulation of cell cycle regulatory proteins FOXM1, E2F8, HMGB2, and BRIP1 and the upregulation of immunomodulatory protein IVNS1ABP.Additionally, downregulation of proinflammatory regulators IRF7, EZH2, HDGF, and EIF2AK2 was observed when comparing hlfMf+LPS to uMf+LPS, highlighting that hLF induces transcriptional unresponsiveness towards proinflammatory LPS-stimuli (Figure 3B).To better understand the functional implications of hLF-mediated transcriptional regulation, the top-five regulators per pathway found to be enriched in hlfMf were retrieved.This analysis revealed the importance of BRIP1, BRCA2, BARD1, ASH2L, and BCLAF1 as functional regulators of cell-cycle progression and CBX5, ATF6B, BHLHE40, EAF1, and ABTB1 as mediators of IL-4/IL-13-like effects (Figure 3C).Because RegEnrichper defaultinfers a regulatory network on all significantly expressed genes, irrespective of their logarithmic fold change value (logFC), a protein-protein interaction network (PPI) for genetic regulators of hLF-induced functions and differentially expressed genes that displayed a log2-fold change of 0.57 or higher was constructed.Although functional regulators interacted with only 27 proteins directly, network analysis revealed high connectivity with all differentially expressed genes identified in our experimental model (Figure 3D).
In concert, these results highlight the tight connection between cell-cycle regulators and functional heterogeneity in hlfMf and underline their importance in the development of a quiescent state that remains transcriptionally irresponsive to immunogenic stimuli.

HLF-induced quiescent macrophages display a tissue-homeostatic interactome
As our initial analyses pointed towards induction of a quiescent M2-like state, we aimed to explore whether this homeostatic phenotype also extends to the interactome of hLF-treated macrophages.Harnessing ImmuneDBs data on immunologically relevant genes, Immports cytokine registry data, and information on subcellular protein localization available from Human Protein Atlas, membrane protein, and cytokine gene abundances were computed for each experimental condition.Global changes in cytokine and membrane protein gene expressions were evident for all immunologically implicated enriched Reactome pathways (Figure 4A).Upon closer examination, hlfMf displayed modified expression of PLAUR, ICAM1, ITGB3, and ITGA3, arguing for changes in cell adhesion and matrix interaction properties.Moreover, together with the altered presence of CD180, Ly9, LAT, and PLSCR1, these changes suggested modifications in phagocytotic and efferocytotic functions.Complementing this homeostatic interactome are the increased expression of SIGLEC7, SDC4, S100A9, and TNFRSF9, proteins that have been ascribed antiinflammatory properties (Figure 4B).Regarding soluble interactions, upregulation of homeostatic and gut protective cytokines IL-10, TNFSF14, and CXCL8 was seen in macrophages treated with hLF.Moreover, hlfMf displayed an increased expression of cytokines involved in regulating the proliferation and activity of mucosal innate lymphoid cells.Contradictory at first glance, hLF also mediated the expression of pro-inflammatory molecules OSM, CCL7, CXCL5, IL1B, and CCL3.Yet globally, hlfMf+LPS displayed substantial decreases in the expression of pro-inflammatory molecules when compared to uMf+LPS (Figure 4C).To additionally validate our findings, we employed ANOVA with posthoc Tukey-correction to exclude a solely TLR4 dependent reduction of LPS-signaling in hlfMf and statistically support the increases observed in IL-10, CCL22 and TNFSF15 productioncytokines detrimental to intestinal homeostasis (Figure 4D).Collectively, these results argue for the induction of a quiescent, tissue-homeostatic interactome in hLF-treated neonatal macrophages.Moreover, the near complete abrogation of LPS-induced effects suggests that hLF efficiently drives the cellular communication of neonatal macrophages towards tolerance.

HLF increases physical cell-cell interaction propensity and attenuates LPSinduced cytokine crosstalk
Due to the marked changes hlfMf displayed in membrane protein and cytokine expression, we next evaluated their global propensity to interact with other immune cells.Thus, we combined the previously Functional enrichment analysis unravels cell cycle regulation as a driver of hLF-mediated quiescence.Functional Enrichment Analysis was performed using ClusterProfiler.(A) Biological theme comparison between treatment conditions depicted as a dot plot.Upregulated pathways are highlighted in red, downregulated pathways marked blue.(B, C) Chord diagrams showing the involvement of genes with logFC-values higher than 2.5 and lower than -2.5 in pathways discovered during functional enrichment analysis.Graphs correspond to differential gene expression matrices hlfMf vs. uMf (B) and hlfMf+LPS vs. uMf+LPS (C), respectively.computed membrane protein and cytokine expression data with Innate DB's manually curated annotation of immunologically relevant physical protein-protein interactions, cytokine-receptor interactions stored in the KEGG pathway database, and normalized gene expression values available from the Monaco immune cell gene data provided by the Human Protein Atlas.
hlfMf displayed a markedly increased probability for physical cell-cell interactions when compared to uMf and uMf+LPS.These physical interactions were biased towards granulocytes, regulatory Tcells, and myeloid dendritic cells (Figure 5A)although to a markedly lesser extent than soluble interactions (Figure 5B).Indeed, treatment with hLF strongly increased the capability for soluble interaction with neutrophilic granulocytes when compared to uMf, arguing for extensive crosstalk between these cell types upon hLF exposure.Moreover, large differences between uMf+LPS and hlfMf+LPS were observable for both physical and soluble interaction propensities.Detailed analysis of interactome differences between these two conditions revealed differential expression of CD9, IL7R, ICAM1, TGFBR1, and CD300LB on macrophages as drivers of hLFinduced alterations in physical cell-cell interactions (Figure 5C).Regarding cytokines, downregulation of chemoattractants CCL8, CXCL10, CCL19, CCL7, and CXCL9amongst othersmediated the decreased probability for soluble interaction observable in hlfMf +LPS, when compared to uMf+LPS (Figure 5D).Highlighting our previous findings, upregulation of the propensity for physical cell-cell interaction and concomitant downregulation of LPS-mediated soluble interactions argue for hLF-induced polarization of neonatal macrophages into a tissuehomeostatic phenotype.

Discussion
In our experimental model, differentiation of macrophages in the presence of hLF induced a distinct transcriptional program capable of repressing changes in gene expression provoked by LPS stimulation.HLF's functional signature involved changes in cell cycle regulation, induction of IL-4/IL-13-like signaling, and modified expression of molecules known to mediate extracellular matrix interactions, efferocytosis, and inflammation.Interestingly, in terms of global immune function and cellular interactome, hLF exhibited a 'Janus-faced' character.This duality was characterized by the induction of a basal interaction-prone phenotype incapable of mounting adequate immune synergisms upon TLR4-engagement by LPS.Congruent with previously published data by our group, these findings argue for the polarization of monocyte-derived macrophages into a quiescent state upon treatment with hLF.The results underline the physiological and trajectorial complexity of human lactoferrin as a soluble modulator of macrophage function at the peripheral site of infection and a promotor of immunehomeostasis and tissue integrity within the intestine upon birth.
It Is well established that commensal colonization of the neonatal gut poses an immunological challenge to a hitherto naïve immune system.Thus, it is very likely that physiological mechanisms exist that prevent the development of excessive inflammation, disruption of intestinal barrier function, and onset of diseases such as necrotizing enterocolitis (NEC).Macrophages play a key role in TLR4-mediated induction of intestinal inflammation upon challenge with pathogen-associated molecular patterns (PAMPs) such as LPS (37).In this regard, their polarization towards pro-inflammatory M1-and tissuehomeostatic M2-phenotypes has been shown to be associated with the development of NEC, and modulation proposed as a possible treatment strategy for excessive gut-inflammation (38,39).As blood monocytes that migrate into the developing gut (40,41) encounter abundant hLF from colostrum, we aimed to investigate whether hLF modulates the function of these newly differentiating macrophages to prevent excessive gut inflammation upon LPS challenge, the main PAMP associated with gramnegative bacteria.
hlfMf displayed cell cycle arrest in the G2/M phasea condition recently shown to promote an M2-like phenotype and tissue-homeostatic functions (36).Interestingly, this state has been associated with enhanced expression of FN1, a molecule also markedly upregulated in our hlfMf (36).This finding aligns well with the downregulation of gene-regulatory proteins BRCA2 and Propensity for soluble and physical cell-cell interaction is markedly altered upon hLF-treatment.Cell-cell interaction probability was manually computed using data deriving the publicly available databases ImmPort, Human Protein Atlas, ImmuneDB, and KEGG.Radar charts depicting cell-cell interaction probability in arbitrary values between differentially treated macrophages in comparison to uMf were generated for physical (A) and soluble (B) cell-cell interactions.Moreover, bar charts visualizing the mean expression values of the most important and differentially regulated interaction molecules between hlfMf + LPS and uMf + LPS contributing to physical (C) or soluble (D) cellular crosstalk were created.Bars are ordered and color-coded according to the number of cells respective genes mediate interactions with, denoted as "interaction count".
Complementing its gut-protective role, macrophages differentiated in the presence of hLF displayed enhanced expression of IL-10, an interleukin robustly associated with gut epithelial barrier restoration, suppressor of intestinal inflammation, and mitigator of NEC development (51, 52).Moreover, hlfMf likely induces Th2-immunity via expression of CCL22, a molecule shown to promote chemotaxis of CCR4-positive Th2-skewed lymphocytes (53), and TNFSF15, a cytokine relevant to the expansion of innate lymphoid cells type 2 (ILC2) (54).In the context of gut immunity, Th2-skewed lymphocytes are known to produce cytokines involved in the maintenance of gut-epithelial barrier homeostasis and mitigation of inflammation (55, 56).Interestingly, TNFSF15 has also been shown to trigger the release of IL-22 by ILC3 (57).As reduction of IL-22 producing RORgt + NKp46 + ILC3 has recently been linked to the development of NEC, hLF-induced TNFSF15 secretion in macrophages might convey an important gutprotective role (58).
Underlining hLF's 'Janus-faced' character, microarray analysis also revealed pro-phagocytic signatures via upregulation of PLAUR, LAT, and downregulation of CD180 (59-61).While in the context of gut homeostasis this function may aid the clearance of apoptotic cells and mitigate inflammation, upregulation of these molecules might also reflect hLF's function as a soluble mediator in the periphery.This theory is also supported by hLF-induced priming of macrophages for cytokine-induced immune interactions through enhanced IL1R1 expression and elevated transcription of proinflammatory molecules OSM, IL-1B, CCL3, CCL7, and CXCL5 (62)(63)(64)(65).
Although contradictory at first glance, hLF-induced priming of macrophages for pro-inflammatory activity and concomitant global transition towards a tissue homeostatic phenotype seems physiologically reasonable.Considering the high amount of hLF synthesized by granulocytes at the site of infection (5), hLFinduced upregulation of transcripts relevant to phagocytosis, proinflammatory cytokine signaling, and cell migration may aid pathogen clearance.Consequently, as inflammation and tissue damage progress, a more global transcriptional program drives macrophages into a state of immunological anergy and homeostatic functionas represented by a slow, cell-cycle specific transcriptional transition towards an M2 phenotype (36).The observed transcriptional response is also in line with evidence linking hLF-function to basal TLR-4 activation via its carbohydrate chains and suppression of TLR4 signaling upon endotoxemia through its peptide moiety (66).To specifically delineate the molecular mechanisms that govern LPS irresponsiveness upon hLF exposure in human macrophages, future studies would benefit from investigating the persistence of immunological effects upon withdrawal of hLF from culture medium and incubation with different hLF-moieties.
The observed dual function of hLF is also consistent with its ontogenetic aspect.Given the high abundance of hLF in colostrum, breastfeeding and uptake of this nutrient lead to uniquely elevated concentrations within the gut.This primes intestinal macrophages towards tolerance, induces the migration and proliferation of ILCs and Th2-skewed lymphocytes, and prohibits the instantaneous development of gut inflammation.The near-total abrogation of LPS-induced changes on the transcriptome of hlfMf encountered in all experiments also supports this notion.Thus, based on our data and available literature we propose a mechanistic framework of homeostatic hlfMf-function within the neonatal gut (Figure 6).
As for study limitations, although the data have been quality controlled ahead of analyses to account for unspecific signals ( 16), the sample size (n = 4) does not allow correction for donor-specific effects.The potential influence of donor-variation on experimental results is also depicted in Figure 1E, where some data-points lie outside the respective confidence intervals.Future studies would benefit from including a larger number of biological replicates to allow for even more stringent exclusion of experimental outliers and determination of potential donor-specific effects.Additionally, while our analysis shows a clear abrogation of TLR4 signaling upon differentiation of macrophages in the presence of hLF, the experimental setup does not consider stimulation of other innate signaling pathways or synergistic signaling effects elicited by whole bacteria.Therefore, even though E.coli has been shown to be a highly abundant strain during early colonialization upon vaginal delivery (67) and LPS arguably serves as a model agonist for TLR engagement by this bacteria (68), future studies would benefit from also investigating transcriptional responses after stimulation with gram-positive bacteria such as Staphylococci and Enterococci to provide a more holistic immunological framework for hLF-function.
Nevertheless, our study provides compelling evidence for the role of hLF as a soluble modulator of macrophage activity.The substantive transcriptional effects hLF exerted on neonatal macrophages, including the near-complete abrogation of proinflammatory responses to LPS-treatment, likely reflect the molecule's ontogenetic importance as an immunosuppressive agent within the developing neonatal gut.Thus, exposure of infants to high concentrations of hLF via uptake of colostrum may enable safe intestinal bacterial colonialization and prevent the development of inflammation and disease.Noteworthy, the considerable effects observed in our study not only confer relevance to hLF but also underline the importance of macrophage function in early-life development -marking this immune-subset a promising therapeutic target in the prevention of inflammation and autoimmunity in infants.

1
FIGURE 1Neonatal macrophages differentiated in the presence of hLF reveal a distinct RNA-signature.(A) Cord-blood derived neonatal monocytes were isolated and differentiated into macrophages in the presence or absence of hLF for seven days and stimulated with LPS.RNA was isolated and analyzed using microarray technology (Created with BioRender.com).(B) Bar plot depicting the count of negatively (blue) and positively (red) differentially expressed genes (DEG) between experimental conditions.(C) DEG of all conditions hierarchically clustered using Euclidean distance as a similarity measure, displayed as a heatmap.(D) Venn Diagram of unique and shared DEG between respective treatments.(E) Principal component analysis of macrophage expression profiles.

3
FIGURE 3Gene regulator enrichment analysis identifies key-regulators of hLF-function and reveals their high transcriptomic connectivity.Transcription regulator-target network inference based on weighted gene co-expression analysis was performed using RegEnrich.(A) Heatmap depicting mean expression values of the top-25 transcription regulators per condition, employing Euclidian distance as a similarity measure.(B) Bar chart showing the 20 regulators with the highest RegEnrich score for hlfMf (green) and hlfMf+LPS (blue), respectively.(C) Using the adjacency matrix previously computed to display the influence of gene expression on enriched Reactome pathways, the top-five genetic regulators for each enriched pathway were identified in hlfMf and their contribution to biological function visualized using a Sankey plot.(D) Regulators involved in the control of hLFinduced biological functions were retrieved and a protein-protein interaction network constructed that displays their contribution to the regulation of all DEG found in the dataset.

4 HLF
FIGURE 4HLF-treatment induces a homeostatic interactome in neonatal macrophages.Using the annotated databases ImmPort, Human Protein Atlas and ImmuneDB, DEG of cytokines and membrane proteins were retrieved from the dataset.(A) A heatmap depicting DEG of cytokines and immunologically important membrane proteins was generated using Euclidian distance as a similarity measure for immunologically relevant enriched pathways of all experimental conditions.A similar visualization was performed for all membrane protein-(B) and cytokine-(C) related DEG found in the dataset, irrespective of enriched pathways.(D) Gene expression values of TLR4, CCL22, IL-10 and TNFSF15 were plotted as boxplots and significance indicated with lines and asterisks for p < 0.05 (*), and p < 0.01 (**), respectively.