new insights into the systemic effects of oral lactoferrin

Draft

New Insights into the Systemic Effects of Oral Lactoferrin: Transcriptome Profiling

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2020-0069.R1

Manuscript Type: Article

Date Submitted by the Author: 12-May-2020

Complete List of Authors: Kruzel, Marian; UTHealth McGovern Medical School, Olszewska, Paulina; Medical University of Lodz Faculty of Medicine, Department of Pharmaceutical ChemistryPazdrak, Barbara; University of Texas MD Anderson Cancer CenterKrupinska, Anna; Wroclaw Medical University Faculty of Dentistry, Prosthetic DentistryActor, Jeffrey; UTHealth McGovern Medical School,

Keyword: Lactoferrin, Immune Response, Transcriptome Profiling, Inflammation

Is the invited manuscript for consideration in a Special

Issue? :Lactoferrin 2019

https://mc06.manuscriptcentral.com/bcb-pubs

Biochemistry and Cell Biology

Draft

New Insights into the Systemic Effects of Oral Lactoferrin: Transcriptome Profiling

5Marian L. Kruzel1, Paulina Olszewska2, Barbara Pazdrak3, Anna M. Krupinska4 Jeffrey K. Actor1

1. McGovern Medical School at Houston, Houston, Texas2. Medical University of Lodz, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Drug Analysis

10 and Radiopharmacy, Lodz, Poland3. MD Anderson Cancer Center, Houston, Texas4. Medical University of Wroclaw, Poland

15

20Corresponding author: Marian L. Kruzel, Ph.D.University of Texas, McGovern Medical School at HoustonDepartment of Integrative Biology and Pharmacology

25 7505 Fannin Street, Third Floor 313Houston, TX 77054Phone: 832 419 3693; e-mail: [email protected]

30

Running Title: Systemic Effects of Oral Lactoferrin

35

Keywords: Lactoferrin, Immune Responses, Transcriptome Analysis, Inflammation

40 This work was supported in part by NIH Grant 1R42-AI117990.

Conflict of Interest/Disclosures:

MLK is a founder of PharmaReview Corporation and a donor of rhLF for this study. Potential conflicts of 45 interest are managed by documentation registered between PharmaReview Corporation and the University of

Texas McGovern Medical School Conflicts of Interest and Commitment Committee.

50

Page 1 of 19



Draft

Abstract55

The immunomodulatory nature of lactoferrin (LF) derives from its ability to bridge innate and adaptive immunity

in obtaining physiological equilibrium. LF is an attractive molecule for treatment of diseases that compromise

immune homeostasis. Oral delivery is a preferable method for LF administration; however, its bioavailability is

60 affected by protein degradation and absorption. The aim of this study was to evaluate the systemic effects of

oral and intravenous (IV) administered recombinant human LF (rhLF) on blood cell transcriptome profiling.

Rats were administered with a single dose of rhLF by gavage or IV. The transcriptome profiles from control and

rhLF-treated rats after 3h, 6h and 24h were analyzed by Clariom D microarray. The results showed

differentially expressed genes in response to IV as well as oral administered rhLF including coding and

65 noncoding RNAs. Moreover, a comparison of the differentially expressed genes between oral and IV after 6h

revealed that a majority (72.8%) of altered genes in response to oral rhLF administration was common with IV

treatment. The pathway profiles showed similarities in up-regulation of specific genes involved in oxidative

stress and inflammatory responses for both routes of treatments. These findings provide evidence of the

systemic signal transduction effects of orally administered rhLF.

70

75

80

Page 2 of 19



Draft

Introduction

85 Lactoferrin (LF), an iron-binding glycoprotein is an essential component of the innate network maintaining

immune homeostasis in mammals. It is synthesized by exocrine glands and neutrophils providing a first line

defense mechanism to combat against microbial infections and trauma sequelae in humans (Lonnerdal and

Iyer 1995) (Sanchez et al. 1992) (Baynes and Bezwoda 1994) (Baveye et al. 1999; Kruzel and Zimecki 2002),

with LF receptors likely regulating pathogenic protective activities (Morgenthau et al. 2013). Accordingly, LF is

90 present in fluids where pathogens are first encountered, such as in tears, saliva, vaginal fluids, semen (Van der

Strate et al., 2001), nasal and bronchial secretions, and gastrointestinal fluids (Oztas et al., 2005). The highest

concentrations of LF are found in human colostrum (6-7 g/L) and breast milk (1-3 g/L) (Liao et al. 2011)

(Montagne et al. 2001). Although there are many bioactive compounds in human breast milk, LF is considered

as the major component related to the establishment and maintenance of immune homeostasis in infants.

95 Indeed, it is well accepted that breastfeeding is vital not only for the calories contained within mother’s milk, but

for the growth promotion of certain probiotic strains (Oda et al. 2014), and development of infant’s brain and

cognition (Chen et al. 2015; Li et al. 2019; Wang 2016) is clearly attributed to LF. Recently the American

Academy of Pediatrics reaffirmed its recommendation of exclusive breastfeeding for approximately 6 months,

followed by continued breastfeeding as complementary foods are introduced (Johnston et al. 2012).

100 The properties of LF are well established and derive mostly from observations of the endogenous molecule to

control physiological equilibrium in vivo (Actor et al. 2009; Kruzel et al. 2017; Kruzel et al. 2000; Kruzel et al.

2007). Many researchers are challenged by the fact that the effects of exogenous LF should be dissected from

the endogenous LF that is intrinsically present in mammals. Although it is valid concern, in most cases, the

effects of exogenous LF are reported as global responses in reference to non-treated controls. LF is highly

105 homologous across mammalian species, including bovine versus human. Bovine lactoferrin (bLF) shares

~70% amino acid sequence homology with human milk-derived lactoferrin (hLF) with very similar functionalities

(Rosa et al. 2017). In fact, based on bioequivalence of bLF with its human counterpart, the Food and Drug

Administration (FDA) has recently determined bovine milk-derived LF as safe for use in infant formula (general

recognized as safe-GRAS Notice 465 and 669). Moreover, bLF is also known as a nutritional supplement to

110 maintain physiological homeostasis in adults (Dix and Wright 2018).

The mechanism of action by which systemic effects of orally administered LF function is not fully understood,

despite decades of research. The lack of progress in this research is mostly due to limited ability to study the

mechanisms of digestion, distribution, and metabolism directly in human infants; hence, various animal models

are used as a proxy, including the piglet model as the most suitable model for human infants (Alizadeh et al.

115 2016; Donovan 2016; Miller and Ullrey 1987; Montagne et al. 2001; Moughan et al. 1992a; Moughan et al.

1992b). The primary consideration however is that orally administered LF is a substrate for gastrointestinal

proteases, with targeted digestion in the stomach (Brines and Brock 1983). Like all other stomach degraded

proteins, LF is absorbed in a form of amino acids by facilitative diffusion or active transport, although some di-

and tripeptides may survive cytosolic hydrolysis and be transported intact across the basolateral membrane.

Page 3 of 19



Draft

120 However, there is no strong scientific evidence that large proteins are absorbed by healthy intestinal epithelia

and pass intact into the hepatic portal system (Miner-Williams et al. 2014). Recently, it has been reported that

sublingual administered bLF can be detected in the brain in rats (Hayashi et al. 2017). Nevertheless, the

transport of larger intact macromolecules across the intestinal endothelium remains a controversial issue

despite emerging science on food-derived bioactive peptides. Indeed, the pharmacokinetic studies on LF

125 given orally show little, if any, presence of LF immunological epitopes in circulation as measured by ELISA (Kirkpatrick et al. 2019). Accordingly, the concept of signal transduction mechanism for systemic effects of

orally administered LF seems to be the most plausible pathway. It is accepted in the scientific community that

LF given orally interacts with the specific receptors on gut epithelial cells to trigger signaling pathways that may

spread systemic effects (Ashida et al. 2004). Moreover, it has been shown that under certain circumstances LF

130 can pass through the digestive tract to the feces without being absorbed or digested (Troost et al. 2002).

Several laboratories have also demonstrated that LF given orally is not toxic for animals or humans (Johnston

et al. 2015). The safety of bLF was evaluated in rats and the NOAEL (No Observed Adverse Effect Level) was

estimated to be in excess of 2,000 mg/kg/day (Yamauchi et al. 2000).

However, the effect of oral administered LF on gene expression has never been fully investigated, although

135 gene alteration by LF has been studied by numerous investigators in different cell cultures (Blais et al. 2014;

Frioni et al. 2014; Jiang and Lonnerdal 2014; Valenti and Vogel 2014). Transcriptome profiling has been one of

the most utilized approaches to investigate human disease on the molecular levels and to identify biomarkers

and therapeutic targets. The aim of the current study was to perform a comparison analysis of the blood cell

transcriptome profiles from control rats and rats treated with rhLF using Clariom D microarray. This genome-

140 wide methodology of total RNA analysis enables quantification of gene expression levels, splice isoforms, and

noncoding RNA to advance our understanding of mechanisms underlying the biological activity of LF. Notably,

we used rhLF expressed in Chinese Hamster Ovary (CHO) cells allowing mammalian glycosylation of the

protein, a post-translational modification required for proper biological functions of LF in humans (Kruzel et al.

2013). It is critically important of understanding the systemic biological effects transduced by rhLF to optimize

145 routes of administration for targeting specific diseases and disorders. Finally, and of equal importance, no

networks were identified which indicate induction of any type of systemic toxicity.

The goal of this article is to review evidence that systemic effects of orally delivered LF are driven by signal

transduction mechanisms in a similar way as that seen for IV administration. The experiments here are

designed to test whether rhLF differentially alters the gene expression levels of multiple transcriptome

150 networks when given orally or via IV administration.

Page 4 of 19



Draft

Materials and Methods155

Human Lactoferrin

Recombinant human lactoferrin (rhLF) was supplied by PharmaReview Corporation, Houston, TX, USA. The

rhLF was expressed in Chinese Hamster Ovary (CHO) cells followed by the scale up production and

purification described by Kruzel et al., 2013 (Kruzel et al. 2013). The lyophilized powder of rhLF (<15% iron

160 saturated; <0.5 endotoxin units/mg) was freshly prepared by dissolving in PBS buffer, pH 7.2.

Chemicals and reagents

Unless otherwise mentioned, all the chemicals and reagents were purchased from Sigma‐Aldrich Inc. (St.

Louis, MO, USA) and were of the highest purity available.

Animals and treatment protocols

165 Six male Sprague Dawley rats (Harlan Laboratories, Indianapolis, IN) (350–400 g) were catheterized (jugular

vein) and divided into 2 groups of 3 animals each. Animals from Group 1 were given a single intravenous (tail

vein) dose of rhLF at 5 mg/kg and animals from Group 2 were treated with a single oral (gavage) dose of rhLF

at 5 mg/kg in PBS buffer (pH 7.2) as previously established (Zhao et al. 2020). Blood from the animals in each

group was collected from the IV catheter prior to dosing (control) and at 3h, 6h and 24h after rhLF

170 administration. A heparinized saline flush of 0.3 mL was administered after each blood draw. All procedures

were approved by the local Institutional Animal Care and Use Committee (IACUC #1943) and were consistent

with the National Institutes of Health guidelines.

RNA isolation

100 L of whole blood samples were immediately transferred to RNeasy Protect Animal Blood Tubes (Qiagen

175 MD, USA Cat, No. 76544) containing a stabilizer for cellular RNA. Total RNA was then purified using RNeasy

Protect Animal Blood Kit (Qiagen MD, USA; Cat. No. 73224) according to manufacturer’s instructions. The kit

provides a complete solution for the purification of high-quality RNA. RNA concentration was quantified using a

NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific MA, USA) at 260 nm absorbance. The

260/280 nm absorbance ratio was used to assess the purity of isolated RNA (ratio: 2.01-2.03).

180 Clariom D microarray and data analysis

Applied BiosystemsTM Clariom D Rat microarray was performed using the High Throughput (HT) WT PLUS Kit

(ThermoFisher, CA, USA) with automated RNA preparation of samples for whole transcriptome expression

analysis with GeneChip™ Whole Transcript (WT) Expression Array (ThermoFisher, CA, USA). Clariom D is

based on a photolithographic microarray platform that layers oligonucleotide sequences onto a glass substrate

185 formatted cartridge. Reverse transcription is initiated at the poly-A tail, as well as throughout the entire length

of RNA, to capture both coding and multiple forms of noncoding RNA. Complementary RNA (cRNA)

amplification is achieved using low-cycle PCR followed by linear amplification with T7 in vitro transcription (IVT)

technology. The cRNA is then converted to biotinylated double-stranded cDNA (ds-cDNA) hybridization targets

Page 5 of 19



Draft

for unbiased coverage of the transcriptome. The hybridization was performed with the Gene TitanTM

190 hybridization kit (ThermoFisher, CA, USA) according to manufacturer’s direction. Once hybridized, the

cartridge was scanned with the GeneChip Scanner 3000 7G System (Affymetrix CA, USA). Data files were

generated and processed with the Affymetrix software and analyzed with the Applied Biosystems™

Transcriptome Analysis Console 4.0.2 software (all from ThermoFisher, CA, USA). A threshold of 200 signal

intensity units was used. Gene expression levels in blood cells obtained from rhLF-treated rats at each time

195 point were compared to the corresponding gene expression levels from controls (pre-dose) animals in a pair-

wise fashion. The identified differentially expressed genes with increased or decreased expression levels ≥ 2-

fold compared to controls for oral and IV treatment were selected for further analysis.

Statistical analysis

The analysis of microarray data for differential gene expression was performed by the Transcriptome Analysis

200 Console 4.0.2 software using the Linear Models for Microarray and RNA-Seq Data method (LIMMA). For

statistical analysis a One-Way ANOVA test was used to compare differences in gene expression between two

groups with p value < 0.05. Genes that displayed an expression level at least 2-fold different in rhLF-treated

sample versus control sample were carried forward in the analysis. The cell signaling pathway analysis was

selected from the WikiPathways database and limited to inflammatory response pathways (WikiPathways.org)

205 in rats (Rattus norvegicus).

210

215

220

225

Page 6 of 19



Draft

Results and Discussion

rhLF Induces Changes in Gene Expression

To evaluate the systemic effects of intravenous and oral administrated rhLF, transcriptome profiling of blood

cells isolated from pre-dose (control) rats and after 3h, 6h, and 24h of treatment with rhLF was performed

230 using the Clariom D assay. The sequences on the Clariom D arrays are based on coding and noncoding

sequences obtained from 16 different databases. This high-density array contains millions of copies of DNA

oligonucleotide probes that are designed to bind specific sequences of target RNA. Thus, the Clariom D

platform provides full coverage of the transcribed genome including gene- and exon-level expression profiles

as well as the ability to detect alternative splicing of coding and long noncoding RNAs (lncRNAs).

235 A graphic representation of genes differentially expressed in response to IV or oral administrated rhLF after 6h

compared with the control samples is presented in Fig 1. The results showed up-regulation of the similar

percentage of genes from coding sequence by oral and IV administrated LF relative to control (58.49% and

57.79%, respectively). This differs from the 33.66% of genes from coding sequences, which were down-

regulated by oral treatment as compared to 23.11% by IV treatment. Of interest, the percentage of down-

240 regulated genes from noncoding sequences was higher in response to IV than oral treatment (36.99% and

21.53%, respectively). Furthermore, the up-regulation of genes from noncoding sequences was similar for both

treatments (16.25% for IV versus 18.61% for oral). The number of noncoding sequences affected by rhLF

treatment is relatively high when compared to coding sequences. Recently, the implementation of next

generation sequencing technologies identified distinct lncRNAs in human that account for a large majority of all

245 RNA sequences transcribed across the human genome (Moore and Uchida 2020). The function of lncRNA is

steadily being uncovered, with fewer than 200 characterized so far but tens of thousands already identified. Evidence suggests that lncRNAs are functional as a modulator of transcription and translation, therefore; they

are essential regulators not only of gene expression but also proteins (Moore and Uchida 2020). For example,

some IncRNAs can mediate the sequestration of free miRNAs. Importantly, lncRNAs have also been identified

250 as valuable sources of diagnostic biomarkers such as PCA3 for diagnosis of prostate cancer and potential

therapeutic targets (Moranova and Bartosik 2019). Our results demonstrated for the first time that rhLF induced

the changes in transcriptome of IncRNA sequences in the rat, providing a novel mechanism of regulation of

transcription as well as translation reported in human.

255 Route Dependent Differential Gene Expression

To identify differential expressed genes for each route of treatment, the blood cell transcriptome results

obtained from control (pre-dose) rats were compared with the transcriptome data from rats treated with either

IV or oral rhLF for each time point of treatment. As shown in Fig 2A, 2237 of 3337 genes differentially

expressed were up-regulated in response to 3h of IV treatment, while 1100 were down-regulated. In

260 comparison, 1133 transcripts were up-regulated in response to 3h of oral treatment, out of 1964, while 831

were down-regulated. After 6h, 4865 (IV) and 2387 (oral) differentially expressed genes were identified, with

similar relative differences between the up- and down-regulated activities. At the 24h time point, 2141 genes

Page 7 of 19



Draft

were up- and 1535 were down-regulated following IV treatment as compared to 1537 up-regulated genes and

1030 down-regulated genes after oral treatment. Overall, the responses to IV administrated rhLF were

265 associated with more pronounced alteration of transcriptome at all time points examined when compared with

orally delivered rhLF. In addition, we observed that the number of differentially expressed genes reached the

maximum levels at 6h after IV treatment and gradually declined by 24h. In contrast, the number of differentially

expressed genes in response to oral administrated rhLF were slightly increased during time course of 24h

treatment (Fig 2A).

270 To determine whether oral administrated rhLF induces the systemic effects similar to IV treatment, an

additional analysis was performed of the identified differentially expressed genes. The genes that were

differentially expressed in response to IV treatment were directly compared with the genes that were

differentially expressed in response to oral administration for each time point of treatment duration (Fig. 2B).

For example, a comparison analysis of 6h treatment revealed that 72.8% differentially expressed genes (1738

275 genes out of 2387 total) in response to oral rhLF administration was common with IV treatment (Fig. 2B). In

addition, 649 genes were specifically altered by oral administrated rhLF and 3127 genes were affected only in

response to IV treatment (Fig. 2B).

Together, these results showed that IV as well as oral administrated rhLF altered transcriptome profiles of the

blood cells including coding and noncoding RNA species and a majority of the changes in transcriptome

280 induced by oral administration of rhLF are similar to IV responses.

To gain insight into the molecular mechanisms by which rhLF may exert its complex biological effects, specific

pathways of differentially expressed genes in response to IV and oral treatments were further analyzed.

Examples of LF effects on gene modulation within these pathways are presented as direct comparison of

genes that were differentially expressed by IV versus oral administration using WikiPathways database

285 (http://wikipathways.org) for biological pathways in rats (Rattus norvegicus). The data depicted in Fig 3 was

generated based on differential gene expression after 6 h for rhLF given IV versus oral administration using

database from the Cytokine and Inflammatory Response Pathway. Both treatments produced remarkably

similar responses as illustrated in Fig 3. A significant up-regulation in interleukin-1β (IL-1), transforming growth

factor beta 1 (TGFβ1), and colony stimulating factor 1 (CSF-1) was identified for both IV and oral treatments as

290 compared to control. These genes are important for clonal expansion of T cells as well as for macrophage

inflammatory response activities. It is worth mentioning that the upregulation of these genes was sustained

through 24 h, although the intensity gradually diminished (data not shown). These observations are in line with

our data and general understanding that exogenous LF, given IV or orally, mimics a natural in vivo response of

immune system during any insult-induced activation of neutrophils and massive release of endogenous LF

295 (Kruzel et al. 2017).

It is known that LF protects against oxidative stress-induced mitochondrial dysfunction and DNA damage

(Kruzel et al. 2010). LF has the ability to control cellular oxidative stress by virtue of iron sequestration. Earlier

studies reported that LF up-regulates oxidative stress related genes in Caco-2 cells (Ashida et al. 2004; Blais

et al. 2014; Kruzel et al. 2010; Kruzel et al. 2013). Therefore, it was interesting to compare IV versus oral rhLF

Page 8 of 19



about:blank

Draft

300 effects on rat’s blood cells transcriptome using an Oxidative Stress Pathway. Fig 4 illustrates rhLF effects on

suppression and/or activation of oxidative stress related genes following IV and oral rhLF treatments of rats.

The similarities between IV and oral gene expression responses are prominent, however the intensity of

expression response indicates different levels of gene activation. Both treatments were effective in up-

regulation of Mitogen-Activated Protein Kinase 14 (MAPK14), a major stress regulator that also controls

305 activation of nuclear factor-kappa B (NFB), FOS Proto-Oncogene transcription factor (FOS), and JUNB Proto-

Oncogene transcription factor (JUNB). Of interest, a repression of reactive oxygen species (ROS) production is

similarly affected in both routes of rhLF administration by activation of two major oxidase inhibitors,

Cytochrome B-245 Alpha Chain (CYBA) and Xanthine dehydrogenase (XDH) as compared with control.

Furthermore, suppression of major genes within the Oxidative Stress Pathway was observed for both

310 treatments.

Not all pathways examined showed similar event trajectories of matching gene regulation. Specific differences

were identified when the inflammatory response pathways were investigated. Evidence supports that LF plays

a key role in the development of acute and chronic inflammatory responses (Kruzel and Zimecki 2002). To

investigate how the route of rhLF administration may affect early inflammatory responses the transcriptome

315 results were applied to the Inflammatory Response Pathway database. Fig 5 illustrates the genes of early

responses to inflammation that were differentially affected by 6h rhLF treatment (IV versus oral). In both cases

three genes were significantly upregulated: Tumor Necrosis Factor Receptor Superfamily Member 1α

(TNFrsf1α), Tumor Necrosis Factor Receptor Superfamily Member 1β (TNFrs1β), and Fibronectin 1 (FN1). The

up-regulation of TNF receptors by 6h LF treatment may facilitate the binding of TNFα and activation of NFB

320 pathways which trigger inflammation and leads to the production of a variety of immune mediators (Liu et al.

2017). Clearly, the difference in response to IV versus oral treatment is illustrated by down-regulation of CD40

ligand by oral treatment and up-regulation of CD80 by IV treatment. In addition, there was significant down-

regulation of IL5 receptor following IV treatment, and no such effect was observed by oral treatment. It is

generally accepted that CD40-CD40L interactions are important for stimulation of CD4 T-cell effector

325 functions. Therefore, these results suggest possible reduction of cell-to-cell interactions when rhLF is given

orally. On the other hand, up-regulation of CD80 following IV treatment may facilitate T cell proliferation and

production of inflammatory mediators in general.

Conclusions330

In conclusion, this is the first report to provide direct comparison of oral and IV administrated rhLF on the

transcriptome profile analysis of blood cells to evaluate the systemic effects of oral rhLF treatment in rats.

These results demonstrated that both treatments induced changes in coding and noncoding RNAs. Moreover,

comparison of the differential gene expression showed that oral and IV administrated rhLF altered expression

335 of common genes as well as distinct genes. Notably, the majority of modified genes in response to oral

treatment were also altered by IV administration. It is important to emphasize that additional genome analysis

including other signaling pathways needs to be performed to gain insights into pathway crosstalk; this would

Page 9 of 19



Draft

potentially provide clinically relevant implications. The next step would be to validate which changes in

transcriptome mediated by rhLF are translated into biologically active proteins using proteomic analysis.

340 Nevertheless, it appears that oral administration rhLF may be an alternative, at least in some cases, to

intravenous administration. Importantly, a high level of bioequivalence was demonstrated for these two routes

of administration. This may change a common perception of bioavailability that is limited to measuring the level

of compound physically crossing the gut-blood barrier without a consideration of systemic effects due to the

epithelial cell receptor binding and signal transduction. LF is relatively large protein (~80 kD) that is unlikely to

345 directly cross that barrier, but as demonstrated in this report, it shows biological activity at the level of gene

regulatory pathways in blood cells. These studies confirm that oral delivered LF can display systemic biological

activity by inducing signal transduction mechanisms, which lead to similar activities defined by IV administered

agent.

350 Figure Legends

Fig 1. Overall representation of the blood cell transcriptome profiles in response to intravenous and oral administrated rhLF in rats. The percentage of indicated groups of coding and noncoding RNAs that were differentially expressed in blood cells after 6h treatment with IV (left) and oral rhLF (right) compared with control. Graphs show up-regulated (top) and down-regulated (bottom) genes for each route of administration.

355Fig 2. Differential gene expression in the blood cells in response to intravenous and oral administrated rhLF in rats. A. Analysis of differentially expressed genes in blood cells after 3h, 6h and 24h treatment with IV and oral rhLF compared with control. The numbers of total, up-regulated and down-regulated differentially expressed genes are shown. B. The venn diagrams show a comparison analysis of the differentially expressed

360 genes in response to IV and oral rhLF administration for identification of common altered genes after 3h, 6h and 24h treatment. The number of common (overlap) and distinct genes that were differentially expressed in response to IV and oral rhLF treatment are indicated.

Fig 3. Cytokines and inflammatory response related molecular networks affected by IV and oral rhLF 365 treatment of rats. Comparison of relative ≥ 2-fold changes in inflammatory response genes were identified

after IV treatment (left panel) compared to oral treatment with rhLF (right panel). The nodes are colored red to indicate up-regulation, green to indicate down-regulation and gray to indicate nodes which did not reach the threshold for expression level of at least 2-fold change. Genes identified include Interleukin-1β (IL-1), Transforming growth factor beta 1 (TGF β 1), and Colony Stimulating Factor 1 (CSF-1). Solid arrows indicate

370 direct interaction effects on pathways; dashed arrows indicate indirect effects. Data shown for 6h post treatment.

Fig 4. Oxidative stress related molecular network affected by IV and oral rhLF treatment of rats. Comparison of relative ≥ 2-fold changes in inflammatory response genes were identified after IV treatment (left

375 panel) compared to oral treatment with rhLF (right panel). Xanthine dehydrogenase (XDH), Mitogen-Activated Protein Kinase 14 (MAPK14), Microsomal glutathione S-transferase 1 (MGST1), Cytochrome B-245 Alpha Chain (CYBA), nuclear factor-kappa B (NFB), FOS Proto-Oncogene transcription factor (FOS), JUNB Proto-Oncogene transcription factor (JUNB), Thioredoxin Reductase (TXNRD1). The nodes are colored red to indicate up-regulation, green to indicate down-regulation and gray to indicate nodes that did not reach the

380 threshold for 2-fold change in expression level. Data shown for 6h post treatment.

Fig 5. Inflammatory Response related molecular networks affected by IV and oral rhLF treatment of rats. Comparison of relative ≥ 2-fold changes in inflammatory response genes were identified after IV treatment (left panel) compared to oral treatment with rhLF (right panel). Tumor Necrosis Factor Receptor

385 Superfamily Member 1 (TNFRS 1), Tumor Necrosis Factor Receptor Superfamily Member 1β (TNFRSF 1β),

Page 10 of 19



Draft

Fibronectin 1 (FN1), Interleukin 5 Receptor Subunit Alpha (IL5Rα), T-Lymphocyte Activation Antigen CD80 (CD80), CD40 Ligand (CD40LG). The nodes are colored red to indicate up-regulation, green to indicate down-regulation and gray to indicate nodes which did not reach the threshold for expression level 2-fold change. Data shown for 6h post treatment.

390Acknowledgements

This work was given in part at the 14th International Conference on Lactoferrin Structure, Function and Applications, held in Lima, Peru (2019) (Kruzel 2019).

395

References

Actor, J.K., Hwang, S.A., and Kruzel, M.L. 2009. Lactoferrin as a natural immune modulator. Curr Pharm Des 400 15(17): 1956-1973. doi:10.2174/138161209788453202.

Alizadeh, A., Akbari, P., Difilippo, E., Schols, H.A., Ulfman, L.H., Schoterman, M.H., et al. 2016. The piglet as a model for studying dietary components in infant diets: effects of galacto-oligosaccharides on intestinal functions. The British journal of nutrition 115(4): 605-618. doi:10.1017/S0007114515004997.

405Ashida, K., Sasaki, H., Suzuki, Y.A., and Lonnerdal, B. 2004. Cellular internalization of lactoferrin in intestinal

epithelial cells. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 17(3): 311-315. doi:10.1023/b:biom.0000027710.13543.3f.

410 Baveye, S., Elass, E., Mazurier, J., Spik, G., and Legrand, D. 1999. Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clinical chemistry and laboratory medicine 37(3): 281-286. doi:10.1515/CCLM.1999.049.

Baynes, R.D., and Bezwoda, W.R. 1994. Lactoferrin and the inflammatory response. Adv Exp Med Biol 357: 415 133-141. Available from

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7762425 [accessed.

Blais, A., Fan, C., Voisin, T., Aattouri, N., Dubarry, M., Blachier, F., et al. 2014. Effects of lactoferrin on 420 intestinal epithelial cell growth and differentiation: an in vivo and in vitro study. Biometals : an

international journal on the role of metal ions in biology, biochemistry, and medicine 27(5): 857-874. doi:10.1007/s10534-014-9779-7.

Brines, R.D., and Brock, J.H. 1983. The effect of trypsin and chymotrypsin on the in vitro antimicrobial and 425 iron-binding properties of lactoferrin in human milk and bovine colostrum. Unusual resistance of human

apolactoferrin to proteolytic digestion. Biochimica et biophysica acta 759(3): 229-235. doi:10.1016/0304-4165(83)90317-3.

Chen, Y., Zheng, Z., Zhu, X., Shi, Y., Tian, D., Zhao, F., et al. 2015. Lactoferrin Promotes Early 430 Neurodevelopment and Cognition in Postnatal Piglets by Upregulating the BDNF Signaling Pathway

and Polysialylation. Molecular neurobiology 52(1): 256-269. doi:10.1007/s12035-014-8856-9.

Dix, C., and Wright, O. 2018. Bioavailability of a Novel Form of Microencapsulated Bovine Lactoferrin and Its Effect on Inflammatory Markers and the Gut Microbiome: A Pilot Study. Nutrients 10(8).

435 doi:10.3390/nu10081115.

Page 11 of 19



about:blank

about:blank

Draft

Donovan, S.M. 2016. The Role of Lactoferrin in Gastrointestinal and Immune Development and Function: A Preclinical Perspective. The Journal of pediatrics 173 Suppl: S16-28. doi:10.1016/j.jpeds.2016.02.072.

440 Hayashi, T., To, M., Saruta, J., Sato, C., Yamamoto, Y., Kondo, Y., et al. 2017. Salivary lactoferrin is transferred into the brain via the sublingual route. Bioscience, biotechnology, and biochemistry 81(7): 1300-1304. doi:10.1080/09168451.2017.1308241.

Frioni, A., Conte, M.P., Cutone, A., Longhi, C., Musci, G., di Patti, M.C., et al. 2014. Lactoferrin differently 445 modulates the inflammatory response in epithelial models mimicking human inflammatory and

infectious diseases. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 27(5): 843-856. doi:10.1007/s10534-014-9740-9.

Jiang, R., and Lonnerdal, B. 2014. Transcriptomic profiling of intestinal epithelial cells in response to human, 450 bovine and commercial bovine lactoferrins. Biometals : an international journal on the role of metal ions

in biology, biochemistry, and medicine 27(5): 831-841. doi:10.1007/s10534-014-9746-3.

Johnston, M., Landers, S., Noble, L., Szucs, K., and Viehmann, L. 2012. Section on Breastfeeding: Breastfeeding and the use of human milk. Pediatrics 129(3): e827-841. doi:10.1542/peds.2011-3552.

455Johnston, W.H., Ashley, C., Yeiser, M., Harris, C.L., Stolz, S.I., Wampler, J.L., et al. 2015. Growth and

tolerance of formula with lactoferrin in infants through one year of age: double-blind, randomized, controlled trial. BMC pediatrics 15: 173. doi:10.1186/s12887-015-0488-3.

460 Kamemori, N., Takeuchi, T., Sugiyama, A., Miyabayashi, M., Kitagawa, H., Shimizu, H., et al. 2008. Trans-endothelial and trans-epithelial transfer of lactoferrin into the brain through BBB and BCSFB in adult rats. The Journal of veterinary medical science 70(3): 313-315. doi:10.1292/jvms.70.313.

Kirkpatrick, T., Firley, W., and Kruzel, M.L. 2019. An LC-MS/MS Method for Quantifying Lactoferrin in 465 Plasma. In The 14th International Conference on Lactoferrin Structure, Function and Applications,

Lima, Peru.

Kruzel, M.L., and Zimecki, M. 2002. Lactoferrin and immunologic dissonance: clinical implications. Archivum immunologiae et therapiae experimentalis 50(6): 399-410. Available from

470 http://www.ncbi.nlm.nih.gov/pubmed/12546066 [accessed.

Kruzel, M.L., Zimecki, M., and Actor, J.K. 2017. Lactoferrin in a context of inflammation-induced pathology. Frontiers in immunology 8: 1438.

475 Kruzel, M.L., Harari, Y., Chen, C.Y., and Castro, G.A. 2000. Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice. Inflammation 24(1): 33-44. doi:10.1023/a:1006935908960.

Kruzel, M.L., Actor, J.K., Boldogh, I., and Zimecki, M. 2007. Lactoferrin in health and disease. Postepy 480 higieny i medycyny doswiadczalnej 61: 261-267. Available from

http://www.ncbi.nlm.nih.gov/pubmed/17507874 [accessed.

Kruzel, M.L., Actor, J.K., Radak, Z., Bacsi, A., Saavedra-Molina, A., and Boldogh, I. 2010. Lactoferrin decreases LPS-induced mitochondrial dysfunction in cultured cells and in animal endotoxemia model.

485 Innate immunity 16(2): 67-79. doi:10.1177/1753425909105317.

Page 12 of 19



about:blank

about:blank

Draft

Kruzel, M.L., Actor, J.K., Zimecki, M., Wise, J., Ploszaj, P., Mirza, S., et al. 2013. Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression. Journal of biotechnology 168(4): 666-675. doi:10.1016/j.jbiotec.2013.09.011.

490Kruzel, M.L.H., S-A., Olszewska, P.,Actor J.K. . 2019. Systemic effects of oral lactoferrin. In The 14th

International Conference on Lactoferrin Structure, Function and Applications, Lima, Peru.

Li, F., Wu, S.S., Berseth, C.L., Harris, C.L., Richards, J.D., Wampler, J.L., et al. 2019. Improved 495 Neurodevelopmental Outcomes Associated with Bovine Milk Fat Globule Membrane and Lactoferrin in

Infant Formula: A Randomized, Controlled Trial. The Journal of pediatrics 215: 24-31 e28. doi:10.1016/j.jpeds.2019.08.030.

Liao, Y., Alvarado, R., Phinney, B., and Lonnerdal, B. 2011. Proteomic characterization of human milk whey 500 proteins during a twelve-month lactation period. Journal of proteome research 10(4): 1746-1754.

doi:10.1021/pr101028k.

Liu, T., Zhang, L., Joo, D., and Sun, S.C. 2017. NF-kappaB signaling in inflammation. Signal transduction and targeted therapy 2. doi:10.1038/sigtrans.2017.23.

505Lonnerdal, B., and Iyer, S. 1995. Lactoferrin: molecular structure and biological function. Annual review of

nutrition 15: 93-110. doi:10.1146/annurev.nu.15.070195.000521.

Miller, E.R., and Ullrey, D.E. 1987. The pig as a model for human nutrition. Annual review of nutrition 7: 361-510 382. doi:10.1146/annurev.nu.07.070187.002045.

Miner-Williams, W.M., Stevens, B.R., and Moughan, P.J. 2014. Are intact peptides absorbed from the healthy gut in the adult human? Nutrition research reviews 27(2): 308-329. doi:10.1017/S0954422414000225.

515 Montagne, P., Cuilliere, M.L., Mole, C., Bene, M.C., and Faure, G. 2001. Changes in lactoferrin and lysozyme levels in human milk during the first twelve weeks of lactation. Advances in experimental medicine and biology 501: 241-247. doi:10.1007/978-1-4615-1371-1_30.

Moore, J.B.t., and Uchida, S. 2020. Functional characterization of long noncoding RNAs. Current opinion in 520 cardiology. doi:10.1097/HCO.0000000000000725.

Moranova, L., and Bartosik, M. 2019. Long Non-Coding RNAs - Current Methods of Detection and Clinical Applications. Klinicka onkologie : casopis Ceske a Slovenske onkologicke spolecnosti 32(Supplementum 3): 65-71. doi:10.14735/amko20193S65.

525Morgenthau, A., Pogoutse, A., Adamiak, P., Moraes, T.F., and Schryvers, A.B. 2013. Bacterial receptors for

host transferrin and lactoferrin: molecular mechanisms and role in host-microbe interactions. Future microbiology 8(12): 1575-1585. doi:10.2217/fmb.13.125.

530 Moughan, P.J., Birtles, M.J., Cranwell, P.D., Smith, W.C., and Pedraza, M. 1992a. The piglet as a model animal for studying aspects of digestion and absorption in milk-fed human infants. World review of nutrition and dietetics 67: 40-113. doi:10.1159/000419461.

Moughan, P.J., Birtles, M.H., Cranwell, P.D., Smith, W.C., and Pedraza, M. 1992b. The piglet as a model for 535 studying aspects of digestion and absorption in milk-fed human infants. World review of nutrition and

dietetics 67: 40-113.

Page 13 of 19



Draft

Oda, H., Wakabayashi, H., Yamauchi, K., and Abe, F. 2014. Lactoferrin and bifidobacteria. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 27(5): 915-922.

540 doi:10.1007/s10534-014-9741-8.

Rosa, L., Cutone, A., Lepanto, M.S., Paesano, R., and Valenti, P. 2017. Lactoferrin: A Natural Glycoprotein Involved in Iron and Inflammatory Homeostasis. International journal of molecular sciences 18(9). doi:10.3390/ijms18091985.

545Sanchez, L., Calvo, M., and Brock, J.H. 1992. Biological role of lactoferrin. Arch Dis Child 67(5): 657-661.

Available from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1599309 [accessed.

550Troost, F.J., Saris, W.H., and Brummer, R.J. 2002. Orally ingested human lactoferrin is digested and secreted in

the upper gastrointestinal tract in vivo in women with ileostomies. The Journal of nutrition 132(9): 2597-2600. doi:10.1093/jn/132.9.2597.

555 Valenti, P., and Vogel, H.J. 2014. Lactoferrin, all roads lead to Rome. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 27(5): 803-806. doi:10.1007/s10534-014-9787-7.

Wang, B. 2016. Molecular Determinants of Milk Lactoferrin as a Bioactive Compound in Early 560 Neurodevelopment and Cognition. The Journal of pediatrics 173 Suppl: S29-36.

doi:10.1016/j.jpeds.2016.02.073.

Yamauchi, K., Toida, T., Nishimura, S., Nagano, E., Kusuoka, O., Teraguchi, S., et al. 2000. 13-Week oral repeated administration toxicity study of bovine lactoferrin in rats. Food and chemical toxicology : an

565 international journal published for the British Industrial Biological Research Association 38(6): 503-512. doi:10.1016/s0278-6915(00)00036-3.

Zhao, X., Kruzel, M.L., Ting, S.-M., Sun, G., Savitz, S.I., and Aronowski, J. 2020. Optimized lactoferrin as a highly promising treatment for intracerebral hemorrhage: Pre-clinical experience. Journal of Cerebral

570 Blood Flow and Metabolism. doi:10.1177/0271678X20925667.

575

Page 14 of 19



about:blank

about:blank

Draft

Fig. 1

Page 15 of 19



Draft

3h

6h

24h

IV ORAL

IV ORAL

IV ORAL

Fig. 2

A B

Page 16 of 19



Draft

Fig. 3

Page 17 of 19



Draft

Fig. 4

Page 18 of 19



Draft

Fig. 5

Page 19 of 19



new insights into the systemic effects of oral lactoferrin

Documents