Human breast milk-derived exosomes attenuate lipopolysaccharide-induced upregulation of CD40 and NLRP3 in microglia

doi:10.21203/rs.3.rs-4818836/v1

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Human breast milk-derived exosomes attenuate lipopolysaccharide-induced upregulation of CD40 and NLRP3 in microglia

https://doi.org/10.21203/rs.3.rs-4818836/v1

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Microglia mediate the immune response in the central nervous system to many insults, including lipopolysaccharide (LPS), a bacterial endotoxin that initiates neuroinflammation in the neonatal population, especially preterm infants. The synthesis of the proinflammatory proteins CD40 and NLRP3 depends on the canonical ΝF-κΒ cascade as the genes encoding CD40 and NLRP3 are transcribed by the phosphorylated ΝF-κΒ p50/p65 heterodimer in LPS-induced microglia. Exosomes, which are nanosized vesicles (40–150 nm) involved in intercellular communication, are implicated in many pathophysiological processes. Human breast milk, which is rich in exosomes, plays a vital role in neonatal immune system maturation and adaptation. Activated microglia may cause brain-associated injuries or disorders; therefore, we hypothesize that human breast milk-derived exosomes (HBME) attenuate LPS-induced activation of CD40 and NLRP3 by decreasing p38 MAPK and ΝF-κΒ p50/p65 activation/phosphorylation downstream of TLR4 in murine microglia (BV2). We isolated purified HBME and characterized them using nanoparticle tracking analysis, transmission electron microscopy, fluorescence-activated cell sorting, and western blots. Analysis of BV2 microglia exposed to LPS and HBME indicated that HBME modulated the expression of signaling molecules in the canonical ΝF-κΒ pathway, including MyD88, ΙκΒα, p38 MAPK, NF-κB p65, and their products CD40, NLRP3, and cytokines IL-1β and IL-10. Thus, HBME have great potential for attenuating CD40 and the NLPR3 inflammasome signaling in the microglial response to LPS.

breast milk

exosomes

BV2 microglia

neonatal neuroinflammation

lipopolysaccharide

ΝFκΒ

CD40

NLRP3

IL-1β

IL-10

Microglia, the resident immune cells of the central nervous system (CNS), are vital for axonal growth, immune surveillance, and maintenance of the neuronal circuitry. However, under hyperinflammatory conditions, they have deleterious effects on the surrounding brain tissue, thereby contributing to neuroinflammatory diseases. Microglia are the first responders to CNS injury and infiltration by foreign bodies. The activated microglia protect against various brain insults, but they also induce injury as they initiate phagocytosis by secreting multiple toxic cytokines, chemokines, and reactive oxygen intermediates (1, 2). Neonates, especially preterm infants, are highly susceptible to infection, which results in a hyperinflammatory response. The Gram-negative bacteria that often cause these infections induce a dysregulated microglial response compared to the more regulated activation response from other inducers (3). Microglia in the preterm infant brain respond to both infectious and sterile potentiators of neuroinflammation (4, 5), leading to neurodevelopmental damage that predisposes the infant to learning disorders, autism, schizophrenia, and epilepsy, among other disorders (6–8).

Lipopolysaccharide (LPS) is a potent Gram-negative bacterial endotoxin that drives systemic inflammation in neonates and is an adjuvant for an adaptive immune response (9, 10). In response to LPS-induced endotoxemia, various types of brain cells synthesize cytokines. Subsequently, peripheral granulocytes invade the CNS through the compromised blood-brain barrier, resulting in immunoreactivity and injury in the affected brain area (1, 11–14). A similar process is implicated in necrotizing enterocolitis (NEC), a severe and often fatal preterm neonatal gut disease characterized by widespread intestinal ischemic necrosis (15). NEC has been linked directly to toll-like receptor 4 (TLR4)-mediated microglial activation and is associated with neuroinflammation and neuronal cell death. NEC and neuroinflammation are initiated primarily by the binding of LPS to TLR4 on the intestinal epithelial surface and microglia, respectively (Fig. 1). Typically, cell activation leads to the signaling of the nuclear factor κ light-chain enhancer of activated B cells (ΝF-κΒ) pathway, which causes the release of proinflammatory chemokines and cytokines, such as interleukin (IL)-1β, IL-6, IL-8, IL-18, and tumor necrosis factor-alpha (TNF-α). This sustained inflammation compromises the blood-brain barrier, resulting in the leakage of inflammatory markers and LPS into the CNS (16, 17). The activation of the inhibitor of the nuclear factor-κB kinase (IKK) complex and the p38 mitogen-activated protein kinase (MAPK) leads to ΝF-κΒ p50/p65 heterodimer signaling. IKK and MAPK are activated downstream of TLR4 and phosphorylate the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκΒα). IκΒα is then targeted for proteasomal degradation, which releases the cytoplasmic ΝF-κΒ heterodimer for phosphorylation and translocation to the nucleus, where it produces several proinflammatory mediators (18, 19).

The p38 MAPK further potentiates ΝF-κΒ signaling by translocating to the nucleus where it 1) activates the mitogen-activated protein kinase-activated protein kinase 2 (MK2), which produces proinflammatory mediators, including IL-1β, nitric oxide (NO), TNF-α, and IL-6 (20, 21); 2) activates mitogen- and stress-activated kinase (MSK)1/2, which initiates and prolongs ΝF-κΒ p65 subunit activation (22, 23); and 3) enhances ΝF-κΒ binding through modification of certain promoter regions (21, 24–26). The TLR4 adaptor protein myeloid differentiation primary response 88 (MyD88) acts directly upstream of the IKK kinase complex and p38 MAPK to ensure their activation (27–29). MyD88 is critical for LPS-induced signaling to produce many factors, such as IL-1β, TNF-α, and IL-6 (9, 10, 30). The cluster of differentiation 40 (CD40) receptor and the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), which are implicated in surmounting neuroinflammatory effects (31, 32), are key products of LPS-induced ΝF-κΒ activation in microglia.

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on the surface of microglia, macrophages, B cells, dendritic cells, endothelial cells, and tumor cells. The binding of CD40 to its ligand, CD154 (CD40L), is essential for an effective immune response. Contact of the CD40-CD40L complex with MHC-II, CD80, and CD86 results in the secretion of many of the cytokines previously mentioned, macrophage inflammatory protein 1 (MIP-1), and toxic free radicals (33). The best-studied pathways signaling this adaptive immune response include the canonical and noncanonical ΝF-κΒ pathways that are secondary to the recruitment of proteins to the CD40 cytoplasmic domains. First, LPS-induced glial/macrophage activation enhances CD40 expression by activating the signal transducer and activator of transcription (STAT)-1α and ΝF-κΒ transcription factors through the innate immune response (31). Several immune and cellular functions are inactive without this CD40-CD40L interaction. Activated T cells are the major carriers of CD40L, but other immune and nonimmune cells carry CD40L (34). CD40 is implicated in many human diseases, especially autoimmune diseases (31), and blocking the CD40-CD40L interaction with antibodies provided benefits in autoimmune animal studies and human trials, suggesting the importance of the CD40-CD40L interaction in maintaining immune homeostasis (31, 35–37). The CD40-CD40L interaction results in further secretion of proinflammatory cytokines by microglia that may promote neuroinflammatory-induced disease progression. Resting microglia show low expression of markers of antigen presentation, including CD40 (31), but after activation, microglia undergo various morphological modifications to express cell surface markers necessary for antigen presentation (38). Microglial CD40 signaling is important in the progression of neurodegenerative diseases, including vascular/ischemic disease and Alzheimer’s disease (39, 40).

NLRP3 is important for proinflammatory cytokine secretion and, like CD40, has been implicated in inflammatory diseases, autoimmune diseases, neurodegeneration, and traumatic brain injury. An increase in NLRP3 expression directly amplifies NLRP3 inflammasome activation. Microglia are first primed by pathogen-associated molecular patterns (PAMPs) (e.g., LPS) or cytokines (e.g., IL-1β) to produce NLRP3 and precursors for caspase-1, IL-1β, and IL-18. A secondary signal, such as another PAMP or a damage-associated molecular pattern (DAMP), facilitates the oligomerization of inactive NLRP3, which recruits other inflammasome components, including caspase-1, NIMA-related kinase 7 (NEK7), and apoptosis-associated speck-like protein containing CARD (ASC) (41–43). The active inflammasome uses NLRP3 as a cytoplasmic pathogen recognition sensor (44) and caspase-1 as the effector, which cleaves pro-IL-1β and pro-IL-18 into their active forms in preparation for secretion from the cell (41–43). Inflammasome activation results from self-recognizing patterns, such as the high mobility group box 1 (HMGB1) protein (44, 45), and does not require an external molecular pattern for activation. LPS stimulation of microglia also promotes the packaging of IL-1β into exosomes, which are secreted from the cell as a result of NLRP3 and ASC functions. These exosomes, which have major histocompatibility complex (MHC) class II receptors, have immunomodulatory effects in addition to the effects of their cytokine cargo (46, 47).

Exosomes are 40–150 nm extracellular vesicles (EVs) derived from mammalian breast milk, blood, urine, semen, saliva, and cerebrospinal fluid (48) that function in intercellular communication and in immunological and pathophysiological processes (1, 2, 48, 49). They reflect the composition and physiology of their cellular origin, including the proteins, lipids, and nucleic acids they carry (50). Their low immunogenicity is an advantage in the immature and sensitive immune system of preterm infants (51). Their lipid bilayer allows them to survive transport across the blood-brain barrier and into the parenchyma and spinal cord (52, 53). Their integrity, separation, and functionality are well preserved after isolation and storage at − 80^οC for several months (54), making them a viable option for therapeutic and diagnostic applications.

Exosomes derived from breast milk modulate the immune response (55). We hypothesize that human breast milk-derived exosomes (HBME) attenuate LPS-induced CD40 and NLRP3 expression in BV2 microglial cells and attenuate microglial activation via inhibition of the TLR4-induced ΝF-κΒ pathway. Thus, we determined the effects of HBME on the NF-κΒ signaling cascade to identify specific molecular targets for neuroinflammatory intervention. We propose that the anti-inflammatory effect of HBME attenuates microglial activation, thus serving as a potential treatment for neuroinflammation.

Human breast milk collection

Human breast milk (HBM) was collected at the University of Alabama at Birmingham (UAB) Regional Intensive Care Unit. Breastmilk collection was approved by the UAB Institutional Review Board Protocol N160203002. Samples were recovered from patient feedings that were not completely consumed and would have been discarded. The initial experiments used individual samples; however, to improve consistency, we later pooled donor milk, which was pasteurized using the Holder technique (56). Exosomes in the milk samples were isolated and purified for immediate use or stored at − 80℃.

Extracellular vesicle isolation and purification

HBM was diluted 1:10 in sterile phosphate-buffered saline (PBS), centrifuged at 300 × g (1,300 revolutions per minute [rpm]) for 10 minutes, and the supernatant was centrifuged again at 2600 × g (3,900 rpm) for another 10 minutes. The supernatant was filtered through a sterile 0.22 µm filter using a 10 mL syringe and centrifuged at 20,000 × g (10,800 rpm) for 45 min in an SW41T1 swinging bucket rotor at 4°C using a Beckman Coulter Optima ™ L-70K Ultracentrifuge. The resulting supernatant was further centrifuged at 110,000 × g (32,000 rpm) for 70 min at 4°C using a Beckman Coulter Optima ™ L-70K Ultracentrifuge to collect the EV particles in the pellet, which were resuspended in sterile PBS.

Nanoparticle tracking analysis

HBME particle size and concentration were determined by nanoparticle tracking analysis (NTA). The samples were diluted in 1× PBS in a 1-mL disposable syringe. The samples were analyzed using the Nano-Sight NS 300 Sub-Micron Particle Imaging System (Malvern Instruments, Inc., Malvern, UK) and NTA v3.0 software. In this system, a laser light source illuminated the exosome particles, which were then analyzed based on Brownian motion by a semiconductor camera. For each reading frame, the mean values were recorded and analyzed.

Transmission electron microscopy

Transmission electron microscopy (TEM) was used to confirm the size and structure of EVs in the exosome samples. Carbon film-coated mesh copper EM-grids were glow discharged at 50 mA for 20 sec before loading 7 µL of the exosome suspension on the grid and incubating for 1 min at room temperature (RT). Samples were stained immediately with 7 µL of filtered uranyl acetate (UA) solution on the surface of the EM-grid. After 15 sec, the excess UA solution was removed, and samples were observed within 24 h under the TEM Tecnai 120kV (FEI, Hillsboro, OR) at 80 kV and compared to the negatively stained grids. Digital images were captured with a BioSprint 29 CCD camera (AMT, Woburn, MA).

Fluorescence-activated cell sorting

For ImageStream analysis, isolated HBME were stained with APC-CD81, FITC-CD63, and PE-CD9 antibodies with the respective isotype controls in 100 µL of 1× fluorescence-activated cell sorting (FACS) buffer and analyzed by ImageStream (BioLegend CA, USA).

Microglia cell culture and treatment with LPS and/or HBME

BV2 microglial cells, which are derived from C57/BL6 fetuses and pups and immortalized by v-raf/v-myc carrying J2 retrovirus (57), were grown in Roswell Park Memorial Institute (RPMI) 1640 media, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.5 µg/mL amphotericin B (Gibco, Thermo Fisher Scientific, Rockford, IL, USA). Glial cells at 70–80% confluency were transferred to 6-well plates with or without 1 µg/mL of LPS and 10 µg/mL of HBME for 0, 0.25, 0.5, 1, or 24 h.

Cell viability

Cell viability was measured by trypan blue staining in the Invitrogen Countess 2 automated cell counter. In brief, 10 µl of 0.4% trypan blue solution was mixed with 10 µl of the sample, and the device was used to measure cell number and viability in 10 µL of the mixture.

mRNA sequencing (mRNA-seq) analysis

RNA was extracted from microglial cells using 0.75 mL Trizol LS reagent (ThermoFisher Scientific, USA) per 0.25 mL of BV2 cell pellet. Samples were homogenized, centrifuged at 12000 × g for 5 min at 4 ºC, and incubated for 2–5 min after adding 0.2 mL of chloroform. The aqueous phase was collected after centrifugation at 12000 × g for 15 min at 4 ºC. RNA in the pellet was washed in 75% ice-cold ethanol diluted in DEPC water for 5 min at 7500 × g at 4 ºC and quantified by Nanodrop. RNA samples from treatment and control groups were tested for quality and processed for mRNA-seq, bioinformatics, and pathway analysis by Novogene NC, USA (https://en.novogene.com). Briefly, 1 µg of RNA was used to generate sequencing libraries on the Illumina HiSeq 2500 platform. A cutadapter software tool was used to remove the adapter sequences. Paired-end clean reads were aligned to the mouse genome (mm10) using the Spliced Transcripts Alignment to a Reference (STAR) software. The mapped reads were generated by FeatureCounts. Differential expression analysis was performed by edgeR (thresholds: fold change > 1.5, P < 0.05).

RT-PCR assay

RNA was extracted from BV2 cells using Direct-zolTM RNA MiniPrep (cat#R2052, ZYMO Research) or Trizol method and treated with DNase I and proteinase K to purify the RNA. Then, 500 ng of total RNA was reverse transcribed into cDNA using the PrimeScriptTMRT Reagent kit (cat#RR037Q, Takara). Quantitative real-time PCR was performed using Lunar Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA). Changes in relative expression were calculated by the 2^ΔΔCt method, normalized to GAPDH. Primer sequences are as follows: CD40, F: 5′-TTG TTG ACA GCG GTC CAT CTA-3′; R: 5′-GCC ATC GTG GAG GTA CTG TTT-3′; GAPDH, F: 5′-AGG TCG GTG TGA ACG GAT TTG-3′; R: TGT AGA CCA TGT AGT TGA GGT CA-3′.

Western blot (WB)

Protein was extracted from cells in Pierce RIPA buffer (Thermo Scientific, USA) with proteinase and phosphatase inhibitors (Millipore Sigma, Burlington, MA, USA) as well as 0.1 mM dithiothreitol and 0.1 mM ethylenediamine tetraacetic acid (Thermofisher Scientific, USA). For WB analysis, protein concentrations were estimated using the bichloroacetic acid (BCA) protein assay kit (Thermofisher Scientific, USA). About 10–25 µg of protein from HBME and BV2 microglia lysates were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels at 100V until the samples ran to the bottom of the resolving gel. The gels were subsequently electroblotted onto nitrocellulose (Thermofisher Scientific, Rockford, IL, USA; GVS, Bologna, Italy), polyvinylidene fluoride, or polyvinylidene difluoride (PVDF) (Millipore Sigma, Burlington, MA, USA) membranes at 15 volts for 30 minutes. Membranes were blocked with 5% non-fat milk (SKU: 30620074-1, BioWorld, Dublin, OH, USA) and detected by overnight incubation with an appropriate dilution of the following primary antibodies: anti-CD9, anti-CD63, anti-CD81, anti-GAPDH (Thermo Scientific, Rockford, IL, USA); anti-ΝF-κΒ p65, anti-p-ΝF-κΒ p65, anti-CD40, anti-ΙκΒα, anti-p38 MAPK, anti-p-p38 MAPK, anti-p44/42 MAPK, anti-p-p44/42 MAPK, anti-α/β tubulin (Cell Signaling Technology, Danvers, MA, USA); and anti-IL-1β, anti-ΝLRP3, anti-IL-10 (Abcam, Waltham, MA, USA). Membranes were then incubated with a goat anti-rabbit, goat anti-rat, (Thermo Scientific, Rockford, IL, USA) or horse anti-mouse (Cell Signaling Technology, Danvers, MA, USA) horseradish peroxidase (HRP)-conjugated secondary antibody in 2% non-fat milk (SKU: 30620074-1, BioWorld, Dublin, OH, USA) for 2 h. Membranes were washed with 1× Tris-buffered saline with 0.1% Tween® 20 (TBST) between each incubation step. Protein expression was detected by a chemiluminescent substrate (Cat #: XR92, Alkali Scientific, Fort Lauderdale, FL, USA; Millipore Sigma, Burlington, MA; ) followed by exposure of the membrane to film that was developed in a film processor (SRX 101-A, Konica Minolta Medical and Graphics, INC, Tokyo, Japan).

Enzyme-linked immunosorbent assay (ELISA)

Secreted mouse IL-1β and IL-10 from BV2 microglia were measured in cell medium supernatants using DuoSet ELISA kits (R&D Systems). Absorbance was determined at 450 nm with subtraction of a 570 nm reference wavelength using the Epoch™ microplate reader (BioTek Instruments Inc.).

Statistical analysis

The difference between two groups was analyzed using the student’s t-test. For multi-group comparisons, one-way ANOVA was used with the Fisher least significant difference test for post-hoc analysis. Statistical significance was set to P < 0.05. Analysis was performed with Microsoft Excel and GraphPad Prism. Data are presented as mean ± standard error of the mean (SEM).

Characterization of HBME

HBME characterized by NTA and TEM ranged in diameter from 50–150 nM (Fig. 2A), which is within the range reported previously (55). Immunoblot and ImageStream analysis showed exosome markers CD9, CD63, and CD81 on isolated particles (Fig. 2B–D).

HBME increase the viability of LPS-induced BV2 microglia

BV2 microglia were incubated with LPS (1 µg/mL) and HBME (10 µg/mL) for 24 h. LPS treatment decreased microglial viability compared to the control (P = 0.006), which was largely restored by HBME (Fig. 3A; P = 0.0109). Our data suggest an increase in LPS-induced microglia after HBME treatment. Although there were fewer microglia after LPS induction, there was likely a higher percentage of activated cells, which would account for the increase in proinflammatory markers.

HBME attenuate CD40 expression in LPS-induced BV2 microglia

The mRNA-seq analysis of cells incubated with LPS for 24 h identified 156 genes that were downregulated and 17 genes that were upregulated compared to the control (Fig. 3B). The addition of HBME to LPS-stimulated cells led to the downregulation of 136 genes and the upregulation of 25 genes (Fig. 3C). Among the 14 genes that we selected for further analysis, CD40 plays an important role in microglia-mediated hyperinflammatory disease, and its expression in LPS-induced microglia was greatly affected by HBME (Fig. 3D). We confirmed that HBME downregulated the mRNA (Fig. 3E; P = 0.0432) and protein expression (Fig. 3F; P = 0.0093) of the gene encoding CD40 compared to LPS treatment alone by RT-qPCR and WB analysis, respectively.

HBME modulate intracellular signaling pathways in LPS-induced BV2 microglia

We showed that HBME treatment increases the expression of MyD88 and ΙκΒα while decreasing the activation of p38 MAPK and NF-κB p65 in microglia simultaneously stimulated with LPS. MyD88 expression decreased in LPS-induced microglia compared to the control (P = 0.037) but increased after treatment with HBME for 1 h compared to the LPS-treated group (P = 0.0238). Interestingly, after LPS-induced microglia were treated with HBME, MyD88 expression exceeded the control (P = 0.0905) and microglia treated with HBME alone (P = 0.0983) (Fig. 4A). ΙκΒα expression decreased in LPS-induced microglia compared to the control after 1 h (P = 0.0071) and 24 h (P = 0.0092) but increased after treatment with HBME for 1 h and 24 h compared to the LPS-treated group (Fig. 4B; P = 0.0131 and P = 0.0063, respectively). Treatment of microglial cells with HBME decreased the LPS-induced phosphorylation of p38 MAPK after 15 min (Fig. 4C; P = 0.0393) and NF-κB p65 after 1 h (Fig. 4D; P = 0.0084).

HBME decrease the expression of intracellular inflammatory markers in LPS-induced BV2 microglia

The decrease in transcription factors NF-κB p50/p65 and p38 MAPK after treatment of LPS-induced BV2 microglia with HBME for 24 h also affected expression of the CD40, NLRP3, IL-1β, and IL-10 effector molecules. Expression of CD40, NLRP3, and IL-1β increased after LPS treatment but decreased after the addition of HBME (Fig. 5A–C; P = 0.026, P = 0.0365, and P = 0.031). In contrast, expression of IL-10 in LPS-induced BV2 microglia decreased compared to the control (P < 0.0001) and increased after treatment with HBME (Fig. 5D; P = 0.0177).

HBME alter the secretion of cytokines from LPS-induced BV2 microglia

Cytokine IL-1β promotes inflammatory signaling by activating NF-κB, much like LPS (58), whereas IL-10 is an essential suppressor of inflammatory pathways (59). It has been shown to inhibit NF-κB and p38 MAPK transcriptional activity (60, 61). It is both induced by STAT3 and in turn initiates immunosuppression by activating STAT3-dependent anti-inflammatory signaling (62–65). STAT3 also decreases the binding of IL-1β to its receptor by inducing the expression of a decoy protein that competes for receptor binding (66). LPS typically increases the expression of IL-10 in immune cells, but mRNA processing steps may delay this response (67). IL-10 release from immune cells exposed to LPS can be upregulated by changing the cells’ metabolic profile and environment (68–70). Using ELISA, we demonstrated that treatment of BV2 microglial cells with HBME reduced LPS-mediated IL-1β secretion but increased IL-10 secretion from microglia into the medium (Fig. 6; P = 0.0301 and P = 0.0178, respectively). These results demonstrate the anti-inflammatory effects of HBME.

Our results indicate that HBME inhibit the expression of LPS-induced proinflammatory proteins in BV2 microglia. As preterm neonates are very vulnerable, we need biologically safe therapeutics to combat common neonatal pathologies. Human breast milk protects against NEC (71, 72), and HBME decrease the deleterious effects of an induced NEC-like disease in mouse pups (73, 74). Bovine milk exosomes carry signaling proteins for immune cell receptors, phagocytosis, and cytotoxicity (75), and HBME are likely to have the same properties. We demonstrated that HBME protect intestinal epithelial cells exposed to hydrogen peroxide-induced oxidative stress (76). Additionally, IL-10 deficiency has been linked to inflammatory gut disease in mice (3). As intestinal diseases such as NEC often lead to neurodevelopmental delays in preterm neonates (77, 78), we investigated the neuroprotective potential of exosomes. In animal models, the decreased brain myelination and brain volume associated with brain injury, which result from microglial activation and neuronal cell death, occur secondary to NEC (16, 17). As both exosomes and microglia regulate the immune response and inflammation, we targeted microglia.

Among the many genes showing LPS-induced changes in expression, the gene encoding CD40 was the most interesting because upregulation of the CD40 receptor amplifies the immune cell response in human microglia (79), exaggerating the neuroinflammatory response and potentially increasing autoinflammatory disease (80, 81). The role of microglia in CNS disorders such as multiple sclerosis and Alzheimer’s disease (82, 83) prompted our interest in the regulation of CD40 expression in these cells. The expression of the CD40 receptor on the surface of microglia orchestrates peripheral leukocyte infiltration and retention in the CNS (84). Compared to the adult brain, the neonatal immune system is immature, but it mounts a robust response, especially to the LPS endotoxin (6–8, 51); thus, CD40 may play a key role in this process. Our results on the effects of HBME on microglial cell survival and CD40 expression (Fig. 3) led us to investigate the intracellular pathway(s) affected by HBME that underlie this change in CD40 expression.

LPS induces the binding of transcription factor NF-κB p50/p65 to the promotor of the gene encoding CD40 in microglial nuclei via the TLR4 pathway (31, 85, 86). We demonstrated that HBME inhibited the activation of the p65 component in the p50/p65 heterodimer by decreasing the expression and activation of MyD88, ΙκΒα, and p38 MAPK, which are key molecules upstream of the TLR4 pathway (18, 19, 30). Without activation of MyD88, TLR4 will not activate, and the IKK complex and p38 MAPK will also fail to activate, thereby arresting the NF-κB pathway (21, 22). We consistently found a decrease in MyD88 expression in LPS-stimulated microglia, in contrast to the increase seen in previous studies (30, 87, 88). HBME not only restored the protein level but also increased expression above the control. The difference in MyD88 expression in our study may be due to the shorter time of exposure compared to previous studies. Although one study was performed with BV2 microglia, the investigators used Dulbecco’s modified Eagle’s medium vs. the RPMI 1640 medium used here. There may also be a difference in the reactivity of the epitope recognized by different antibodies used or from the LPS purification method (88). The differences may reflect negative feedback through ubiquitination of MyD88, leading to receptor internalization/downregulation and/or proteasomal degradation after LPS treatment. The tumor necrosis factor α-induced protein 3 (TNFAIP3), more commonly known as A20, is a well-characterized deubiquitinase with E3 ligase functionality that inhibits NF-κB signaling by tagging several molecules for ubiquitination along the pathway (89–92), and MyD88 may be one of these targeted molecules. However, the targeting of specific molecules may depend on laboratory conditions, methods, and materials. Since other pathways activated by secreted, proinflammatory cytokines, such as IL-1β and interferon-β (IFN-β), are established in the environment (31), the TLR4/MyD88 axis may not be required to maintain the inflammatory response. The increase in MyD88 expression in microglia treated with HBME alone and the further increase in LPS-induced microglia treated with HBME may be due to the ability of some proteins carried by exosomes to alter the signaling of immune cell receptors directly (75). How this leads to the downregulation of downstream molecules requires further investigation.

IκΒα is also targeted for proteasomal degradation, as we propose for MyD88 in LPS-induced BV2 microglia. Our data indicated that HBME directly and indirectly stabilized IκΒα in the cytoplasm, thereby increasing its adherence to p50/p65 compared to the untreated LPS group (Fig. 4B) (18, 19). Decreased p38 MAPK expression after HBME treatment (Fig. 4C) will decrease activation of its downstream kinases, MK2 and MSK1/2, thereby decreasing NF-κB p50/65 functionality and proinflammatory cytokine production. MSK1/2 independently promotes the expression of anti-inflammatory cytokines IL-10 and the IL-1 receptor antagonist (IL-1ra) and promotes the dephosphorylation of p38 through a negative feedback loop after its initial proinflammatory effects (93). MSK1/2 is inhibited by MK2 (22), which may account for the increase in intracellular and secreted IL-10 (Fig. 5D; Fig. 6B). Activation of IκΒα by p38 MAPK will also be mitigated when MK2 inhibits MSK1/2 (18, 19). HBME may initiate a switch in MSK1/2 and inhibit p38 MAPK directly to promote its anti-inflammatory effects in BV2 microglia more quickly. These changes caused by HBME culminate in the decreased phosphorylation of NF-κB p65 (Fig. 4D), and the activation of the p65 component is largely responsible for the transcriptional activity of the p50/p65 heterodimer (94).

Neuroinflammation is mediated by the secretion of proinflammatory cytokines, especially the potent IL-1β (58), from various cells in the brain, particularly microglia and invading immune cells. HBME treatment of BV2 microglia decreased the production and secretion of IL-1β (Fig. 5C; Fig. 6A), inducing microglial hyperactivation because IL-1β stimulates microglial signaling through its receptor along with LPS-TLR4 signaling (58). IL-1β must be activated to cause cellular changes, and the NLRP3 inflammasome activates IL-1β. The decrease in NLRP3 and IL-1β expression observed in microglia (Fig. 5B, C) suggests that NLRP3 inflammasome activation also decreased.

IL-10 counters proinflammatory effector molecules (62, 68); thus, a deficiency in this anti-inflammatory cytokine is linked to chronic inflammatory conditions, such as inflammatory bowel diseases, enteropathies, and autoimmune disorders, and to exacerbation of chronic organ conditions such as liver and kidney disease, and hypersensitive disorders, such as asthma (4). Recombinant IL-10 was used to treat autoimmune and chronic inflammatory diseases in mouse and human trials, respectively (95, 96). T cells are an abundant source of IL-10, and their response is decreased by IL-10 (3, 97). IL-10 may also decrease CD40L presentation to LPS-induced microglia that are highly expressing CD40 because T cells commonly express CD40L (98). Although most immune cells secrete IL-10 during inflammation, a delayed response results in significant brain insult before IL-10 can counter those effects (67). Here, we demonstrated that HBME induced the production and secretion of IL-10 by microglia (Fig. 5D; Fig. 6B). Co-culture studies with microglia and T cells would provide further insight into the role of IL-10 in HBME-mediated anti-inflammatory effects.

The temporal sequence of gene expression and enzyme activation is critical in transcriptional programs, but the transcription of genes is often transient. For example, LPS-induced activation of NF-kB p50/p65 via MyD88-dependent pathways leads to its association with the CD40 promoter while p38 MAPK’s activation promotes MK2 to become associated with the IL-1β promoter region (18–21, 99, 100). Thus, the expression and activity of pathway molecules were affected within 1 h of LPS induction and HBME treatment. The response of IκΒα to LPS and HBME treatment at both 1 h and 24 h suggests either a wide timeframe for expression of this molecule or a biphasic response due possibly to its role in different pathways, such as tumor suppressor p53 (101). Thus, it is critical to identify a rapid response for the treatment of neonates. This study is limited by the genotypic/phenotypic differences common in in vitro studies and immortalized cell lines. However, as we studied cells derived from mouse fetuses/pups (57), our results may have phenotypic relevance to the preterm human population we aim to help with this study. These results must be confirmed in human microglia and animal studies, and further studies are needed to develop HBME as a novel and safe therapy for preterm neonates.

We demonstrated that HBME decreased the production of the proinflammatory mediators CD40, NLRP3, and IL-1B by inhibiting the TLR4/MyD88/NF-kB signaling pathway in LPS-induced BV2 microglia. Treatment of neonates requires a rapid response to an infectious insults; thus, this study provides evidence of the potential of HBME to regulate the microglial response to LPS in vitro. More studies need to be performed to examine this effect in animals and human.

CNS

central nervous system

LPS

lipopolysaccharide

NEC

necrotizing enterocolitis

TLR4

toll-like receptor 4

NF-kB

nuclear factor κ light-chain enhancer of activated B cells

interleukin

TNF-α

tumor necrosis factor-alpha

IKK

inhibitor of nuclear factor-κB kinase

MAPK

mitogen-activated protein kinase

ΙκΒα

nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha

MK2

mitogen-activated protein kinase-activated protein kinase 2

MSK

mitogen- and stress-activated kinase

MyD88

myeloid differentiation primary response 88

cluster of differentiation

NLRP3

nucleotide-binding oligomerization domain-like receptor protein 3

CD40L

CD40 ligand

MIP-1

macrophage inflammatory protein 1

TRAF

tumor necrosis factor receptor-associated factor

STAT

signal transducer and activator of transcription

PAMP

pathogen-associated molecular pattern

DAMP

damage-associated molecular pattern

NEK7

NIMA-related kinase 7

ASC

apoptosis-associated speck-like protein containing CARD

HMGB1

high mobility group box 1

extracellular vesicle

HBME

human breast milk-derived exosomes

HBM

human breast milk

PBS

phosphate-buffer saline

NTA

nanoparticle tracking analysis

TEM

transmission electron microscopy

uranyl acetate

RPMI

Roswell Park Memorial Institute

FACS

fluorescence-activated cell sorting

STAR

Spliced Transcripts Alignment to a Reference

BCA

bichloroacetic acid

western blot

HRP

horseradish peroxide

ELISA

enzyme-linked immunosorbent assay

TNFAIP3

tumor necrosis factor α-induced protein 3

IFN-b

interferon-b

Ethics approval and consent to participate were not needed. BV2 cells were a generous gift from Dr. Harald Neumann at the University of Bonn LIFE and Brain Center in Bonn, Germany.

Consent for publication: Not applicable

Availability of data and materials: Data and materials are in our laboratory and available upon request.

Competing interests: The authors declare that they have no competing interests.

Funding: This work was supported by the National Institutes of Health (1R15DA045564-01) and # 5R25MH080661-13/2004445114.

Authors’ contributions: OTA, SK, BS, and QH contributed to the experimental design and writing the manuscript. OTA, SK, YC, and BT executed the experiments.

Acknowledgments: We thank the University of Alabama's high-resolution imaging service center for assistance with NTA and TEM.

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Human breast milk-derived exosomes attenuate lipopolysaccharide-induced upregulation of CD40 and NLRP3 in microglia

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Version 1

Abstract

Figures

Introduction

Materials and methods

Human breast milk collection

Extracellular vesicle isolation and purification

Nanoparticle tracking analysis

Transmission electron microscopy

Fluorescence-activated cell sorting

Microglia cell culture and treatment with LPS and/or HBME

Cell viability

mRNA sequencing (mRNA-seq) analysis

RT-PCR assay

Western blot (WB)

Enzyme-linked immunosorbent assay (ELISA)

Statistical analysis

Results

Characterization of HBME

HBME increase the viability of LPS-induced BV2 microglia

HBME attenuate CD40 expression in LPS-induced BV2 microglia

HBME modulate intracellular signaling pathways in LPS-induced BV2 microglia

HBME decrease the expression of intracellular inflammatory markers in LPS-induced BV2 microglia

HBME alter the secretion of cytokines from LPS-induced BV2 microglia

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1