Dendrimer-Based N-Acetyl Cysteine Maternal Therapy Ameliorates Placental Inflammation via Maintenance of M1/M2 Macrophage Recruitment

Abstract

Intrauterine inflammation (IUI) is the primary cause of spontaneous preterm birth and predisposes neonates to long-term sequelae, including adverse neurological outcomes. N-acetyl-L-cysteine (NAC) is the amino acid L-cysteine derivative and a precursor to the antioxidant glutathione (GSH). NAC is commonly used clinically as an antioxidant with anti-inflammatory properties. Poor bioavailability and high protein binding of NAC necessitates the use of high doses resulting in side effects including nausea, vomiting, and gastric disruptions. Therefore, dendrimer-based therapy can specifically target the drug to the cells involved in inflammation, reducing side effects with efficacy at much lower doses than the free drug. Towards development of the new therapies for the treatment of maternal inflammation, we successfully administered dendrimer-based N-Acetyl Cysteine (DNAC) in an animal model of IUI to reduce preterm birth and perinatal inflammatory response. This study explored the associated immune mechanisms of DNAC treatment on placental macrophages following IUI, especially on M1/M2 type macrophage polarization. Our results demonstrated that intraperitoneal maternal DNAC administration significantly reduced the pro-inflammatory cytokine mRNA of Il1β and Nos2, and decreased CD45⁺ leukocyte infiltration in the placenta following IUI. Furthermore, we found that DNAC altered placental immune profile by stimulating macrophages to change to the M2 phenotype while decreasing the M1 phenotype, thus suppressing the inflammatory responses in the placenta. Our study provides evidence for DNAC therapy to alleviate IUI via the maintenance of macrophage M1/M2 imbalance in the placenta.

Keywords: DNAC; M1/M2; macrophage; maternal therapy; placental inflammation.

FIGURE 1

Measurements of inflammatory cytokine expression at mRNA level in placenta following exposure to intrauterine inflammation and maternal Dendrimer-based N-Acetyl Cysteine (DNAC) treatment. (A) Study Strategy. At embryonic (E) day 17, pregnant CD-1 mice underwent a mini-laparotomy in the lower abdomen for intrauterine injection of lipopolysaccharide (LPS). One hour later, dams received intraperitoneally injection of DNAC or phosphate-buffered saline (PBS). At 6 h post injection (hpi) dams were sacrificed. Placentas were harvested from each dam and quantitative polymerase chain reaction (QPCR) was performed to evaluate the effect of DNAC treatment. The mRNA levels of Il1β (B), Il10 (C) and Nos2 (D) were statically compared between groups. PBS + PBS, n = 8; LPS + PBS, n = 8; LPS + DNAC, n = 7; PBS + DNAC, n = 6. *p < 0.05 **p < 0.01 ***p < 0.001. Data were reported as Mean ± SEM.

FIGURE 2

Placenta leukocytes infiltration following exposure to intrauterine inflammation and maternal Dendrimer-based N-Acetyl Cysteine (DNAC) treatment. Lipopolysaccharide (LPS) or phosphate‐buffered saline (PBS) was injected intrauterine at embryonic day 17 (E17). DNAC was injected intraperitoneally 1 h post inflammation (hpi). Placentas were collected at 3, 6 and 12 hpi. (A) a. All captured placental cells were distinguished based on properties of side scatter area (SSC-A) versus forward scatter area (FSC-A) generally, by using polygon/rectangular/oval gates. b. Debris and doublets were excluded by gating on side scatter height (SSC-H) versus SSC-A to further delaminate singlets. c. Placental leukocytes were gated sequentially on CD45 ⁺ versus SSC-A properties. (B) The ratio of CD45⁺ leukocytes infiltrated in the placenta were statically compared between groups. PBS + PBS, n = 16; LPS + PBS, n = 16; LPS + DNAC, n = 15; PBS + DNAC, n = 12. *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001. Data were reported as Mean ± SEM.

FIGURE 3

Placenta M1 and M2 phenotype macrophage polarization following exposure to intrauterine inflammation and maternal dendrimer-based N-Acetyl Cysteine (DNAC) treatment. At embryonic (E) day 17, pregnant CD-1 mice underwent a mini-laparotomy in the lower abdomen for intrauterine injection of lipopolysaccharide (LPS). One hour later, dams received intraperitoneally injection of DNAC or phosphate-buffered saline (PBS). Placentas were collected at 6 hpi after LPS or PBS injection. (A) The placental macrophages were further identified by F4/80+ and CD11b + properties on placental leukocytes. (B) M2 macrophages was identified by CD163+ and side scatter sequentially on CD45⁺ CD11b⁺ F4/80⁺ macrophages. The ratios of total macrophages (C), M1 (D) and M2 (E) macrophages infiltrated in the placenta were statically compared between groups. PBS + PBS, n = 8; LPS + PBS, n = 8; LPS + DNAC, n = 7; PBS + DNAC, n = 6. *p < 0.05; **p < 0.01. Data were reported as Mean ± SEM.

FIGURE 4

Immunohistochemical staining (IHC) of M1 and M2 macrophages in the placenta following exposure to intrauterine inflammation and maternal dendrimer-based N-Acetyl Cysteine (DNAC) treatment. At embryonic (E) day 17, pregnant CD-1 mice underwent a mini-laparotomy in the lower abdomen for intrauterine injection of lipopolysaccharide (LPS). One hour later, dams received intraperitoneally injection of DNAC or phosphate-buffered saline (PBS). Placentas were collected at 6 hpi after LPS or PBS injection. Placentas were stained with anti-NOS2 (red, a marker for M1 macrophages) and anti-CD163 (green, a marker for M2 macrophage) antibodies. DAPI (blue) staining identified the nuclei. (A) Representative image of IHC. NOS2+ (B) and CD163+ (C)cells were quantified in the labyrinth of the placenta and were statically compared between groups. Images are ×40 magnification, and scale bars represent 50 μm n = 5 for each group. *p < 0.05 **p < 0.01. Data were reported as Mean ± SEM.