MAC mediates mammary duct epithelial cell injury in Plasma cell mastitis and Granulomatous mastitis

doi:10.21203/rs.3.rs-1543228/v1

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MAC mediates mammary duct epithelial cell injury in Plasma cell mastitis and Granulomatous mastitis

https://doi.org/10.21203/rs.3.rs-1543228/v1

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Plasma cell mastitis (PCM) and granulomatous mastitis (GM) are common inflammatory nonbacterial mastitis (NBM). However, the pathogenesis of NBM is still unclear.

Methods. In this study, we statistically analyzed the pathological features of PCM and GM using pathological HE staining and tissue transmission electron microscopy. The levels of MAC (C5b-9n), P-selectin, E-selectin, and ICAM-1 were detected through IHC, WB, ELISA, and qPCR. The expression level and location of MAC were observed by tissue immunological electron microscopy. In addition, exosomes were isolated from tissues, identified using transmission electron microscopy, and the densities were detected by Nano-FCM. Finally, the expression intensity of MAC in exosomes was detected by flow cytometry and immunoelectron microscopy.

Results. The damage and apoptosis of mammary duct epithelial cells are the common pathological features of PCM and GM. MAC is primarily located in the cell membrane of mammary ductal epithelial cells and is significantly expressed in PCM and GM. The density of exosomes in PCM and GM tissues was elevated, and MAC was highly expressed in exosomes. In addition, the expression of P-selectin, E-selectin, and ICAM-1 in PCM and GM was significantly higher than in the normal group.

Conclusion. We found severe damage of the mammary duct epithelial cells in PCM and GM tissues, which was verified by relevant pathological methods. Earlier studies demonstrated that MAC is highly expressed in PCM and GM tissues and exosomes seem to play a very important role in the understanding of MAC. Furthermore, MAC is involved in inflammatory infiltration and lesion of mammary duct epithelial cells upregulated by P-selectin, E-selectin, and ICAM-1. These findings provide new insights into PCM and GM molecular mechanisms with novel therapeutic advances.

Plasma cell mastitis

Granulomatous mastitis

inflammation

MAC(C5b-9n)

Nonbacterial mastitis (NBM) is a mammary gland disease, common in clinics with Plasma cell mastitis (PCM) and Granuloma mastitis (GM). PCM is characterized by the accumulation of plasma cells and lymphocytes around dilated ducts [1, 2]. Typically, PCM occurs in young and middle-aged females at nonpregnant or nonlactating stages [3]. GM has granulomatous structures formed in the breast lobules accompanied by many inflammatory cells [4, 5]. GM mainly occurs in women of childbearing age, 30–45 years old, with breastfeeding an essential medical history [4, 6]. PCM and GM presented the same clinical manifestations like localized redness, mass, and sinus formation on the affected breast. Although drug therapy and surgical treatment have been reported, the treatment outcome is poor, mainly due to postoperative recurrence and breast deformation [5, 6]. In addition, there is a lack of cell lines and animal models [7]. Although the incidence of PCM and GM has been increasing in recent years [1, 2], the pathogenesis of PCM and GM remains unclear [7, 8].

Membrane attack complex (MAC) is the biological complement lytic effector of the standard three complement activation pathway [9]. C5b can stably bind to C6 to form C5b6 at the end of the complement activation pathway, spontaneously binding to C7 to form C5b67 [10]. In this complex, C7 is initially inserted into the lipid bilayer of the target cell membrane. Then C8 binds to C5b67 with a high affinity on the insertion membrane to form stable and deeply inserted C5b678 [10]. Finally, the complex combines with 12 ~ 18 C9 molecules to create C5b6789n, the membrane attacking complex [10]. Under electron microscopy, MAC appeared as a hollow C9 polymer, traversing the lipid bilayer membrane of target cells and forming a transmembrane channel with an inner diameter of about 11nm [11, 12]. The medium allows the free flow of water, ions, and small soluble molecules across the membrane. At the same time, the intracellular colloid osmotic pressure is higher than the extracellular one with a heavy flow of a large amount of water, leading to intracellular osmotic pressure reduction, cell swelling, and eventual rupture (i.e., cell lysis) [11, 12].

Under electron microscopy, exosomes originate from endosomal vesicles and appear cup-shaped at 20–100 nm [13]. Recent studies have depicted a novel means of exosome-mediated cell-to-cell communication for regulating inflammation [14, 15] and autoimmune diseases like arthritis [16, 17]. However, there is a disagreement about the differential diagnosis of PCM and GM [6]. Previous studies identified differential genes of PCM in 30 pathways using RNA-SEQ bioinformatics analysis [7]. The involvement of the C3 and C5 pathways in the inflammatory progression of PCM and GM requires verification. In this study, we investigated the pathological characteristics of PCM and GM and the role of MAC during the injury mechanism of mammary ductal epithelial cells.

2.1 Study population

We randomly selected 80 patients with the histological diagnosis of PCM and GM from the Department of Pathology, Shengli Oilfield Central Hospital, Shandong, China, between January 2011 and October 2021. The inclusion criteria for PCM and GM patients were: (I): newly diagnosed and histologically confirmed PCM and GM; (II): no evidence or history of cancer; and (III): the baseline information is complete and available. The exclusion criteria included a history of other breast diseases and recent relapses (< 3 months). Therefore, 40 normal breast tissue samples were collected as NC groups (normal breast tissue obtained from peritumoral tissue from fibroadenoma surgery patients).

2.2 Sample collection and processing

For subsequent experiments, fresh tissue samples were obtained from PCM and GM patients who underwent surgical treatment between August 2021 and December 2021. The diseased tissue samples were collected under sterile conditions, and contaminants such as fat were removed. The samples were washed in physiological saline and immediately stored at − 80°C until further analysis. Normal breast tissue samples were excised from the breast tissue of patients with breast fibroadenoma. The tissue adjacent to the fibroadenoma was considered a control sample (NC group). Meanwhile, blood samples (5 ml) were collected from these patients before discharge from the hospital and centrifuged at 3000 g for 15 min. The resulting supernatants were stored at − 80°C. Inclusion criteria for the patients were: (1) patients with plasma cell mastitis, granulomatous mastitis, or fibroadenoma of the breast diagnosed through pathology; (2) no history of any breast disease, family history of breast cancer, or previous malignant tumor; (3) complete patient information.

2.3 Pathological HE staining

The paraffin sections were dewaxed and hydrated using xylene and gradient alcohol. Then, the fragment was stained with hematoxylin in a staining flask for 10 min to stain the nuclei. After that, hydrochloric acid and ethanol droplets were added to the tissue parts for differentiation for about 30 s. Later, the fragments were immersed in tap water for 15 min. After staining with eosin solution for 1 min, they were soaked in tap water for 3 min. Finally, after dehydration with gradient alcohol followed by transparent treatment with xylene, the sheets were sealed with neutral resin and observed under a microscope. Three separate pathologists analyzed the pathological diagnosis and characteristics of the sections under double-blinded conditions.

2.4 Pathological evaluation of mammary duct epithelial cells

We scored pathological HE sections for inflammatory infiltration and mammary duct epithelial cell damage based on the renal injury and lung injury score (LISs) to grade the mammary duct epithelial cell injury [18]. The severity of the mammary ductal epithelial injury depended on the percentage of mammary ductal epithelial necrosis and inflammatory cell infiltration. The degree of mammary duct epithelial cell injury was graded as follows: 0 = 0% lesion area, 1 = 0–25% lesion area, 2 = 25–50% lesion area, 3 = 50–75% lesion area, and 4 = above 75% lesion area. Then, ten high-powered areas were randomly selected. The mean value was the pathological score in each sample.

2.5 Transmission Electron Microscopy Processing and Observation

The ultrastructure of PCM, GM, and normal breast tissue was observed by transmission electron microscopy. Fresh PCM, GM, and normal breast tissue were isolated and quickly inserted into a 2.5% glutaraldehyde fixed solution precooled at 4°C. Then it was stored in a refrigerator at 4°C for 36 h, and the 2.5% glutaraldehyde fixed solution was kept clear. After that, it was fixed with osmium tetroxide, stained with uranyl acetate, dehydrated with ethanol, and embedded with epoxy resin. Later, ultrathin sections were prepared, stained with 2% uranium dioxide acetate and lead citrate, and examined using HT7700 transmission electron microscopy (Hitachi, Japan). Finally, two Professors observed and analyzed the ultrastructure using a transmission electron microscope in a blinded manner.

2.6 Tissue immunoelectron microscopy

The expression of MAC in human breast tissue was detected by immunoelectron microscopy.

Ultrathin sections were incubated at 4%PFA + 0.01%GA/0.1M PB for two hours and then incubated at 4%PFA/0.1M PB overnight under 4°C. Ultrathin sections were washed and dehydrated with an alcohol gradient (30%, 50%, 70%, 95%, 100%, 100%) at 4° for one hour each. The ultrathin sections were first incubated with antibodies against MAC (1:400; Bioss, bs-2673R) overnight at 4°C. Next, the sections were incubated with secondary antibodies at room temperature for 20 min and 37°C drying box temperature for one hour. Next, the nickel mesh was dyed in 2% uranium acetate saturated alcohol solution without light for 8 min. After washing with alcohol and ultra-pure water, the ultrathin sections were placed on the mesh plate and observed with a JEOL 1011 electron microscope.

2.7 Immunohistochemistry staining

The expression of MAC, P-selectin, E-selectin, and ICAM-1 in the PCM, GM, and NC groups were detected using immunohistochemistry. First, 4-µm paraffin sections were dewaxed in xylene, submitted to heat antigen retrieval under high pressure, and treated with 3% H₂O₂ hydrogen peroxide. After washing, the sections were incubated using first antibodies against MAC (1:500; Bioss, bs-2673R), P-selectin (1:1000; Proteintech, 60322–1-ig), E-selectin (1:300; Proteintech; 20894–1-AP) and ICAM-1 (1:500; Proteintech, 60299–1-ig), respectively, overnight at 4°C inside a moist chamber. Next, the sections were further incubated with horseradish peroxidase-labeled secondary antibody (Boster, SA1022) for 30 min after being washed with PBS. Subsequently, all the sections were visualized with the DAB kit (KeyGEN BioTECH, KGP1045–20). Then, the sections were counter-stained with hematoxylin, dehydrated, sealed, and observed under a light microscope. The company supplied positive control. In the negative control, the primary antibody was replaced with PBS.

Three experienced pathologists, blinded from the patients and their clinical outcomes, independently examined the immunoreactivity. Five regions were randomly selected within each section; the percentage of positive cells and staining intensity were considered in each region. The staining intensities were graded as 0 (negative), 1 (weak), 2 (middle) or 3 (strong) and the percentage of positive cells were graded as 1 (≤ 25%), 2 (26%-50%), 3 (51%-75%) or 4 (≥ 76%). The final immunohistochemical score was the product of staining intensity and the percentage of positive cells. High expression was the final immunohistochemical score ≥ 4.

2.8 Western blotting analysis

The level of MAC were detected using Western blotting. The human breast tissues were completely homogenized under a liquid nitrogen drip, and the nuclear and cytoplasmic proteins were extracted with a protein extraction kit (Sangon Biotech, C500005–0010). The protein concentrations were determined by the BCA protein assay kit (Solarbio, PC0020–500). Equal amounts of protein were loaded within each lane, separated by 10% SDS-PAGE, and transferred to various PVDF membranes (Roche, 03010040001). The membranes were then blocked with 5% nonfat milk at room temperature for two hours after shaking and incubated overnight with primary antibodies against MAC(C5b-9) (1:500; Bioss, bs-2673R) and GAPDH (1:20000; Proteintech; 10494–1-AP) at 4°C. After washing, the membranes were incubated overnight with secondary antibodies (1:10000; Proteintech, SA00001–2) at room temperature for two hours. Finally, PVDF membranes were stained with the ECL reagent to reveal the nuclear and cytoplasmic proteins. The gray value of the protein in PVDF membranes was detected using the ImageJ software.

2.9 Quantitative real-time PCR

We detected the expression of MAC, P-selectin, E-selectin, and ICAM-1 in PCM, GM, and normal breast tissues using quantitative real-time PCR. Fresh tissues were obtained, and their total RNA was extracted using TRIzol (B610409–0100, Sangon Biotech) by following the manufacturer’s directions. RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The amplification protocol used included: initial denaturation at 95°C for 5 min, followed by 45 cycles, at 95°C for 15 sec, 56°C for 30 sec, and 72°C for 20 sec. Relative expression of the target genes was normalized to GAPDH levels, and the ΔΔCt method was used to calculate relative expression levels. The primer sequences used are listed as follows.

C5b-9(forward:5′-TTTGCAGCTTAGGTCCGAGG-3′;reverse:5′-CTGTGTCTGCTCTGGGTTCC-3′).

P-selectin(forward:5′-TGGACCAACCCTGTTTCCTG-3′;reverse:5′-AGAGATGCCACCTGCTTTCC-3′).

selectin(forward:5′-ATGTGAAGCTGTGAGATGCG-3′;reverse:5′-CCACTGCAGCTCATGTTGAT-3′).

ICAM-1(forward:5′-TCTTCCTCGGCCTTCCCATA-3′;reverse:5′-AGGTACCATGGCCCCAAATG-3′).

GAPDH(forward:5′-AGAAGGCTGGGGCTCATTTG-3′;reverse:5′-AGGGGCCATCCACAGTCTTC-3′).

2.10 Enzyme-Linked Immunosorbent Assay (ELISA)

Blood was centrifuged at 4000 g for 15 min, and subsequently, the serum was collected for the assay. The supernatants were collected to measure C5b and C5b-9n (MAC). C5b and C5b-9n (MAC) serum levels were quantified using the human complement fragment 5b (C5b) ELISA kit following the instructions of the manufacturer (EK12057, Signalway Antibody) and the human membrane attack Complex (MAC) using the ELISA kit with instructions from the manufacturer (MM-1095H1, MEIMIAN).

2.11 Extraction and identification of tissue exosomes

The PCM, GM, and the normal breast tissues were separately homogenized at medium speed on ice with physiological saline at a ratio of 1:3. The filtrate was centrifuged to remove the tissue fragments at 1200 g for 5 min. The clear liquid was left in the middle after the supernatant was centrifuged at 4800 g for 10 min. The supernatant was filtered using a 40 µm filter. Then, the filtrate was centrifuged to remove the debris at 20000 g for one hour. Next, the supernatant was filtered with a 0.2 µm filter and centrifuged at 100000 g for one hour to remove the impurities. Then, the precipitates were retained, mixed with PBS, and centrifuged at 100000 g for two hours to obtain the exosomes. The exosomes extracted from the PCM, GM, and the normal breast tissues were renamed PCM/Exo, GM/Exo, and NC/Exo. Exosomes were analyzed through transmission electron microscopy and ZetaView® NanoFCM technique using the Particle Metrix (Malvern, Worcestershire, UK) for identification and characterization.

2.12 Immunoelectron microscopy of exosomes

We analyzed the exosomes labeled with C5b-9 by transmission electron microscopy. First, exosomes were fixed with 2% paraformaldehyde and 5% glutaraldehyde for 15 min. The samples were sealed with 1% BSA for 30 min and washed three times with PBS. A droplet of the sample was floated over the carbon support membrane for 10 min at room temperature. The C5b-9 antibodies (1:20 dilution) were added to the sample at room temperature for one hour and then at 4°C for 36 h. The secondary antibodies (1:30 dilution, Abcam, ab270700) were added after being washed with PBS for one hour. The sample was fixed on the carbon support membrane using 2% hydrogen peroxide acetate for 1 min. After drying, the grids were examined with a JEOL 1011 electron microscope being operated at 80 kV.

2.13 Flow cytometry of exosomes

The expression of C5b-9 labeled exosomes was detected using flow cytometry. Exosomes (30 µg) were supplemented with 10 µL beads and rotated at room temperature for 15 min. Then, the exosomes were diluted with 1 mL PBS, rotated for 2–4 h at room temperature, and centrifuged at 14800 g for 1 min. C5b-9 (5 µL) was then added to exosomes (95 µL) and rotated overnight at 4°C using the DH-II rotary mixer. After that, the exosomes were washed in PBS and centrifuged at 14800 g for 1 min. Fluorescent II antibody (5 µL) was then added to exosomes (95 µL) and kept at 4°C for 30–60 min, washed in PBS, and centrifuged at 14800 g for 1 min. Then, the samples were resuspended in PBS, examined with LSR II Flow Cytometer (Beckman-Coulter), and the data obtained were graphically represented with FlowJo software.

2.14 Ethics statement

This research was approved by the Research Ethical Committee of the Shengli Oilfield Central Hospital, Ethical Committee Number: Q/ZXYY-ZY-YWB-LL202207Each participant provided signed informed consent prior to participation in the present study.

2.15 Statistical Analysis

All statistical analyses were performed using the SPSS Statistics 20 software. Data were represented in the form of mean ± standard deviation. Normally distributed data were analyzed with one-way analysis of variance (ANOVA) and not normally distributed ones with the Mann-Whitney t-test. P < 0.05 was considered to be statistically significant.

3.1 The injury of mammary duct epithelial cells is a common pathologic feature of PCM and GM

Fig. 1. shows the typical pathological characteristics of PCM, GM, and the normal breast tissues. Normal mammary gland tissues are composed of independently existing mammary lobules. The mammary ducts in the lobules are arranged neatly, without inflammatory cell infiltration and damage to the duct epithelial cells (Fig.1-A1). Ductal epithelial cells of the breast were placed within a double layer of tubes. The inner cells were glandular epithelial cells, and the outer ones were myoepithelial cells (Fig. 1-A2). Extensive inflammatory cells around the dilated duct are a typical histopathological feature of PCM, and a high percentage of plasma cells have diagnostic values (Fig.1-B1). Inflammatory infiltration caused injury and apoptosis of mammary duct epithelial cells, which showed typical nuclear pyknosis, nuclear fragmentation, and nucleolysis, the reddish phagosome found in PCM. Usually, it engulfs apoptotic cell fragments (Fig.1-B2). Typical pathological features of GM (Fig.1-C1) are granulomatous structures and lipid vacuoles. In the high background, the remaining ductal epithelial breast cells showed classic signs of apoptosis, as seen in PCM (Fig.1-C2). Inflammatory cell infiltration and mammary duct epithelial cell injury were elevated in the PCM and GM groups than the control group (*P＜0.01)(Fig.1-D). However, the inflammatory cell infiltration and mammary duct epithelial cell injury of the GM group were apparent compared with the PCM group (*P＜0.01)(Fig.1-D).

3.3 Damage of mammary ductal epithelial cells under transmission electron microscopy

Fig. 2. shows the ultrastructural changes of PCM, GM, and the normal breast tissues. Within normal mammary ducts, the epithelial cells were closely arranged without infiltration of the inflammatory cells. The capillaries were observed between fibrous tissues, and different white blood cells were observed in the blood vessels (Fig.2-A1). The ultrastructure of PCM showed that the epithelial cells of mammary ducts were disordered and depicted different morphological changes, accompanied by many inflammatory cells (Fig.2-A2). The ultrastructure of GM was very similar to PCM (Fig.2-A3). The ultrastructure of normal mammary duct epithelial cells was intact, and the intercellular links were well developed, along with gap links and tight junctions. The intracellular staining was homogeneous, and the cell and nuclear membranes were intact without damage. There were poorly developed microvilli on the surface of the cell membrane (Fig.2 B1-B2). PCM and GM ductal epithelial cells had similar configuration changes, including connections with the microvilli on the cell membrane surface. There was a noticeable increase in cell differences, lysosomes, the nucleus, irregular shapes, increased heterochromatin, and chromatin edge set. Some cells with prominent nucleoli and nuclear pulp ratio also increased, along with abnormal chromatin phenomenon (Fig.2 C1-C2,Fig.2 D1-D2). Mammary ductal epithelial cells within the PCM and GM tissues showed typical damage and apoptosis, including cell and nuclear membrane deterioration, chromatin shrinkage, and fragmentation (Fig.2 E1-E2). In addition, a large number of exosomes were found in the PCM and GM tissues but rarely within normal breast tissues (Fig.2-F2 and Fig.2-F3); however, no exosomes were found in normal breast tissue (Fig.2-F1).

3.3 The expression of MAC was significantly increased in PCM and GM

Figure 3 shows the increased expression of MAC and its related molecules in PCM and GM tissues.We observed the high expression of MAC in PCM, GM and NC groups cells with tissue immunoelectron microscopy (Fig.3 A1-A3). The more the black particles, the higher was the positive charge. The black particles indicated that MAC was located in the cytoplasm and membrane. As shown in the figure, the expression of MAC in PCM and GM groups was significantly increased than in the NC group (Fig.3-B) (*P＜0.05). Representative micrographs of immunohistochemical staining are illustrated in Fig.3-C MAC immunoreactivities were detected in epithelial cells, plasma cells, macrophages, lymphocytes, and neutrophils. In addition, immunohistochemical staining scores were analyzed using ANOVA or Welch’s ANOVA test followed by a posthoc test. The results showed that MAC immunoreactivities were significantly elevated in the PCM and GM than in the NC groups (Fig.3-D) (both *P＜0.05). However, there was no statistical significance between the PCM and GM groups (Fig.3-D) (*P＞0.05). C5b and C5b-9n (MAC) expression in the supernatants of peripheral blood in the PCM, GM, and NC group was detected using ELISA. Compared with the NC group, C5b and C5b-9n(MAC) expression in the PCM and GM groups was significantly increased (Fig.3-E) (*P＜0.05). The mRNA levels of MAC in PCM and GM groups were significantly increased than in the NC group (*P＜0.001). Representative Western blot data are illustrated in Fig. 3G, with quantitation and comparison by ANOVA or Welch’s ANOVA test followed by the posthoc test in Fig.3-H. Specifically, Western blot results indicated that MAC was significantly expressed in most PCM and GM samples. In addition, the protein contents of MAC were no observable statistical difference between the PCM and GM groups (*P＞0.05).

3.4 The concentration of exosome increased significantly in PCM and GM

The transmission electron microscopy analysis showed that the exosomes isolated from PCM, GM, and the normal breast tissues were morphologically homogeneous. They ranged from 30 to 150 nm in size, with a standard round or cup-shaped appearance (Fig.4A1-A3). The particle size distribution and concentration of PCM/Exo, GM/Exo, and NC/Exo were detected using the Nano-FCM (N30E). The exosome concentration was 4.68 × 10¹⁰ particles/mL in PCM/Exo, 3.99 × 10¹⁰ particles/mL in GM/Exo, and 1.07 × 10¹⁰ particles/mL in NC/Exo (Fig.4-B). There was a statistically significant difference in the concentration of exosomes (Fig.4 -C) (*P＜0.05) . The average particle size of exosomes was 70.05 nm, 75.26 nm, and 76.27 nm in PCM/Exo, GM/Exo, and NC/Exo (Fig.4-B), respectively, and there was no statistically significant difference in the particle size (*P＞0.05).

3.5 Elevated MAC amounts in exosomes

Overrepresented MAC in PCM/Exo, GM/Exo and NC/Exo were confirmed using immune electron microscopy (Fig.4 D1-D3). There was a statistically significant difference in the PCM/Exo and GM/Exo compared with NC/Exo (Fig.4-E)(*P＜0.05). The levels of MAC in exosomes were further detected by Western blotting (Fig.4-F). Furthermore, the results indicated that the protein level of MAC was significantly upregulated in PCM/Exo and GM/Exo than in NC/Exo (Fig.4-G, Both* P＜0.05). These results suggest that exosomes derived from the tissues of PCM and GM contain concentrated MAC, which could be related to the MAC removal process.

Furthermore, we assessed the levels of exosomal MAC in breast tissue by flow cytometry analysis to explore the predictive value of MAC for investigating the injury to breast ductal epithelial cells in PCM and GM patients. The results showed that exosomal MAC in the breast tissue of PCM and GM was significantly higher than in the NC group (Fig.4-H and Fig.4-I, Both *P＜0.05).

3.6 P-selectin, E-selectin, and ICAM-1 were highly expressed in PCM and GM

Representative micrographs of immunohistochemical staining are illustrated in Fig.5-A. P-selectin, E-selectin, and ICAM-1 are mainly found in the nucleus and membrane of the epithelial cells, macrophages, neutrophils, and lymphocytes. Compared with the PCM and GM group, the immunoreactivities of P-selectin, E-selectin, and ICAM-1 were weaker and detected only in epithelial cells. Immunohistochemical staining scores were analyzed by ANOVA or Welch’s ANOVA test followed by the posthoc test (Fig.5-B). The results showed that the immunoreactivities of P-selectin, E-selectin, and ICAM-1 were significantly elevated in macrophages, neutrophils, and lymphocytes in the PCM and GM group compared with NC cases (both P＜0.0001). There were no statistical differences in P-selectin, E-selectin, and ICAM-1 between the PCM and GM groups (p＞0.05).

Furthermore, we assessed the mRNA levels of P-selectin, E-selectin, and ICAM-1 in breast tissue by Q-PCR. The mRNA levels of P-selectin, E-selectin, and ICAM-1 in PCM and GM groups were significantly increased compared with the NC group (*P＜0.001)(Fig.5-C).

PCM and GM are the two common diseases of non-lactation mastitis with similar clinical manifestations and poor therapeutic outcomes. Due to the lack of cell lines and animal models, the mechanism of PCM and GM publication is unclear [7, 19]. Therefore, pathological diagnosis is the only criterion for differentiating the two diseases [4, 20]. The pathological feature of PCM involves the infiltration of a large number of inflammatory cells, particularly plasma cells, around dilated breast ducts [2]. Granulomatous lobule formation is a pathological feature of GM, characterized by lipid vacuoles, microabscesses, and Langerhans giant cells [4]. Our results further confirmed the pathological diagnosis of the two diseases. In previous studies, mammary duct injury has been highlighted in the pathogenesis of NBM without much attention. The normal ducts are arranged within a double tubular structure, with an inner glandular epithelium serving as a secretory layer and an outer myoepithelial barrier. Emad et al. inferred that myoepithelial breast cells are barriers in breast lesions, especially in the differential diagnosis of ductal carcinoma in situ and invasive mastitis [21]. Researchers have observed that the parenchyma involved was characterized by missing acinar structures and damaged ducts [22, 23]. Wolfrum et al. suggested that this could be a response of inflammatory autoimmunity towards epithelial cell injury [4]. The damage of mammary duct epithelial cells leads to the extravasation of glandular secretions into connective tissue lobules, causing local inflammatory lesions [6, 24]. Our experimental results confirmed that mammary ductal epithelial cells showed damage and apoptosis in PCM and GM tissues. The deterioration of mammary ductal epithelial cells was observed in pathological HE, and cell damages were observed under tissue transmission electron microscopy. This phenomenon needs to be understood because ductal epithelial cell damage causes the mammary gland tissue to lose its essential structure and function.

The membrane attack complex (MAC) acts as a trigger factor of the inflammatory response as the final product of the complement activation pathway [10]. It is involved in the injury and apoptosis of various cells [25]. Under the electron microscope, MAC is a hollow C9 polymer crossing the lipid bilayer membrane of target cells and forms a transmembrane channel with an inner diameter of ~ 11nm [11, 12, 26]. Studies have shown that MAC is formed on the cell membrane surface, resulting in osmotic pressure imbalance on both sides of the cell membrane, ultimately causing cell damage [27]. Furthermore, MAC deposited on the cell surface resulted in changes in intracellular ion activity and activated the intracellular signal transduction process, leading to the generation of various bioactive substances and cytokines [28, 29]. These effects promote local tissue hyperplasia and repair and aggravate local inflammatory lesions [28]. Hughes et al. found that the apoptosis of renal tubule skin cells with complement deposition was significantly enhanced and dependent on MAC [30]. We established that MAC was localized to the epithelial membrane cells through immunoelectron microscopy and found that the expression of MAC was significantly increased in PCM and GM, which could be an essential reason for the injury of mammary duct epithelial cells. As MAC was formed in endothelial cells, P-selectin was produced after fusing intracytoplasmic particles with the cell membrane, which enhanced the adhesion of neutrophils and monocytes to release inflammatory mediators upregulating E-selectin and ICAM-1 [30]. We also observed the high expression of P-selectin, E-selectin, and ICAM-1 in PCM and GM using immunohistochemistry and PCR, synergistic with MAC to cause inflammatory infiltration and damage to the mammary duct epithelial cells.

Exosomes in PCM and GM tissues increased significantly than in the normal group, verified by tissue transmission electron microscope and particle size analysis. Exosomes are regarded as secretory vesicles involved in intercellular communication [14, 15]. One of the dual roles of exosomes is the proinflammatory mechanism. Paracrine signaling has an integral role in the inflammatory cell recruitment and secretion of proinflammatory factors [31]. Wang et al. [7] found that exocrine bodies could be involved in paracrine/paracrine secretion of acinar epithelial cells and ducts in PCM. Moreover, our previous study also confirmed that exosome-mediated secretion of complement C3a and C5a were involved in the inflammatory infiltration of NBM. The other role is the protective effect of exosomes on mammary duct epithelial cells. Cunningham et al. depicted that nucleated cells defend themselves from complement lysis by removing the MAC deposited on the cell membrane through vesiculation or endocytosis [32]. This study demonstrated that MAC was highly expressed on exosomes, having an essential role in removing the MAC deposited on cell membranes and protecting the mammary duct epithelial cells from injury.

This study had certain limitations. First, we did not conducted any animal and cell experiments, as animal models are controversial and no cell experiments on NBM have been reported. In addition, there was no control group for acute mastitis, because acute mastitis is not NBM, and this paper mainly studied the specific causes of the injury of ductal epithelial cells in PCM and GM tissues. Thirdly, we intended to observe the stereoscopic structure of MAC through tissue transmission electron microscopy and negative staining sample preparation, but due to the large serum sample size, we were unable to obtain a large amount of peripheral blood serum with informed consent.

To the best of our knowledge, this is the first study to describe the mechanisms by which MAC mediates PCM and GM mammary duct epithelial cell injuries. However, several studies have assessed the role of MAC in renal tubular duct epithelial cell injury. The pathogenesis of NBM is unclear, and the clinical treatment is not uniform due to the lack of cell lines and animal models. We hypothesize that the structural and functional integrity of mammary ductal epithelial cells has a vital role in breast disease. The current study suggests that mammary duct epithelial cell injury is an important pathological feature of NBM. Further studies on the pathogenesis of NBM are required for a better understanding of the disease.

Acknowledgements

We thank Xiao-yang Xu made the professional pathological assistance, including diagnosis and immunohistochemical analysis of PCM and GM.

Funding

This work was supported by National Natural Science Foundation of China (No. 81902702), Natural Science Foundation of Shandong Province (No.ZR2017LH072 and ZR2017MH033), National Key Research and Development Project (No. 2018YFC0114705), The Special Funds for The Qilu Health and Health Leading Talents Cultivation Project (Wangxiaohong).

Availability of data and materials

All data generated or analysed during this study are included in this published article. And all the data is real and available.

Authors' contributions

Xiao-qiang Li and Di-wen Sun designed the study and performed editing and critical revision of this manuscript. Xiao-qiang Li, and Hao-jie Zhang performed the experiments and analyzed the data. Peng-peng Ding and Xiang-sheng collected the samples. Xi-chao Wang and Qing-ao Bu classified the samples. All authors read and approved the final manuscript. Haojie Zhang and Peng-peng Ding contributed equally to this work.

Ethics approval and consent to participate

The present study was approved by the was obtained from Ethics Committee of Shengli Oilfield Central Hospital (Dongying, China; approval; 2021‑08‑01). Written informed consent was obtained from patients prior to enrolment. Ethics Committee of Shengli Oilfield Central Hospital agree to submit this article.

Competing interests

The authors declare no competing interests. This work was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part.

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MAC mediates mammary duct epithelial cell injury in Plasma cell mastitis and Granulomatous mastitis

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Study population

2.2 Sample collection and processing

2.3 Pathological HE staining

2.4 Pathological evaluation of mammary duct epithelial cells

2.5 Transmission Electron Microscopy Processing and Observation

2.6 Tissue immunoelectron microscopy

2.7 Immunohistochemistry staining

2.8 Western blotting analysis

2.9 Quantitative real-time PCR

2.10 Enzyme-Linked Immunosorbent Assay (ELISA)

2.11 Extraction and identification of tissue exosomes

2.12 Immunoelectron microscopy of exosomes

2.13 Flow cytometry of exosomes

2.14 Ethics statement

2.15 Statistical Analysis

3 Results

3.1 The injury of mammary duct epithelial cells is a common pathologic feature of PCM and GM

3.3 Damage of mammary ductal epithelial cells under transmission electron microscopy

3.3 The expression of MAC was significantly increased in PCM and GM

3.4 The concentration of exosome increased significantly in PCM and GM

3.5 Elevated MAC amounts in exosomes

3.6 P-selectin, E-selectin, and ICAM-1 were highly expressed in PCM and GM

4 Discussion

5. Conclusion

Declarations

References

Additional Declarations

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