Transforming growth factor-β1 is associated with inflammatory resolution via promoting M2c macrophage polarization in lung injury mice

doi:10.21203/rs.3.rs-3334233/v1

Download PDF

Research Article

Transforming growth factor-β1 is associated with inflammatory resolution via promoting M2c macrophage polarization in lung injury mice

https://doi.org/10.21203/rs.3.rs-3334233/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Macrophage polarization is vital for the resolution of inflammation and tissue injury. Here, we investigated the role of transforming growth factor (TGF)-β1 on macrophages polarization. In the mouse model of ventilation-induced lung injury, high tidal volume-induced injury and inflammation were resolved 3 days post-ventilation (PV3d) to PV10d, with increased elastic fiber, proteoglycan, and collagen, as well as higher TGF-β1 level. M1 increased temporarily, whereas M2a and M2c began to increase from PV3d. In vivo upregulating or neutralizing TGF-β1 expression indicated that an appropriate expression of TGF-β1 is required to resolve lung injury and inflammation via regulating macrophage polarization. M1 are the main pro-inflammatory macrophages to induce pulmonary edema and inflammation. Inhibition of M1 to M2a is associated with pulmonary injury and inflammation, but M2b is contributed to the attenuation of serum interleukin (IL)-1β and pulmonary IL-6 levels. M2c contributed to the attenuation of serum and pulmonary IL-6 levels as well as serum tumor necrosis factor-α levels. TGF-β1 is a crucial factor to promote M2b and M2c polarization with the reduction of pulmonary IL-6 levels. Together, secretion of TGF-β1-mediated macrophage polarization plays a crucial role in the resolution of inflammatory lung injury.

TGF-β1

Macrophages polarization

M2c macrophages

Inflammation

Ventilator-induced lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are among the most common and deadly clinical syndromes. They are induced by severe shock, trauma and infection (especially during the COVID-19 pandemic), with a mortality of 35–45%[1, 2]. ARDS leads to an acute respiratory failure by apoptosis or pyroptosis, uncontrolled immune cell infiltration, and the development of fibrosis[3]. Mechanical ventilation is the main respiratory support therapy of ARDS, which also triggers ALI or aggravates the original injury (ventilator-induced lung injury, VILI)[3, 4]. Several studies have highlighted a critical role of Toll-like receptors 4 and 9 signaling pathway or NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome on alveolar macrophages in VILI[5–8]. However, whether macrophage polarization contributes to the resolution of lung injury and inflammation are not well understood.

Macrophages responding to persistent inflammation can exist in two distinct states, with the phenotype spectrum of classical M1 macrophages and alternative M2 macrophages coordinating this cell-fate decision process. Wound-healing M2 macrophages are non-homogeneous and can be compartmentalized into three major subsets, with M2a serving as a profibrotic and wound-healing function, M2b for regulation of the immune and inflammatory response, and M2c that possess pro-phagocytotic, profibrotic, anti-inflammatory and wound-healing functions[9, 10]. Moreover, M2c is the major macrophages to anti-inflammation and tissue repair by secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β1 during inflammation and immune responses[11].

In rats VILI model, macrophages were polarized to M1 with the upregulated expression levels of Notch pathwayrelated proteins, and the inhibition of Notch pathway reduced the proportion of M1 macrophages and inflammatory responses[12, 13]. Promoting phenotypic transformation of alveolar macrophages from M1 to M2 would alleviate lung injury and apoptosis of VILI by regulating miR-20b expression[14]. Knowing the kinetic and mechanism of sub-type of M2 macrophages responding to persistent inflammation may be a prospective direction to promote the resolution of lung injury and inflammation.

TGF-β1 not only maintains immune homeostasis via involving from the development and maturation of immune cells, but also regulates physiological to excessive pathological repair[15]. Src is crucial in mediating the TGF-β1-induced epithelial-mesenchymal transition (EMT) during the fibro-proliferative phase of ARDS, which was inhibited by nintedanib to reduce ventilation-augmented EMT[16]. The persistent polarization toward M2 macrophages was associated with EMT during the course of ventilation-induced pulmonary fibrosis through activation of epithelial TGF-β1/Smad2/3 signaling[17]. However, TGF-β1 overexpressing mesenchymal stem cells (MSCs) showed the well therapeutic effects on organ injury and inflammation resulting in reduced macrophage infiltration and induced macrophage transformation[18]. TGF-β1 requires liberation from its inactive latent forms (i.e., large or small latent complex) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors, which in turn activate SMAD2/3[19]. Here we focused on the role of macrophage polarization on the resolution of inflammation and lung injury, which would be explored through overexpression and inhibition of TGF-β1 in the VILI mice.

Mice

Male or female C57BL/6 mice, aged 4–6 weeks and weighted 25 ± 5 g, were purchased from the Animal Center of Guangxi Medical University (Nanning, China). Mice were kept in the air-filtered room with ad libitum access to food and water at a room temperature of 20–25 ºC with 50–70% humidity.

Mouse experiments

Mice were fasting and no drinking from 8 hours before experiments, and anesthetized by intraperitoneal injection of 60 mg/kg sodium pentobarbital, as well as administered 1/3 of the first dose to maintain anesthesia every 45 min. These mice were received oral endotracheal intubation and then allowed to breathe spontaneously (CON) or aided by ventilation with room air by a ventilator (TOPO, Kent Scientific, South Elgin, IL, USA). The respiratory rate was 80 bpm and the inspiration to expiration ratio was kept at 1:1 without positive end-expiratory pressure. High tidal volume (HTV) and normal tidal volume (NTV) were defined as 20 or 7 mL/kg, respectively. Ventilated mice were randomly allocated to undergo periods of recovery of 0 hour (end of ventilation, EOV), 4 hour (post-ventilation 4 hour, PV4h), 8 hour (post-ventilation 8 hour, PV8h), 1 day (post-ventilation 1 day, PV1d), 3 day (post-ventilation 3 day, PV3d), 7 day (post-ventilation 7 day, PV7d), and 10 day (post-ventilation 10 day, PV10d) subgroups. There were even numbers of male and female animals in each group (n = 4). After 4-hours (hrs) of ventilation, mice in the EOV subgroup were sacrificed. Mice in other subgroups, with recovery of consciousness and breathing spontaneously, were extubated for anesthesia resuscitation, and sacrificed at 4, 8, and 24 hrs and 3, 7, and 10 days after extubation, whereas mice in the CON group were sacrificed at 10 days after extubation. Bronchoalveolar lavage fluid (BALF), blood serum, and lung tissues was collected and stored at -80 ˚C except for the right-upper and middle lobes, which were obtained for frozen sectioning, transmission electron microscope (TEM) examination and wet/dry (W/D) ratio calculation.

Upregulation and neutralization of TGF-β1

Mice in the rTGF-β group were injected with carrier-free recombinant mouse TGF-β1 (rTGF-β1; 763104, Biolegend, CA, USA) via caudal vein before ventilation to up-regulate the level of TGF-β1[20, 21]. Another mice in the nTAb group were injected intravenously with 200 µg/kg InVivoMab anti-mouse TGF-β1 (nTAb; BE0057-5MG, BioXCell, NH, USA) to neutralize TGF-β1 before ventilation[22]. The rTGF-β1 and nTAb were both diluted to 50 µL normal saline, hence the vehicle group of mice was injected with 50 µL normal saline intravenously. In each treatment, mice were divided into seven subgroups including EOV, PV4h, PV8h, PV1d, PV3d, PV7d, and PV10d.

Pulmonary edema, permeability and cytokines measurement

The right upper lobe was wiped with filter paper to remove external water and obtain the weighed weight (W). The lung tissues were dried at 60˚C for 48 h and the dry weight (D) was obtained. The ratio of W/D weight was calculated as an index of pulmonary edema. For evaluating the changes in pulmonary permeability, the total protein of BALF was assessed by a BCA Protein Assay Kit (23225, ThermoFisher Scientific, MA, USA), and infiltration of total cells was counted by a hemocytometer. IL-1β, IL-6, tumor necrosis factor (TNF)-α, and TGF-β1 levels in plasma, BALF, or total lung homogenates were determined by enzyme-linked immunosorbent assay kits (CUSABIO, Wuhan, China).

Histological study

Right lower lung lobes were fixed with 4% paraformaldehyde (30525-89-4, Sigma-Aldrich, AR, USA) and embedded in the Tissue-Tek OCT compound (4583, Sakura, Tokyo, Japan). The pathological score of lung injury was estimated on hematoxylin and eosin stained sections by two authors independently according to previously reported criteria[23]. Some lung samples were cut for TEM analysis to observe the ultrastructural changes of type II alveolar epithelial cells (AEC-IIs). To observe the changes in pulmonary fibrosis, the lung tissues were embedded with paraffin and stained with Movat Russell modified pentachrome according to the manufacturer’s instructions. The nucleus and elastic fiber are dyed deep purple to black, collagen protein and reticular fiber are dyed red, proteoglycan is stained to blue, cellulose is stained to dark red, and cardiac muscle or smooth muscle are bright red.

Single-cell dissociation for flow cytometry (FCM)

To prepare a single cell suspension from left upper lung lobes, it was digested with 10 mM HEPES RPMI (22400105, ThermoFisher Scientific, MA, USA) including 0.1 mg/mL Dispase II (4942078001-1g, Roche, Basel, Switzerland), 2000U/mL DNase I (10104159001, Roche, Basel, Switzerland), and 0.2% collagenase (038-22361, Wako, Osaka, Japan). For a single cell suspension from the spleen, put it in a cell staining buffer (420201, BioLegend, CA, USA) and ground by the rough surface of the glass slide. After red cell lysis (11814389001, Roche, Basel, Switzerland) and washing twice, cells were passed through a 70-µm strainer and incubated with TruStain FcX™ PLUS antibody (S17011E, BioLegend, CA, USA) to block Fc-receptors. Cells were next incubated with surface antibodies on ice for 20 minutes in the dark. After fixation and permeabilization, cells were incubated with intracellular antibodies. The detailed information on surface and intracellular antibodies is shown in Table 1. Cells were analyzed using a Beckman Coulter CytoFLEX FLOW CYTOMETRY.

Table 1

Flow cytometry antibodies
Type of cells	Antibodies	Catalog number	Clone	Manufacturer
Surface antibodies (1:1000)
	CD3-FITC	100204	17A2	Biolegend
	CD5-PE	100607	53 − 7.3	Biolegend
	CD19-APC	152409	1D3/CD19	Biolegend
	Ly6G/Ly6C-APC	108411	RB6-8C5	Biolegend
M1	F4/80-APC	123116	BM8	Biolegend
	CD40-PerCP/Cy5.5	124624	3/23	Biolegend
M2a, M2c	CD163-PE	12-1631-82	TNKUPJ	eBioscience
	CD206-APC	141708	C068C2	Biolegend
Intracellular antibodies (1:1000)
M1	iNOS-PE	696806	W16030C	Biolegend
M2a	Arg1-PE/Cy7	25-3697-82	A1exF5	eBioscience
	CD68-PerCP/Cy5.5	137010	FA-11	Biolegend
M2b	IL-10-PerCP/Cy5.5	505028	JES5-16E3	Biolegend
	TNF-α-APC/Cy7	506344	MP6-XT22	Biolegend
	iNOS-PE	696806	W16030C	Biolegend
	Arg1-PE/Cy7	25-3697-82	A1exF5	eBioscience
M2c	TGF-β(LAP)-PE	563143	TW7-16B4	BD Biosciences
	CD68-PerCP/Cy5.5	137010	FA-11	Biolegend

Immunostaining

Frozen tissue sections were dried in air for 30 min and then fixed with 3.7% paraformaldehyde for 15 min and ice-cold 100% methanol for 15 min. After blocking with phosphate-buffered saline containing 3% bovine serum albumin (SRE0096, Sigma-Aldrich, AR, USA), 3% goat serum (16210064, Gibco, CA, USA), 0.2% Triton X-100 (X100, Sigma-Aldrich, AR, USA), and 0.02% NaN3 (S2002, Sigma-Aldrich, AR, USA), the slides were incubated with primary antibodies and related secondary antibodies. The detailed information on primary and secondary antibodies is shown in Table 2. The sections were treated with ProLong®Gold Antifade Reagent with 4’, 6-diamidino-2-phenylindole (DAPI) (8961S, Cell Signaling Technology, MA, USA), and analyzed by multiplex confocal microscopy (LSM980, Zeiss, German).

Table 2

Immunostaining antibodies for immunofluorescence
Targets	Antibodies	Catalog number	Dilution	Manufacturer
M1	Alexa Fluor 488 anti-mouse F4/80	123119	1:100	Biolegend
	Anti-iNOS antibody	DF0199	1:200	Affinity
	Alexa Fluor 549 Anti-Rabbit IgG H&L antibody	A-11037	1:500	Invitrogen
M2a	Anti-CD163 antibody	DF8235	1:200	Affinity
	Alexa Fluor 549 Anti-Rabbit IgG H&L antibody	A-11037	1:500	Invitrogen
	Anti-Arg-1antibody	sc-166920	1:500	Santa Cruz
	Dylight® 550 Anti-mouse IgG H&L antibody	Ab96880	1:500	PythonBio
M2b	Anti-iNOS antibody	DF0199	1:200	Affinity
	Alexa Fluor 549 Anti-Rabbit IgG H&L antibody	A-11037	1:500	Invitrogen
	Anti-Arg-1antibody	sc-166920	1:500	Santa Cruz
	Dylight® 550 Anti-mouse IgG H&L antibody	Ab96880	1:500	PythonBio
M2c	Anti-CD163 antibody	DF8235	1:200	Affinity
	Alexa Fluor 549 Anti-Rabbit IgG H&L antibody	A-11037	1:500	Invitrogen
	Purified anti-mouse LAP (TGF-β1)	141402	1:200	Biolegend
	Dylight® 550 Anti-mouse IgG H&L antibody	Ab96880	1:500	PythonBio
α-SMA	α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb	19245	1:500	CST
Vim	Recombinant Anti-Vimentin antibody [EPR3776]	ab92547	1:500	Abcam
CoL1	Anti-Collagen I antibody	ab21286	1:500	Abcam

Immunoblotting

Left lower lung lobes were homogenized in RIPA lysis buffer (20–188, Sigma-Aldrich, AR, USA) adding Protease Inhibitor Tablet (11836170001, Roche, Basel, Switzerland) and PhosphoSTOP Phosphatase Inhibitor Tablet (4906845001, Roche, Basel, Switzerland) using a tissue homogenizer. A total of 50–75 µg proteins were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies (listed in Table 3) and Alexa Fluor® 750-conjugated IgG secondary antibodies (Abcam, Cambridge, UK). Relative band densities of the various proteins were measured from scanned films using Odyssey® CLX (LI-COR, USA).

Table 3

Immunostaining antibodies
Targets	Antibodies	Catalog number	Dilution	Manufacturer
TGF-β1	Anti-TGF beta 1 antibody [EPR18163]	ab179695	1:1000	Abcam
NLRP3	NLRP3 (D4D8T) Rabbit mAb	15101	1:1000	CST
NF-κB	NF-κB p65 (D14E12) XP® Rabbit mAb	8242	1:1000	CST
IL-1β	IL-1β (D6D6T) Rabbit mAb (Mouse Specific)	31202	1:1000	CST
TNF-α	TNF-α (D2D4) XP® Rabbit mAb (Mouse Specific)	11948	1:1000	CST
E-cad	Anti-E Cadherin antibody [4A2]	ab231303	1:1000	Abcam
α-SMA	α-Smooth Muscle Actin (D4K9N) XP® Rabbit mAb	19245	1:1000	CST
Vim	Recombinant Anti-Vimentin antibody [EPR3776]	ab92547	1:1000	Abcam

Bioinformatics

The expression matrix of all target genes was uploaded to the Database for Annotation, Visualization and Integrated Discovery (DAVID) to obtain the Gene Ontology (GO) enrichment analysis, including biological processes, cellular components, and molecular functions. All target genes were submitted to DAVID to perform the GO enrichment and KEGG pathway analysis with count > 5 and P < 0.01. The STRING database was used to assess the interactive relationships of all target genes.

Statistical analysis.

Data were analyzed using StataCorp LP software (STATA Institute, Inc., Collgege Station, TX, USA) and performed using Graphpad Prism 9 (San Diego, CA, USA). Intergroup differences were assessed for significance using non-parametric analyses, and pair-wise comparisons were assessed using Turkey tests. Pearson Correlation analyses between every two factors were performed. A two-tailed P < 0.05 was defined as statistically significant.

HTV induced lung injury and inflammation.

We observed the resolution of HTV-induced lung injury and inflammation. Mice were challenged with 4 hours NTV or HTV and extubated to observe the lung injury and inflammation at different times (Fig. 1A). There was one mouse died in the PV1d subgroup of HTV group, and the probability of survival between the NTV and HTV group was lack of statistical difference (P = 0.3173) (Fig. 1B). In the subgroups of EOV, PV4h, PV8h, PV1d, and PV3d, the W/D ratios in the HTV group were significantly higher than that in the NTV group (Fig. 1C). Compared with the NTV group, different degrees of lung tissue injury occurred from EOV to PV1d with a higher pathological score in HTV-treated mice (Fig. 1D). The microscopic images of CON group showed generally normal structure of pulmonary alveoli, uniform thickness of alveolar wall, no or less blood and inflammatory cells infiltrated into lung interstitium and alveolar spaces (Fig. 1E). The microstructures of AEC-IIs of control mice included clear boundary of nucleus, evenly distributed chromatin, oval or round lamellar corpuscles and mitochondria with uniform texture and density distribution, complete membrane structure, and clearly visible microvilli (Fig. 1F).

Injured lung tissues showed different degrees of thickening of alveolar walls, intra-alveolar exudation, and extravasated blood, accumulation of inflammatory cells into the lung interstitium and alveolar spaces, as well as the formation of hyaline membranes (Fig. 1G). AEC-IIs in HTV-treated mice also showed different degrees of disrupted cytoplasmic and nuclear structure, increased mitochondrial swelling and cavitating changes in the lamellar corpuscles, as well as cell membrane discontinuities (Fig. 1H). However, according to the strength of the changing trend in AEC-IIs, the sequences were PV4h, PV8h, PV1d, and EOV. Compared with the NTV group, AEC-IIs in the PV4h subgroup of HTV group showed most pyknotic nucleus, small-round mitochondria and phagocytic vesicles; AECs- II in the PV7d subgroup of HTV group showed the fragmented chromatin, increased microvesicles and fissional mitochondria; and AEC-IIs in the PV7d subgroup of HTV group represented the oval nucleus with minority chromatin margination, increased phagocytic vesicles and small mitochondria (Fig. 1I).

HTV significantly increased total protein levels in BALF at EOV, PV1d and PV3d, with persistent cell accumulation in BALF from EOV to PV1d (Supplemental Fig. 1A, B). The serum and pulmonary IL-1β at EOV and PV8h in the HTV group were both higher in comparison with the mice of the NTV group (Supplemental Fig. 1C). Compared with the NTV group, IL-6 and TNF-α levels in serum in the HTV group were both elevated from EOV to PV1d; however, IL-6 levels in BALF in the EOV, PV4h and PV1d subgroups of HTV-treated mice were higher. TNF-α levels in BALF in the HTV group were elevated from EOV to PV7d (Supplemental Fig. 1D, E). As for the changes in these inflammatory factors, the serum and pulmonary IL-1β levels, and pulmonary TNF-α levels gradually decreased from EOV to PV10d. However, the serum and pulmonary IL-6 levels and serum TNF-α increased the most in the PV1d subgroup. We also observed that elastic fiber, proteoglycan, and collagen in lung tissue began to increase from PV3d (Supplemental Fig. 1F).

TGF-β1 is overexpressed during the resolution of lung injury and inflammation

TGF-β1 expression in BALF of HTV-treated mice remained higher than NTV group during the period from EOV to PV3d and PV10d whereas the serum level of TGF-β1 in HTV-treated mice was only higher at PV4h (Fig. 2A). Compared with NTV group, HTV lungs showed higher expressions of TGF-β1 latent and monomer at EOV and during the period from PV8h to PV10d. In the HTV-treated mice, the expression of TGF-β1 latent and monomer was gradually increased from the EOV subgroup, and peaked at PV8h subgroup; there were no statistical differences noted on the expression of TGF-β1 latent and monomer between EOV, PV1d, PV3d, and PV10d subgroups. In the PV7d subgroup of NTV and HTV group, the expression of TGF-β1 monomer were both significantly lower than other subgroups except for CON subgroup (Fig. 2B). CD5 + CD19 + B cells are defined as a subset of regulatory B cells producing TGF-β1, hence we analyzed the relative expression of TGF-β1 on CD5 + CD19 + B cells[24]. Compared with the CON and NTV_PV1d group, the proportion of TGF-β1 + macrophages and neutrophils in the HTV_PV1d group were significantly increased, and the proportion of TGF-β1 + neutrophils in the NTV_PV1d group was higher than that in the CON group (Fig. 2C, D and G). The proportion of TGF-β1 + CD3 + T cells and TGF-β1 + CD5 + CD19 + B cells in the NTV_PV1d group were both higher than that in the CON and HTV_PV1d group, and the proportion of TGF-β1 + CD3 + T cells and TGF-β1 + CD5 + CD19 + B cells between the CON and HTV_PV1d group were lack of statistical differences (Fig. 2C and E-F). This suggests that upregulated TGF-β1 expression may be a reason for the attenuation of lung injury and inflammation.

Macrophage polarization resolves inflammatory lung injury in VILI mice

Whether macrophage phenotype transition was sufficient to resolve HTV-induced inflammatory lung injury was determined. We observed increased M1 macrophages (F4/80 + iNOS + CD40+) in the EOV, PV4h, PV1d, and PV3d subgroups of HTV-treated mice. The proportion of M1 macrophages peaked at PV4h and PV1d subgroups, which was significantly decreased from PV4h compared with NTV-treated mice (Fig. 3A). Next, we found reduced M2a (CD68⁺CD206⁺CD163⁺Arg-1⁺) in the EOV and PV4h subgroups but a higher percentage of M2a from PV1d to PV7d in HTV-treated mice (Fig. 3B). HTV mice only showed a higher percentage of M2b subsets (IL-10⁺TNF-α⁺iNOS⁺Arg-1⁺) in the PV8h subgroup (Fig. 3C). We also observed significantly decreased M2c (CD68⁺CD206⁺CD163⁺TGF-β1⁺) in the PV4h subgroup but a higher percentage of M2c in the PV3d and PV7d subgroups of HTV-treated mice (Fig. 3D). The ratios of M1/M2a, M1/M2b, and M1/M2c were used to evaluate the efficacy of macrophage polarization. We observed markedly increased M1/M2a, M1/M2b, and M1/M2c ratios in the EOV and PV4h, subgroups of HTV-treated mice (Fig. 3E-G). Furthermore, we found the accumulation of M1 (F4/80 + iNOS+), M2a (CD163⁺Arg-1⁺), and M2c (CD163⁺TGF-β1⁺) macrophages in the lung tissues in the PV3d subgroup, and the infiltration of M2b (Arg-1⁺ iNOS⁺) macrophages in the lung tissues in the PV8h subgroup of HTV-treated mice (Fig. 3H, I). Figure S2 shows the chart of FCM analysis with statistical differences in the proportion of M1, M2a, M2b, and M2c subsets. These results suggest that M1 was increased at the acute phase, whereas the M2a and M2c begin to increase from PV3d to PV7d, which may be the time point to initiate lung tissue repair.

TGF-β1 is essential for the resolution of lung injury and inflammation.

Given the potential role of TGF-β1 during the resolution of HTV-induced lung injury and inflammation, we next sought to determine whether TGF-β1 help regulates this multifaceted process of tissue repair and inflammation resolution (Fig. 4A). Administration of rTGF-β1 showed lower W/D ratios in the PV4h and PV8h subgroups, and W/D ratios in the PV7d and PV10d subgroups were higher in the mice treated with nTAb (Fig. 4B). Notably, the rTGF-β1 treatment attenuated the lung injury with a decreased pathological score in the EOV and PV4h subgroups, but nTAb treatment aggravated the lung injury such that it was more severe at EOV and PV4h than in the vehicle and rTGF-β1 group. The nTAb induced significant lung injury at PV3d, PV7d, and PV10d (Fig. 4C, G, H).

Compared with the vehicle group, serum IL-1β levels in the PV4h and PV8h subgroups of the mice treated with nTAb were higher; serum IL-1β levels in the EOV and PV4h subgroups of the mice treated with nTAb were higher than that in the mice treated with rTGF-β1. Serum IL-6 levels in the PV4h, PV8h, PV3d, PV7d, and PV10d subgroups of the mice treated with nTAb were both significantly higher than that in the vehicle and rTGF-β groups. However, serum IL-6 levels in the EOV subgroups was lower whereas serum IL-6 levels in the PV7d and PV10d subgroups of the mice treated with rTGF-β1 were elevated compared with the vehicle group. In addition, serum TNF-α levels in the EOV, PV1d, PV3d, PV7d, and PV10d subgroups of the mice treated with nTAb were significantly higher than that in the other two groups; administration of rTGF-β1 resulted in lower serum TNF-α levels in the EOV, PV4h, and PV8h subgroups compared with the vehicle group (Fig. 4D). Up-regulation of TGF-β1 significantly decreased the pulmonary level of IL-1β, IL-6, and TNF-α before PV8h but depletion of TGF-β1 showed the high pulmonary level of IL-1β, IL-6 and TNF-α in the EOV subgroup. The mice treated with rTGF-β1 showed higher pulmonary IL-1β levels from PV1d to PV10d compared with the other two groups (Fig. 4E). As unexpected, a single injection of rTGF-β1 increased the serum TGF-β1 levels in the EOV subgroup but upregulated TGF-β1 expression in lung tissue in the EOV, PV4h, PV7d, and PV10d subgroups; nTAb treatment decreased the serum and pulmonary levels of TGF-β1 in the EOV and PV4h subgroups (Fig. 4F).

We next examined the expression of inflammatory proteins after the treatment of rTGF-β1 and nTAb. In mice injected with rTGF-β1, the expressions of NLRP3, NF-kB/p65, and IL-1β were decreased with TNF-α expression being higher in the acute phase. We detected the inhibition of TNF-α but upregulation of NLRP3 and IL-1β during the advanced stage. By contrast, the nTAb treatment resulted in the decreased expression of NLRP3 and NF-kB/p65 but the higher expression of IL-1β and TNF-α in the acute phase (Supplemental Figure S3A, B). AEC-IIs in the mice treated with nTAb showed different degrees of increased mitochondrial swelling and cavitating changes in the lamellar body, as well as cell membrane discontinuities (Supplemental Figure S3C).

TGF-β1 is required to regulate macrophages polarization

Because TGF-β1 is the common cytokines to regulate the differentiation of immune cells, we next sought to determine whether TGF-β1 plays a critical role in regulating macrophage polarization. Compared with the vehicle group, M1 proportions in the EOV, PV4h, PV1d and PV3d subgroups in the rTGF-β1 treated mice were significantly lower but the PV7d subgroup showed a higher proportion of M1. However, nTAb-treated mice showed higher proportions of M1 in the EOV, PV1d, PV3d, and PV7d subgroups compared with the rTGF-β group. Administration of nTAb also induced the highest proportions of M1 in the PV3d and PV7d subgroups compared with the other two groups (Fig. 5A). In the PV4h and PV8h subgroups, the proportions of M2a were significantly increased in the rTGF-β group. Interestingly, administration of nTAb induced the higher proportions of M2a in the PV1d, PV3d, and PV10d subgroups compared with the other two groups (Fig. 5B). In the PV8h and PV7d subgroups, mice treated with rTGF-β1 showed increased percentages of M2b, but the nTAb-treated mice showed the lowest percentages of M2b in the PV4h, PV8h, PV1d, and PV3d subgroups compared with the other two groups (Fig. 5C). Compared with the vehicle group, the percentages of M2c in the PV8h and PV7d subgroups of the mice treated with rTGF-β1 were elevated, but the percentages of M2c in the PV1d subgroups of the mice treated with nTAb were higher while the M2c macrophages were significantly decreased in the PV3d and PV7d subgroups (Fig. 5D). However, the rTGF-β1 treatment resulted in decreased M1/M2a in the PV84h subgroup but elevated M1/M2a in the PV7d subgroup, as well as reduced M1/M2c in the EOV, PV4h, PV1d, and PV10d subgroups compared to the vehicle group. Treatment of nTAb increased the M1/M2a ratio at PV3d, M1/M2b and M1/M2c ratio from PV3d to PV7d compared with the other two groups (Fig. 5E).

We next analyzed the correlation between each macrophage subset and found that M1 was positively correlated with M1/M2a, M1/M2b, and M1/M2c ratios (Fig. 5F); M2a were positively correlated with M2b and M2c.M2b was positively correlated with M2c but negatively correlated with the M1/M2b ratio (Fig. 5G-H). M2c was negatively correlated with the M1/M2b ratio too (Fig. 5I). Additionally, the M1/M2a ratio was positively correlated with the M1/M2b and M1/M2c ratios, and the M1/M2b ratio positively correlated with the M1/M2c ratio (Fig. 5J). Together, these results indicate that rTGF-β1 treatment can elevate the expression of M2a, M2b and M2c in the acute phase (only at PV4h and PV8h) but especially promote the polarization from M1 to M2c during the period of at EOV, PV4h, PV1d, and PV10d. Treatment of nTAb temporarily inhibited the TGF-β1 expression that were up-regulated in the resolution phase of VILI, with positive polarization of M2a in from PV1d to PV10d, however, negative induction of M2b from PV4h to PV7d as well as M2c at PV3d and PV7d.

TGF-β1 is crucial factor to promote M2a and M2c polarization during the resolution of lung injury and inflammation.

The expression of the TGF-β1 monomer was positively correlated with the total TGF-β1 level in lung tissues, M2b, and M2c (Fig. 6A). We also showed that serum TGF-β1 level negatively correlated with pulmonary IL-6 level but the pulmonary TGF-β1 level is negatively associated with pulmonary IL-6 level (Fig. 6B).M1 positively correlated with the W/D ratio, serum IL-1β levels, and serum and pulmonary TNF-α levels (Fig. 6C); but M2c was negatively associated with serum IL-6 levels (Fig. 6D). M1/M2a ratio positively correlated with the W/D ratio, pathological score, serum IL-1β and IL-6 levels, as well as serum and pulmonary TNF-α levels (Fig. 6E). The M1/M2b ratio was positively associated with serum IL-1β and pulmonary IL-6 levels (Fig. 6F), and M1/M2c ratio was positively correlated with the W/D ratio, serum and pulmonary IL-6 level, as well as serum and pulmonary TNF-α levels (Fig. 6G). W/D ratio and pathological scores were both positively correlated with serum IL-1β and IL-6 levels and serum and pulmonary TNF-α levels (Fig. 6H, I). Taken together, these results show that TGF-β1 is a crucial factor to promote M2b and M2c polarization with the regulation of pulmonary IL-6 levels. F4/80 + iNOS + CD40 + M1 subsets are the main pro-inflammatory macrophages to induce pulmonary edema and inflammation. Inhibition of M1 to M2a macrophages is associated with pulmonary edema, injury, and inflammation, but M2b macrophage polarization may be contributed to the attenuation of serum IL-1β and pulmonary IL-6 levels. M2c and its polarization contributed to the attenuation of serum and pulmonary IL-6 levels as well as serum TNF-α levels.

Overexpressed TGF-β1 induces the process of EMT.

TGF-β1 has previously been demonstrated to be essential for inducing the fibrogenic and developmental EMT, which is associated with the mechanism of pulmonary, kidney, and other tissue fibrosis[25]. The elevated mechanical tension of VILI activates a TGF-β1 signaling loop in type II alveolar epithelial cells to drive the progression of lung fibrosis[26]. We observed that rTGF-β1 injection elevated the expression of the TGF-β1 monomer in lung tissues at EOV, PV8h, PV7d, and PV10d, whereas the lower level of the TGF-β1 monomer at PV4h and from PV1d to PV3d. The nTAb treatment inhibited the expression of TGF-β1 in the acute phase but were both abnormally up-regulated at PV10d. Our data showed that TGF-β1 plays an anti-inflammatory and anti-fibrotic role in the acute phase, but the aberrant overexpression of TGF-β1 promotes EMT with downregulation of E-cad but high expression of Vim and α-SMA (Figure S4A-D).

We analyzed the biological enrichment of Tgfb1, Vim, Cdh1 (E-cad), Acta2 (α-SMA), Nlrp3, Nfkb1, Il1b, and Tnfa, and found that these genes had direct interactions (Figure S4E). These genes are enriched in the biological process such as response to other organisms, external stimuli and organonitrogen compounds; cellular components as identical protein binding, enzyme binding, protein binding, and cytokines activity; molecular functions including autophagosome and phagophore assembly site membrane (Figure S4F). These genes are enriched in the following KEGG pathways: NOD-like receptor signaling pathway, MAPK signaling pathway, and other inflammatory diseases (Figure S4G). Administration of rTGF-β1 induced higher expression of Vim, α-SMA and TGF-β1 protein in the EOV subgroup compared with other two groups, but the nTAb injection promoted the expression of Vim, α-SMA and TGF-β1 protein in the PV7d and PV10d subgroups in comparison with other two groups (Figure S5A-B). As for the expression of CoL1 protein, it was highly expressed in the EOV subgroup of Vehicle group compared with the control mice. Administration of nTAb induced up-regulation of CoL1 protein in the PV1d and PV3d subgroups compared with other three groups (Figure S5C). Overall, these data indicate a complicated role of TGF-β1 in tissue repair and fibrosis, likely by alleviating lung injury and inflammation in the acute phase. Preternaturally elevating TGF-β1 aggravates lung injury, inflammation, and fibrosis during VILI resolution.

To observe the changes and mechanisms of VILI, we used HTV with 20 mL/kg to enlarge the lung injury and inflammation due to inappropriate administration of the ventilator. According to a previous study[23], exposure for 4.0 hours resulted in lung injury and inflammation via activation of the TLR9/MyD88/NF-κB pathway by mitochondrial DNA. In the present study, we observed the resolution of lung injury and inflammation after 4 hours of aggravated VILI. The lung injury and inflammation had begun to subside at PV3d with decreased W/D ratio, pathological injury, and lower inflammatory factors. Elastic fiber, proteoglycan, and collagen in lung tissue began to increase from PV3d, which is the apparent time point to initiate lung tissue repair.

TGF-β1 is produced by multiple lineages of leukocytes, stromal cells, and epithelial cells to regulate pulmonary inflammation and fibrosis[11, 15, 25]. The polymorphisms of the TGF-β1 gene are associated with lung function decline, Pseudomonas aeruginosa infection, and inflammatory cytokines[27]. TGF-β1 is also a potential marker to distinguish COVID-19 patients with pulmonary involvement and indicate disease severity[28]. We found that serum and pulmonary TGF-β1 levels were both negatively associated with pulmonary IL-6 levels. Some studies have reported that IL-6 activates the JAK2/STAT3 axis to induce TGF-β1 secretion and directly regulates tissue repair and EMT[29, 30]. IL-6 and TGF-β1 can activate DNA methyltransferases1 to regulate the expression of E-cad, providing a potential therapeutic candidate for benign prostatic hyperplasia[31]. Hence, TGF-β1 may be coordinated with IL-6 to promote the resolution of lung injury and inflammation.

TGF-β1 and its pathway is the potential target for the treatment of ALI/ARDS. For example, TGF-β1-dependent production of IL-6 by alveolar macrophages prevents ALI development in influenza A virus-infected mice that are heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator[32]. In lipopolysaccharide-related ALI, IL-17-mediated collagen I and TGF-β1 were both upregulated by activating NLRP3 inflammasome signaling at least in part[33]. Alveolar epithelial cells used TGF-β1 from alveolar macrophages to regulate insulin-like growth factor − 1 production resulting in the attenuation of endogenous inflammatory signals during alveolar inflammation[34]. Besides, TGF-β1/Smads pathway had proved to be the canonical mechanism of fibrosis process in ALI/ARDS[35–37]. How to perform the immune regulation and tissue repair of TGF-β1 but inhibit or eliminate its profibrotic function would be the novel direction in the resolution of ALI.

Many immune and non-immune cells can produce TGF-β1 that must be activated to exert functional effects under specific conditions[38]. M2 macrophages can secrete large amounts of TGF-β1, which in turn promote the differentiation of M2 macrophages, especially M2c[11]. TGF-β1 secreted by MSCs promotes lipopolysaccharide-stimulated macrophage polarization toward M2, reduces inflammation, and improves the phagocytic ability through the Akt/FoxO1 pathway[39]. Appropriate secretion of TGF-β1 is required to resolve lung injury and inflammation. Preternaturally elevating TGF-β1 would aggravate lung injury and inflammation, induce EMT, and disadvantage tissue repair in the resolution phase of VILI consistent with a previous study[40].

Macrophage polarization is classically defined by integrated multiple signals, such as microbes or damaged tissues, that are controlled by epigenetic and cell survival pathways, the tissue microenvironment, and extrinsic factors, such as microbial products and cytokines released in inflammation[41]. Additionally, macrophage polarization is implicated as being selectively re-programmed via plasticity[42, 43]. Studies have shown that M1 is mainly involved in the acute phase, and M2 is associated with resolving phases of inflammation in ALI, indicating that macrophage polarization is found in the inflammation process of ALI[44]. During the rehabilitation phase of ALI/ARDS, M2 macrophages enhance the expression of IL-10, TGF-β1-induced matrix-associated protein BIG-H3, and insulin-like growth factor 1, and suppress the production of pro-inflammatory cytokines. This process promotes tissue repair and reduces alveolar epithelial cell damage after inflammation[42, 44]. As expected, we observed that M1 macrophages were increased temporarily, whereas the M2a and M2c macrophages begin to increase from PV3d, which is associated with the appropriate secretion of TGF-β1.

We also found that the M2a macrophage is the main wound-healing macrophage to decrease W/D ratio and pathological score with reduction of serum IL-1β and IL-6 levels as well as serum and pulmonary TNF-α levels. M2b and M2c macrophages contributed to the attenuation of serum IL-1β levels and pulmonary IL-6 levels. The M2a macrophage is particular phenotype to promote granuloma resolution in 60-day Mmp12KO mice, but the M2c macrophage is activated at 10 days when granulomas form[45]. M2a alveolar macrophages with IL-33 production promote lung defense against the Pneumocystis murina infection[46]. However, there is another viewpoint: IL-10-producing M2c macrophages are more potent than M2a macrophages to protect against lung injury and subsequent fibrosis[47]. Shifting M2a and M2c macrophages to M2b macrophages is a potential therapy to suppress asthma development by suppressing the type 2 response[48]. Single rTGF-β1 treatment does not elevate the expression of M2a and M2c subsets but promotes macrophage polarization from M1 to M2a and M2c; inhibition of TGF-β1 temporarily inhibits M2c induction, which is up-regulated in the advanced stages of VILI.

The effect of TGF-β1 was associated with macrophage polarization. Wang Y and his colleagues found that TGF-β1 from MSCs-secreted extracellular vesicles promotes M2 polarization of macrophages through modulation of the miR-132/Mycbp2/TSC2 axis[49]. TGF-β1 from bone marrow MSCs derived exosomes carried highly expressed miR-135b to promote M2 polarization of synovial macrophages through targeting MAPK6[50]. TGF-β1 overexpressing MSCs also promoted the transformation of pro-inflammatory macrophages to pro-resolution macrophages in inflammatory environment. Here, we found that the appropriate secretion of TGF-β1 is required for the specific M2a and M2c macrophage polarization to resolve lung injury and inflammation; nevertheless, abnormally elevated TGF-β1 induces the EMT resulting in aggravation of lung injury and limiting tissue repair. However, there were some limitations in this study, such as not performing adoptive transfer experiments for the M2a or M2c macrophages, and using Tgfb1 knockout mice to identify the role of endogenous TGF-β1 because of lengthy breeding. The mechanisms of the TGF-β1 and IL-6 interaction warrant further study. Moreover, the quantitative data were not reported on lung fibrosis valuation obtained by Movat Russell modified pentachrome staining, and the correlation between these data and TGF-β1 needs us further study.

Taken together, the results show that ventilation-associated lung injury and inflammation began to resolve from PV3d with increased elastic fibers, proteoglycans, and collagen. The appropriate secretion of TGF-β1 is required for M2a and M2c macrophage polarization, which contribute to withholding the inflammation cascade and lung injury in the acute phase.

The authors have no financial conflicts of interest to declare.

Ethic Approval

The study protocol was approved by the Animal Care and Use Committee of Guangxi Medical University (KY-2022-288).

Competing interests

The authors declare no conflict of interest.

Author’s contribution

L.H.P. designed research and revised the paper; J.R. wrote and revised the paper; J.R., X.L.X., and X.T.L. performed research; S. H., J.L.M. and H.J.D. analyzed data; Z.K.H. drew figures.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81970078), Guangxi Anesthesiology Clinical Medicine Research Center Construction Project (scientific foundation of Guangxi No: 2022AC04002), Youth Science Foundation of Guangxi Medical University (GXMUYSF202120), and Innovation Project of Guangxi Graduate Education (YCBZ2021043).

Data availability statement

Not applicable.

Perez-Fernandez XL, Sabater-Riera J&Fuset-Cabanes M. COVID-19 ARDS: getting ventilation right. Lancet (London, England) 399, 22 (2022)
Meyer NJ, Gattinoni L&Calfee CS. Acute respiratory distress syndrome. Lancet (London, England) 398, 622–37 (2021)
Villar J, Zhang H&Slutsky AS. Lung Repair and Regeneration in ARDS: Role of PECAM1 and Wnt Signaling. Chest 155, 587 – 94 (2019)
Nieman GF, Gatto LA, Andrews P, Satalin J, Camporota L, Daxon B, et al. Prevention and treatment of acute lung injury with time-controlled adaptive ventilation: physiologically informed modification of airway pressure release ventilation. Ann Intensive Care 10, 3 (2020)
Yu Z, Wang T, Zhang L, Yang X, Li Q&Ding X. WISP1 and TLR4 on Macrophages Contribute to Ventilator-Induced Lung Injury. Inflammation 43, 425 – 32 (2020)
Su K, Bo L, Jiang C, Deng X, Zhao YY, Minshall RD, et al. TLR4 is required for macrophage efferocytosis during resolution of ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 321, L787-L801 (2021)
Zhang S, Zhu L, Dai H&Pan L. Silencing ROCK1 ameliorates ventilator-induced lung injury in mice by inhibiting macrophages' NLRP3 signaling. Int Immunopharmacol 101, 108208 (2021)
Chen J, Li D, Huang Z, Zheng C, Lin Q, Feng N, et al. Role of Mitophagy-based TLR9 Signal Pathway in Neonatal Ventilator-induced Lung Injury. Cell Mol Biol (Noisy-le-grand) 68, 103 – 10 (2022)
Vannella KM&Wynn TA. Mechanisms of Organ Injury and Repair by Macrophages. Annu Rev Physiol 79, 593–617 (2017)
Abdelaziz MH, Abdelwahab SF, Wan J, Cai W, Huixuan W, Jianjun C, et al. Alternatively activated macrophages; a double-edged sword in allergic asthma. Journal of translational medicine 18, 58 (2020)
Dees C, Potter S, Zhang Y, Bergmann C, Zhou X, Luber M, et al. TGF-beta-induced epigenetic deregulation of SOCS3 facilitates STAT3 signaling to promote fibrosis. J Clin Invest 130, 2347–63 (2020)
Yin D, Wang W, Han W&Fan C. Targeting Notch-activated M1 macrophages attenuate lung tissue damage in a rat model of ventilator induced lung injury. International journal of molecular medicine 44, 1388–98 (2019)
Huang H, Feng H&Zhuge D. M1 Macrophage Activated by Notch Signal Pathway Contributed to Ventilator-Induced Lung Injury in Chronic Obstructive Pulmonary Disease Model. The Journal of surgical research 244, 358 – 67 (2019)
Liu Y, Tang G&Li J. Long non-coding RNA NEAT1 participates in ventilator-induced lung injury by regulating miR-20b expression. Molecular medicine reports 25, (2022)
Batlle E&Massague J. Transforming Growth Factor-beta Signaling in Immunity and Cancer. Immunity 50, 924 – 40 (2019)
Li LF, Kao KC, Liu YY, Lin CW, Chen NH, Lee CS, et al. Nintedanib reduces ventilation-augmented bleomycin-induced epithelial-mesenchymal transition and lung fibrosis through suppression of the Src pathway. J Cell Mol Med 21, 2937–49 (2017)
Wang L, Zhang Y, Zhang N, Xia J, Zhan Q&Wang C. Potential role of M2 macrophage polarization in ventilator-induced lung fibrosis. Int Immunopharmacol 75, 105795 (2019)
Liu F, Xie J, Zhang X, Wu Z, Zhang S, Xue M, et al. Overexpressing TGF-beta1 in mesenchymal stem cells attenuates organ dysfunction during CLP-induced septic mice by reducing macrophage-driven inflammation. Stem Cell Res Ther 11, 378 (2020)
Zhao B, Xu S, Dong X, Lu C&Springer TA. Prodomain-growth factor swapping in the structure of pro-TGF-beta1. J Biol Chem 293, 1579–89 (2018)
Zhang XY, Liu ZM, Zhang HF, Li YS, Wen SH, Shen JT, et al. TGF-beta1 improves mucosal IgA dysfunction and dysbiosis following intestinal ischaemia-reperfusion in mice. J Cell Mol Med 20, 1014–23 (2016)
Taylor RA, Chang CF, Goods BA, Hammond MD, Mac Grory B, Ai Y, et al. TGF-beta1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage. J Clin Invest 127, 280–92 (2017)
Lee KM, Stott RT, Zhao G, SooHoo J, Xiong W, Lian MM, et al. TGF-beta-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance. Eur J Immunol 44, 1728–36 (2014)
Lin JY, Jing R, Lin F, Ge WY, Dai HJ&Pan L. High Tidal Volume Induces Mitochondria Damage and Releases Mitochondrial DNA to Aggravate the Ventilator-Induced Lung Injury. Front Immunol 9, 1477 (2018)
Liu ZQ, Wu Y, Song JP, Liu X, Liu Z, Zheng PY, et al. Tolerogenic CX3CR1 + B cells suppress food allergy-induced intestinal inflammation in mice. Allergy 68, 1241–8 (2013)
Su J, Morgani SM, David CJ, Wang Q, Er EE, Huang YH, et al. TGF-beta orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1. Nature 577, 566 – 71 (2020)
Wu H, Yu Y, Huang H, Hu Y, Fu S, Wang Z, et al. Progressive Pulmonary Fibrosis Is Caused by Elevated Mechanical Tension on Alveolar Stem Cells. Cell 180, 107 – 21 e17 (2020)
Trojan T, Alejandre Alcazar MA, Fink G, Thomassen JC, Maessenhausen MV, Rietschel E, et al. The effect of TGF-beta1 polymorphisms on pulmonary disease progression in patients with cystic fibrosis. BMC pulmonary medicine 22, 183 (2022)
Laloglu E&Alay H. Role of transforming growth factor-beta 1 and connective tissue growth factor levels in coronavirus disease-2019-related lung Injury: a prospective, observational, cohort study. Rev Soc Bras Med Trop 55, e06152021 (2022)
Wang B, Liu T, Wu JC, Luo SZ, Chen R, Lu LG, et al. STAT3 aggravates TGF-beta1-induced hepatic epithelial-to-mesenchymal transition and migration. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 98, 214 – 21 (2018)
Li H, Yang P, Wang J, Zhang J, Ma Q, Jiang Y, et al. HLF regulates ferroptosis, development and chemoresistance of triple-negative breast cancer by activating tumor cell-macrophage crosstalk. J Hematol Oncol 15, 2 (2022)
Xu H, Chen Y, Chen Q, Xu H, Wang Y, Yu J, et al. DNMT1 regulates IL-6- and TGF-beta1-induced epithelial mesenchymal transition in prostate epithelial cells. Eur J Histochem 61, 2775 (2017)
Woods PS, Tazi MF, Chesarino NM, Amer AO&Davis IC. TGF-beta-induced IL-6 prevents development of acute lung injury in influenza A virus-infected F508del CFTR-heterozygous mice. Am J Physiol Lung Cell Mol Physiol 308, L1136-44 (2015)
Kim DI, Song MK&Lee K. Diesel Exhaust Particulates Enhances Susceptibility of LPS-Induced Acute Lung Injury through Upregulation of the IL-17 Cytokine-Derived TGF-beta1/Collagen I Expression and Activation of NLRP3 Inflammasome Signaling in Mice. Biomolecules 11, (2021)
Mu M, Gao P, Yang Q, He J, Wu F, Han X, et al. Alveolar Epithelial Cells Promote IGF-1 Production by Alveolar Macrophages Through TGF-beta to Suppress Endogenous Inflammatory Signals. Front Immunol 11, 1585 (2020)
Wang L, Liu J, Xie W, Li G, Yao L, Zhang R, et al. miR-425 reduction causes aberrant proliferation and collagen synthesis through modulating TGF-beta/Smad signaling in acute respiratory distress syndrome. International journal of clinical and experimental pathology 12, 2604–12 (2019)
Xu Y, Zhai D, Goto S, Zhang X, Jingu K&Li TS. Nicaraven mitigates radiation-induced lung injury by downregulating the NF-kappaB and TGF-beta/Smad pathways to suppress the inflammatory response. Journal of radiation research 63, 158 – 65 (2022)
Yao Y, Yuan Y, Lu Z, Ma Y, Xie Y, Wang M, et al. Effects of Nervilia fordii Extract on Pulmonary Fibrosis Through TGF-beta/Smad Signaling Pathway. Front Pharmacol 12, 659627 (2021)
Travis MA&Sheppard D. TGF-beta activation and function in immunity. Annu Rev Immunol 32, 51–82 (2014)
Chapman HA, Wei Y, Montas G, Leong D, Golden JA, Trinh BN, et al. Reversal of TGFbeta1-Driven Profibrotic State in Patients with Pulmonary Fibrosis. The New England journal of medicine 382, 1068–70 (2020)
Liu F, Qiu H, Xue M, Zhang S, Zhang X, Xu J, et al. MSC-secreted TGF-beta regulates lipopolysaccharide-stimulated macrophage M2-like polarization via the Akt/FoxO1 pathway. Stem Cell Res Ther 10, 345 (2019)
Murray PJ. Macrophage Polarization. Annu Rev Physiol 79, 541 – 66 (2017)
Cheng P, Li S&Chen H. Macrophages in Lung Injury, Repair, and Fibrosis. Cells 10, (2021)
Wang S, Liu R, Yu Q, Dong L, Bi Y&Liu G. Metabolic reprogramming of macrophages during infections and cancer. Cancer letters 452, 14–22 (2019)
Chen X, Tang J, Shuai W, Meng J, Feng J&Han Z. Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome. Inflammation research: official journal of the European Histamine Research Society [et al] 69, 883 – 95 (2020)
Ogburn D, Bhalla S, Leffler N, Mohan A, Malur A, Malur AG, et al. The M2a Macrophage Phenotype Accompanies Pulmonary Granuloma Resolution in Mmp12 Knock-Out Mice Instilled with Multiwall Carbon Nanotubes. International journal of molecular sciences 22, (2021)
Nelson MP, Christmann BS, Werner JL, Metz AE, Trevor JL, Lowell CA, et al. IL-33 and M2a alveolar macrophages promote lung defense against the atypical fungal pathogen Pneumocystis murina. J Immunol 186, 2372–81 (2011)
Tang L, Zhang H, Wang C, Li H, Zhang Q&Bai J. M2A and M2C Macrophage Subsets Ameliorate Inflammation and Fibroproliferation in Acute Lung Injury Through Interleukin 10 Pathway. Shock 48, 119 – 29 (2017)
Kang H, Bang JY, Mo Y, Shin JW, Bae B, Cho SH, et al. Effect of Acinetobacter lwoffii on the modulation of macrophage activation and asthmatic inflammation. Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology 52, 518–29 (2022)
Wang Y, Han B, Wang Y, Wang C, Zhang H, Xue J, et al. Mesenchymal stem cell-secreted extracellular vesicles carrying TGF-beta1 up-regulate miR-132 and promote mouse M2 macrophage polarization. J Cell Mol Med 24, 12750–64 (2020)
Wang R&Xu B. TGF-beta1-modified MSC-derived exosomal miR-135b attenuates cartilage injury via promoting M2 synovial macrophage polarization by targeting MAPK6. Cell Tissue Res 384, 113–27 (2021)

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Transforming growth factor-β1 is associated with inflammatory resolution via promoting M2c macrophage polarization in lung injury mice

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Mice

Mouse experiments

Upregulation and neutralization of TGF-β1

Pulmonary edema, permeability and cytokines measurement

Histological study

Single-cell dissociation for flow cytometry (FCM)

Immunostaining

Immunoblotting

Bioinformatics

Statistical analysis.

Results

TGF-β1 is overexpressed during the resolution of lung injury and inflammation

Macrophage polarization resolves inflammatory lung injury in VILI mice

TGF-β1 is required to regulate macrophages polarization

Discussion

Declarations

Funding

Data availability statement

References

Additional Declarations

Supplementary Files

Status:

Version 1