Mechanics-activated Fibroblasts orchestrating the plasticity of group 2 innate lymphoid cells propel the progression of silicosis

doi:10.21203/rs.3.rs-3457730/v1

Download PDF

Article

Mechanics-activated Fibroblasts orchestrating the plasticity of group 2 innate lymphoid cells propel the progression of silicosis

https://doi.org/10.21203/rs.3.rs-3457730/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Exposure to crystalline silica particle leads to silicosis characterized as progressive fibrosis. Fibroblasts are described as vital effector cells in fibrogenesis. Emerging studies identified immune sentinel role of fibroblasts in chronic disease, while their immune-modulatory role in silicosis remained elusive. Herein, we confirmed that a conversion of ILC2 to ILC1 closely involved in silicosis was mediated by activated fibroblast via IL-18. Mechanistically, Notch3 signaling in mechanics-activated fibroblasts modulated IL-18 production. The mice specific knockout Notch3 in fibroblast exerted retardatory progression of pulmonary fibrosis that tightly linked to attenuated conversion of ILCs. Our results indicated that the activated-fibroblast in silicotic lung served as a regulator of ILC2-ILC1 conversion that associated with silicosis progression via Notch3-IL-18 axis. The findings broadened the cognitive boundaries of the immune regulation of silicosis, also provide potential therapeutic targets in treating lung fibrotic diseases.

Health sciences/Pathogenesis/Immunopathogenesis/Innate immunity

Health sciences/Medical research/Experimental models of disease

Sustained activation of fibroblasts to produce collagen-rich extracellular matrix is a hallmark of fibrosis that drives silicosis. While the immunomodulatory role of fibroblast was ignored. Our research identified and described fibroblasts were responsible for modulating ILCs conversion within silicosis. Nevertheless, based on regulating ILC2 remodeling via IL-18, Notch3 signaling links a previously unknown immunological role of fibroblasts to pathogenesis of fibrotic progression. Our data expand the knowledge about fibroblasts as both crucial effector cells of fibrogenesis and immuno-regulators in silicosis, as well as providing a potential molecular basis of Notch3 for targeted silicotic therapeutics.

Silicosis is an ancient and potentially fatal pneumoconiosis caused by exposure to respirable crystalline silica (CS) particles. Silicosis is historically a disease closely related to mining, and often found among miners. However, failure to recognize and control the risk associated with silica exposure in contemporary work practices such as sandblasting denim jeans and manufacturing of artificial stone benchtops has led to re-emergence of silicosis around the world (1, 2). In the United States over 1.7 million workers are exposed to CS in the general, maritime and construction industries. It is estimated that 3–5 million workers in Europe and Australia have been exposed to silica (1, 3). While the exposure is much more common in low- and middle-income countries (4). Though it is known that exposure to CS particles trigger silicosis, the mechanisms underlying disease pathology remain poorly understood. The burden of CS-associated disease remains high, while treatment options are limited.

Fibrogenesis is a hallmark of silicosis, in which fibroblast activation and proliferation play dominant roles. While fibroblasts were classically thought of as ‘immune neutral’ cells, whose functions were mainly focused on the construction of tissue and remodeling of the ECM, it is now clear that fibroblasts play a multifaceted role in health and disease, especially in the setting of chronic inflammation (5). Fibroblasts emerge as key immune sentinel cells in activating and modulating immune responses, in inflammatory diseases. Specifically, fibroblasts take on different roles as inflammatory cells themselves, recruiting leukocytes, enabling chronic inflammation in tissues (6). Our previous study indicated fibroblasts could mediate the polarization and recruitment of interstitial macrophages (7), highlighting the immunomodulatory role of fibroblasts in silicosis. As major proliferating stromal cells in silicosis, the critical immunomodulatory role of fibroblasts was underestimated.

The pulmonary immunity plays an active part in the pathophysiology of silicosis. The roles of myeloid cells, specifically macrophages, and lymphocytes, especially T cells, in silicosis, have been widely studied (8, 9), while that of innate lymphoid cells (ILCs) remains scarcely explored. ILCs are a heterogeneous family of innate lymphocytes that, unlike T or B lymphocytes, lack surface expression of antigen-specific receptors, who are enriched at mucosal barrier are predominantly tissue-resident, and dramatically impact tissue local immunity, inflammation, and tolerance (10). Lung ILC cells were defined as a population of non–T cells, non–natural killer (NK) cells that lacked expression of lineage markers (Lin⁻) but exerted high CD90 (Thy-1) expressions. Within the local tissue, ILCs not only establish a first-line defense system against infections but also orchestrate tissue homeostatic functions through interactions with other tissue-resident cells, such as epithelial cells, stromal cells, or neurons. Although ILCs are important in immuno-logical protection in many infectious disease models, they are paradoxically also capable of driving immunopathology, including recently reported in fibrogenesis (11).

Three distinct ILC populations have been described that manifested various pattern of cytokine production and functions mirroring those of CD4 T helper (Th) cell (12). Group 2 ILCs (ILC2s) take the dominant part in the pulmonary ILC pools and produce type II cytokines (IL-4, IL-5, and IL-13). While group 1 ILCs (ILC1s) are characterized by their capacity to produce IFN-γ. They depend on the transcription factor T-bet and exerted T-bet − dependent non-cytotoxic effects. The main cytokine produced by group 3 ILCs (ILC3s) is IL-17, which depend on the transcriptional factor retinoic acid receptor-related orphan receptor-γt (RORγt). However, ILC subsets are not stable, they also have the ability to adapt to the local microenvironment by changing these profiles, which were termed as the plasticity of ILCs (13). The plasticity of ILCs enables them to adapt to changing local tissue conditions, which may be essential to tune responses to different pathogenic stimuli. In some circumstances, the conversion of ILC cells mediated the pathology of disease. For example, rheumatoid arthritis stimulation in the presence of IL-1β and IL-23 were reported to drive ILC1 plasticity and conversion to ILC3s (14), but also, in other contexts, some ILC3 converted to ILC1 (15). Some ILC2s exhibited the ability to produce IL-17 and expressed transcription factor RORγt, that drives IL-17 expression (16). Owing to the plasticity of ILCs, ILC1 can also arise from either ex-ILC2 or ex-ILC3, which down-regulate GATA3 or RORγt, respectively, and up-regulated T-bet, and IFN-γ production. With regards to COPD, an increase in the frequency of ILC1 cells was correlated with disease progression, which was confirmed as the result of direct conversion from lung resident ILC2 cells (17). As for silicosis, it is unclear if ILCs respond to cues in the fibrotic milieu to alter phenotype and reshape their functions.

Fibrogenesis in different tissue types shares common pathological abnormalities, including the activation of fibroblast, and the polarization of immune cells. Here we tightened fibroblast activation with immune cell polarization, demonstrating that activated fibroblast regulated ILC cell polarization and conversion within silicosis. Mechanistically, Notch3-IL-18 axis in activated-fibroblast modulated ILCs conversion. The mice specific knockout Notch3 in fibroblast exerted retardatory progression of pulmonary fibrosis that tightly linked to attenuated conversion of ILCs. These results advance our understanding of fibroblast’s immunomodulatory role, as well as defining a pathway controlling the conversion of ILC cells in the fibrotic lung. Therefore, targeting fibroblast immune-modulatory signaling Notch3 could be a promising strategy to postpone the progression of fibrogenesis.

1. CS particles in the lung activated ILC cells and altered the phenotype of ILCs.

In the silicotic lung, we observed increased ILC cells at both percentage and cell number levels (Fig. 1a,b, Fig. S1a), implying its causal relationship to fibrogenesis. Cytokines in the immuno-microenvironment affected ILC functions, we further checked cytokine receptor expressions on the ILCs. Apparently, ILCs in the silicotic lung expressed analogic levels of CD127 (IL-7R), but decreased CD25 (IL-2Rα) and ST-2 (IL-33R) compared to those from saline-treated lung, implying decreased responsiveness to cytokines IL-2 and IL-33 (Fig. 1c). Notably, we noticed a substantially increased IL-18Rα levels in the ILCs from fibrotic lung, suggesting the ILC cells increased sensitiveness to IL-18 (Fig. 1d), while IL-17RB (receptor to IL-25) was not induced on the ILCs from silicotic lung (Fig.S1b). We also discovered increased CD69 and CD103, as well as elevated CXCR6 expressions in the ILCs (Fig. 1e), manifesting enhanced immobilizations in the fibrotic lung. The chemokine receptor CCR9, expressed by ILC precursors, was not affected, implying the ILCs were not recruited from the circulation(Fig.S1c). To gain more insights, we further checked effector molecule levels on the ILCs. Notably, ILCs from fibrotic lung expressed elevated MHC-II, indicating a lifting role in antigen-presenting control of adaptive immunity (Fig. 1f). Besides, ILCs expressed increased levels of ICOS (Inducible T-cell costimulator) (Fig. 1g), that promoting the survival and cytokine productions (18). Moreover, NRP-1 (Neuropilin-1) was elevated in the fibrotic ILCs (Fig. 1h), supporting the idea that NRP-1 enhancing ILC activation mediates pulmonary fibrogenesis (19). The decreased KLRG1 (Killer cell lectin-like receptor subfamily G member 1) levels suggested the ILCs in fibrotic lung were newly expanded and activated (Fig. 1i). Some inhibitory molecules, including PD-1, PD-L1, and CD73, but not TIGIT, LAG3, and CD39, were augmented in the ILCs from fibrotic lung (Fig. 1j, Fig. S1d), suggesting the activated ILCs could negatively regulate the immune microenvironment. Remarkably, we detected the ILCs in the silicotic lung expressed decreased IL-13 but increased IFN-γ (Fig. 1k), which was contradictory to the notion that ILC2 was the dominant subset within the lung, indicating the ILC components in the lung were affected by invaded CS particles. Collectively, these data indicated that ILCs in the silicotic lung were activated, while the constitutions of ILCs in the fibrotic lung were affected by CS particles.

2. The phenotype of ILCs in the silicotic lung was shifted from ILC2 to ILC1.

We next sought to explore whether the invaded particles affected the constitution of pulmonary ILCs. Basically, ST-2⁺ ILC2 cells take a dominant part of ILCs in the lung at physiological state. Strikingly, we noticed the proportion of ILC2 decreased while the percentage of NKp46⁺ILC1 gradually increased along with the progression of silicosis (Fig. 2a, b, Fig. S2a). To consolidate the findings, we checked fate-determine transcriptional factors T-bet and Gata-3 expressions which echoed our previous results (Fig. 2c). The ratio of ILC1 to ILC2 in silicotic lung augmented along with fibrosis progression (Fig. 2d). Furthermore, we utilized Rag1^−/− mice, who lacks T and B cells but not ILCs, establishing silicosis model. Intriguingly, the shifted ILCs from group 2 to group 1 were recurrence in the Rag1^−/− mice (Fig. 2e), indicating the altered ratio of ILC2 and ILC1 in silicosis progression was independent of adaptive immunity. Additionally, we utilized another common pulmonary-fibrosis model that was induced by bleomycin (Fig. 2f), proving there was still a shift from ILC2 to ILC1 (Fig. 2g). suggesting that the phenotype changes in ILCs were related to the progression of pulmonary fibrosis but not specifically in silicosis.

Next, we sought to explore the reason for the altered ILCs. Significantly, the decreased ILC2 cells were not due to cell death (Fig. S2b), or egress into the hilar lymph nodes (Fig. S2c). Furthermore, with the help of in vivo labelling, we demonstrated the alteration was not related to circulatory cell replenishment (Fig. S2d). Taking the notion that elevated IL-18Rα expression in the ILCs, and its causal relation to ILC1 function, we next sought to explore its expressions on ILC subsets. We proved the NKp46⁺ILC1 expressed high IL-18Rα, which increased along with the pathogenesis (Fig. S2e). Interestingly, we discovered the emergence of IL-18Rα⁺ST-2⁺ cells in the silicotic lung, but not in physiological condition, and expanded with silicosis progression (Fig. 2h). The increased IL-18Rα⁺ ILC cells were not attributed to increased circulating ILC cells (Fig. S2f). Strikingly, the loss of ST2 on ILCs was strongly correlated with a parallel increase in the frequency of the IL-18Rα⁺ ILCs (Fig. 2i), as well as NKp46⁺ ILC1 (Fig. S2g). The characterization of the IL-18Rα⁺ST-2⁺ ILC cells manifested high expressions of tissue-resident markers CD69, CXCR6, and CD103, but few were labeled with circulating CD45 antibodies, suggesting that the IL-18Rα⁺ILC2 were from tissue local ILC2 pools (Fig. 2j). Additionally, the result from BrdU labeling ruled out the possibility of ILC1 proliferation in fibrotic lung (Fig S2h). Collectively, these data demonstrated there were major functional and phenotypic changes in lung-resident ILCs with silicosis progression, characterized by a shift from ILC2 to ILC1.

3. IL-18 mediated conversion of pulmonary ILC2 to ILC1 in silicotic lung.

The strong negative correlation between ST2-expressing and IL-18Rα⁺ ILCs within silicosis progression indicated the ILC1 cells might emerge from local ILC2. Taking the notion that ILC cells exerted plasticity in some circumstances, we next did cell transfer experiments aiming to explore whether these ILC1s were converted from the local ILC2s. The congenic mice (CD45.1/2) were intranasally treated with IL-33 expanding lung ILC2 cells that were sorted by the FACS method (Fig. S3a). The sorted ILC2s were transferred into Rag1^−/− mice (CD45.2/2), who were in the initial stage of fibrogenesis (Fig. 3a). The transferred ILCs, readily identifiable by CD45.1, infiltrated into the lungs of recipient mice (Fig. S3b). Though the sorted ILC2s were ST-2⁺ and NKp46^–, they were converted into NKp46⁺ ST-2– phenotype and expressed high levels of IL-18Rα (Fig. 3b), implying the critical role of IL-18 in mediating ILCs conversion. We further checked the transcriptional factor expressions in the transferred cells. Significantly, the ILC2 cells downregulated ST-2 expressions and correlated striking upregulation of T-bet, the ILC1-specific transcriptional factor. Besides, the converted ILCs expressed high IL-18Rα while downregulated Gata-3 (Fig. 3c). Collectively, these results indicated that ILC2s were converted into ILC1 cells in silicosis.

To consolidate the results of ILCs conversion, we generated ST-2-eGFP reporter mice by inserting the eGFP gene into the exon of Il1rl1 (encoding ST2, a direct target of Gata-3, commonly used to label ILC2s) (Fig. 3d), in which the eGFP⁺ ILC cells were bonafide ILC2s. We also identified the ST2 (eGFP)⁺ cells were almost exclusively labeled with ST2 fluorescent antibodies that decreased with the progression of silicosis (Fig. S3c). By checking IL-18Rα expression on ILCs of the ST2-eGFP mice, we found it emerged on eGFP⁺ cells (Fig. 3e). Notably, we noticed a significant leaning shift from the ST-2 (eGFP)⁺ to IL-18Rα⁺ ILCs (Fig. 3e), recommending a conversion of ILC2 to ILC1. Apparently, the IL-18Rα expressions on ST-2 (eGFP)⁺ IL-18Rα⁺ ILC cells were lower than those on ST2 (eGFP)^– IL-18Rα⁺ ILCs (Fig. 3f). Additionally, we explored the receptor for IL-12 that were related to ILC conversion but found few ILCs expressed IL-12Rβ2 (Fig. 3g), suggesting a weak relation to ILC conversion in silicotic lungs. To directly prove our hypothesis that IL-18 mediates ILCs conversion in silicosis, we introduced IL-18 knockout mice and checked the ratio of ILCs in the fibrotic lung. Expectedly, mice deficient in IL-18 exerted attenuated ILC2 to ILC1 conversion (Fig. 3h). In synthesis, these data demonstrated that the ILC1 cells in the fibrotic lung were derived from the local ILC2 pool rather than the outgrowth of existing ILC1 cells, which were mediated by IL-18, but its cellular origin still needs to be determined.

4. Mechanics-activated fibroblast secreting IL-18 modulated the conversion of ILC2 to ILC1.

We next sought to explore the cellular origin of IL-18 that mediated ILC conversions in fibrotic lungs. It was commonly believed that IL-18 was tightly linked to pyroptotic macrophages. To test the hypothesis, we applied clodronate liposomes (CL) depleting macrophages within silicosis progression (Fig. S4a, b) and checked the ratio of ILCs in the lung. Interestingly, macrophage depletion did not attenuate pulmonary ILCs conversion (Fig. 4a), implying other cellular sources mediating the effects. Various sources of IL-18 have been identified, including leukocytes and stromal cells (20). To further investigate whether leukocyte-derived IL-18 regulated ILCs conversion, we constructed bone marrow (BM) chimera using wildtype and IL-18^–/– mice (Fig. S4c).

By using the chimera mice with silicosis model, we found hematopoietic cell–derived IL-18 did not boost ILC1s in the lung (Fig. S4d). Thus, IL-18 from non-hematopoietic cells (stromal cells) would be necessary for the ILCs conversion.

Taking the notion that fibroblasts actively proliferated in fibrosis progression and secreted cytokines modulating the immune microenvironment, we speculated that fibroblasts might control the ILC conversion. With immunofluorescence (IF) staining, we observed that ILC2 cells (Lin^–Gata-3⁺) were clustered around or interacted with the activated fibroblasts (Fig. 4b), highly recommending that fibroblast modulated the ILCs conversion. Further, we prospectively detected whether activated fibroblast secreting IL-18 with an in vitro model that matrix stiffness-activated fibroblasts, which mimic fibroblast activation in the silicotic lung and avoid the effects of exogenous cytokines (Fig. S4e). Notably, the stiff matrix-cultured fibroblasts exerted a sharp increase of pro-IL-18 in the cells (Fig. 4c). Moreover, mature form IL-18 was also increased in the culture medium (Fig. 4d).

To directly certify that the activated fibroblast modulates the conversion of ILC2 to ILC1, we did some in vitro experiments. First, we cultured sorted pulmonary ILC2 with conditional medium (CM) from different stiffness-treated fibroblasts (Fig. 4e). A high portion of NK1.1⁺ ILCs was detected in the ST-2⁺ILC2s cultured with CM from high stiff-activated fibroblasts, while the ratio of ST-2⁺NK1.1– was decreased. Accordingly, the number of ST-2⁺NK1.1⁺ ILCs increased (Fig. f, g), implying a potential for the CM mediating ILCs conversion. To consolidate these findings, we further introduced a trans-well cocultured system of fibroblasts with ILC2 (Fig. 4h). After 36h co-culture, we observed a lift ratio of NKp46⁺ ILC1 and a decreased ST-2⁺ ILC2 (Fig. 4i, j), which was analog to the CM-cultured ILC2s. Strikingly, when did the co-cultured experiments for 60h, we discovered a boosted ratio of NKp46⁺ILC1s (Fig. 4k, l), indicating that activated fibroblasts modulated ILCs conversion in a time-dependent manner. Collectively, we demonstrated that IL-18 derived from the activated fibroblast would modulate the conversion of ILC2 to ILC1.

5. Mechanics-activated fibroblast secreting IL-18 was dependent on the Notch3 signaling pathway.

To gain insights into the mechanism that fibroblast secreting IL-18, we performed RNA-Seq to the primary lung fibroblasts cultured on matrix gels, identified 1281 upregulated and 936 downregulated transcriptions in stiff matrix cultured fibroblast (Fig. 5a). By Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to upregulated genes, we determined multiple signaling pathways implicated, including HIF-1 and PI3K-Akt and rheumatoid arthritis-related signaling, cytokine-cytokine receptor interaction, etc. (Fig. b). Moreover, Gene ontology (GO) analysis showed significant enrichment in inflammatory response and wound healing, implying fibroblast’s critical role in immuno-regulation (Fig. c). Further, by protein-protein interaction analysis, we predicted that Notch signaling would be involved in process (Fig. 5d). Given that Notch3 in fibroblast recently emerged as a novel mediator in several disease pathogenesis, including fibrogenesis and rheumatoid arthritis (21, 22), also, the molecule reported to modulate fibroblast activation and survival (21), we then focused on Notch3 signaling in the fibroblasts. To gain insights about Notch3 in activated fibroblast, we first did immunoblot and proved a sharply increased Notch3 intracellular domain (Notch3ICD) in the activated fibroblast nuclei (Fig. 5e). Consistently, increased nuclear Notch3ICD localization were observed, further confirming that high mechanics activated Notch3 signaling in the fibroblasts (Fig. 5f). To directly explore the relationship between Notch3 and IL-18 expression in fibroblasts, we generated fibroblast-specific Notch3 knock-out mice, by crossing Notch3^fl/fl mice to Col1a2-creERT mice (Fig. Sa, b), and sorted the pulmonary fibroblasts, repeated previous in vitro experiments. IF staining demonstrated Notch3 abrogating decreased pro-IL-18 expressions in stiff matrix-activated fibroblasts (Fig. 5g). Accordingly, the bioactive form IL-18 in the culture medium was not elevated in the Notch3 abrogating fibroblast though stimulated by high mechanics (Fig. 5h). In synthesis, these data proved that the Notch3 signaling was necessary for the mechanics-activated fibroblasts secreting IL-18.

6. Notch3 signaling in fibroblasts mediated the ILCs conversion that associated with attenuated fibrotic phenotype.

Now that we proved that Notch3 mediates IL-18 production in mechanics-activated fibroblasts in vitro, we next attempted to explore whether it modulated ILCs conversion in vivo. Consistent with the previous notion, we observed upregulated Notch3 expressions in the activated fibroblasts from silicotic lungs (Fig. 6a). Further, we used fibroblast-specific Notch3 knockout mice and treated with tamoxifen (TAM), which allowed us to disrupt Notch3 in fibroblasts at a specific time schedule. First, TAM was applied from Week 3 to 4 after CS treatment, when relays of acute inflammation and fibrogenesis (Fig. 6b). We prospectively analyzed the ratio of ILC1/ILC2 in the fibrotic lung at the times of Week 8 to 10 but did not get a significant altered ILC ratios (Fig. c-e). Unexpectedly, with fibrosis progression, we determined the ratio of ILC1 to ILC2 was apparently decreased in Notch3 knockout mice at Week 12 to 14 (Fig. 6c-e), demonstrating fibroblast would affect ILCs conversion dependent on Notch3. Further, taking the notion that fibroblasts proliferated actively in fibrogenesis, we tried to apply TAM from Week 8 to 9, expecting to gain more insights about its role at the fibrogenesis stage (Fig. 6f). By flow analysis to the ILCs, we noticed a restored ILC2 proportion, notwithstanding the ratio of ILC1 was not appreciably impacted by the Notch3 ablation at the time phase (Fig. 6g-i). Collectively, these results indicated that Notch3 signaling in fibroblast was essential for ILCs conversion in silicosis, but in a time-dependent manner.

We further aimed to explore whether the reduced ILCs conversion was linked to attenuated fibrotic phenotype in Notch3 cKO mice. Histological analysis with H&E and Masson staining demonstrated the Notch3^fl/fl Colla2-Cre mice with reduced ILC1/ILC2 ratio exerted relatively attenuated cellular inflammation and collagen deposition (Fig. 6j, k). demonstrating Notch 3 signaling in fibroblast controlled ILCs conversion and fibrogenesis. The results were consolidated by qPCR analysis of the fibrotic-related gene expressions in the lung (Fig. 6l). Conversely, in the mice with unaffected ILC ratios, we did not observe apparently alleviated fibrogenesis (Fig. S6a). Furthermore, by monitoring the tidal volume (TV) and breathe frequency (f) of the mice, we extended our conclusion that the attenuated fibrosis phenotype with fast recovered pulmonary functions (Fig. S6b). Taken together, these data indicated fibroblast controlled ILC2 to ILC1 conversion in the fibrotic lung which was dependent on the Notch3 signal. The lack of Notch3 in fibroblast confers protection to the lung not only by a more preserved morphology with decreased collagen deposition but also by a fast recovery of lung function.

Tissue-resident innate lymphoid cells (ILCs) are critical in regulating tissue homeostasis and inflammatory responses. We aimed to depict phenotypes of pulmonary ILCs in silicotic mice and, unexpectedly, discovered a shift of group 2 to group 1 in ILC cells with the progression of silicosis, further proving the shift was due to conversions within ILC subsets that were mediated by IL-18. We also demonstrated that activated fibroblast modulated ILC conversions. Besides, the loss of Notch3, the mechanosensitive receptor in fibroblasts, parallel to blocking ILC remodeling, also impeded the fibrosis progression (Fig. 7). These data expand our knowledge about fibroblasts as both crucial effector cells of fibrogenesis and immune regulators of local immune homeostasis.

In the current study, we unexpectedly discovered the conversion of ILC2 to ILC1 cells in silicosis but refrained from delving into the functional role of the converted ILC1. Research about ILC1s mainly focused on mucosal inflammation that is exclusively relevant to the capacity of producing IFN-γ. However, recent research highlighted an unexpected role for ILC1 in tissue remodeling, emphasizing transforming growth factor β1 (TGF-β1) rather than IFN-γ. ILC1s secrete TGF-β1 and express matrix metalloproteinase 9 (MMP9) that drives matrix softening and stiffening, modulating balanced matrix degradation and deposition (23). Analogously, a recent RNA-seq dataset confirmed increased TGF-β1 and MMP9 expressions in ILC1 of myocardial infarction patients (24). In parallel, adipose ILC1s exerted a characterization that promotes tissue fibrogenesis through activation of TGF-β1 signaling and increasing macrophages in an IFN-γ-dependent fashion (25). These data provide insights into ILC1’s function in fibrogenesis. However, considering the difference in tissue micro-environments, as well as the heterogeneous phenotype and transcriptional profile of the isolated ILC1s in different tissues, future studies about the role of ILCs in pulmonary fibrosis would attract wide attention.

ILC functions were affected by the local cytokine milieu, among which the IL-1 family was tightly linked to ILC2 to ILC1 conversion (26). IL-18 together with IL-12 were reported to control ILC2-ILC1 conversion in mouse model of chronic obstructive pulmonary disease (COPD) (17). Whereas, in silicosis, no upregulated IL-12Rβ but a substantial IL-18Rα expression was detected. By utilizing IL-18 knockout mice, we substantiated the pivotal role of IL-18 in ILCs conversion. Pyroptotic macrophages were tightly linked to IL-18 production since its biological form was executed by caspase 1 (27). However, in the current study, we refuted that macrophage or even hematopoietic cell-derived IL-18 mediated the ILC conversions. Noteworthy, the cellular source of IL-18 extends beyond leucocytes, as the stromal cells served as a crucial cellular source, mediating the pathogenesis of diseases (20, 28). Analog to our study, there were reports indicating it was the lung fibroblasts but not macrophage upregulated IL-18 controlling the function of CD8⁺ T cells in COPD disease (29). Notably, different cellular contexts of cytokine in the tissue microenvironment played various regulatory roles in disease progression (30). For instance, IL-33 serves a proinflammatory role when derived from epithelia but exerts an immunosuppressive function when came from dendritic cells (30). Nevertheless, in our study, experiments using myeloid cell and fibroblast-specific IL-18 knockout mice would provide more insights into its role in silicosis.

As key effector cells in fibrogenesis, fibroblasts get activated in response to injury, proliferate, and migrate, promoting extracellular matrix deposition (21, 31). Meanwhile, tissue-resident fibroblasts have emerged as key immune sentinel cells, garnering significant attention. Analog to myeloid cells, fibroblasts possess the ability to initiate pro-inflammatory signaling pathways to facilitate leukocyte recruitment and regulate their activity (32). NLRP3 inflammasome activation in fibroblast could trigger the release of IL-18 and other pro-inflammatory cytokines, which are linked to tissue damage (33). Moreover, the PDGFRα⁺ fibroblast in the lung and intestine expressed IL-33, which was crucial for maintaining ILC2s within the niche of the microenvironment (34, 35), highlighting the notion that fibroblast could sustain ILCs viability. Herein, we proved the spatial correlation between fibroblasts and ILC2 in silicotic lungs, confirming the capacity of activated fibroblasts modulating ILCs conversion by IL-18. Even so, the immunoregulatory role of fibroblasts in chronic inflammatory diseases is still worthy of more studies.

Notch acts as a novel activator of the NLRP3 inflammasome leading to chronic tissue damage and fibroblast differentiation. Previous studies focused on the interaction of Notch1 signaling and cytokine production in fibroblasts (36), while both Notch1 and Notch3 have been previously associated with lung injury (37). The crosstalk between Notch1 and Notch3 may exist, as it has been suggested in kidney and pancreatic damage (38–40). Particularly, Notch3 signaling has only recently emerged as a novel mediator in tissue pathology (21, 22, 39). Accordingly, a recent publication showed that Notch3 is upregulated at the transcriptional level in the human fibrotic lung, specifically in fibroblasts (41). The involvement of Notch3 in fibroblast activation has gained significant attention due to its reported role in regulating the development of lung fibrosis. Notch3 deficiency in fibroblasts attenuates pulmonary fibrosis and impedes lung function decline (21). However, Notch3 deficiency did not affect fibroblast proliferation directly. Rather, it regulated the survival of fibroblast, indicating that Notch3 affected fibroblast viability (21). Herein, we extended our knowledge that Notch3 promoted fibroblast expressing pro-inflammatory cytokine IL-18, that linked with pyroptotic cell death. In the complex environment of a tissue such as the lung, Notch3 appears to have a multifunctional role. However, the research about Notch3 were all based on whole-body gene knockout mice (21, 22, 39). By utilizing fibroblast-specific Notch3 cKO mice, our study advances the previous understanding of Notch signaling by identifying Notch3 as a critical receptor in fibroblast pathologic expansion as well as immunomodulatory function in silicosis. Additionally, our recent publication evidenced that Notch 3 signaling in myeloid cells was necessary for interstitial macrophage recruitment that promotes silicosis progression (42). These results provide a molecular basis that modulation of Norch3 signaling can be therapeutically targeted in silicosis.

Mechanical hallmarks of fibrotic microenvironments are both outcomes and causes of fibrosis progression (43). Most studies overlook the significant changes in the biomechanical microenvironment within the fibrotic tissue. It remains elusive how the cells and mechanical cues interact during fibrosis progression. Notch signaling is activated with the protease hydrolysis site exposure triggered by the pulling force of the binding ligand (44). Notably, extrinsic pull could activate Notch receptors directly (45), providing insights into how the extracellular matrix activated Notch3 in fibroblasts. Pathological cyclic stretch transmits pathological mechanical cues through Notch3. Previously, we demonstrated that mechanics could activate fibroblast through CD44-RhoA-YAP signaling that promoted the progression of silicosis (46). Blocking the pathway ameliorated silicosis as well as significantly affected the lung tissue immune microenvironment, highlighting the notion that mechanical affected the function of immune cells. Fibrogenesis in different tissue types shares common pathological abnormalities, including fibroblast activation and immune cells polarization. While, whether mechanotransduction directly affecting immune cell function and polarization would attract more attention in the future.

In conclusion, we discovered a novel regulatory role of fibroblast in modulating ILC conversion and provided potential therapeutic molecular targets in treating silicosis.

Mice

C57BL/6JGpt mice (Strain NO.N000013), B6-Rag1-KO mice (Strain NO.T004753), Il1rl1-EGFP mice (Strain NO.T055284), Il18-KO mice (Strain NO.T052436), Notch3-flox mice (Strain NO.T007271) were purchased from GemPharmatech (Nanjing, China). CD45.1 congenic mice (C57BL/6JGpt-Ptprcem1Cin(p.K302E)/Gpt, straining No. T054816). Col1a2 Cre-ERT mice (B6.Cg-Tg(Col1a2-cre/ERT, -ALPP)7Cpd/J) were purchased from Shanghai Model Organisms Center, Inc. All the mice used were on C57BL/6J background and age, sex matched. Littermate mice were used when possible. The mice were housed in a specific pathogen-free (SPF) facility in individually ventilated cages on a 12-hour day-night cycle with ad libitum access to food and water. The experiments were approved by IACUC (CMU2020349) of China Medical University, which complies with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Generation of ST2-GFP reporter mice

A targeting construct was injected into murine blastocysts containing an upstream 4-kb short homology arm followed by a flippase (FLP) recombinase target (FRT)-flanked puromycin resistance cassette nestled in intron 10, then exon 11 of the Il1rl1 gene where the stop codon was deleted and immediately followed by a fusion sequence of the T2A self-cleaving peptide fused to enhanced green fluorescent protein (eGFP) followed by a new stop codon.

Crystalline silica particles

CS particles were purchased from the U.S. Silica Company (Frederick, MD, USA). The characteristics of crystalline silica particles are described in detail (7, 46). Briefly, the size distribution of CS particles was 97% < 5 µm in diameter and 80% < 3 µm in diameter, with a median diameter of 1.4 µm. CS particles were acid washed in 1 M HCl, autoclaved for 1 h and allowed to dry, and then suspended in sterile saline. The suspension was autoclaved and sonicated for 10 min before use.

Murine model of silicosis and other treatments

The mouse model of silicosis was established according to our previously published method (7, 42, 46). To establish silicosis model, mice were treated with 3.0 mg/50 µL CS suspension by intratracheal installation after anesthetization with pentobarbital sodium (30 mg/kg, Sigma-Aldrich) intraperitoneally. Mice in the control group were instilled with the same amount of sterile saline. The mouse model of bleomycin (BLM)-induced lung fibrosis was constructed by non-invasive endotracheal intubation of BLM (2U/kg body weight, Hanhui pharmaceuticals). The same amount of sterile saline was applied as control. To induce Notch3 knockout in fibroblast, Tamoxifen (MCE) dissolved in corn oil (MCE) was injected intraperitoneally at the indicated time with a daily dose of 0.1g/kg body weight for 7 days.

Flow cytometry

Flow cytometry analysis was performed according to the guideline (16, 17). Dead cells were excluded from analysis using Living/Dead cell viability dye Aqua (Invitrogen). Flow antibodies were purchased from BioLegend, eBioscience, or Miltenyi, which were listed in the Table below.

For surface antigens staining, cells were incubated with fluorescently labeled antibodies at 4℃ for 30 min. For transcription factors staining, cells were first stained for cell-surface markers, then fixed and permeabilized using the FoxP3 staining buffer kit (FoxP3 staining kit, eBioscience) and stained with indicated antibodies to transcriptional factors. For intracellular cytokine staining, cells were stimulated with leukocyte stimulation cocktail containing PMA, ionomycin, brefeldin A, and monensin (Invitrogen) for 3–4 h before being surface stained, fixed, permeabilized and subsequently intracellular staining. The antibodies detecting transcriptional factors or cytokines were applied and incubated at 4℃ overnight. Flow cytometry data were acquired by BD FACS Celesta (BD Bioscience, San Jose, CA, USA). FACS data were analyzed with Flowjo 10.6 software.

ILC2 cells enrichment and sorting from mouse lung

Pulmonary ILC2s were enriched by magnetic-activated cell sorting (MACS) and sorted by fluorescence-activated cell sorting (FACS). The mice were treated intranasally with rIL-33 (Novoprotein) every other day expanding ILC2s. For MACS sorting, EasySep™ Mouse ILC2 Enrichment Kit were used (Stemcell technologies). The purification ILC2 population was > 95%. For FACS sorting, ILC2 cells were sorted as CD45⁺Lin^–CD3^–CD90.2⁺ST2⁺ cells by BD FACSAria Ⅲ. Details methods are provided in SI files.

Primary fibroblasts isolation

Lung tissue was harvested and minced into 1 mm³ pieces, and then cultured on tissue culture flasks with Dulbecco’s modified Eagle’s medium (DMEM, Vivacell) containing 20% fetal bovine serum (FBS, Vivacell), 100 U/ml penicillin/streptomycin (Sigma-Aldrich), 1mM sodium pyruvate (Sigma-Aldrich) and 1x non-essential amino acids (Sigma-Aldrich) to obtain fibroblasts, using a “walk out” approach to culture adherent lung stromal cells. Cells were passed when confluent after harvest with trypsin-EDTA. Adherent cells were selected by repeated subcultures. Confluent cells at passages 4–6 were used for experiments.

2D cell culture and treatment

The 2D Col-gel was acquired from Bioruo (Beijing, China). Cells were cultured at 37℃ with 5% CO₂. MLg2908 cells (ATCC) were grown in EMEM (Vivacell) supplemented with 10% FBS (Vivacell) and primary lung fibroblasts were cultured in DMEM (Vivacell) supplemented with 20% FBS (Vivacell), both containing 100 U/ml penicillin/streptomycin (Sigma-Aldrich), 1mM sodium pyruvate (Sigma-Aldrich) and 1x non-essential amino acids (Sigma-Aldrich). As for 2D cell culture, MLg2908 cells or primary mouse lung fibroblasts after overnight serum deprivation (EMEM or DMEM without FBS) were seeded on coverslips or plastic dished coated with either 1 Kappa (soft) or 25 Kappa (stiff) polyacrylamide hydrogels with sterile collagen to create a uniform, thin layer of ECM protein according to the manufacturer’s protocol. After 24 or 48 hours, cells and cultured medium were collected separately for subsequent analysis.

ILC2 cells enrichment and sorting from mouse lung

Pulmonary ILC2s were enriched by magnetic-activated cell sorting (MACS) and sorted by fluorescence-activated cell sorting (FACS). Prior to MACS/FACS sorting, the mice (CD45.1/2) were treated intranasally with 0.5µg rIL-33 (Novoprotein) in PBS every other day for three times expanding ILC2s. For MACS sorting, ILC2 isolation kits were used (Stemcell technologies). The sorting was performed according to the manufacturer’s instructions. Briefly, lung single-cell suspensions were stained with lineage antibodies in MACS buffer, and then incubated with anti-biotin microbeads. MS columns were used to deplete lineage-positive cells. For ILC2 enrichment, the depletion was repeated twice and yielded > 95% pure ILC2 populations. For FACS sorting, ILC2 cells were sorted as CD45⁺Lin^–CD3^–CD90.2⁺ST2⁺ cells by BD FACSAria Ⅲ.

Lung ILC2s culture and treatment

C-RPMI used for in vitro ILC2 culture was RPMI 1640 (Vivacell) supplemented with 10% FBS (Vivacell), 100 U/ml penicillin/streptomycin (Sigma-Aldrich), 1mM sodium pyruvate (Sigma-Aldrich) and 1x non-essential amino acids (Sigma-Aldrich), containing 10 ng/ml IL-2 (Novoprotein) and 10 ng/ml IL-7 (Novoprotein).

For conditional culture experiment, sorted lung ILC2s by MACS were cultured with conditioned media from the primary lung fibroblast stimulated on 2D Col-gel supplemented with the same amount of fresh c-RPMI. After 36 hours, lung ILC2s were collected to perform flow cytometry analysis.

For co-culture experiment, the transwell culture system of 0.4µm aperture (Labselect) were used. The primary lung fibroblasts were stimulated on 2D Col-gel at the bottom. 24 hours later, the sorted lung ILC2s by MACS were added to the upper compartment of the transwell system. After co-cultured for 36 hours or 60 hours, lung ILC2s were collected to perform flow cytometry analysis.

Bulk RNA sequencing and gene expression analysis

Total RNA was extracted from isolated primary lung fibroblasts cultured on soft (~ 1kPa) or stiff (~ 25kPa) matrix gels using Trizol (Vazyme). Bulk RNA sequencing was performed by BGI Global Genomic Services. The sequencing data was filtered with SOAPnuke (v1.5.6) and the subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com). Expression level of gene was calculated by RSEM (v1.3.1) and differential expression analysis was performed using the DESeq2(v1.4.5) with P value ≤ 0.05. To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expression gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test. The significant levels of terms and pathways were corrected by P value with a rigorous threshold (P value ≤ 0.05). Images were produced by Dr.Tom from BGI (a web-based multi-omics visualization tool: https://biosys.bgi.com/#/report/login).

Immunofluorescence staining

Paraffin-embedded lung sections of 5 µm were deparaffinized. Heat-mediated antigen retrieval was performed by using Citrate Antigen Retrieval Solution (Beyotime). Sections were blocked in 5% BSA for 1 h and treated with 0.25% Triton X-100 for 15 min, then stained with primary antibodies(Lin, Miltenyi; αSMA, Proteintech; Gata3-e660, eBioscience) diluted in PBS containing 1% FBS overnight at 4°C. After washing, lung sections were incubated with fluorochrome-conjugated secondary antibody (CraLite 594-Phalloidin, Proteintech) for 1 h, washed again, incubated with the other fluorochrome-conjugated secondary antibody (AF488 streptavidin, Biolegend) to bind to biotin for 1 h, and stained with DAPI for 5 min.

As for fibroblast immunofluorescent staining, collected cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, washed again, and blocked in 5% BSA for 30 min. For intracellular antibody staining, cells were permeabilized with 0.25% Triton X-100 for 15 min. Primary and secondary antibody incubation were performed for overnight at 4°C and 60 min at room temperature, respectively.

Images were acquired using an Olympus confocal microscope.

Immunoblot analysis

Cells and lung tissues were homogenized in a tissue grinder with radioimmunoprecipitation assay (RIPA) buffer (Beyotime). Protein levels were measured by BCA protein assay (Beyotime). Protein samples (30µg) were separated on a 8%-12% Tris-Glycine Gels and electroblotted onto Immobilon-P Transfer Membrane (Merck Millipore). The membrane was blocked for 1 hour at room temperature with 5% milk, and then probed with 1:500-1:1000 diluted antibodies to determine the corresponding protein levels. After three washing steps (10 min each), the levels of protein were detected using secondary antibodies (1:2000 dilution in 5% milk in PBS containing 0.1% Tween-20 for 1h) linked to horseradish peroxidase. The bound complexes were detected by the ChemiDoc Touch Imaging System (BIO-RAD) using ECL western blotting detection reagents (Absin). Protein band intensities were quantified using ImageJ 1.44p software.

Elisa analysis

The amounts of IL-18 in the indicated in vitro culture supernatants were assessed using Elisa kit (Cat: DY7625, R&D) according to the manufacturer’s instructions. Absorbance was measured at 450 nm and 570 nm by a 96-well multimode plate reader.

Histological analysis

When indicated, one lobe per lung was collected for histology and stored in paraformaldehyde 4% buffered in PBS. Lungs were embedded in paraffin and cut into 4-µm sections for hematoxylin and eosin (H&E) and Masson’s trichrome staining. The staining was performed according to the manufacturer’s protocol. Histology pictures were acquired on an Aperio ScanScopeand and selected with ImageScope.

Quantitative PCR (q-PCR) analysis

Total RNA was extracted from treated cells using Trizol (Life Technologies) according to the manufacturer's protocol, and reverse transcribed into cDNA with the PrimeScript RT kit (Vazyme). Equivalent amounts of each cDNA sample were added to a SYBR Green Master Mix Kit (Vazyme). The primers used were

Collagen-1a1-F: GCTCCTCTTAGGGGCCACT;

Collagen-1a1-R: CCACGTCTCACCATTGGGG;

Fibronectin-F: GCAGTGACCACCATTCCTG;

Fibronectin-R: GGTAGCCAGTGA-GCTGAACAC.

The mRNA levels of Fibronectin, and Collagen-1a1 were determined using the 2 ^–ΔΔCT method.

Measurement of pulmonary function

Body weight as well as tidal volume and breathing frequency of the indicated mice were measured before the mice sacrificed. We used tidal volume and breathing frequency relative to body weight to describe the mice pulmonary function. DSI Buxco® FinePointe™ NAM Non-Invasive Airway Mechanics and Buxco FinePointe Non-Invasive Airway Mechanics (NAM) (DSI, Wilmington, NC) was used for measurement and record.

Clodronate liposomes treatment

For circulating macrophage depletion experiment, mice were treated i.p. 200µl of clodronate liposome (Yeasen, Shanghai, China) for the first time 7 weeks after CS treated. Later, mice were treated i.p. 100µl of clodronate liposome every other day. For alveolar macrophage depletion experiment, mice were treated i.n. 10µl of clodronate liposome for one time 7 weeks after CS treated. Control mice were received the same treated with equal amounts of PBS. Mice were killed 12 weeks after CS treated.

Generation of bone marrow chimeras

Femurs and tibias from donor 6- to 8-week-old WT and IL-18^–/– mice were removed aseptically, and BM was flushed using sterile cold PBS supplemented with 2mM EDTA. 6- to 8-week-old recipient C57BL/6J mice were irradiated with 5.5Gy dose of γ-radiation. Then bone marrow chimeras were established by intravenously transferring 5×10⁶ bone marrow cells isolated from donor WT and IL-18^–/– mice into irradiated recipient C57BL/6J mice. One week later, the mouse model of silicosis was established on bone marrow chimeras as described above. Mice were maintained on autoclaved water supplemented with antibiotics (trimethoprim/sulfamethoxazole; GLPBIO) for 4 weeks. Mice were killed 4–5 weeks after CS treated.Statistical analysis

All data was presented as the mean ± SD. A two-tailed Student’s t test for paired or unpaired data was applied for comparisons between 2 groups. For multigroup comparisons, we used one-way ANOVA with the Tukey’s test. Data were analyzed with GraphPad Prism Software (version 9.0, GraphPad Software Inc.). P value < 0.05 was considered to denote statistical significance. P values were indicated on the graph.

Statistical analysis

Data availability

All data generated or analyzed during this study are included in this article and its Supplementary Information.

Acknowledgments

Funding: Research reported in this publication was supported by the National Natural Science Foundation of China (No. 82173490 to Chao Li).

Author Contributions: Y.H., C.L. designed research; Y.H., H.Y., Y.Y., F.Y., Y.C., X.W., H.M. performed research; Y.H., C.L. analyzed data; Y.H., C.L. wrote the paper. J.C., C.L. supervised the study. All authors commented and approved the manuscript.

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

R. F. Hoy, D. C. Chambers, Silica-related diseases in the modern world. Allergy 75, 2805–2817 (2020).
H. Barnes, N. S. L. Goh, T. L. Leong, R. Hoy, Silica-associated lung disease: An old-world exposure in modern industries. Respirology 24, 1165–1175 (2019).
S. Si et al., The Australian Work Exposures Study: Prevalence of Occupational Exposure to Respirable Crystalline Silica. Ann Occup Hyg 60, 631–637 (2016).
N. Sharma, D. Kundu, S. Dhaked, A. Das, Silicosis and silicotuberculosis in India. Bull World Health Organ 94, 777–778 (2016).
S. Davidson et al., Fibroblasts as immune regulators in infection, inflammation and cancer. Nat Rev Immunol 21, 704–717 (2021).
K. Wei, H. N. Nguyen, M. B. Brenner, Fibroblast pathology in inflammatory diseases. J Clin Invest 131 (2021).
Y. You, H. Yuan, H. Min, C. Li, J. Chen, Fibroblast-derived CXCL14 aggravates crystalline silica-induced pulmonary fibrosis by mediating polarization and recruitment of interstitial macrophages. J Hazard Mater 460, 132489 (2023).
S. Lo Re, D. Lison, F. Huaux, CD4 + T lymphocytes in lung fibrosis: diverse subsets, diverse functions. J Leukoc Biol 93, 499–510 (2013).
F. Puttur, L. G. Gregory, C. M. Lloyd, Airway macrophages as the guardians of tissue repair in the lung. Immunology and Cell Biology 97, 246–257 (2019).
E. Vivier et al., Innate Lymphoid Cells: 10 Years On. Cell 174, 1054–1066 (2018).
C. S. Klose, D. Artis, Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nat Immunol 17, 765–774 (2016).
D. E. Cherrier, N. Serafini, J. P. Di Santo, Innate Lymphoid Cell Development: A T Cell Perspective. Immunity 48, 1091–1103 (2018).
S. M. Bal, K. Golebski, H. Spits, Plasticity of innate lymphoid cell subsets. Nat Rev Immunol 20, 552–565 (2020).
J. H. Bernink et al., Interleukin-12 and – 23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria. Immunity 43, 146–160 (2015).
C. Vonarbourg et al., Regulated expression of nuclear receptor RORγt confers distinct functional fates to NK cell receptor-expressing RORγt(+) innate lymphocytes. Immunity 33, 736–751 (2010).
Y. Huang et al., IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential 'inflammatory' type 2 innate lymphoid cells. Nat Immunol 16, 161–169 (2015).
J. S. Silver et al., Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs. Nat Immunol 17, 626–635 (2016).
H. Maazi et al., ICOS:ICOS-ligand interaction is required for type 2 innate lymphoid cell function, homeostasis, and induction of airway hyperreactivity. Immunity 42, 538–551 (2015).
J. Zhang et al., Neuropilin-1 mediates lung tissue-specific control of ILC2 function in type 2 immunity. Nat Immunol 23, 237–250 (2022).
R. Nowarski et al., Epithelial IL-18 Equilibrium Controls Barrier Function in Colitis. Cell 163, 1444–1456 (2015).
L. Vera et al., Notch3 Deficiency Attenuates Pulmonary Fibrosis and Impedes Lung-Function Decline. Am J Respir Cell Mol Biol 64, 465–476 (2021).
K. Wei et al., Notch signalling drives synovial fibroblast identity and arthritis pathology. Nature 582, 259–264 (2020).
G. M. Jowett et al., ILC1 drive intestinal epithelial and matrix remodelling. Nat Mater 20, 250–259 (2021).
J. Li, J. Wu, M. Zhang, Y. Zheng, Dynamic changes of innate lymphoid cells in acute ST-segment elevation myocardial infarction and its association with clinical outcomes. Sci Rep 10, 5099 (2020).
H. Wang et al., Adipose group 1 innate lymphoid cells promote adipose tissue fibrosis and diabetes in obesity. Nat Commun 10, 3254 (2019).
G. T. Belz, ILC2s masquerade as ILC1s to drive chronic disease. Nat Immunol 17, 611–612 (2016).
M. Y. Hachim, B. A. Khalil, N. M. Elemam, A. A. Maghazachi, Pyroptosis: The missing puzzle among innate and adaptive immunity crosstalk. J Leukoc Biol 108, 323–338 (2020).
A. Jarret et al., Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity. Cell 180, 813–814 (2020).
J. H. Yun et al., Hedgehog interacting protein-expressing lung fibroblasts suppress lymphocytic inflammation in mice. JCI Insight 6 (2021).
L. Y. Hung et al., Cellular context of IL-33 expression dictates impact on anti-helminth immunity. Sci Immunol 5 (2020).
C. E. Barkauskas, P. W. Noble, Cellular mechanisms of tissue fibrosis. 7. New insights into the cellular mechanisms of pulmonary fibrosis. Am J Physiol Cell Physiol 306, C987-996 (2014).
S. Bhattacharyya et al., TLR4-dependent fibroblast activation drives persistent organ fibrosis in skin and lung. JCI Insight 3 (2018).
Ø. Sandanger et al., The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia-reperfusion injury. Cardiovasc Res 99, 164–174 (2013).
M. W. Dahlgren et al., Adventitial Stromal Cells Define Group 2 Innate Lymphoid Cell Tissue Niches. Immunity 50, 707–722 e706 (2019).
S. Wirtz, A. Schulz-Kuhnt, M. F. Neurath, I. Atreya, Functional Contribution and Targeted Migration of Group-2 Innate Lymphoid Cells in Inflammatory Lung Diseases: Being at the Right Place at the Right Time. Front Immunol 12, 688879 (2021).
S. Lee et al., Contribution of Autophagy-Notch1-Mediated NLRP3 Inflammasome Activation to Chronic Inflammation and Fibrosis in Keloid Fibroblasts. Int J Mol Sci 21 (2020).
D. Zong, R. Ouyang, J. Li, Y. Chen, P. Chen, Notch signaling in lung diseases: focus on Notch1 and Notch3. Ther Adv Respir Dis 10, 468–484 (2016).
S. Djudjaj et al., Notch-3 receptor activation drives inflammation and fibrosis following tubulointerstitial kidney injury. J Pathol 228, 286–299 (2012).
P. Kavvadas et al., Notch3 orchestrates epithelial and inflammatory responses to promote acute kidney injury. Kidney Int 94, 126–138 (2018).
H. Y. Song, Y. Wang, H. Lan, Y. X. Zhang, Expression of Notch receptors and their ligands in pancreatic ductal adenocarcinoma. Exp Ther Med 16, 53–60 (2018).
P. A. Reyfman et al., Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis. Am J Respir Crit Care Med 199, 1517–1536 (2019).
H. Yuan et al., Crystalline Silica-Induced Proinflammatory Interstitial Macrophage Recruitment through Notch3 Signaling Promotes the Pathogenesis of Silicosis. Environ Sci Technol 57, 14502–14514 (2023).
Y. Long, Y. Niu, K. Liang, Y. Du, Mechanical communication in fibrosis progression. Trends Cell Biol 32, 70–90 (2022).
V. C. Luca et al., Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355, 1320–1324 (2017).
W. R. Gordon et al., Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch. Dev Cell 33, 729–736 (2015).
S. Li et al., Targeting Mechanics-Induced Fibroblast Activation through CD44-RhoA-YAP Pathway Ameliorates Crystalline Silica-Induced Silicosis. Theranostics 9, 4993–5008 (2019).

There is NO Competing Interest.

NatureCommunicationSupplementaryInformation.pdf
Nature Communication Supplementary Information.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

Mechanics-activated Fibroblasts orchestrating the plasticity of group 2 innate lymphoid cells propel the progression of silicosis

Status:

Version 1

Abstract

Figures

Reporting Summary

Introduction

Results

Discussion

Materials

Mice

Generation of ST2-GFP reporter mice

Crystalline silica particles

Murine model of silicosis and other treatments

Flow cytometry

ILC2 cells enrichment and sorting from mouse lung

Primary fibroblasts isolation

2D cell culture and treatment

ILC2 cells enrichment and sorting from mouse lung

Lung ILC2s culture and treatment

Bulk RNA sequencing and gene expression analysis

Immunofluorescence staining

Immunoblot analysis

Elisa analysis

Histological analysis

Quantitative PCR (q-PCR) analysis

Measurement of pulmonary function

Clodronate liposomes treatment

Generation of bone marrow chimeras

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1