IntroductionMyeloid-derived suppressor cells (MDSCs) are elevated in cancer, chronic inflammation and fibrosis. The immune checkpoint molecule B7-H3 (CD276) is upregulated in fibrotic conditions and has been implicated in the expansion and recruitment of MDSCs to the fibrotic lung; however, the underlying mechanism remains unclear.MethodsTo investigate the role of B7-H3 in MDSC induction, myeloid differentiation and lung fibrosis, we employed in vivo B7-H3 blockade, cell type-specific deletion mouse models, exogenous B7-H3 administration, and in vitro bone marrow differentiation assays.ResultsOur results showed that in vivo B7-H3 blockade, and cell type-specific deletion of B7-H3 in collagen-expressing or myeloid cells attenuated lung fibrosis. Reduction of B7-H3 was associated with decreased MDSC expansion and increased numbers of MHCII+ F4/80+ cells. In contrast, exogenous B7-H3 administration in vivo reduced MHCII+ F4/80+ cells in the lung. In vitro, treatment of bone marrow cells with B7-H3 promoted MDSC generation and inhibited myeloid differentiation. Additionally, we found that soluble B7-H3, potentially cleaved from fibroblasts by matrix metalloproteinases (MMPs), contributed to the induction of MDSCs in co-cultured bone marrow cells and the suppression of myeloid cell differentiation. Finally, CD84, a novel immunosuppressive marker for M-MDSCs, expressed on MDSCs was required for B7-H3-mediated enhancement of their immunosuppressive capacity toward T cells.DiscussionTogether, these findings reveal that bleomycin-induced B7-H3 signals to the bone marrow, skewing myeloid differentiation toward an immature MDSC phenotype by inhibiting normal myeloid maturation during fibrosis.

The reactivation of Wnt signaling pathways plays an important role in driving myofibroblast differentiation in fibrotic diseases; however, the mechanism is not clearly understood. In this study, we investigate the role of non-canonical Wnt11 signaling in human lung fibroblasts and its contributions to myofibroblast differentiation. Our results show that components of the non-canonical Wnt pathway are upregulated in bleomycin-induced pulmonary fibrosis and that in vivo depletion of Wnt11 in mouse lung fibroblasts significantly reduces lung fibrosis. Furthermore, co-culture studies using fibroblasts and alveolar type II epithelial cells (AECII) revealed a Wnt11-mediated mechanism that promotes myofibroblast differentiation. Finally, we demonstrate that in human lung fibroblasts, TGFβ can increases Wnt11 transcription by regulating Smad3 binding to the Wnt11 promoter and by modulating Wnt11 promoter activity. Together, these findings identify non-canonical Wnt11 as a regulator of myofibroblast differentiation and lung fibrosis.

Rationale: soluble B7H3 (sB7H3) is known to alter myeloid cell fate by inducing immature myeloid-derived suppressor cell (MDSCs) and promoting immunosuppressive capacity within the lung, thus facilitating the development of idiopathic pulmonary fibrosis (IPF). Emerging evidence suggests an essential role of the CD47/SIRPα axis in wound healing and IPF. Interestingly, anti-B7H3 antibodies may alter CD47/SIRPα in tumor cells. The objective of this study was to explore the potential role of CD47 expression in the myofibroblast differentiation and the effect of B7H3 on Sirpα in myeloid cells during lung fibrosis. Method: Publicly available single-cell RNA sequencing (scRNAseq) data from IPF Cell Atlas was first analyzed for CD47 and Sirpα expression. Their expressions were then investigated in lung and Tert-overexpressed human foreskin fibroblasts in response to TGFβ stimulations. To explore B7H3 impact on CD47/Sirpα function in myeloid cells, we generated a myeloid cell-specific B7H3 KO mouse line (B7H3-LysMCre KO), CD47 and Sirpα levels was measured in BM-derived macrophages isolated from B7H3-LysMCre KO or WT mice stimulated with sB7H3. Finally, Bleomycin-induced mouse lung fibrosis model was used to examine the expression of CD47 and Sirpα in fibrotic lungs, and flow cytometry was used to analyze their expression fibroblast vs. myofibroblast populations. Results: Analysis of scRNAseq data revealed that CD47 expression was induced in myofibroblasts but not in fibroblasts of patient with human IPF. Consistently, CD47 expression was upregulated in α-smooth muscle actin (α-SMA)-expressing myofibroblasts and reduced in α-SMA-negative cell populations in bleomycin-induced lung fibrosis compared to PBS controls. However, in vitro treatment with TGFβ failed to induce CD47 expression in human or mouse fibroblasts. Contrary to the observed TGFβ effect on CD47 expression, TGFβ treatment significantly induced Sirpα mRNA expression in both human and mouse fibroblasts, which was associated with increased levels of α-SMA mRNA in normal BJ cells. Notably, this was inhibited by TERT overexpression in BJ5ta, which exhibited decreased α-SMA mRNA expression. In BM macrophages, sB7H3 significantly increased Sirpα mRNA expression, while B7H3 knockout significantly reduced it. Moreover, in fibrotic lungs, both SIRPα mRNA levels and the proportion of Sirpα-positive cells were increased compared to controls in vivo. Conclusion: These findings suggest that the differential expression patterns of CD47 and Sirpα in response to TGFβ in fibroblasts may play distinct roles during myofibroblast differentiation. Elevated CD47 levels in myofibroblast and sB7H3-induced Siprα expression in BM-derived macrophages may indicate impaired macrophage phagocytosis, potentially leading to the persistence of myofibroblasts via CD47/Siprα axis.

Recently discovered heterogeneous myeloid-derived suppressor cells (MDSCs) are some of the most discussed immunosuppressive cells in contemporary immunology, especially in the tumor microenvironment, and are defined primarily by their T cell immunosuppressive function. The importance of these cells extend to other chronic pathological conditions as well, including chronic infection, inflammation, and tissue remodeling. In many of these conditions, their accumulation/expansion correlates with disease progression, poor prognosis, and reduced survival, which highlights the potential of how these cells may be used in a clinical setting as both prognostic factor and therapeutic target. In healthy individuals, these cells are usually not present in the circulation. Therefore, monitoring this cell population is of potential clinical significance, and utility in basic research. However, these cells have a complex phenotype without one single marker of sufficient specificity for their identification. Flow cytometry is a powerful tool allowing multi-parameter analysis of heterogeneous cell populations, which makes it ideally suitable for the complex phenotypic analysis essential for identification and enumeration of circulating MDSCs. This approach has the potential to provide a novel clinically useful tool for assessment of prognosis and treatment outcomes. The protocol in this chapter describes a flow cytometric analysis to identify and quantify MDSCs from human or mouse whole blood leukocytes and peripheral blood mononuclear cells, as well as a single cell suspension from solid tissue, by using multicolor fluorescence-conjugated antibodies against their surface markers.

Background: The transcription factor CCAAT/enhancer-binding protein beta (C/EBP beta) is implicated in diverse processes and diseases. Its two isoforms, namely liver-enriched activator protein (LAP) and liver-enriched in-hibitor protein (LIP) are translated from the same mRNA. They share the same C-terminal DNA binding domain except LAP has an extra N-terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, LIP can inhibit LAP transcriptional activity even at substoichiometric levels. However, the regulatory mechanism of C/EBP beta gene expression and the LAP: LIP ratio is unclear. Methods: In this study, the C/EBP beta promoter sequence was scanned for conserved P53 response element (P53RE), and binding of P53 to the C/EBP beta promoter was tested by Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation assay. P53 over-expression and dominant negative P53 expression plasmids were transfected into rat lung fibroblasts and tested for C/EBP beta gene transcription and expression. Western blot analysis was used to test the regulation of C/EBP beta LAP and LIP isoforms. Constructs containing the LAP 5'untranslated region (5'UTR) or the LIP 5'UTR region were used to test the importance of 5'UTR in the control of C/EBP beta LAP and LIP translation. Results: The C/EBP beta promoter sequence was found to contain a conserved P53 response element (P53RE), which binds P53 as demonstrated by Electrophoresis Mobility Shift Assay and chromatin immunoprecipitation assays. P53 over-expression suppressed while dominant negative P53 stimulated C/EBP beta gene transcription and expression. Western blot analysis showed that P53 differentially regulated the translation of the C/EBP beta LAP and LIP isoforms through the regulation of eIF4E and eIF4E-BP1. Further studies with constructs containing the LAP 5'untranslated region (5'UTR) or the LIP 5'UTR region showed that the 5'UTR is important in differential control of C/EBP beta LAP and LIP translation. Conclusion: Analysis of the effects of P53 on C/EBP beta expression revealed a novel mechanism by which P53 could antagonize the effects of C/EBP beta on its target gene expression. For the first time, P53 is shown to be a repressor of C/EBP beta gene expression at both transcriptional and translational levels, with a differential effect in the magnitude of the effect on LAP vs. LIP isoforms.

The clinical significance of B7H3 (CD276) and its cleavage product soluble B7H3 (sB7H3) in idiopathic pulmonary fibrosis (IPF) is unknown. Mounting evidence suggests the potential utility of peripheral blood myeloid cell enumeration to predict disease outcome and indicate active lung disease. Here we hypothesized that sB7H3 is involved in regulation of circulating myeloid cells in pulmonary fibrosis. In support of this possibility, both plasma sB7H3 and B7H3 + cells were elevated in IPF patient blood samples, which correlated negatively with lung function. To analyze its function, the effects of sB7H3 on naïve or bleomycin‐treated mice were examined. The results revealed that sB7H3 injection induced an influx of myeloid‐derived suppressor cells (MDSCs) and Ccl2 expression in lung tissue of naïve mice, accompanied by enhanced overall inflammation. Additionally, sB7H3 caused accumulation of MDSCs in bone marrow with increased expression of inflammatory cytokines. Notably, in vitro assays revealed chemotaxis of MDSCs to sB7H3, which was dependent on TLT‐2 (TREML2), a putative receptor for sB7H3. Thus, increased circulating sB7H3 and/or B7H3 + cells in IPF patient blood samples correlated with lung function decline and potential immunosuppressive status. The correlation of sB7H3 with deterioration of lung function might be due to its ability to enhance inflammation and recruitment of MDSCs into the lung and their expansion in the bone marrow, and thus potentially contribute to IPF exacerbation. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSCs) in cancer, inflammation, and fibrosis, with some of these cells expressing B7H3. We sought to investigate the role of MDSCs in IPF and its potential mediation via B7H3. Here we prospectively collected peripheral blood samples from IPF patients to analyze for circulating MDSCs and B7H3 expression to assess their clinical significance and potential impact on co-cultured lung fibroblasts and T-cell activation. In parallel, we assess MDSC recruitment and potential B7H3 dependence in a mouse model of pulmonary fibrosis. Expansion of MDSCs in IPF patients correlated with disease severity. Co-culture of soluble B7H3 (sB7H3)-treated mouse monocytic MDSCs (M-MDSCs), but not granulocytic MDSCs (G-MDSCs), activated lung fibroblasts and myofibroblast differentiation. Additionally, sB7H3 significantly enhanced MDSC suppression of T-cell proliferation. Activated M-MDSCs displayed elevated TGFβ and Arg1 expression relative to that in G-MDSCs. Treatment with anti-B7H3 antibodies inhibited bone marrow-derived MDSC recruitment into the bleomycin-injured lung, accompanied by reduced expression of inflammation and fibrosis markers. Selective telomerase reverse transcriptase (TERT) deficiency in myeloid cells also diminished MDSC recruitment associated with the reduced plasma level of sB7H3, lung recruitment of c-Kit+ hematopoietic progenitors, myofibroblast differentiation, and fibrosis. Lung single-cell RNA sequencing (scRNA-seq) revealed fibroblasts as a predominant potential source of sB7H3, and indeed the conditioned medium from activated mouse lung fibroblasts had a chemotactic effect on bone marrow (BM)-MDSC, which was abolished by B7H3 blocking antibody. Thus, in addition to their immunosuppressive activity, TERT and B7H3-dependent MDSC expansion/recruitment from BM could play a paracrine role to activate myofibroblast differentiation during pulmonary fibrosis with potential significance for disease progression mediated by sB7H3.

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.

The Wnt/β-catenin pathway initiates a signaling cascade that is critical in cell differentiation and the normal development of multiple organ systems. The reactivation of this pathway has been documented in experimental and human idiopathic pulmonary fibrosis, wherein Wnt/β-catenin activation has been implicated in epithelial-cell repair. Furthermore, the canonical ligand Wnt3a is known to induce myofibroblast differentiation; however, the role of noncanonical Wnt ligands remains unclear. This study showed significantly higher levels of Wnt11 expression in cells from both patients with idiopathic pulmonary fibrosis and bleomycin-treated mice, as well as in TGFβ-treated mouse lung fibroblasts. Moreover, Wnt11 induced myofibroblast differentiation as manifested by increased α-SMA (ACTA2) expression, which was similar to that induced by canonical Wnt3a/β-catenin signaling. Further investigation revealed that Wnt11 induction of α-SMA was associated with the activation of JNK (c-Jun N-terminal kinase)/c-Jun signaling and was inhibited by a JNK inhibitor. The potential importance of this signaling pathway was supported by in vivo evidence showing significantly increased levels of Wnt11 and activated JNK in the lungs of mice with bleomycin-induced pulmonary fibrosis. Interestingly, fibroblasts did not express canonical Wnt3a, but treatment of these cells with exogenous Wnt3a induced endogenous Wnt11 and Wnt5a, resulting in repression of the Wnt3a/β-catenin target gene Axin2. These findings suggested that the noncanonical Wnt induction of myofibroblast differentiation mediated by the JNK/c-Jun pathway might play a significant role in pulmonary fibrosis, in addition to or in synergy with canonical Wnt3a/β-catenin signaling. Moreover, Wnt3a activation of noncanonical Wnt signaling might trigger a switch from canonical to noncanonical Wnt signaling to induce myofibroblast differentiation.

Aging is a risk factor in fibrotic interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF). Cellular senescence is observed in IPF and animal models of lung fibrosis along with telomere dysfunction, although the potential differential pathogenic significance of their cell type specific localization is unknown. Removal of senescent cells ameliorates fibrosis in animal models and are considered as potential therapeutic approaches. However the pathogenic role of aging and/or cellular senescence in the development of lung fibrosis are not fully understood. Declining cellular nicotinamide adenine dinucleotide (NAD+) levels as seen in aging, may impair the activity of senescence suppressor Sirt1, an NAD+‐dependent deacetylase, hence boosting the levels of NAD+ have been proposed to delay or inhibit aging related illnesses and improved longevity. To elucidate the role of NAD+ in cellular senescence in pathogenesis of pulmonary fibrosis, we investigated the impact of increasing or reducing NAD+ levels on bleomycin (BLM)‐induced mouse pulmonary fibrosis by supplementation with nicotinamide mononucleotide (NMN) by i.p injection, or nicotinamide phosphoribosyltransferase (Nampt) deficiency, respectively. The latter was induced by treating transgenic mice bearing the floxed Nampt and Col1a2‐CreERT transgenes with tamoxifen, resulting in mesenchymal cell specific Nampt deletion. The results showed that pre‐treatment of NMN injection before BLM injury significantly reduced lung myofibroblast differentiation as manifested by decreased Acta2 mRNA, which was accompanied by decreased numbers of senescent type II alveolar epithelial cells (SPC+/p16+), and BM‐derived monocytic myeloid cells. In contrast, deletion of Nampt in mesenchymal cells with consequent reduction of NAD+ levels caused increased myofibroblast differentiation, and more severe lung fibrosis measured by lung hydroxyproline and histology after BLM injury when compared to the response in wild type animals. In contrast to wild type cells, Nampt‐deficient lung fibroblasts exhibited elevated expression of p16, and senescence‐associated secretory phenotype (SASP) cytokines, including TNFα, IL‐1β, and IL‐6. Notably enhanced recruitment of BM‐derived myeloid cells into BLM‐injured lung was also observed. These findings suggest that NAD+ may play a significant role in prevention of cellular senescence and protection from pulmonary injury/fibrosis.Support or Funding InformationThis study is supported by NIH grants HL112880 and HL 138417.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with limited therapeutic options. Both innate and adaptive immune mechanisms contribute to fibrogenesis. B7 family member B7H3, identified as important regulator of the immune system, has been implicated in cancer, graft-versus host disease, and allergy-related disease. Increased soluble B7H3 (sB7H3) was associated with enhanced lung fibrosis in mice receiving BLM-primed BM. Elevated sB7H3 in bronchoalveolar lavage fluid was associated with acute exacerbation IPF. To investigate whether induced sB7H3 could be used as a biomaker for IPF diagnosis and/or prognosis, plasma sB7H3 from control subjects and IPF patients was measured by ELISA. Its correlation with peripheral blood immune cells, lung function, and drug treatments were evaluated in this study. The results showed that the sB7H3 level was significantly elevated in IPF patients. This induction was associated with increased circulating B7H3+ cells, and significantly correlated with increased granulocytic myeloid-derived suppressive cells (G-MDSCs). This suggests a potential regulatory role of the induced B7H3 in the immune response in IPF. The plasma sB7H3 was negatively correlated with lung function measured as diffusing capacity of the lungs for carbon monoxide (DLCO), indicating the potential utility of plasma sB7H3 as an indicator of disease severity. The slightly elevated sB7H3 in the pirfenidone and/or nintedanib treated IPF patients were not significantly different relative to controls, which was in contrast to the significant elevation noted for the untreated patients. These findings suggest that sB7H3 may be a useful biomarker for clinical assessment of prognosis and response to drug treatment.

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

Mutations in the genes encoding telomerase reverse transcriptase (TERT) and telomerase's RNA components as well as shortened telomeres are risk factors for idiopathic pulmonary fibrosis, where repetitive injury to the alveolar epithelium is considered a key factor in pathogenesis. Given the importance of TERT in stem cells, we hypothesized that TERT plays an important role in epithelial repair and that its deficiency results in exacerbation of fibrosis by impairing this repair/regenerative process. To evaluate the role of TERT in epithelial cells, we generated type II alveolar epithelial cell (AECII)-specific TERT conditional knockout (SPC-Tert cKO) mice by crossing floxed Tert mice with inducible SPC-driven Cre mice. SPC-Tert cKO mice did not develop pulmonary fibrosis spontaneously up to 9 months of TERT deficiency. However, upon bleomycin treatment, they exhibited enhanced lung injury, inflammation, and fibrosis compared with control mice, accompanied by increased pro-fibrogenic cytokine expression but without a significant effect on AECII telomere length. Moreover, selective TERT deficiency in AECII diminished their proliferation and induced cellular senescence. These findings suggest that AECII-specific TERT deficiency enhances pulmonary fibrosis by heightening susceptibility to bleomycin-induced epithelial injury and diminishing epithelial regenerative capacity because of increased cellular senescence. We confirmed evidence for increased AECII senescence in idiopathic pulmonary fibrosis lungs, suggesting potential clinical relevance of the findings from our animal model. Our results suggest that TERT has a protective role in AECII, unlike its pro-fibrotic activity, observed previously in fibroblasts, indicating that TERT's role in pulmonary fibrosis is cell type-specific.

Rationale: The impact of lung insult on the bone marrow (BM) and subsequent disease is unknown. Objectives: To study alterations in the BM in response to lung injury/fibrosis and examine their impact on subsequent lung insult. Methods: BM cells from control or bleomycin-treated donor mice were transplanted into naive mice, which were subsequently evaluated for bleomycin-induced pulmonary fibrosis. In addition, the effect of prior bleomycin treatment on subsequent fibrosis was examined in wild-type and B7H3-knockout mice. Samples from patients with idiopathic pulmonary fibrosis were analyzed for potential clinical relevance of the findings. Measurements and Main Results: Recipient mice transplanted with BM from bleomycin-pretreated donors showed significant exacerbation of subsequent fibrosis with increased B7H3(+) cell numbers and a T-helper cell type 2-skewed phenotype. Pretreatment with a minimally fibrogenic/nonfibrogenic dose of bleomycin also caused exacerbation, but not in B7H3-deficient mice. Exacerbation was not observed if the mice received naive BM cell transplant after the initial bleomycin pretreatment. Soluble B7H3 stimulated BM Ly6C(hi) monocytic cell expansion in vitro and caused similar expansion in the lung in vivo. Notably, soluble B7H3 was elevated in plasma of patients with idiopathic pulmonary fibrosis and in BAL fluid in those with acute exacerbation. Finally, ST2 deficiency diminished the bleomycin-induced B7H3 and IL-13 upregulation, suggesting a role for type 2 innate lymphoid cells. Conclusions: Pulmonary fibrosis caused significant alterations in BM with expansion and activation of monocytic cells, which enhanced fibrosis when transplanted to naive recipients with potential mediation by a novel role for B7H3 in the pathophysiology of pulmonary fibrosis in both mice and humans.

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5‐bisphosphate (PIP2)‐binding protein, myristoylated alanine‐rich C‐kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25‐mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α‐smooth muscle actin (α‐SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)‐trisphosphate pools and AKT activity occurred in MPS‐treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin‐treated mice in vivo. Surprisingly, we found that MPS peptide decreases α‐SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.—Yang, D. C., Li, J.‐M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.‐H. Tackling MARCKS‐PIP3 circuit attenuates fibroblast activation and fibrosis progression. FASEB J. 33, 14354‐14369 (2019). www.fasebj.org

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective curative therapy. Recruitment of bone marrow-derived myeloid cells is implicated in lung fibrosis by promoting fibrosis via paracrine mechanisms. Recent evidence shows increased circulating myeloid-derived suppressor cells (MDSC) in IPF patients. More recently, the importance of MDSC is suggested by the observation of MDSC accumulation in IPF lungs. The objective of this study was to assess the accumulation/expansion of MDSC in bleomycin-induced mouse model of pulmonary fibrosis and human IPF, and investigate their potential role in lung myofibroblast differentiation. Flow cytometry analysis showed that MDSCs in lung were increased in a mouse model of lung fibrosis, as well as in peripheral blood samples from IPF patients. The increase of the monocytic subtype of MDSC (M-MDSC) was greater than that for the granulocytic subtype (G-MDSC) in the peripheral blood samples from patients with IPF. In vitro co-culture of normal mouse lung fibroblasts with sorted bone marrow (BM)-derived MDSCs using trans-well inserts revealed paracrine activation of lung fibroblasts by activated M-MDSC, but not GMDSC, as manifested by significantly elevated mRNA levels of α-smooth muscle actin and TGFβ mRNAs. Both quiescent M-MDSC and G-MDSC failed to stimulate fibroblast activation or myofibroblast differentiation. Interestingly, the level of TGFβ mRNA in M-MDSC was higher than G-MDSC. These findings indicated that in pulmonary fibrosis, BM-derived MDSCs were mobilized and recruited to the lung, and the recruited M-MDSC subpopulation subsequently played a paracrine role by activating resident lung fibroblasts and myofibroblast differentiation.