FSTL1 aggravates OVA-induced inflammatory responses by activating the NLRP3/IL-1ß signaling pathway in mice and macrophages.

OBJECTIVE: Asthma, a well-known disease with high morbidity, is characterized by chronic airway inflammation. However, the allergic inflammation mechanisms of follistatin-like protein 1 (FSTL1) have not been elucidated. This study aims to investigate the effects of FSTL1 in ovalbumin (OVA)-induced mice and macrophages on nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3)/interleukin-1ß (IL-1ß) signaling pathway. METHODS: Mice were randomly divided into control-WT, OVA-WT, control-Fstl1±, OVA-Fstl1±. Histological changes were assessed by HE and PAS staining. The protein levels of Muc-5AC, FSTL1, NLRP3, and IL-1ß in lung tissue were detected by immunohistochemistry and ELISA. The bronchoalveolar lavage fluid (BALF) in mice and human serum samples were detected by ELISA. Then, mice were grouped into control, FSTL1, MCC950 + FSTL1 to further investigate the relationship between FSTL1 and NLRP3/IL-1ß. Alveolar macrophage cells (MH-S cells) were separated into control, OVA, FSTL1, OVA + FSTL1, OVA + siNC, OVA + siFSTL1, MCC950, and FSTL1 + MCC950 groups to explore the effect of FSTL1 on the NLRP3/IL-1ß signaling. The protein expression of NLRP3 and IL-1ß in MH-S cells was detected by Western blot analysis. RESULTS: The present results uncovered that Fstl1± significantly ameliorated OVA-induced Muc-5AC production and mucus hypersecretion. Fstl1± was also found to decrease the production of inflammatory cytokines and inflammatory cell infiltration in OVA-induced asthmatic mice. Meanwhile, the serum concentrations of FSTL1 and IL-1ß were higher in  asthma subjects than the health subjects, and Fstl1± ameliorated the production of NLRP3 and IL-1ß in OVA-induced asthmatic mice. Furthermore, mice by injected FSTL1 substantially stimulated the expression of NLRP3 and IL-1ß, while pretreatment with MCC950 in mice significantly weakened the production of NLRP3 and IL-1ß induced by injection FSTL1. Pretreatment with siFSTL1 or MCC950 significantly reduced the production of NLRP3 and IL-1ß induced by OVA or FSTL1 in MH-S cells. CONCLUSIONS: The study results showed that FSTL1 played an important role in allergic airway inflammation by activating NLRP3/IL-1ß. Hence, inhibition FSTL1 could be applied as a therapeutic agent against asthma.

Circular RNA hsa_circ_0004812 impairs IFN-induced immune response by sponging miR-1287-5p to regulate FSTL1 in chronic hepatitis B.

BACKGROUND: The present study aims to explore the functions of circular RNA hsa_circ_0004812 in chronic hepatitis B (CHB) and its underlying molecular mechanisms. METHODS: The expression of circular RNA (circRNA)_0004812 was examined using qRT-PCR and Western blot in blood and liver tissues from CHB patients and healthy volunteers. In the in vitro study, the expression levels of circular RNA hsa_circ_0004812, miR-1287-5p, interferon (IFN)-α, IFN-ß were determined using qRT-PCR and Western blotting in HBV-infected hepatoma cells, respectively. Luciferase and biotin pull-down assays were used to investigate the interactions between miR-1287-5p and circ_0004812. RESULTS: Levels of circ_0004812 were upregulated in CHB patients and HBV-infected hepatoma cells. Knockdown of circ_0004812 increased the expression of IFN-α and IFN-ß in HBV-infected Huh7 cells. MiR-1287-5p was identified as a target of circ_0004812 whose overexpression inhibited the expression of miR-1287-5p. Additionally, circ_0004812 promoted the expression of Follistatin-related protein (FSTL) 1 through inhibiting miR-1287-5p. Circ_0004812/miR-1287-5p/FSTL1 axis regulated HBV-induced immune suppression. CONCLUSION: Circ_0004812 was identified as a potential target for CHB infection. Circ_0004812 promoted the expression of FSTL1 by inhibiting miR-1287-5p.

Fstl1 Promotes Asthmatic Airway Remodeling by Inducing Oncostatin M.

Chronic asthma is associated with airway remodeling and decline in lung function. In this article, we show that follistatin-like 1 (Fstl1), a mediator not previously associated with asthma, is highly expressed by macrophages in the lungs of humans with severe asthma. Chronic allergen-challenged Lys-Cre(tg) /Fstl1(Δ/Δ) mice in whom Fstl1 is inactivated in macrophages/myeloid cells had significantly reduced airway remodeling and reduced levels of oncostatin M (OSM), a cytokine previously not known to be regulated by Fstl1. The importance of the Fstl1 induction of OSM to airway remodeling was demonstrated in murine studies in which administration of Fstl1 induced airway remodeling and increased OSM, whereas administration of an anti-OSM Ab blocked the effect of Fstl1 on inducing airway remodeling, eosinophilic airway inflammation, and airway hyperresponsiveness, all cardinal features of asthma. Overall, these studies demonstrate that the Fstl1/OSM pathway may be a novel pathway to inhibit airway remodeling in severe human asthma.

Follistatin-like protein 1 enhances NLRP3 inflammasome-mediated IL-1ß secretion from monocytes and macrophages.

Follistatin-like protein 1 (FSTL-1) is overexpressed in a number of inflammatory conditions characterized by elevated IL-1ß. Here, we found that FSTL-1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL-1 to IL-1ß secretion, WT and FSTL-1-deficient mice were injected with LPS. While LPS induced IL-1ß in the sera of WT mice, it was low or undetectable in FSTL-1-deficient mice. Monocytes/macrophages, a key source of IL-1ß, do not normally express FSTL-1. However, FSTL-1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL-1 at sites of inflammation. In vitro, intracellular FSTL-1 localized to the mitochondria. FSTL-1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL-1ß secretion. FSTL-1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus-mediated overexpression of FSTL-1 in mouse paws led to activation of the inflammasome complex and local secretion of IL-1ß and IL-1ß-related proinflammatory cytokines. These results suggest that FSTL-1 may act on the NLRP3 inflammasome to promote IL-1ß secretion from monocytes/macrophages.

Circ_0001490/miR-579-3p/FSTL1 axis modulates the survival of mycobacteria and the viability, apoptosis and inflammatory response in Mycobacterium tuberculosis-infected macrophages.

BACKGROUND: Macrophages play an important role in the host immune response against mycobacterial infection, and this process is regulated by various factors, including circular RNAs (circRNAs). We intended to explore the role of circ_0001490 in tuberculosis (TB) using Mycobacterium tuberculosis (M.tb)-infected THP-1 macrophages. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to measure RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to analyze the viability of THP-1 macrophages. Flow cytometry was performed to analyze the apoptosis rate of THP-1 macrophages. Enzyme-linked immunosorbent assay (ELISA) was conducted to assess the release of inflammatory cytokines. Colony-forming unit (CFU) assay was conducted to analyze the survival of M.tb in THP-1 macrophages. Intermolecular target interaction was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0001490 expression was down-regulated in the serum samples of TB patients and M.tb-infected THP-1 macrophages. Circ_0001490 overexpression suppressed M.tb survival and promoted the viability and inflammatory response of THP-1 macrophages. Circ_0001490 interacted with microRNA-579-3p (miR-579-3p), and circ_0001490 overexpression-induced protective effects in M.tb-infected THP-1 macrophages were largely overturned by the overexpression of miR-579-3p. miR-579-3p interacted with the 3' untranslated region (3'UTR) of follistatin-like protein 1 (FSTL1). FSTL1 silencing largely overturned miR-579-3p knockdown-induced effects in M.tb-infected THP-1 macrophages. Circ_0001490 acted as miR-579-3p sponge to up-regulate FSTL1 in THP-1 macrophages. CONCLUSION: In conclusion, our results demonstrated that circ_0001490 suppressed M.tb survival and promoted the viability and inflammatory response of M.tb-infected THP-1 macrophages partly by regulating miR-579-3p/FSTL1 axis.

Blocking the FSTL1-DIP2A Axis Improves Anti-tumor Immunity.

Immune dysfunction is a strong factor in the resistance of cancer to treatment. Blocking immune checkpoint pathways is a promising approach to improve anti-tumor immunity, but the clinical efficacies are still limited. We previously identified follistatin-like 1 (FSTL1) as a determinant of immune dysfunction mediated by mesenchymal stromal/stem cells (MSCs) and immunoregulatory cells. Here, we demonstrate that blocking FSTL1 but not immune checkpoint pathways significantly suppresses cancer progression and metastasis in several mouse tumor models with increased MSCs. Expression of DIP2A (the receptor of FSTL1) in tumor cells is critical for FSTL1-induced immunoresistance. FSTL1/DIP2A co-positivity in tumor tissues correlates with poor prognosis in NSCLC patients. Thus, breaking the FSTL1-DIP2A axis may be a useful strategy for successfully inducing anti-tumor immunity.

Cardiokines as Modulators of Stress-Induced Cardiac Disorders.

Almost 30 years ago, the protein, atrial natriuretic peptide, was identified as a heart-secreted hormone that provides a peripheral signal from the myocardium that communicates to the rest of the organism to modify blood pressure and volume under conditions of heart failure. Since then, additional peripheral factors secreted by the heart, termed cardiokines, have been identified and shown to coordinate this interorgan cross talk. In addition to this interorgan communication, cardiokines also act in an autocrine/paracrine manner to play a role in intercellular communication within the myocardium. This review focuses on the roles of newly emerging cardiokines that are mainly increased in stress-induced cardiac diseases. The potential of these cardiokines as clinical biomarkers for diagnosis and prognosis of cardiac disorders is also discussed.

Leukocyte Beta-Catenin Expression Is Disturbed in Systemic Lupus Erythematosus.

Wnt/ß-catenin signaling is relatively understudied in immunity and autoimmunity. ß-catenin blocks inflammatory mediators and favors tolerogenic dendritic cell (DC) phenotypes. We show here that leukocytes from lupus-prone mice and SLE patients express diminished ß-catenin transcriptional activity, particularly in myeloid cells, although other leukocytes revealed similar trends. Serum levels of DKK-1, an inhibitor under transcriptional control of Wnt/ß-catenin, were also decreased in lupus-prone mice. Surprisingly, however, preemptive deletion of ß-catenin from macrophages appears to have no effect on lupus development, even in mice with varying genetic loads for lupus. Although myeloid-specific loss of ß-catenin does not seem to be important for lupus development, the potential role of this transcription factor in other leukocytes and renal cells remain to be elucidated.

Follistatin-like protein 1 and its role in inflammation and inflammatory diseases.

Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein produced mainly by cells of mesenchymal origin. FSTL1 has been shown to play an important role during embryogenesis; FSTL1-deficient mice die at birth from multiple developmental abnormalities. In the last decade, FSTL1 has been identified as a novel inflammatory protein, enhancing synthesis of proinflammatory cytokines and chemokines by immune cells in vitro and in vivo. FSTL1 mediates proinflammatory events in animal models of inflammatory diseases, particularly in collagen-induced arthritis in mice. FSTL1 is elevated in various inflammatory conditions and decreased during the course of treatment. FSTL1 may therefore be a valuable biomarker for such diseases. Moreover, a variety of experiments suggest that targeting of FSTL1 may be useful in the treatment of diseases in which inflammation plays a central role.

Targeting FSTL1 prevents tumor bone metastasis and consequent immune dysfunction.

Bone metastasis greatly deteriorates the quality of life in patients with cancer. Although mechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatin-like glycoprotein secreted by Snail(+) tumor cells, which metastasize frequently to bone. We found that FSTL1 plays a dual role in bone metastasis-in one way by mediating tumor cell invasion and bone tropism but also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45(-)ALCAM(+) cells derived from bone marrow. CD45(-)ALCAM(+) cells induced bone metastasis de novo, but they also generated CD8(low) T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interference-mediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAM(+) cells and CD8(low) T cells. These effects were accompanied by heightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAM(+) cells increased with FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Our findings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bone metastasis, which remains a major challenge in patients with cancer.

Follistatin-related protein/follistatin-like 1 evokes an innate immune response via CD14 and toll-like receptor 4.

Follistatin-related protein (FRP)/follistatin-like 1 (FSTL1) has multi-specific binding nature especially with TGF-ß superfamily proteins, and thereby modulates organ development. However, its function in immune systems remains unclear. Previously, we reported FRP interacts with CD14, which is known to mediate toll-like receptor 4 (TLR4) signaling. Here, we investigated whether FRP activates TLR4 signaling. Recombinant FRP induced interleukin 6 or interleukin 8 production from target cells in a CD14- and TLR4-dependent manner. Moreover, similar to lipopolysaccharide (LPS), FRP induced tolerance to the second LPS stimulation. FRP has the function of evoking innate immune responses as one of the endogenous TLR4 agonists.

Follistatin-like protein 1 is elevated in systemic autoimmune diseases and correlated with disease activity in patients with rheumatoid arthritis.

INTRODUCTION: Follistatin-like protein 1 (FSTL1) is a proinflammation mediator implicated in arthritis in rodent animal models. The present study is aimed at assessing FSTL1 levels in systemic autoimmune diseases and correlating them with disease activity in patients with rheumatoid arthritis (RA). METHODS: Serum FSTL1 levels from 487 patients with systemic autoimmune diseases and 69 healthy individuals were measured by enzyme-linked immunosorbent assay (ELISA). FSTL1 expression in synovial fluid (SF) and synovial tissues (STs) was determined by ELISA, immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and western blot analysis in RA patients and trauma controls. FSTL1 levels in fibroblast-like synoviocytes (FLSs) from RA patients were determined by real-time PCR and western blot analysis. RESULTS: Serum FSTL1 levels were significantly elevated in patients with RA, ulcerative colitis, systemic lupus erythematosus, Sjögren's syndrome (SS), systemic sclerosis and polymyositis/dermatomyositis. Serum FSTL1 levels in the RA and secondary SS patients were substantially higher than those in other patients. Serum FSTL1 levels were increased in early RA, rheumatoid factor (RF)- and anti-cyclic citrullinated peptide antibody (ACPA)-negative patients compared to healthy controls. Moreover, serum FSTL1 concentrations were significantly higher in long-standing RA patients than in early RA patients and in the RF- and ACPA-positive RA patients than in RF- and ACPA-negative RA patients. Elevated FSTL1 levels in the STs and SF of RA patients were also observed. FSTL1 levels in serum were markedly higher than those in SF in RA patients. The strongest FSTL1 staining was detected in the cytoplasm of synovial and capillary endothelial cells from RA synovium. Furthermore, FSTL1 was induced in FLSs by inflammatory mediators. Importantly, serum FSTL1 levels were correlated with several important biologic and clinical markers of disease activity, including erythrocyte sedimentation rate, C-reactive protein, RF, ACPA, swollen joint count, patient global visual analogue scale score and Disease Activity Score 28 in the adult RA patient population. Notably, serum FSTL1 levels were significantly diminished following successful treatment and clinical improvement. CONCLUSIONS: Elevated FSTL1 levels reflect not only joint diseases but also inflammation and tissue degradation in systemic autoimmune diseases. Serum FSTL1 levels may thus serve as a serological inflammatory marker of disease activity in RA patients.

[Characterization of follistatin-related protein from the hard tick Haemaphysalis longicornis].

We designed the primers based on the sequence of the follistatin-related protein from Haemaphysalis longicornis Okayama strain accessed in GenBank. We cloned a gene encoding follistatin-related protein by RT-PCR, and the length cDNA is 814 bp, encoding a deduced protein of 289 amino acids. The alignment with the sequence of follistatin-related protein from the H. longicornis Okayama strain showed that the percent of nucleotide sequence and amino acid sequence is 97.8% and 99%, respectively. The expected size of GST-fused recombinant protein was 57 kD. We purified the recombinant protein through MagneGST protein purification system. Western blotting revealed that stronger reaction happened with the antiserum against eggs, but not clear with antisera against other developmental stages.

Follistatin-like protein-1 is a novel proinflammatory molecule.

While analyzing gene expression in collagen-induced arthritis, we discovered that a poorly characterized gene, follistatin-like protein 1 (FSTL-1), is highly overexpressed in mouse paws during early arthritis, especially at the interface of synovial pannus and eroding bone. In this study, we show that FSTL-1 is a novel proinflammatory molecule with a previously unrecognized role in inflammation. Transfection of FSTL-1 into macrophages and fibroblasts leads to up-regulation of proinflammatory cytokines, including IL-1beta, TNF-alpha, and IL-6. Overexpression of FSTL-1 in mouse paws by gene transfer results in severe paw swelling and arthritis.