Regnase-1 and Roquin Regulate a Common Element in Inflammatory mRNAs by Spatiotemporally Distinct Mechanisms.

Regnase-1 and Roquin are RNA binding proteins essential for degradation of inflammation-related mRNAs and maintenance of immune homeostasis. However, their mechanistic relationship has yet to be clarified. Here, we show that, although Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure, they function in distinct subcellular locations: ribosome/endoplasmic reticulum and processing-body/stress granules, respectively. Moreover, Regnase-1 specifically cleaves and degrades translationally active mRNAs and requires the helicase activity of UPF1, similar to the decay mechanisms of nonsense mRNAs. In contrast, Roquin controls translationally inactive mRNAs, independent of UPF1. Defects in both Regnase-1 and Roquin lead to large increases in their target mRNAs, although Regnase-1 tends to control the early phase of inflammation when mRNAs are more actively translated. Our findings reveal that differential regulation of mRNAs by Regnase-1 and Roquin depends on their translation status and enables elaborate control of inflammation.

Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation.

Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase that destabilizes a set of mRNAs, including Il6 and Il12b, through cleavage of their 3' UTRs. Although Regnase-1 inactivation leads to development of an autoimmune disease characterized by T cell activation and hyperimmunoglobulinemia in mice, the mechanism of Regnase-1-mediated immune regulation has remained unclear. We show that Regnase-1 is essential for preventing aberrant effector CD4(+) T cell generation cell autonomously. Moreover, in T cells, Regnase-1 regulates the mRNAs of a set of genes, including c-Rel, Ox40, and Il2, through cleavage of their 3' UTRs. Interestingly, T cell receptor (TCR) stimulation leads to cleavage of Regnase-1 at R111 by Malt1/paracaspase, freeing T cells from Regnase-1-mediated suppression. Furthermore, Malt1 protease activity is critical for controlling the mRNA stability of T cell effector genes. Collectively, these results indicate that dynamic control of Regnase-1 expression in T cells is critical for controlling T cell activation.

Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz.

ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

TAK1-binding proteins (TAB)2 and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including Toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to lysine 63-linked polyubiquitin chain through TAK1-binding proteins (TAB) 2 and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and mRNA levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in TLR signaling pathway.

Pattern recognition receptors and inflammation.

Infection of cells by microorganisms activates the inflammatory response. The initial sensing of infection is mediated by innate pattern recognition receptors (PRRs), which include Toll-like receptors, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors. The intracellular signaling cascades triggered by these PRRs lead to transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. However, aberrant activation of this system leads to immunodeficiency, septic shock, or induction of autoimmunity. In this Review, we discuss the role of PRRs, their signaling pathways, and how they control inflammatory responses.

Regnase-1-related endoribonucleases in health and immunological diseases.

Dynamic changes in gene expression are key factors in the development and activation of immune cells. RNA metabolism is one of the critical steps for the control of gene expression. Together with transcriptional regulation, mRNA decay by specific ribonucleases (RNases) plays a vital role in shaping gene expression. In addition to the canonical exoribonuclease-mediated mRNA degradation through the recognition of cis-elements in mRNA 3' untranslated regions by RNA-binding proteins (RBPs), endoribonucleases are involved in the control of mRNAs in immune cells. In this review, we gleam insights on how Regnase-1, an endoribonuclease necessary for regulating immune cell activation and maintenance of immune homeostasis, degrades RNAs involved in immune cell activation. Additionally, we provide insights on recent studies which uncover the role of Regnase-1-related RNases, including Regnase-2, Regnase-3, and Regnase-4, as well as N4BP1 and KHNYN, in immune regulation and antiviral immunity. As the dysregulation of immune mRNA decay leads to pathologies such as autoimmune diseases or impaired activation of immune responses, RNases are deemed as essential components of regulatory feedback mechanisms that modulate inflammation. Given the critical role of RNases in autoimmunity, RNases can be perceived as emerging targets in the development of novel therapeutics.

Androgens Alleviate Allergic Airway Inflammation by Suppressing Cytokine Production in Th2 Cells.

Asthma is more common in females than males after adolescence. However, the mechanism of the sex bias in the prevalence of asthma remains unknown. To test whether sex steroid hormones have some roles in T cells during development of asthma, we analyzed airway inflammation in T cell-specific androgen receptor (AR)- and estrogen receptor (ER)-deficient mice. T cell-specific AR-deficient male mice developed severer house dust mite-induced allergic airway inflammation than did control male mice, whereas T cell-specific ERα- and ERß-deficient female mice exhibited a similar degree of inflammation as for control female mice. Furthermore, administration of dihydrotestosterone reduced cytokine production of Th2 cells from control, but not AR-deficient, naive T cells. Transfer of OT-II transgenic AR-deficient Th2 cells into wild-type mice induced severer allergic airway inflammation by OVA than transfer of control Th2 cells. Gene expression profiling suggested that the expression of genes related with cell cycle and Th2 differentiation was elevated in AR-deficient Th2 cells, whereas expression of dual specificity phosphatase (DUSP)-2, a negative regulator of p38, was downregulated. In addition, a chromatin immunoprecipitation assay suggested that AR bound to an AR motif in the 5' untranslated region of the Dusp2 gene in Th2 cells. Furthermore, the Dusp2 promoter with a wild-type AR motif, but not a mutated motif, was transactivated by dihydrotestosterone in a reporter assay. Finally, forced expression of DUSP-2 by retrovirus vector reduced IL-4 expression in Th2 cells. Thus, these results suggest that androgen signaling suppresses cytokine production of Th2 cells by inducing DUSP-2, explaining, in part, the sex bias of asthma after adolescence.

Levels of environmental contamination with SARS-CoV-2 in hospital rooms and salivary viral loads of patients with coronavirus disease 2019.

BACKGROUND: Clarifying the presence of viable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rather than SARS-CoV-2 viral RNA in inpatient rooms is important for infection control of coronavirus disease 2019 (COVID-19). In this study, we investigated levels of viral RNA and viable virus on environmental surfaces and in patient saliva. METHODS: Environmental samples from 23 sites in hospital rooms were collected every other day until patient discharge. Saliva specimens and samples from the inner surface of patient masks were also collected. Additionally, environmental samples were collected from 46 sites in hospital rooms on discharge day. The samples were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and plaque assays. RESULTS: The 10 enrolled cases were classified as mild COVID-19, and patients were discharged after 6-9 days. The viral RNA was detected in 12.4% (105/849) of serially collected environmental samples during hospitalization, whereas viable virus was detected only in 0.47% (4/849), which were from sinks and tap levers. Although all patients recovered, three cases retained viable virus in the last saliva specimen collected. In the 15 discharged rooms, viral RNA was detected in 6.6% (45/682) of the samples, and viable virus was detected in only one sample from the sink. CONCLUSIONS: Although environmental surfaces surrounding patients with COVID-19 were frequently contaminated with viral RNA, the presence of viable virus was rare and limited only to areas around sinks. These results suggest that contact infection risk via fomites in hospital rooms is extremely rare.

RNA Metabolism Governs Immune Function and Response.

Inflammation is a complex process that protects our body from various insults such as infection, injury, and stress. Proper inflammation is beneficial to eliminate the insults and maintain organ homeostasis, however, it can become detrimental if uncontrolled. To tightly regulate inflammation, post-transcriptional mechanisms governing RNA metabolism play a crucial role in monitoring the expression of immune-related genes, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6). These mechanisms involve the coordinated action of various RNA-binding proteins (RBPs), including the Regnase family, Roquin, and RNA methyltransferases, which are responsible for mRNA decay and/or translation regulation. The collaborative efforts of these RBPs are essential in preventing aberrant immune response activation and consequently safeguarding against inflammatory and autoimmune diseases. This review provides an overview of recent advancements in our understanding of post-transcriptional regulation within the immune system and explores the specific roles of individual RBPs in RNA metabolism and regulation.

Regnase-1 Prevents Pulmonary Arterial Hypertension Through mRNA Degradation of Interleukin-6 and Platelet-Derived Growth Factor in Alveolar Macrophages.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a type of pulmonary hypertension (PH) characterized by obliterative pulmonary vascular remodeling, resulting in right-sided heart failure. Although the pathogenesis of PAH is not fully understood, inflammatory responses and cytokines have been shown to be associated with PAH, in particular, with connective tissue disease-PAH. In this sense, Regnase-1, an RNase that regulates mRNAs encoding genes related to immune reactions, was investigated in relation to the pathogenesis of PH. METHODS: We first examined the expression levels of ZC3H12A (encoding Regnase-1) in peripheral blood mononuclear cells from patients with PH classified under various types of PH, searching for an association between the ZC3H12A expression and clinical features. We then generated mice lacking Regnase-1 in myeloid cells, including alveolar macrophages, and examined right ventricular systolic pressures and histological changes in the lung. We further performed a comprehensive analysis of the transcriptome of alveolar macrophages and pulmonary arteries to identify genes regulated by Regnase-1 in alveolar macrophages. RESULTS: ZC3H12A expression in peripheral blood mononuclear cells was inversely correlated with the prognosis and severity of disease in patients with PH, in particular, in connective tissue disease-PAH. The critical role of Regnase-1 in controlling PAH was also reinforced by the analysis of mice lacking Regnase-1 in alveolar macrophages. These mice spontaneously developed severe PAH, characterized by the elevated right ventricular systolic pressures and irreversible pulmonary vascular remodeling, which recapitulated the pathology of patients with PAH. Transcriptomic analysis of alveolar macrophages and pulmonary arteries of these PAH mice revealed that Il6, Il1b, and Pdgfa/b are potential targets of Regnase-1 in alveolar macrophages in the regulation of PAH. The inhibition of IL-6 (interleukin-6) by an anti-IL-6 receptor antibody or platelet-derived growth factor by imatinib but not IL-1ß (interleukin-1ß) by anakinra, ameliorated the pathogenesis of PAH. CONCLUSIONS: Regnase-1 maintains lung innate immune homeostasis through the control of IL-6 and platelet-derived growth factor in alveolar macrophages, thereby suppressing the development of PAH in mice. Furthermore, the decreased expression of Regnase-1 in various types of PH implies its involvement in PH pathogenesis and may serve as a disease biomarker, and a therapeutic target for PH as well.

The IκB kinase complex regulates the stability of cytokine-encoding mRNA induced by TLR-IL-1R by controlling degradation of regnase-1.

Toll-like receptor (TLR) signaling activates the inhibitor of transcription factor NF-κB (IκB) kinase (IKK) complex, which governs NF-κB-mediated transcription during inflammation. The RNase regnase-1 serves a critical role in preventing autoimmunity by controlling the stability of mRNAs that encode cytokines. Here we show that the IKK complex controlled the stability of mRNA for interleukin 6 (IL-6) by phosphorylating regnase-1 in response to stimulation via the IL-1 receptor (IL-1R) or TLR. Phosphorylated regnase-1 underwent ubiquitination and degradation. Regnase-1 was reexpressed in IL-1R- or TLR-activated cells after a period of lower expression. Regnase-1 mRNA was negatively regulated by regnase-1 itself via a stem-loop region present in the regnase-1 3' untranslated region. Our data demonstrate that the IKK complex phosphorylates not only IκBα, thereby activating transcription, but also regnase-1, thereby releasing a 'brake' on IL-6 mRNA expression.

PIN and CCCH Zn-finger domains coordinate RNA targeting in ZC3H12 family endoribonucleases.

The CCCH-type zinc finger (ZnF) containing ZC3H12 ribonucleases are crucial in post-transcriptional immune homoeostasis with ZC3H12A being the only structurally studied member of the family. In this study, we present a structural-biochemical characterization of ZC3H12C, which is linked with chronic immune disorders like psoriasis. We established that the RNA substrate is cooperatively recognized by the PIN and ZnF domains of ZC3H12C and analyzed the crystal structure of ZC3H12C bound to a single-stranded RNA substrate. The RNA engages in hydrogen-bonded contacts and stacking interactions with the PIN and ZnF domains simultaneously. The ZC3H12 ZnF shows unprecedented structural features not previously observed in any member of the CCCH-ZnF family and utilizes stacking interactions via a unique combination of spatially conserved aromatic residues to align the target transcript in a bent conformation onto the ZnF scaffold. Further comparative structural analysis of ZC3H12 CCCH-ZnF suggests that a trinucleotide sequence is recognized by ZC3H12 ZnF in target RNA. Our work not only describes the initial structure-biochemical study on ZC3H12C, but also provides the first molecular insight into RNA recognition by a ZC3H12 family member. Finally, our work points to an evolutionary code for RNA recognition adopted by CCCH-type ZnF proteins.

The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.

Polarization of macrophages to M1 or M2 cells is important for mounting responses against bacterial and helminth infections, respectively. Jumonji domain containing-3 (Jmjd3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in the activation of macrophages. Here we show that Jmjd3 is essential for M2 macrophage polarization in response to helminth infection and chitin, though Jmjd3 is dispensable for M1 responses. Furthermore, Jmjd3 (also known as Kdm6b) is essential for proper bone marrow macrophage differentiation, and this function depends on demethylase activity of Jmjd3. Jmjd3 deficiency affected trimethylation of H3K27 in only a limited number of genes. Among them, we identified Irf4 as encoding a key transcription factor that controls M2 macrophage polarization. Collectively, these results show that Jmjd3-mediated H3K27 demethylation is crucial for regulating M2 macrophage development leading to anti-helminth host responses.

An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence.

Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea-induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes.

Post-transcriptional regulation of immunological responses by Regnase-1-related RNases.

Regulation of messenger RNA (mRNA) decay plays a crucial role in the control of gene expression. Canonical mRNA decay pathways are initiated by deadenylation and decapping and are followed by exonucleolytic degradation. However, recent studies revealed that endoribonucleolytic cleavage also mediates mRNA decay, and both exoribonucleolytic and endoribonucleolytic decay pathways are important for the regulation of immune responses. Regnase-1 functions as an endoribonuclease to control immunity by damping mRNAs. Particularly, Regnase-1 controls cytokines and other inflammatory mediators by recognizing their mRNAs via stem-loop structures present in the 3' untranslated regions. Regnase-1 was found to be critical for human inflammatory diseases such as ulcerative colitis and idiopathic pulmonary fibrosis. Furthermore, a set of Regnase-1-related RNases contribute to immune regulation as well as antiviral host defense. In this review, we provide an overview of recent findings as to immune-related RNA-binding proteins (RBPs) with an emphasis on stem-loop-mediated mRNA decay via Regnase-1 and related RNases and discuss how the function of these RBPs is regulated and contributes to inflammatory disorders.

Double-stranded RNA of intestinal commensal but not pathogenic bacteria triggers production of protective interferon-ß.

The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor by using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered interferon-ß (IFN-ß) production, which protected mice from experimental colitis. The LAB-induced IFN-ß response was diminished by dsRNA digestion and treatment with endosomal inhibitors. Pathogenic bacteria contained less dsRNA and induced much less IFN-ß than LAB, and dsRNA was not involved in pathogen-induced IFN-ß induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and suggest that dsRNA in commensal bacteria contributes to anti-inflammatory and protective immune responses.

The effects of codon bias and optimality on mRNA and protein regulation.

The central dogma of molecular biology entails that genetic information is transferred from nucleic acid to proteins. Notwithstanding retro-transcribing genetic elements, DNA is transcribed to RNA which in turn is translated into proteins. Recent advancements have shown that each stage is regulated to control protein abundances for a variety of essential physiological processes. In this regard, mRNA regulation is essential in fine-tuning or calibrating protein abundances. In this review, we would like to discuss one of several mRNA-intrinsic features of mRNA regulation that has been gaining traction of recent-codon bias and optimality. Specifically, we address the effects of codon bias with regard to codon optimality in several biological processes centred on translation, such as mRNA stability and protein folding among others. Finally, we examine how different organisms or cell types, through this system, are able to coordinate physiological pathways to respond to a variety of stress or growth conditions.

Cell-Level Analysis Visualizing Photodynamic Therapy with Porphylipoprotein and Talaporphyrin Sodium.

We revealed the difference in the mechanism of photodynamic therapy (PDT) between two photosensitizers: porphylipoprotein (PLP), which has recently attracted attention for its potential to be highly effective in treating cancer, and talaporphyrin sodium (NPe6). (1) NPe6 accumulates in lysosomes, whereas PLP is incorporated into phagosomes formed by PLP injection. (2) PDT causes NPe6 to generate reactive oxygen species, thereby producing actin filaments and stress fibers. In the case of PLP, however, reactive oxygen species generated by PDT remain in the phagosomes until the phagosomal membrane is destroyed, which delays the initiation of RhoA activation and RhoA*/ROCK generation. (4) After the disruption of the phagosomal membrane, however, the outflow of various reactive oxygen species accelerates the production of actin filaments and stress fibers, and blebbing occurs earlier than in the case of NPe6. (5) PLP increases the elastic modulus of cells without RhoA activity in the early stage. This is because phagosomes are involved in polymerizing actin filaments and pseudopodia formation. Considering the high selectivity and uptake of PLP into cancer cells, a larger effect with PDT can be expected by skillfully combining the newly discovered characteristics, such as the appearance of a strong effect at an early stage.

Functional dissection of the KRAS G12C mutation by comparison among multiple oncogenic driver mutations in a lung cancer cell line model.

The development of molecular targeted therapy has improved clinical outcomes in patients with life-threatening advanced lung cancers with driver oncogenes. However, selective treatment for KRAS-mutant lung cancer remains underdeveloped. We have successfully characterised specific molecular and pathological features of KRAS-mutant lung cancer utilising newly developed cell line models that can elucidate the differences in driver oncogenes among tissues with identical genetic backgrounds. Among these KRAS-mutation-associated specific features, we focused on the IGF2-IGF1R pathway, which has been implicated in the drug resistance mechanisms to AMG 510, a recently developed selective inhibitor of KRAS G12C lung cancer. Experimental data derived from our cell line model can be used as a tool for clinical treatment strategy development through understanding of the biology of lung cancer. The model developed in this paper may help understand the mechanism of anticancer drug resistance in KRAS-mutated lung cancer and help develop new targeted therapies to treat patients with this disease.

Profibrotic function of pulmonary group 2 innate lymphoid cells is controlled by regnase-1.

Regnase-1 is an RNase critical for post-transcriptional control of pulmonary immune homeostasis in mice by degrading immune-related mRNAs. However, little is known about the cell types Regnase-1 controls in the lung, and its relevance to human pulmonary diseases.Regnase-1-dependent changes in lung immune cell types were examined by a competitive bone marrow transfer mouse model, and group 2 innate lymphoid cells (ILC2s) were identified. Then the associations between Regnase-1 in ILC2s and human diseases were investigated by transcriptome analysis and a bleomycin-induced pulmonary fibrosis mouse model. The clinical significance of Regnase-1 in ILC2s was further assessed using patient-derived cells.Regnase-1-deficiency resulted in the spontaneous proliferation and activation of ILC2s in the lung. Intriguingly, genes associated with pulmonary fibrosis were highly upregulated in Regnase-1-deficient ILC2s compared with wild-type, and supplementation of Regnase-1-deficient ILC2s augmented bleomycin-induced pulmonary fibrosis in mice. Regnase-1 suppresses mRNAs encoding transcription factors Gata3 and Egr1, which are potent to regulate fibrosis-associated genes. Clinically, Regnase-1 protein levels in ILC2 negatively correlated with the ILC2 population in bronchoalveolar lavage fluid. Furthermore, idiopathic pulmonary fibrosis (IPF) patients with ILC2s >1500 cells·mL-1 peripheral blood exhibited poorer prognosis than patients with lower numbers, implying the contribution of Regnase-1 in ILC2s for the progression of IPF.Collectively, Regnase-1 was identified as a critical post-transcriptional regulator of the profibrotic function of ILC2s both in mouse and human, suggesting that Regnase-1 may be a novel therapeutic target for IPF.