WO2022020544A1

WO2022020544A1 - Method of treating an inflammatory condition

Info

Publication number: WO2022020544A1
Application number: PCT/US2021/042686
Authority: WO
Inventors: Asrar B. Malik; Bisheng ZHOU; Jalees Rehman
Original assignee: The Board Of Trustees Of The University Of Illinois
Priority date: 2020-07-23
Filing date: 2021-07-22
Publication date: 2022-01-27

Abstract

Disclosed are methods for stimulating the transition of macrophages into anti-inflammatory macrophages and treating an inflammatory condition such as an acute inflammatory lung condition with a Rspondin3 agonist,

Description

METHOD OF TREATING AN INFLAMMATORY CONDITION

Introduction

[0001] This application claims the benefit of priority from U.S. Provisional Application Serial Number 63/055,454, filed July 23, 2020, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant number HL045638, HL060678, HL007829, HL118068, and HL090152 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background

[0003] Macrophages in all tissues exhibit remarkable phenotypic plasticity, characterized by transitioning into distinct phenotypes with specific functions in response to microenvironmental cues. Infection and injury drive the generation of pro-inflammatory phenotypes, whereas tissue niche signals can induce the switch of tissue macrophages toward anti-inflammatory and pro-reparative phenotypes to facilitate the resolution of inflammation. Thus, the orchestration of pro- and anti-inflammatory macrophage phenotypes governs the fate of organs during inflammation and injury. In the lung, deregulation of macrophages is a leading cause of an unrestrained inflammatory response to bacterial and viral infection and is a critical factor in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) including its most severe manifestations involving cytokine storms that have been described in COVID-19.

[0004] Macrophage reprogramming requires tight regulation of gene expression governed by epigenetic programs and transcriptional regulation (Lawrence & Natoli (2011) Nat. Rev. Immunol. 11:750-761; Satoh, et al. (2010) Nature Immunology 11:936-944). Also, studies have identified metabolic adaptation as a critical hallmark and prerequisite for regulating the macrophage phenotype (Phan, et al. (2017) Immunity 46:714-729; Artyomov, et al. (2016) Semin Immunol 28:417-424). Local microenvironmental cues generated by tissue cells are increasingly recognized as important determinants of resident macrophage identity, phenotype, and function (Amit, et al. (2016) Nat. Immunol. 17:18-25; Colegio, et al. (2014) Nature 513:559-563; Svedberg, et al. (2019) Nature immunology 20:571-580). Resident macrophages are highly heterogeneous as they occupy distinct tissue niches and hence exhibit the phenotype and function that is imprinted by niche-derived signals which trigger specific differentiation programs (Amit, et al. (2016) Nat. Immunol. 17:18-25; Russell & Bell (2014) Nat. Rev. Immunol. 14:81-93; Galli, et al. (2011) Nat. Immunol. 12:1035-1044).

[0005] Macrophages represent the most abundant immune cells in the healthy lung, consisting of two types of tissue resident macrophages that are characterized by their localization: alveolar macrophages (AM), which populate alveoli and airways, and interstitial macrophages (IM), which reside in lung parenchyma. Lung IMs are less well understood but findings suggest that they are critical for maintaining lung homeostasis. The vascular endothelial cells (ECs) lining all blood vessels serve as conduits for blood and tissue nutrient delivery but also constitute a niche for lung macrophages. How the lung endothelial niche regulates lung macrophage plasticity is not known. Nor is there an understanding of the factors essential for host-defense and tissue repair. Summary of the Invention

This invention provides methods for treating an inflammatory condition, e.g._f a cardiovascular disease, cancer, inflammatory lung condition or autoimmune disease; or stimulating the transition of macrophages (e.g., interstitial macrophages) into anti-inflammatory macrophages using an effective amount of a Rspondin3 agonist. In some embodiments, the inflammatory lung condition is acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS/VILI-induced ALI, trauma-induced acute lung injury (TIALI) and brain injury, or radiation-induced lung injury. In other embodiments, the Rspondin3 agonist is a Rspondin3 polypeptide or Lgr4-binding fragment thereof, or polynucleotide encoding the same.

Brief Description of the Drawings

[0006] FIG. 1 shows survival curves for WT and Rspo3^EC-/- mice with or without rRspondin3 i.v. during endotoxemia conditions (n = 16 mice for each group).

Detailed Description of the Invention

[0007] Macrophages demonstrate remarkable plasticity that is essential for host-defense and tissue repair. The tissue niche imprints macrophage identity, phenotype, and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages has actively been investigated. The lung is a highly vascularized organ and replete with a large population of resident macrophages. It has now been found that in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3 which activates b-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation . Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti^¬ inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury. Accordingly, the present invention is based upon the targeting of the Rspondin3-Wnt signaling pathway to modulate macrophages, in particular to stimulate transition of macrophages from an Ml phenotype to M2 phenotype, thereby facilitating or enhancing resolution of inflammation and reducing inflammatory injury, in particular acute inflammatory lung injury.

[0008] Accordingly, the present invention provides methods for using one or more Rspondin3 agonists to stimulate the transition of macrophages, in particular, interstitial macrophages, into anti-inflammatory macrophages and treating a condition associated with inflammation, in particular an acute inflammatory condition, preferably an acute inflammatory lung condition. For the purposes of this invention, the terms "treating," "treatment," "to treat," "alleviating" or "to alleviate" refer to the amelioration, elimination, lessening or resolution of symptoms, clinical signs, or underlying pathology of an inflammatory condition or disorder on a temporary or permanent basis. Thus, a subject in need of treatment can include those already with the condition or disorder; those prone to have the condition or disorder; those at risk of developing the condition or disorder; and those in whom the condition or disorder is to be prevented. By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on. As used herein, phrases such as "a subject in need thereof" and "an animal in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of a Rspondin3 agonist.

[0009] Ideally, treating provides a detectable improvement or a detectable change consistent with improvement that occurs in a subject or in at least a minority of subjects, e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range between any two of these values. Such improvement or change may be observed in treated subjects as compared to subjects not treated with a Rspondin3 agonist, where the untreated subjects have, or are subject to developing, the same or similar injury/condition, disease, symptom, or the like. Amelioration of the condition or assay parameter may be determined subjectively or objectively, e.g., via self- assessment by a subject(s), by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., a quality of life assessment, a slowed progression of a disease(s) or condition(s), a reduced severity of a disease(s) or condition(s), or a suitable assay(s) for the level or activity(ies) of a biomolecule(s), cell(s), by detection of respiratory or inflammatory disorders in a subject, and/or by modalities such as, but not limited to photographs, video, digital imaging and pulmonary function tests. Amelioration may be transient, prolonged or permanent, or it may be variable at relevant times during or after a Rspondin3 agonist is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within timeframes described infra, or about 12 hours to 24 or 48 hours after the administration or use of a Rspondin3 agonist to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more after a subject(s) has received such treatment.

[0010] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described _.herein effective to achieve a particular biological result. Such results may include, but are not limited to, the increased level, expression, or activity of Rspondin3 or increased signaling from the LGR4 (Leucine-rich repeat-containing G-protein coupled receptor 4) receptor in IMs, resolution of an inflammatory response, stimulation of the transition of macrophages into antiinflammatory macrophages, amelioration of symptoms and/or treatment of an inflammatory condition. It is emphasized that a therapeutically effective amount will not always be effective in treating a condition, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. For convenience only, exemplary dosages, drug delivery amounts, therapeutically effective amounts and therapeutic levels are provided herein with reference to adult human subjects. Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject and/or condition.

[0011] Rspondins (roof plate-specific spondins, Rspos), are cysteine-rich secreted glycoproteins which control a variety of cellular and tissue functions. In mammals, Four Rspondins (Rspol to 4) show high structural similarity and 60% sequence homology. They all contain four distinct domains: a putative signal peptide domain, a cysteine-rich furin-like (FU) domain, a thrombospondin (TSP) type I repeat domain and a basic amino acid-rich (BR) domain (Kazanskaya, et al. (2004) Dev. Cell 7:525-534). The FU domains amplify the Wnt ligand- dependent activation of canonical Wnt signaling. After the identification of Lgr4/5/6 as mediators of Wnt and Rspo signaling, crystal structure analysis confirmed that one of FU domains of the Rspos binds to Lgr receptors. The other FU domain binds to the cell-surface transmembrane E3 ubiquitin ligase Znrf3/Rnf43, which antagonizes Wnt signaling by ubiquitinating Frizzled receptors followed by endocytosis of Wnt receptor complex.

[0012] The amino acid and nucleic acid sequences of human Rspondin3 are known in the art and respectively available under GENBANK Accession Nos. NM_032784.5 (SEQ ID NO:1) and NP_116173.2 (SEQ ID NO:2). Orthologs of human Rspondin3 are also known in in the art and include, but are not limited to, those listed the following table.

*GENBANK Accession Number.

[0013] The Furin domain of human Rspondin3 has the amino acid sequence:

(SEQ ID NO:9).

[0014] The Thrombospondin domain of human Rspondin3 has the amino acid sequence:

(SEQ ID NO:10). [0015] The region of basic amino acid-rich repeats of human Rspondin3 has the amino acid sequence:

(SEQ ID NO:11).

[0016] For the purposes of this invention, a Rspondin3 agonist refers to a regulator, effector, or modulator of Rspondin3 that promotes resolution of an inflammatory response by stimulating the transition of macrophages into anti-inflammatory macrophages. As used herein, the terms "regulators" or "effectors" or "modulators" of Rspondin3 are used interchangeably herein and any of the above may be used to refer to antibodies, peptides, polypeptides, aptamers, low molecular weight organic or inorganic molecules and other sources of potentially biologically active materials capable of modulating Rspondin3 polypeptide signal transduction or capable of modulating Rspondin3 polypeptide activity or capable of modulating Rspondin3 expression to promote resolution of an inflammatory response. Said regulators, effectors or modulators can be naturally occurring or synthetically produced. Rspondin3 agonists may directly bind to agonize, activate, or stimulate the activity of Rspondin3 or may mimic the activity of Rspondin3, e.g., by binding to and activating the Lgr4 receptor.

[0017] In some aspects, the Rspondin3 agonist is a Rspondin3 polypeptide or Lgr4-binding fragment thereof. Suitable Rspondin3 polypeptides that can serve as Rspondin3 agonists in the methods of this invention include those provided herein as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. It has been shown that truncated Rspondin proteins without the basic amino acid-rich repeats and truncated Rspondin proteins without the basic amino acid-rich repeats and without the thrombospondin domain, can still bind to Lgr proteins (US 2014/0044713 Al). Accordingly, in some embodiments, the Rspondin3 fragment does not comprise at least part of the Thrombospondin domain and/or at least part of the regions of basic amino acid-rich repeats. In some embodiments, the Rspondin3 fragment does not comprise the Thrombospondin domain and/or the region of basic amino acid-rich repeats. In some embodiments, the Rspondin3 agonist is the Rspondin3 fragment represented by SEQ ID NO:9, SEQ ID NO:12 or SEQ ID NO:13.

[0018] A Rspondin3 polypeptide or Lgr4-binding fragment thereof may have more than 70, 80, 90 or 99% identity to SEQ ID NOs:2, 4, 6, 8, 9, 12, or 13. The Rspondin3 polypeptide or Lgr4-binding fragment thereof may consist of SEQ ID N0s:2, 4, 6, 8, 9, 12, or 13. Alternatively, the Rspondin3 polypeptide or Lgr4-binding fragment comprises at least 50, at least 60, at least 70, at least 80 or at least 90 consecutive amino acids of SEQ ID NOs:2, 4, 6, 8, 9, 12, or 13. In some embodiments the Rspondin3 polypeptide or Lgr4- binding fragment comprises or consists of less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10 consecutive amino acids of SEQ ID NOs:2, 4, 6, 8, 9, 12, or 13 or of sequences with more than 70, 80, 90 or 99% identity to any one of SEQ ID NOs:2, 4, 6, 8, 9, 12, or 13.

[0019] Sequence identity between polypeptide sequences is preferably determined by pairwise alignment algorithm using the Needleman-Wunsch global alignment algorithm (Needleman & Wunsch (1970) J. Mol. Biol. 48:443-453), using default parameters (e.g., with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix) . This algorithm is conveniently implemented in the needle tool in the EMBOSS package (Rice, et al. (2000) Trends Genet. 16:276-277). Sequence identity should be calculated over the entire length of the polypeptide sequence of the invention.

[0020] The Rspondin3 polypeptides and fragments of the present invention can be recombinant, natural, or synthetic polypeptides and fragments. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.

[0021] The Rspondin3 polypeptides and fragments can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties can improve the solubility, the biological half-life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0022] Exemplary protein modifications include, but are not limited to, glycosylated, phosphorylated, sulfated, glycosylated, animated, carboxylated, acetylated and PEGylation. For example, the C-terminal may be modified with amidation, addition of peptide alcohols and aldehydes, addition of esters, and addition of thioesters. The N- terminal and side chains may be modified by PEGylation, acetylation, formylation, addition of a fatty acid, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tetrabutyoxycarbonyl and addition of 3-mercaptopropyl, acylation (e.g., lipopeptides), biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimido group, chelating moieties, chromophores, and fluorophores. [0023] The Rspondin3 polypeptide or fragment thereof may be conjugated to a fatty acid, e.g., it may be myristoylated. For example, a fatty acid may be conjugated to the N-terminus of the polypeptide or fragment, such fatty acids include caprylic acid (C8), capric acid (CIO), lauric acid (C12), myristic acid (C14), palmitic acid (C16) or stearic acid (C18), etc. Furthermore, cysteines can be palmitoylated. [0024] The polypeptide or fragment may be conjugated or linked to another peptide, such as a carrier peptide. The carrier peptide may facilitate cell-penetration, such as antennapedia peptide, penetratin peptide, TAT, transportan or polyarginine.

[0025] The polypeptide or fragment may be cyclic, e.g., it may be cyclized by adding a single or multiple disulfide bridges, adding a single or multiple amide bonds between the N- and C-terminus, head-to-tail cyclization, side chain cyclization (e.g., lactam bridge, thioester), hydrocarbon- stabled peptides.

[0026] The polypeptide or fragment may be labeled with an isotope, e.g., ¹⁵N or ¹³C, FITC, conjugated to a carrier protein, conjugated to an imaging agent, fused to FRET substrates with a fluorophore/quencher pair, subjected to peptide-DNA conjugation, peptide-RNA conjugation, and peptide-enzyme labeling.

[0027] The polypeptide or fragment may be within a fusion protein such as fused to a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability or to increase half-life, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from peptide or the invention, a chemotherapeutic agent, an antibody, or protein for tissue specific targeting. Fusions can be made either at the amino terminus or at the carboxy terminus of the polypeptide. The fusion proteins may be direct with no linker or adapter molecule or indirect using a linker or adapter molecule. A linker or adapter molecule may be one or more amino acid residues, typically up to about 20 to about 50 amino acid residues. A linker or adapter molecule may also be designed with a cleavage site for a protease to allow for the separation of the fused moieties. For example, the polypeptide or fragment may be fused to one or more domains of an Fc region of human IgG to increase the half-life of the polypeptide or fragment or the addition of a Fab variable domain to shorten the half-life of the polypeptide or fragment.

[0028] The isolated Rspondin3 polypeptides and fragments described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable- transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest (Rspondin3 or Lgr4-binding fragment thereof). Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof .

[0029] The Rspondin3 polypeptide or fragment can be purified by any suitable method known in the art including, e.g., affinity chromatography, ion exchange chromatography, filter, ultrafiltration, gel filtration, electrophoresis, salting out, dialysis, and the like. When the polypeptide or fragment of the invention is produced in the form of a fusion protein, the fusion moiety (or tag) can optionally be cleaved off using a protease before further analysis. [0030] In some embodiments, the Rspondin3 agonist of the invention is not an Rspondin3 polypeptide. In some embodiments, the Rspondin3 agonist of the invention is not a fragment of the Rspondin3 polypeptide. In other embodiments, the Rspondin3 agonist is an agonistic antibody. In further embodiments, the Rspondin3 agonist is a nucleotide sequence encoding Rspondin3 or an aptamer.

[0031] As used herein, the term "antibody" or "antibodies" includes but is not limited to recombinant polyclonal, monoclonal, chimeric, humanized, or single chain antibodies or fragments thereof including Fab fragments, single chain fragments, and fragments produced by an Fab expression library.

[0032] In some aspects, the present invention provides isolated nucleic acid molecules encoding a Rspondin3 polypeptide or Lgr4-binding fragment thereof as a Rspondin3 agonist. The nucleic acid molecules may be, e.g., DNA molecules or RNA molecules. Suitable Rspondin3 nucleic acid molecules preferably have a nucleotide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98% or 99% identical to a polynucleotide consisting of, or consisting essentially of the polynucleotide sequence of a Rspondin3 polynucleotide sequence as set forth herein, or fragments thereof. To obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the amino- or carboxy- terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0033] Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli),

[0034] Rspondin3 nucleic acid molecules for use in the methods of this invention can be used as is (naked DNA or mRNA molecules), incorporated into vectors, or encapsulated into nanoparticles or liposomes to facilitate delivery. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted such as by standard molecular cloning techniques. Another type of vector is a viral vector (e.g., retrovirus, replication defective retrovirus, adenovirus, replication defective adenovirus, and adeno-associated virus) . Such vectors are known in the art and used in conventional gene therapy applications.

[0035] In one embodiment of the invention, the agonist is an aptamer. As used herein, the term "aptamer" refers to strands of oligonucleotides (DNA or RNA) that can adopt highly specific three-dimensional conformations. Aptamers are designed to have high binding affinities and specificities towards certain target molecules, including extracellular and intracellular proteins.

[0036] Once produced and isolated/purified, the Rspondin3 agonist of the invention can be used as is or formulated in a pharmaceutical composition containing a pharmaceutically acceptable excipient. Pharmaceutical compositions provided herein can be specially formulated for intravenous administration in solid or liquid form or for intravenous injection. Optimal pharmaceutical compositions can be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, Id.

[0037] The Rspondin3 agonist can be incorporated in a conventional systemic dosage form, such as an injectable formulation. The dosage forms may also include the necessary physiologically acceptable carrier material, excipient, lubricant, buffer, surfactant, antibacterial, bulking agent (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite) or the like.

[0038] The primary carrier or excipient in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable carrier or excipient may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral-buffered saline or saline mixed with serum albumin are further exemplary vehicles. Pharmaceutical compositions can include Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor. Pharmaceutical compositions of the invention may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, Id.) in the form of a lyophilized cake or an aqueous solution.

[0039] Administration routes for the Rspondin3 agonist, or pharmaceutical compositions of the invention include injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. Compositions may be administered by bolus injection or continuously by infusion, or by implantation device. Compositions also can be administered locally via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

[0040] The Rspondin3 agonist compositions of the invention can be delivered parenterally. When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the Rspondin3 agonist in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the Rspondin3 agonist is formulated as a sterile, isotonic solution, appropriately preserved. Preparation can involve the formulation of the Rspondin3 agonist into injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which may then be delivered via a depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. Implantable drug delivery devices may be also used to introduce the Rspondin3 agonist.

[0041] The compositions may also be formulated for inhalation. In these embodiments, the Rspondin3 agonist is formulated as a dry powder for inhalation, or inhalation solutions may also be formulated with a propellant for aerosol delivery, such as by nebulization. Pulmonary administration is further described in WO 1994/020069, which describes pulmonary delivery of chemically modified proteins.

[0042] The compositions of the invention can be delivered through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The Rspondin3 agonist of the invention that is administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. A capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the Rspondin3 agonist of the invention disclosed herein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed. [0043] Compositions of the invention may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0044] Provided herein is a method of treating an inflammatory condition, e.g., a condition characterized by excessive, aberrant, or unregulated inflammation as evidenced by elevated levels of pro-inflammatory markers. Examples of such conditions include, e.g., cardiovascular disease, cancer, inflammatory lung condition or autoimmune disease. In some aspects, the cancer can be adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, Castleman's Disease, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer, non-melanoma skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

[0045] Autoimmune diseases treated by the methods of the invention include, without limitation, acute disseminated encephalomyelitis, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Bullous pemphigoid, Celiac disease, Crohn's disease, dermatomyositis, diabetes mellitus type 1, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Idiopathic thrombocytopenic purpura, Lupus erythematosus, Mixed Connective Tissue Disease, multiple sclerosis, myasthenia gravis, narcolepsy, Pemphigus vulgaris, Pernicious anemia, Polymyositis, Primary biliary cirrhosis, Rheumatoid arthritis, Sjogren's syndrome, Temporal arteritis, Ulcerative Colitis, Vasculitis, or Wegener's granulomatosis.

[0046] In certain aspects, the inflammatory lung condition is acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS/VILI-induced ALI, trauma-induced acute lung injury (TIALI) and brain injury, or radiation-induced lung injury. [0047] The Rspondin3 agonist may be administered as a monotherapy or simultaneously or metronomically with other treatments. The term "simultaneous" or "simultaneously" as used herein, means that the Rspondin3 agonist and other treatment be administered within 48 hours, preferably 24 hours, more preferably 12 hours, yet more preferably 6 hours, and most preferably 3 hours or less, of each other. The term "metronomically" as used herein means the administration of the Rspondin3 agonist at times different from the other treatment and at a certain frequency relative to repeat administration. [0048] The Rspondin3 agonist may be administered at any point prior to another treatment including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, and 1 minute. The therapeutic agent may be administered at any point prior to a second treatment of the Rspondin3 agonist including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours,

14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes,

15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, and 1 minute. [ 0049] The therapeutic agent may be administered at any point after another treatment including about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, and 120 hours. The therapeutic agent may be administered at any point prior after a second treatment of the Rspondin3 agonist including about 120 hours, 118 hours, 116 hours, 114 hours, 112 hours, 110 hours, 108 hours, 106 hours, 104 hours, 102 hours, 100 hours, 98 hours, 96 hours, 94 hours, 92 hours, 90 hours, 88 hours, 86 hours, 84 hours, 82 hours, 80 hours, 78 hours, 76 hours, 74 hours, 72 hours, 70 hours, 68 hours, 66 hours, 64 hours, 62 hours, 60 hours, 58 hours, 56 hours, 54 hours, 52 hours, 50 hours, 48 hours, 46 hours, 44 hours, 42 hours, 40 hours, 38 hours, 36 hours, 34 hours, 32 hours, 30 hours, 28 hours, 26 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours,

15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, and 1 minute. [0050] The method may comprise administering a therapeutically effective amount of a Rspondin3 agonist of the invention to a subject in need thereof. The therapeutically effective amount required for use in therapy varies with the nature of the condition being treated, the length of time desired to stimulate resolution of the inflammatory response, and the age/condition of the patient. In general, however, doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose may be about 0.05 mg/kg to about 10 g/kg per day. The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. Multiple doses may be desired, or required.

[0051] The dosage may be at any dosage such as about 0.05 μg/kg, 0.06 μg/kg, 0.07 μg/kg, 0.08 μg/kg, 0.09 μg/kg, 0.1 μg/kg, 0.2 μg/kg, 0.3 μg/kg, 0.4 μg/kg, 0.5 μg/kg, 0.6 μg/kg, 0.7 μg/kg, 0.8 μg/kg, 0.9 μg/kg, 1 μg/kg, 1.5 μg/kg, 2 μg/kg, 3 μg/kg, 4 μg/kg, 5 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg. [0052] The dosage may be at any dosage such as about 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg, 500 mg/kg, 525 mg/kg, 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650 mg/kg, 675 mg/kg, 700 mg/kg, 725 mg/kg, 750 mg/kg, 775 mg/kg, 800 mg/kg, 825 mg/kg, 850 mg/kg, 875 mg/kg, 900 mg/kg, 925 mg/kg, 950 mg/kg, 975 mg/kg, 1 g/kg, 2 g/kg, 3 g/kg, 4 g/kg, 5 g/kg, 6 g/kg, 7 g/kg, 8 g/kg, 9 g/kg, or 10 g/kg.

[0053] The foregoing may be better understood by reference to the following description and examples, which are presented purely for purposes of explanation and are not intended to limit the scope of the invention.

Example 1 : Materials and Methods

[0054] Mice. C57BL/6J, Lyz2-cre⁺, Rspo3^fl/fl, Ctnnbl^fl/fl, Tet2^fl/fl, TCF/Lef:H2B-GFP transgenic mice, and Rosa26-floxed STOP-Cas9 mice were originally purchased from Jackson Laboratory. VE-cadherin-CreERT2 mice are described in the art (Sorenson, et al. (2009) Blood 113(22):5680-5688). Rspo3^fl/,fl mice were crossed with VE-cadherin-CreERT2 mice to generate VE-cadherin-CreERT2⁺;Rspo3^fl/fl mice (Rspo3^EC-/-), Ctnnbl^fl/fl and Tet2^fl/fl mice were crossed with Lyz2-cre⁺ mice to generate Lyz2-cre⁺;Ctnnbl^fl/fl mice (Ctnnbl^Mφ-/-) and Lyz2-cre⁺;Tet2^fl/fl mice (Tet2^Mφ-/-), respectively. Genotyping of these mice strains were either performed by regular PCR using the recommended primers in Jackson Laboratory website followed by DNA gel imaging or by Transnetyx Inc. (Cordova, TN) using the TAQMAN® probe-based qPCR. All mice were housed in a temperature-controlled specific pathogen-free facility under 12-hour light/dark cycles in the University of Illinois at Chicago Animal Care Facility. Veterinary care and animal experimental procedures were approved by the University of Illinois Animal Care & Use Committee in accordance with the guidelines of the National Institutes of Health. [0055] Cells. Mouse bone marrow-derived macrophages (BMDMs) were isolated and differentiated into mature macrophages according to known methods (Di, et al. (2018) Immunity 49:56- 65 e54). Mouse lung microvascular endothelial cells (ECs) were isolated, purified and cultured according to established methods (Liu, et al. (2019} Nature Communications 10:2126). Mouse lung interstitial macrophages (IMs) and alveolar macrophages (AMs) were isolated by Fluorescence-activated cell sorting (FACS) on the MOFLO® ASTRIOS® cell sorter (Beckman Coulter, Brea, CA) with the strategy described previously (Chakarov, et al. (2019) Science 363: eaau0964). [0056] siRNA Transfection. Mouse Lgr4 siRNA with four different targeting sites as a pool (Dharmacon, Lafayette, CO) and non-targeting pool siRNA (Dharmacon, Lafayette, CO) were transfected into BMDMs using the LIPOFECTAMINE® 3000 reagents (Thermo Fisher Scientific, Waltham, MA) as described (Nepal, et al. (2019) Proc. Natl. Acad. Sci. USA 116:16513- 16518).

[0057] Generation of Macrophage-Specific Lgr4 Knockout (Lgr4^Mφ-/-) Mice. Lgr4^Mφ-/- mice were generated using the CRISPR- Cas9 strategy. Briefly, a transgenic mouse strain with Cas9- specific expression in myeloid cells under control by Lyz2- cre⁺ was generated by crossing Rosa26-floxed ST0P-Cas9 mice with Lyz2-cre⁺ mice as described (Nepal, et al. (2019) Proc. Natl. Acad. Sci. USA 116:16513-16518). Subsequently, single guide RNA targeting three different sites (as a pool) for Lgr4 (IDT#Mm.Cas9.LGR4.1.AA, IDT#Mm.Cas9 .LGR4.1.AB,

IDT#Mm.Cas9.LGR4 .1.AC) were packaged in liposomes using INVIVOFECTAMINE® 3.0 reagent (Thermo Fisher Scientific, Waltham, MA) and delivered into mice via an intravenous tail vein injection (10 nmol per mice), to achieve macrophage uptake by phagocytosis in vivo (Luo, et al. (2018) ACS Nano 12:994-1005) . [0058] Recombinant Proteins. Recombinant murine Rspondin3 was obtained from R&D Systems (Minneapolis, MN). Recombinant Macrophage Colony Stimulating Factor (M-CSF) was obtained from PeproTech (Rocky Hill, NJ),

[0059] Acute Lung Injury Models. Two acute lung injury mice models were used in this study. The first model is the widely used classical lipopolysaccharide (LPS)-induced acute lung injury model in which mice are systemically challenged with sub-lethal LPS (12 mg/kg i.p.; Di, et al. (2018) Immunity 49:56-65 e54; Liu, et al. (2019) Nature Communications 10:2126; Liu, et al. (2019) Nature Communications 10:2126). The second inflammatory lung injury model is the bleomycin model that has also been previously described (Chakarov, et al. (2019) Science 363:eaau0964; Sorensen, et al. (2009) Blood 113:5680-5688). Briefly, mice were anesthetized with isoflurane, their lungs were intubated orally with a 20-gauge Angiocath (BD, Franklin Lakes, NJ), and 0.025 IU bleomycin (Thermo Fisher Scientific, Waltham, MA) in 50 μL of sterile phosphate-buffered saline (PBS) was instilled through the catheter. Mice were sacrificed 5 days (acute lung injury phase) after the instillation of bleomycin for further analysis.

[0060] RNA Isolation and Real-time Quantitative PCR (qPCR). Total RNA from BMDMs or lung ECs was isolated using the PURELINK® RNA kit (Thermo Fisher Scientific, Waltham, MA) and were reverse-transcribed into cDNA using iScript™ reverse transcription supermix (Bio-Rad, Hercules, CA) according to manufacturer's instructions. The cDNA obtained was mixed with PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Waltham, MA) with specific qPCR primers (Table 1) for qPCR on an ABI Prism 7000 system and analyzed with the QuantStudio software vl.3 (Thermo Fisher Scientific, Waltham, MA). The heatmap for the change folds of gene levels were generated by Morpheus (software available at the Broad Institute website).

TABLE 1

[0061] Chromatin Immunoprecipitation (Chip). ChIP was performed with cross-linked chromatin from mouse BMDMs using a SIMPLECHIP® plus sonication Chromatin IP kit (Cell Signaling Technology, Inc., Danvers, MA) with ChIP grade H3K4me3 antibody (Cell Signaling Technology, Inc., Danvers, M) with a dilution ratio of 1:500 according to manufacturer's instructions. qPCR following ChIP were performed with the primers listed in Table 1.

[0062] Lung Vascular Permeability and Inflammation Measurements. Lung vascular permeability were measured by the albumin-Evans blue dye tracer and lung inflammation were evaluated by myeloperoxidase activity as previously described (Di, et al. (2018) Immunity 49:56-65 e54; Liu, et al. (2019) Nature Communications 10:2126).

[0063] Preparation of Mouse Lung Single Cell Suspensions. Mice were anaesthetized with ketamine/xylazine and sacrificed, followed by perfusing with 10 ml of PBS via right ventricle. The lungs were removed and cut into tiny pieces with scissors, followed by incubation with digestion buffer (1 mg/ml of collagenase IV and 0.1 mg/ml DNase I, both from MilliporeSigma, Burlington, MA) in a shaking water bath (37°C) for 30 minutes. The digested lung pieces were passed through a 20-G needle 10 times and filtered by a 40-mih nylon mesh to obtain a single-cell suspension. The remaining red blood cells (RBCs) were lysed using RBC lysis buffer (BioLegend, San Diego, CA). The acquired lung single-cell suspensions were used for applications including, but not limited to, flow cytometry, CyTOF analysis, and cell sorting. [0064] Flow Cytometry. For BMDMs, flow cytometry was performed with anti-mouse CDllb, F4/80, CD64 to gate matured macrophages. Mouse macrophage anti-inflammatory markers CD206, CD301, interleukin (IL)-10, arginase 1 and pro- inflammatory markers CD86, CD80, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) were stained with the fluorophore-coupled antibodies listed in Table 2. For mouse lung macrophages, flow cytometry was performed to identify lung macrophage subpopulations (Misharin, et al. (2013) Am. J. Resp . Cell Mol. Biol. 49:503-510). Briefly, cells were stained with anti-mouse CD45, CDllb, CD64, Ly6G, SiglecF, CD86 and CD206 antibodies listed in Table 2 using an eBioscience™ flow cytometry staining buffer (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Samples were run on a CYTOFLEX ® S Flow Cytometer (Beckman Coulter, Inc., Brea, CA) and data were analyzed by the Kaluza Analysis 2.1 software (Beckman

Coulter, Inc., Brea, CA).

TABLE 2

FITC, Fluorescein isothiocyanate; APC, Allophycocyanin; PE, phycoerythrin; PerCP-Cyanine5.5, Peridinin chlorophyll protein-Cyanine5 .5; Cy7, Cyanine7,

[0065] Cytometry by Time-of-Flight Mass Spectrometry (CyTOF). CyTOF allows for high-dimensional analysis of cell surface markers, cytokines and signaling molecules simultaneously at the single-cell level (Becher, et al. (2014) Nat. Immunol. 15:1181-1189). Thus, CyTOF was used for phenotyping and function assay for the mouse lung myeloid populations including IMs, AMs, monocytes, neutrophils, dendritic cells (DCs) and eosinophils. A panel of metal- labelled antibodies (Table 3) including several markers for macrophages and myeloid cells as well as cytokines and signaling molecules (CD45, CD11b, F4/80, CD64, Ly6C, Ly6G, MerTK, CD24, CD206, SiglecF, CDllc, CD301, Arginase 1, LGR4, b-Catenin, IL-10, iNOS, TNF, CX3CR1, CD80, CD69, CD86, CCR2, CD115, I-A/I-E, BST2, RELMa, CD103, CD3, CD49, TER-119) were used for staining according the FLUIDIGM Inc. recommended protocol. Samples were run on the Helios CyTOF Mass cytometer (FLUIDIGM Inc.) at the flow cytometry core of Research Resources Center of the University of Illinois at Chicago. Data from the CyTOF were analyzed using Cytobank online analysis tool (Cytobank Inc.).

TABLE 3

[0066] Metabolism Assays. Macrophages (BMDMs) prepared from the indicated mouse strains were uniformly plated in XF96 plates overnight and treated with PBS, Rspondin3, LPS alone or LPS and Rspondin3 together for 24 hours. Metabolic profiling was performed with the Seahorse XF96 extracellular flux analyzer (Agilent Technologies, Santa Clara, CA) with the Wave 2.6.1 software (Agilent Technologies, Santa Clara, CA) as previously described (Nelson, et al. (2018) Genes Dev. 32:1035-1044). Experiments were conducted in XF medium (non- buffered DMEM containing 10 mM glucose, 2 mM glutamine, 2 mM sodium pyruvate), with oxygen consumption rate measured basally and in response to sequential addition of oligomycin, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and rotenone plus antimycin. Fuel preference was measured after 1 hour of incubation in Seahorse XF basal medium (Agilent Technologies, Santa Clara, CA) with no fuel substrates and then immediately before the assay supplemented with either 10 mM glucose, 4 mM glutamine, or 10 nM free fatty acids in the form of palmitate bovine serum albumin (BSA). All seahorse XF data were normalized to an equal cell number of 1 mg total protein.

[0067] ELISA. Supernatants were collected from BMDMs under the different conditions indicated, and the cytokines IL-10 and IL-Ib were measured using mouse IL-10 DUOSET® ELISA (R&D Systems (Minneapolis, MN)) or mouse IL-Ib DUOSET® ELISA (R&D Systems (Minneapolis, MN)) respectively; Secreted Rspondin3 in cultured lung ECs were measured using mouse Rspondin3 DUOSET® ELISA (R&D Systems (Minneapolis, MN)) with the supernatants collected. Optical density (OD) data were collected on a Synergy™ HTX multi-mode microplate reader (BioTek, Winooski, VT) with the Gen 5 software (BioTek, Winooski, VT). Cell numbers at each well of the cell culture plate were counted using a CYQUANT® kit (Thermo Fisher Scientific, Waltham, MA). Calculated levels of cytokines were adjusted with the cell number. [0068] Immunoblotting. Protein samples for lung ECs and non- ECs were prepared and western blotting were performed as previously described (Liu, et al. (2019) Nature

Communications 10:2126). Anti-RSP03 polyclonal antibody (ABclonal, Woburn, MA) with a dilution of 1:3000 and anti-p- actin mouse monoclonal antibody (Cell Signaling Technology, Inc., Danvers, MA) with a dilution of 1:5000 was used for immunoblot analyses.

[0069] Angiocrine Factor Profiling. Proteomics-based global secretome analyses were performed by the mass spectrometry core of Research Resources Center in University of Illinois at Chicago using EC-conditioned medium as previously described (Blanco, et al. (2012) Cell Res. 22:1339-1355). [0070] Immunofluorescence Staining and Proximity Ligation Assay (PLA). An anti-TET2 rabbit monoclonal antibody (Cell Signaling Technology, Inc., Danvers, MA) and an anti-β- catenin mouse monoclonal antibody (Thermo Fisher Scientific, Waltham, MA) with a dilution of 1:500 were used as primary antibodies. An AF488-conjugated donkey anti-mouse IgG and an AF594-conjugated goat anti-rabbit IgG were used secondary antibody with a dilution of 1:2000. Immunofluorescence staining was carried out following the Cell Signaling Technology, Inc. immunofluorescence general protocol. Images were taken with the Zeiss LSM880 confocal microscope with Zen software 3.1 (Zeiss, White Plains, NY) and analyzed with Imaged software vl.52p.

[0071] Proximity ligation assay for detecting protein interactions with high specificity and sensitivity were performed with DUOLINK® In Situ Red Starter Kit Mouse/Rabbit (Millipore-Sigma, Burlington, MA) using the above anti-TET2 rabbit monoclonal antibody and anti-p-catenin mouse monoclonal antibody as validated by immunofluorescence staining according to standard protocols (Fredriksson, et al. (2002) Nature Biotechnol. 20:473-477). Briefly, anti-TET2 rabbit monoclonal antibody and anti-p-catenin mouse monoclonal antibody were first used to detect TET2 and b- catenin on macrophage, then a pair of PLA probes (Anti-rabbit Plus and Anti-mouse Minus) were served as secondary antibodies. Subsequently, hybridizing connector oligos join the PLA probes only if they are in close proximity to each other and ligase forms a closed, circular DNA template for generating rolling-circle amplification that gives red fluorescence.

[0072] Assay for Specific Enrichment of 5-hmC DNA Fragments (hMeDIP). To detect the enrichment of 5-hmC DNA fragments, genomic DNA was isolated from macrophages, and 5-hmC DNA immune precipitation (hMeDIP) were performed using a highly specific purified 5-hmC antibody with a dilution ratio of 1:500 contained by hMeDIP kit purchased from ActiveMotif (Carlsbad, CA) as reported (Takayama, et al. (2015) Nature Comm. 6:8219). Real-time quantitative PCR were performed using the 5-hmC antibody precipitated DNA with specific primers on the proximal promoter regions of genes as listed in Table 1.

[0073] a-Ketoglutarate and Succinate Measurements and Glutaminase Activity Assay. α-Ketoglutarate and succinate levels in macrophages were measured by a-Ketoglutarate Colorimetric/Fluorometric Assay Kit (BioVision) and Succinate (Succinic Acid) Colorimetric Assay Kit (BioVision), respectively, as previously described (Liu, et al. (2017) Nat. Immunol. 18:985-994). Glutaminase activity was measured using PICOPROBE® Glutaminase (GLS) Activity Assay Kit (Fluorometric) (BioVision, Milpitas, CA) according to established protocols (Wang, et al. (2010) Cancer Cell 18:207-219) . [0074] 5mC-Hydroxylase TET Activity and JMJD3 Demethylase Activity Assay. 5mC-Hydroxylase TET activity and JMJD3 Demethylase activity were measured using the Epigenase 5mC- Hydroxylase TET Activity Assay Kit (Epigentek) and Epigenase JMJD3 Demethylase Activity kit (Epigentek, Farmingdale, NY), respectively, according to established protocols (Carey, et al. (2015) Nature 518:413-416; Thienpont, et al. (2016) Nature 537:63-68). Briefly, nuclear extracts were prepared from macrophages with EpiQuik Nuclear Extraction Kit (Epigentek, Farmingdale, NY). Five μg of nuclear protein from each sample were used to incubate with the TET or JMJD3 demethylase substrates, respectively. Catalytic activity was measured by the absorbance on a microplate reader (BioTek, Winooski, VT) for 10 minutes at 450 nm with a reference wavelength of 655 nm using Gen5 software (BioTek, Winooski, VT). The TET or JMJD3 activity were represented by OD/min/mg. [0075] Global DNA Hydroxymethylation (5-hmC) Assay. Global DNA hydroxymethylation (5-hmC) levels were measured using the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Kit (Epigentek, Farmingdale, NY) according to known methods (Carey, et al. (2015) Nature 518:413-416). Briefly, genomic DNA were isolated from macrophages from the indicated groups, and 1 μg of DNA was bound to strip-wells that had a high DNA affinity included in the kit. Subsequently, the hydroxymethylated fraction of DNA was detected using a 5-hmC monoclonal antibody-based detection complex in a one-step manner and then quantified colorimetrically by reading the absorbance in microplate spectrophotometer (BioTek, Winooski, VT) at 450 nm with Gen5 software (BioTek, Winooski, VT). 5hmC% were calculated using the formula generated from an accompanied standard curve.

[0076] Statistical Analysis. Statistical significance was analyzed using Prism v.8 (GraphPad Software) with tests indicated. All the experiments were repeated independently at least three times. Sample size and P values were as indicated.

Example 2: Endothelial Cells Instruct Macrophage Phenotypic Transition Via Angiocrine Signaling

[0077] Initially, paracrine factors were analyzed, which were released by ECs and could regulate macrophage transition. Lung ECs were isolated and conditioned medium was collected from either LPS-activated ECs or control ECs, which was added to bone-marrow-derived macrophages (BMDMs). After 24hours, BMDMs were collected for flow cytometry analysis. Macrophages were first gated by CDllb⁺F4/80⁺CD64⁺ and the expression of anti-inflammatory markers (CD206, CD301, arginase 1 and IL-10; Table 4) as well as pro-inflammatory macrophage markers (CD86, CD80, TNF and iNOS; Table 5) was analyzed. It was observed that EC-conditioned medium significantly induced the expression of anti-inflammatory markers while suppressing pro-inflammatory markers and this trend was markedly augmented in ECs activated by the bacterial endotoxin LPS (Tables 4 and 5).

TABLE 4

n=3 samples per group (meanis.d.). Statistical significance was determined by two-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test using GraphPad Prism and individual P values (left to right) are: CD206 (*P=0.0249, †P<0.0001); CD301 (†P<0.0001, †P<0.0001); arginase 1

(†P<0.0001, †P<0.0001); IL-10 (†P<0.0001, †P<0.0001).

TABLE 5

n= 3 samples per group (meanis.d.). Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparisons test using GraphPad Prism and individual P values (left to right) are: CD86 (†P<0.0001, †P<0.0001); CD80 (NS, P= 0.0889, †P<0.0001); TNF (†P<0.0001, †P<0.0001); and iNOS (†P<0.0001, †P<0.0001). NS, not significant.

[0078] The percentages of 'Ml-like' (defined as 'CD86^hiCD206^lo') and 'M2-like' (defined as 'CD206^hiCD86^lo') macrophages (Wang, et al. (2018) Nat. Commun. 9:559) were also quantified and it was found that EC-conditioned medium shifted the balance of macrophage populations toward an M2- like phenotype (Table 6).

TABLE 6

n=3 samples per group (mean ± sd); Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism with individual P values are ***P=0.0006, ****p<0.0001. [0079] A secretome analysis was subsequently conducted to identify candidate proteins mediating the paracrine EC effects on macrophage phenotype transition. This analysis indicated that several proteins were released by ECs following LPS activation (Table 7), with the Wnt signaling activator Rspondin3 clearly ranked as the top secreted EC protein and the release of Rspondin3 was validated by quantitative ELISA but without change on Rspo3 messenger RNA levels. This indicated that angiocrine signals such as Rspondin3 could be regulators for macrophage phenotype transition .

TABLE 7

Mol. Wt., Molecular weight (kD); Quant. Value, Quantitative value. Example 3: Rspondin3 Mediates Interstitial Macrophage

Phenotypic Transition and Prevents Inflammatory Lung Injury [0080] To determine the role of Rspondin3 in regulating macrophage phenotype transition, BMDMs were stimulated with recombinant Rspondin3 protein (40ng/ml) and LPS (100ng/ml) alone or in combination. Flow cytometry analysis demonstrated that Rspondin3 increased the expression of anti-inflammatory markers while concomitantly reducing pro-inflammatory markers in macrophages, whereas LPS strongly induced pro-inflammatory markers and reduced anti-inflammatory markers; crucially, Rspondin3 prevented the generation of LPS-induced pro- inflammatory markers (Table 8).

TABLE 8

n=3 samples per group

. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism and individual P values (left to right) are: CD206 (****P<0. 0001, ****P<0.0001); CD301 (****P<0.0001, ****P< 0.0001); arginase 1 ( ****P<0.0001, ****P<)0;.0001;IL-10 (****P<0.0001, ****P<0.0001) ; CD86 (NS, P= 0.1457, ****P<0.0001); CD80 (NS, P= 0.1576, ****P<0.0001); TNF (****P<0.0001, ****P<0.0001); and iNOS (****P<0.0001, ****P<0.0001). NS, not significant. Comparisons were Rspondin3 to control and LPS+Rspondin3 to LPS.

[0081] It was also found that Rspondin3 induced the release of anti-inflammatory cytokines such as IL-10, IL-4 and TGF-b (in both Rspondin3 and LPS+Rspondin3 groups), while reducing the LPS-mediated release of pro-inflammatory cytokines such as IL-1β, IL-6, TNF, CXCL1, IL-12p70, IL-2 and IL-5 in macrophages (Table 9).

TABLE 9

Data are from three independent experiments.

[0082] Increased expression of multiple anti-inflammatory marker genes (Mrcl, Argl, Chil3, Retnla and Pparg and 1110) and decreased expression of pro-inflammatory marker genes (Cd86, Illb, Tnf, Cxcll, 116 and Nos2) were induced by Rspondin3 (Table 10), underscoring the crucial role of Rspondin3 in promoting a macrophage shift toward an anti- inflammatory phenotype.

TABLE 10

[0083] Furthermore, endotoxemia was induced in wild-type (WT) mice and VE-cadherin-CreERT2⁺;Rspo3^fl/fl mice (herein called Rspo3^EC-/- mice) and lung ECs were isolated from these mice as well as nonendotoxemic control mice. Conditioned medium collected from these ECs showed that Rspondin3 deficiency, specifically in EC medium, prevented the induction of anti-inflammatory markers and suppression of pro-inflammatory markers in macrophages (Table 11); however, EC-macrophage contact was dispensable for these effects (Table 12). This demonstrated the critical role of angiocrine Rspondin3 in mediating the effects of lung ECs on macrophage phenotype transition.

TABLE 11

Data are representative of three independent experiments with n=3 samples per group (mean ± sd), statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism with individual P values (left to right) are: CD206 (****P<0.0001, ns P-0.9405, ***_P=0.0002, ****P<0.0001), CD301 (****p<0.0001, ns P-0.9991, ****P< 0.0001, ****p<0.0001), arginasel ( ****P<0.0001, ns P-0.4164, ****p<0.0001, ****P<0.0001), IL-10 (****p<0.0001, ns P-0.9933, ****p<0.0001, ****P<0.0001}, CD86 (****p<0.0001, _ns p=0.8224, ***P=0.0001, ns P= 0.9075), CD80 (****p<0.0001, ****p<0.0001, ns P-0.7688), TNF (****P<0.0001, ns P-0.4519, **P=0.0061, ns P-0.9997), iNOS (****P<0.0001, ns P-0.9996, ns P-0.6726, ns P>0.9999). WT EC was compared to control; LPS/WT EC was compared to WT EC; EC Rspo3^EC-/- was compared to control; and LPS/EC Rspo3^EC-/- was compared LPS/WT EC (anti- inflammatory) or EC Rspo3^EC-/- (pro-inflammatory).

TABLE 12

Data are representative of three independent experiments with n=3 samples per group (mean+s.d). Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism with individual P values (left to right) are CD206 (****p<0.0001, ns P>0.9999, ns P>0.9999), CD301 (****p<0.0001, ns P-0.9977, ns P-0.9998), arginasel (****P<0.0001, ns P-0.9328, ns P=0.9998), IL-10 ( ****P<0.0001, ns P-0.8989, ns P-0.9393), CD86 (ns P-0.8308, ****P< 0.0001, ns P-0.9600), CD80 (ns P>0.9999, ****p<0.0001, ns P-0.9679), TNF (ns P-0.9997, ****p<o . 0001, ns P-0.9903), iNOS (ns P= 0.9996, ****P<0.0001, ns P= 0.2748). Mφs+WT EC was compared to Mφs only (anti-inflammatory); Mφs+EC Rspo3^EC-/- was compared to Mcps only (anti-inflammatory and pro- inflammatory); Mφs+EC Rspo3^EC-/-/LPS was compared to Mφs+WT EC/LPS (anti-inflammatory and pro-inflammatory); and Mφs+WT EC/LPS was compared to M<ps only (pro-inflammatory).

[0084] The mouse lung has two distinct resident macrophage populations, including AMs and IMs, which are involved in the regulation of lung homeostasis as well as lung injury (Misharin, et al. (2013) Am. J. Resp. Cell Mol. Biol. 49:503- 510). In addition, other myeloid cells, such as monocytes (Mo), including Ly6C⁺ Mo and Ly6C-Mo, neutrophils, dendritic cells (DCs), including CD103⁺ DCs, plasmacytoid DCs and CD11b⁺ DCs and eosinophils also reside in lungs (Becher, et al. (2014) Nat. Immunol. 15:1181-1189). AMs, IMs, and other myeloid lung populations were assessed using cytometry by time-of-flight mass spectrometry (CyTOF), which enabled the detection of more than 30 surface markers and intracellular molecules simultaneously, using metal-labeled antibodies (Table 3) with a myeloid cell-gating strategy. Resident macrophages were identified by

CD45⁺F4/80⁺Ly6G-Ly6C-CD64⁺MerTK⁺ and IMs and AMs were further identified by CDllb⁺SiglecF- and CD11b-SiglecF⁺, respectively. Other myeloid populations were identified as Ly6C⁺ Mo (CD45⁺Ly6G-Ly6C⁺CD11b⁺CD24-MHCII- SiglecF^"CD206^"),

(CD45⁺Ly6G^~Ly6CCD11b⁺CD24-MHCII-SiglecF- CD206-); neutrophils (CD45⁺Ly6G⁺CDllb⁺F4/80-); CD103⁺ DCs

(CD45⁺Ly6G^"CDllc⁺CD11b-CD24⁺CD64^_Ly6C^~SiglecF^”CD103⁺BST2-); plasmacytoid DCs (CD45⁺Ly6G-CD11c⁺CD11b^“CD24⁺CD64-Ly6C⁺

SiglecF-CDl03-BST2⁺); CDllb⁺ DCs (CD45⁺Ly6G-CD11c⁺CD11b⁺

CD24⁺CD64-Ly6C-SiglecF-CDl03-BST2-) and eosinophils

(CD45⁺Ly6G-Ly6C-CDllb⁺CD24⁺SiglecF⁺CDllc-MHCII-CD64-). The absolute number of AMs, IMs, Ly6C⁺ Mo, Ly6C- Mo, neutrophils, CD103⁺ DCs, plasmacytoid DCs, CDllb⁺ DCs and eosinophils in WT and Rspo3^EC-/- mice with or without intravenous (i.v.) administration of Rspondin3 under basal and sublethal LPS challenge conditions (12 mg/kg intraperitoneally (i.p.) for 24 hours or 48 hours} were determined. IMs expanded two-fold to three-fold in response to endotoxemia at 24 hours and 48 hours, respectively, in WT mice, whereas the number of IMs did not change in Rspo3^EC-/- mice. The results thus showed that endothelial Rspondin3 is a prerequisite for the expansion of IMs in response to endotoxemia. In addition, i.v. Rspondin3 was able to partially restore endotoxemia-induced IM expansion in Rspo3^EC-/- mice. However, the number of AMs in WT and Rspo3^EC-/- mice remained the same during the basal state and post-endotoxemia and i.v. Rspondin3 did not change AM numbers. The other myeloid populations, monocytes, DCs and eosinophils were also not affected by Rspondin3. Neutrophils in WT and Rspo3^EC-/- mice remained the same in basal conditions and 48 hours post-LPS challenge but there were more neutrophils in Rspo3^EC-/- mice 24 hours post-LPS, likely reflecting exacerbated inflammatory injury at this time point in the absence of endothelial Rspondin3. Moreover, Rspondin3 activates Wnt signaling in IMs but not other lung myeloid cells .

[0085] The levels of anti-inflammatory markers (CD206, CD301, RELM-α, arginase 1 and IL-10) and pro-inflammatory markers (CD86, CD80, CD69, iNOS and TNF) were also analyzed by CyTOF (Table 13).

TABLE 13

*n=5 mice per group with three independent repeats, shown as fold changes by the mean CyTOF signal intensity normalized to control group

[0086] It was observed that endotoxemia in WT mice significantly increased the expression of pro-inflammatory markers at 24 hours in IMs (Table 13) and that these inflammatory markers were reduced by 50% at 48 hours post-LPS (although the values remained greater than the basal condition) (Table 13). These pro-inflammatory markers in IMs were two-fold to three-fold greater in Rspo3^EC-/- mice than those observed in WT mice at 24 hours after LPS administration and were maintained at these high levels at 48 hours (Table 13). It was found that i.v. Rspondin3 markedly reduced the expression of pro-inflammatory markers in IMs in both WT and Rspo3^EC-/- mice (Table 13). The anti-inflammatory markers (CD206, CD301, RELM-α, arginase 1 and IL-10) increased twofold to four-fold in IMs as compared to the basal condition in response to LPS for 24 hours and continued to increase four-fold to sixteen-fold in response to LPS at 48 hours in WT mice (Table 13). These findings show that the activation of adaptive anti-inflammatory programming of IMs is elicited by endotoxemia. However, in Rspo3^EC-/- mice, deletion of endothelial Rspo3 impaired the induction of these anti- inflammatory markers in IMs at 24 hours after LPS challenge and this impairment became even more evident at 48 hours post- LPS challenge (Table 13). Furthermore, it was observed that the impairment of anti-inflammatory IM programming in Rspo3^EC-/- mice was markedly restored by i.v. Rspondin3 (Table 13) . In contrast to what was observed in IMs, the expression of anti-inflammatory and pro-inflammatory markers in AMs did not differ between WT and mice, as assessed by CyTOF. These data indicated that angiocrine Rspondin3 specifically regulates IMs but not other lung myeloid cells in mice.

[0087] Subseguently, it was determined whether the lung IM phenotype shift induced by endothelial Rspondin3 impacted the extent of inflammatory lung injury. Lung inflammation was assessed by quantifying neutrophil infiltration using the myeloperoxidase (MPO) activity assay (Table 14) as well as by quantifying changes in lung vascular permeability using the albumin-Evans blue dye tracer (Table 15). Deletion of endothelial Rspo3 (Rspo3^EC-/- versus WT) had no effect on lung inflammation at baseline, whereas it markedly enhanced lung inflammatory injury during endotoxemia.

TABLE 14

Data are representative of three independent experiments with five mice per group. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism and individual P values (left to right) are: basal (NS, P=0.9774; NS, P=0.9715); LPS 24 hours (****p<0.0001; ****P<0.0001; ****P<0.0001); LPS 48 hours (****p<0.0001; *P=0.0250; ****P<)0..0001 TABLE 15

Data are representative of three independent experiments with five mice per group). Graphs show raeanls.d, with each dot representing an individual mouse. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test using GraphPad Prism and individual P values (left to right) are: Basal (NS, P=0.9997; NS, P=0.9996); LPS 24 hours (****p<0.0001; ****P<0.0001; ****P<0.0001); LPS 48 hours (****p<0.0001; ****P<0.0001; ****P<0.0001).

[0088] Survival studies also showed that endothelial- specific deletion of Rspo3 significantly increased mortality as compared to control mice (FIG. 1). The i.v. Rspondin3 acted as a therapeutic that attenuated lung inflammatory injury and enhanced survival in endotoxemic Rspo3^EC-/- mice (Table 14, Table 15, and FIG. 1). These data demonstrated that Rspondin3- induced IM phenotypic transition prevents inflammatory lung injury.

[0089] Flow sorting was also used to isolate AMs and IMs and performed gene profiling by quantitative PCR (qPCR). It was observed that induction of the anti-inflammatory genes (Argl, Chil3, Retnla, Mrcl, CleclOa, 1110, Clec7a, Pparg and Tgfbl) was significantly reduced in IMs obtained from endotoxemic Rspo3^EC-/- mice when compared to control mice (Table 16). In contrast, activation of pro-inflammatory genes (Cxcll, Tnf, Nos2, Illb, Cd80, Cd86 and Cd69) was markedly enhanced in IMs from endotoxemic Rspo3^EC-/- mice (Table 16). However, both the expression of anti-inflammatory and pro-inflammatory markers in AMs was not affected by the absence of endothelial Rspondin3 during the basal state or during endotoxemia (Table 16).

TABLE 16

samples per group).

[0090] To establish whether the observed role of endothelial Rspondin3 was also applicable to other forms inflammatory lung injury, a bleomycin model was also used, in which bleomycin is administered intratracheally to induce inflammatory acute lung injury (ALI)(Chakarov, et al. (2019) Science 363: eaau0964). At day 5 post-bleomycin (ALI phase), WT and Rspo3^EC-/- mice were killed, and myeloid cells were analyzed by CyTOF. It was observed that among lung myeloid populations, monocytes, neutrophils, and eosinophils were not affected by bleomycin-induced ALI; DC numbers increased after bleomycin, but no difference was found between WT and Rspo3^EC-/- mice. AMs decreased by 60% after bleomycin administration but there was no difference between WT and Rspo3^EC-/- mice. IMs expanded three-fold in WT mice after bleomycin exposure, whereas IMs in Rspo3^EC-/- mice were unable to mount this expansion response to injury. The antiinflammatory and pro-inflammatory phenotypes for lung macrophages following bleomycin-induced inflammatory lung injury were also analyzed by CyTOF. Endothelial-specific deletion of Rspo3 shifted the lung IM population toward a pro-inflammatory phenotype in response to bleomycin-induced lung injury and thus mirrored the effects seen in the endotoxemia-induced injury. These data indicated that the role of endothelial Rspondin3 as a regulator of an anti- inflammatory lung IM phenotype is a generalizable principle in lung inflammatory injury.

Example 4: LGR4 is Required for Rspondin3~Induced Macrophage Transition

[0091] Rspondin family proteins induce Wnt-β-catenin signaling by binding to members of the leucine-rich repeat- containing G protein receptor (LGR) family (Lgr4, Lgr5 and Lgr6) (de Lau, et al. (2014) Genes Dev. 28:305-316; Glinka, et al. (2011) EMBO Rep. 12:1055-1061; Wang, et al. (2013) Genes Dev. 1339-1344). In the present analysis, it was found that only Lgr4 was highly expressed in macrophages. Depletion of Lgr4 in macrophages abrogated Rspondin3-induced expression of the anti-inflammatory marker CD206 and reversed Rspondin3- induced suppression of the pro-inflammatory marker CD86 (Table 17).

TABLE 17

Data are from three independent experiments with n=3 samples per group. Statistical significance was determined by two- way ANOVA Tukey's multiple comparisons using GraphPad Prism. P values (left to right) are: CD206 siNC ( ****P<0.0001; ****P<0. 0001); CD206 siLrg4 (NS, P=0.9520; NS, P=0.8256); CD86 siNC (*P=0.0286; ****P<0 .0001); CD86 siLrg4 (NS,

P=0.9252; NS, P-0.0619). siNC, nontarget control short interfering RNA.

[0092] It was also observed that Lgr4 depletion dampened Rspondin3-induced gene expression of anti-inflammatory markers and promoted activation of pro-inflammatory genes in BMDMs in response to LPS (Table 18). Furthermore, depletion of Lgr4 augmented the release of pro-inflammatory cytokine IL-lp in BMDMs in response to LPS and prevented the release of anti-inflammatory cytokine IL-10 induced by Rspondin3. These together, indicated the reguisite role of LGR4 in mediating Rspondin3-induced macrophage phenotype transition.

TABLE 18

[0093] It was found that Rspondin3 activated Wnt signaling in BMDMs in a LGR4-dependent manner. To investigate the role of LGR4 in lung macrophages in vivo, its expression was evaluated in IMs, AMs and other myeloid cells using CyTOF. This analysis indicated that LGR4 was highly expressed in IMs (MFI = 1.15) as opposed to AMs (MFI = 0.02) and other myeloid populations. In vivo, with macrophage-specific Lgr4 knockout mice (herein called Lgr4^Mφ-/)-) , it was found that Lgr4^Mφ-/)- mice showed exacerbated inflammatory lung injury responses as compared to WT mice induced by sublethal LPS challenge, as measured by lung MPO activity and albumin-Evans blue dye tracer assessment of lung vascular permeability. These changes could not be rescued by i.v. Rspondin3 as was the case in WT mice. The number of IMs in Lgr4^Mφ-/)- mice also failed to increase in response to endotoxemia by i.v. Rspondin3. Furthermore, expression of anti-inflammatory markers and pro- inflammatory markers in Lgr4^Mφ-/)- mice and control mice during endotoxemia was analyzed by CyTOF. It was found that deletion of Lgr4 prevented the generation of IMs and failed to induce expression of anti-inflammatory markers, which was coupled with marked increases in pro-inflammatory markers in response to endotoxemia; furthermore, these changes were not reversed by i.v. RspondinS. Also, in Lgr4^Mφ-/)- mice, Rspondin3 failed to induce Wnt-β-catenin signaling in IMs as determined by measuring active b-catenin (nonphosphorylated b-catenin). Taken together, these data showed LGR4 is the receptor in IMs required for Rspondin3-mediated reprograming of macrophages and Rspondin3-mediated attenuation of inflammatory lung injury.

Example 5: b-Catenin Signals Rspondin3-Induced Macrophage Transition

[0094] Rspondin family proteins activate both b-catenin- dependent canonical Wnt-β-catenin signaling and b-catenin- independent noncanonical Wnt signaling (Glinka, et al. (2011) EMBO Rep. 12:1055-1061; Scholz, et al. (2016) Dev. Cell. 36:79-93). It was subsequently determined whether b-catenin was essential for Rspondin3-mediated generation of IMs a.nd prevention of inflammatory lung injury. Mice with a genetic deletion of Ctnnbl (encoding b-catenin) in macrophages (Lyz2- cre⁺;Ctnnbl^fl/fl herein called Ctnnbl^Mφ-/-) and Ctnnbl^fl/fl (WT) were generated. Ctnnbl expression was measured in BMDMs prepared from Ctnnbl^Mφ-/-mice and WT mice and active b-catenin was measured in IMs in vivo by CyTOF. It was observed that deletion of Ctnnbl in macrophages abrogated Rspondin3-induced expression of the anti-inflammatory marker CD206 and instead increased expression of the pro-inflammatory marker CD86. It was also found that deletion of Ctnnbl in macrophages suppressed expression of the anti-inflammatory genes induced by Rspondin3 (Mrcl, Argl, Retnla, Chil3) and promoted induction of pro-inflammatory marker genes in BMDMs in response to LPS challenge (Cd86, Illb, Tnf, Cxcll). ELISA confirmed that deletion of Ctnnbl augmented the release of the pro-inflammatory cytokine IL-Ib in BMDMs in response to LPS and prevented the release of anti-inflammatory cytokine IL-10 by Rspondin3. In vivo, the number of IMs in Ctnnbl^Mφ-/- mice failed to increase in response to endotoxemia, and this failure could not be rescued by i.v. Rspondin3 as it was in the WT mice. Furthermore, Ctnnbl^Mφ-/- mice showed a failure to induce expression of anti-inflammatory markers in lung IMs, whereas it concomitantly increased the expression of pro- inflammatory markers in response to endotoxemia; this could not be reversed by i.v. Rspondin3. It was found that Ctnnbl^Mφ-/- mice demonstrated augmented inflammatory lung injury and mortality in response to endotoxemia, which could not be rescued by i.v. Rspondin3. Thus, these results showed that macrophage b-catenin was required for Rspondin3-induced IM phenotype transition and Rspondin3-mediated attenuation of inflammatory lung injury.

Example 6: Rspondin3 Reprograms Metabolism in Macrophages [0095] To address whether the Rspondin3-induced macrophage phenotype transition involved a specific metabolic reprogramming, a metabolic flux assay was carried out to detect changes in the mitochondrial oxygen consumption rate (OCR) and rate of extracellular acidification (ECAR), as measures of OXPHOS and glycolysis, respectively. In BMDMs cultured in full DMEM containing 10mM glucose, 2mM glutamine and 2mM sodium pyruvate, it was observed that Rspondin3 markedly increased mitochondrial respiration as measured by the basal OCR and the spare respiratory capacity (SRC), which is assessed by mitochondrial uncoupling. At the same time, Rspondin3 treatment reduced basal ECAR. It was also found that Rspondin3 treatment of LPS-activated BMDMs was able to reverse the metabolic phenotype of pro-inflammatory macrophages by preventing upregulation of glycolysis and by restoring mitochondrial oxygen consumption. To determine the precise carbon source used by Rspondin3-increased mitochondrial respiration, DMEM containing only glucose, glutamine or free fatty acids as substrates was used for the metabolic assay. This analysis indicated that Rspondin3 failed to increase mitochondrial OCR in macrophages exposed to either glucose or free fatty acids as substrates, whereas Rspondin3 markedly increased OCR with glutamine as the sole substrate. It was also observed that Rspondin3 increased the activity of glutaminase, which is responsible for glutaminolysis and induced gene expression of glutaminase (Gls). Thus, these results showed that the Rspondin3-induced anti-inflammatory switch in macrophages involved increased OXPHOS via glutaminolysis. Next, using macrophages prepared from Lgr4^Mφ-/- mice and Ctnnbl^Mφ-/- mice, it was observed that deletion of Lgr4 or Ctnnbl abrogated Rspondin3-induced changes in OCR and ECAR. Rspondin3-mediated increases in the activity of glutaminase and levels of Gls were prevented in macrophages from Lgr4^Mφ-/- mice and Ctnnbl^Mφ-/- mice. Thus, Rspondin3-mediated reprogramming of macrophages required the activation of glutaminolysis via LGR4/β-catenin signaling. Example 7: Rspondin3 Induces Epigenetic Reprogramming Through <x-Ketoglutarate-TET2 to Activate Anti-Inflammatory Transition

[0096] Glutaminolysis induces the generation of a- ketoglutarate, which serves as the cofactor for DNA hydroxymethylation catalyzed by TET methylcytosine dioxygenase or histone demethylation catalyzed by JMJD3 demethylase (Liu, et al. (2017) Nat. Immunol. 18:985-994; Carey, et al. (2015) Nature 518:413-416; Yang, et al. (2016) Cell Metab. 24:542-554). It was therefore determined whether the Rspondin3-mediated increase of a-ketoglutarate generation induced epigenetic reprogramming in DNA hydroxymethylation or histone methylation. Measurements of TET and JMJD3 activities using nuclear extracts from macrophages with or without Rspondin3 stimulation showed that Rspondin3 increased TET activities, whereas there was no effect on JMJD3 activities. As the TET enzymes induce DNA hydroxymethylation and open chromatin and activate gene expression in the associated loci (Deplus, et al. (2013) EMBO J. 32:645-655), it was determined whether Rspondin3 induced DNA hydroxymethylation in macrophages. Global 5-hydroxymethylcytosine (5hmC) measurements showed that Rspondin3 markedly increased 5hmC levels and prevented LPS-induced 5hmC downregulation in macrophages. Furthermore, 5hmC DNA immunoprecipitation followed by qPCR (hMeDIP-qPCR) used to detect 5hmC enrichment on specific gene loci showed notable enrichment of 5hmC within the proximal promoter regions of anti-inflammatory genes (Mrcl, Argl, Chil3 and Retnla). Chromatin immunoprecipitation (ChIP) with an antibody specific for H3K4me3, the active histone marker for open chromatin regions and transcription active genes, followed with qPCR (ChIP-qPCR) targeting anti- inflammatory gene promoters {Mrcl, Argl, Chil3 and Retnla) also showed that Rspondin3 activated the chromatin state on these gene loci. However, in macrophages obtained from Lgr4^Mφ-/-mice or Ctnnbl^Mφ-/- mice, in which Lgr4 or Ctnnbl was deleted in macrophages, it was observed that Rspondin3 failed to increase α-ketoglutarate levels, TET activity and 5hmC levels as well as activation of anti-inflammatory genes. Thus, the Rspondin3-induced metabolic shift enhanced α- ketoglutarate generation in macrophages, which activated the epigenetic program to catalyze DNA hydroxymethylation by TETs that utilize a-ketoglutarate as a cofactor.

[0097] Using macrophage Tet2 knockout mice (Lyz2- cre⁺;Tet2^fl/fl, herein called Tet2^Mφ-/-), it was observed that the number of IM in Tet2^Mφ-/- mice failed to increase in response to endotoxemia and they could not be rescued by i.v. Rspondin3. Tet2^Mφ-/- mice also showed a failure of IMs to induce expression of anti-inflammatory markers, while markedly increasing expression of pro-inflammatory markers in response to endotoxemia, which could not be rescued by i.v. Rspondin3. Tet2 deletion in macrophages exacerbated inflammatory lung injury induced by endotoxemia, which was not rescued by i.v. Rspondin3. It was observed that Tet2 deletion prevented Rspondin3-induced DNA hydroxymethylation in macrophages. It was also observed that b-catenin translocated to the nucleus of macrophages and markedly increased nuclear TET2/p-catenin interaction as revealed by proximity ligation assays. Therefore, in addition to the role of a-ketoglutarate serving as the cofactor for TET2, direct nuclear β-catenin/TET2 interaction may contribute to epigenetic reprogramming. These data demonstrated that Rspondin3 epigenetically reprogrammed macrophages through TET2-catalyzed DNA hydroxymethylation as a conseguence of enhanced glutamine metabolism, which provided a-ketoglutarate as the necessary cofactor mediating macrophage phenotype transition. [0098] Taken together, these results show that TET2, the primary enzyme catalyzing DNA hydroxymethylation with α- ketoglutarate as cofactor, was essential for reprogramming macrophages and thereby prevented inflammatory lung injury through the activation of Rspondin3-induced metabolic- epigenetic programs in macrophages.

Claims

What is claimed is :

1. A method for treating an inflammatory condition comprising administering to a subject in need thereof an effective amount of a Rspondin3 agonist thereby treating the subject's inflammatory condition.

2. The method of claim 1, wherein the inflammatory condition is a cardiovascular disease, cancer, inflammatory lung condition or autoimmune disease.

3. The method of claim 2, wherein the inflammatory lung condition is acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS/VILI-induced ALI, trauma-induced acute lung injury (TIALI) and brain injury, or radiation-induced lung injury .

4. The method of claim 1, wherein the Rspondin3 agonist is a Rspondin3 polypeptide or Lgr4-binding fragment thereof, or polynucleotide encoding the same.

5. A method for stimulating the transition of macrophages into anti-inflammatory macrophages comprising contacting the macrophages with an effective amount of a Rspondin3 agonist thereby stimulating the transition of the macrophages into anti-inflammatory macrophages.

6. The method of claim 5, wherein the macrophages are interstitial macrophages.

7. The method of claim 5, wherein the Rspondin3 agonist is a Rspondin3 polypeptide or Lgr4-binding fragment thereof, or polynucleotide encoding the same.

8. A method for treating an acute inflammatory lung condition comprising administering to a subject in need thereof an effective amount of a Rspondin3 agonist thereby treating the subject's acute inflammatory lung condition.

9. The method of claim 8, wherein the acute inflammatory lung condition is acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS/VILI-induced ALI, trauma-induced acute lung injury (TIALI) and brain injury, or radiation-induced lung injury.

10. The method of claim 8, wherein the Rspondin3 agonist is a Rspondin3 polypeptide or Lgr4-binding fragment thereof, or polynucleotide encoding the same.