{'fraction_non_alphanumeric': 0.031160963320637017, 'fraction_numerical': 0.01772485979066071, 'mean_word_length': 5.108078402637846, 'pattern_counts': {'":': 0, '<': 13, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Type 2 immunity induced by bladder extracellular matrix enhances corneal wound healing

Extracellular matrix particulate stimulates local and peripheral type 2 immune response to enhance corneal wound healing.


INTRODUCTION
The cornea is the outermost layer of the eye and is therefore suscep tible to trauma and injury. Approximately 3% of all emergency de partment visits are due to eye injury, and most of these involve the cornea (1). When corneal transparency is compromised because of injury and the resulting scar formation, keratoplasty is considered to be the most effective therapy to restore vision. Keratoplasty, or cornea transplant, requires donor tissue and surgery with the asso ciated risks and morbidity. New approaches to improve wound healing and reduce scar formation would obviate the need for corneal trans plantation. The concept of regenerative immunology and targeting immune cells to improve regeneration is being applied to numerous tissues but has not yet been applied to the cornea.
When the cornea is damaged, injured epithelial cells release cyto kines including interleukin 1 (IL1) and tumor necrosis factor- (TNF), which induce stromal cell apoptosis while simultaneously recruiting immune cells such as neutrophils, macrophages/monocytes, and lymphocytes. These immune cells rapidly infiltrate into the limbal stroma, reaching a peak level around 12 hours with continued inflammation lasting more than 1 week (2). These cells produce various cytokines and growth factors such as interleukins and transform ing growth factor- (TGF) (3). Several cell types reside in healthy corneal stroma including quiescent keratocytes, resident immune cells, and nonmyelinating Schwann cells (4). These resident stromal cells are activated by the cytokines and chemokines produced by epithelial cells and immune cells and subsequently proliferate and migrate to the wounded region. At the wound site, they produce extracellular matrix (ECM) to repair the damaged cornea (5,6). The transition of quiescent stromal cells to myofibroblasts is presumably mediated by TGF (3,7). The overproduction of matrix by myo fibroblasts results in an unorganized deposition of ECM proteins such as type III collagen that leads to fibrosis and, eventually, scar formation (8). Crosstalk between immune cells recruited to the wounded tissue, activated resident immune cells, and stromal cells controls the delicate balance between regenerative repair that main tains corneal clarity and scar formation that causes loss of vision. Several approaches are currently used to regulate corneal immune responses to reduce corneal scarring, including conventional steroid treatment and cellular therapies to stimulate stromal regeneration (9,10). Steroids suppress immune responses to decrease inflamma tion and reduce scar formation following corneal injuries. However, prolonged steroid use results in complications including glaucoma and cataracts (11,12). Cornea limbal and stromal stem cell thera pies have produced promising results in animal models and clinical studies (13)(14)(15). However, the stem cell production and cost may limit largescale application.
Tissuederived decellularized ECM is a complex biological bio material that includes structural proteins and biological cues that promote tissue repair. Human and porcine ECM manufactured from a variety of tissue sources such as skin, heart, intestine, bladder, and adipose have shown tissue repair efficacy in preclinical models, clinical testing, and commercial use (16,17). While early studies on ECM scaffolds focused on using the structural cues to promote cell mi gration and stem cell differentiation, more recent investigations re vealed that ECM scaffolds also actively regulate immune responses and create a proregenerative immune environment when implanted to damaged tissue (18,19). These biomaterials actively participate in immune cell recruitment and activation that help direct the tissue repair process (20). For example, Brown et al. (21) identified pro regenerative macrophages that responded to ECM scaffolds and demonstrated that they were required for the scaffolds to promote skeletal muscle regeneration (22). In addition to macrophages, the ECM biomaterials required CD4 + T helper type 2 (T H 2) cells and IL4 production to stimulate tissue repair (23). Porcine urinary bladder-derived matrix (UBM), a commercially available ECM product, increases the number of IL4-producing cell populations recruited into wounded tissues (24,25). The impact of IL4 and type 2 immune responses on corneal wound healing has not yet been in vestigated from an immunoengineering standpoint.
While type 2 cell-mediated immunity emerged as a regulator of wound repair in tissues such as muscle and lung (26,27), there has been minimal application of these therapies in treating corneal wounds and regulating fibrosis in the eye. This gap presents an opportunity to design immunomodulatory therapies to enhance regenerative repair that restores tissue function and, in the cornea, reduce haze formation so that vision is maintained. Our previous study reported that ECM microparticles applied as a wound dressing for injured rabbit corneas reduced the inflammatory reaction and scar forma tion. However, the underlying mechanism of the immune response to ocular injury and repair was yet to be revealed (28). In the current study, we applied different UBM particulate formulations to wounded corneas and investigated the immunological and corneal wound healing responses to the treatment. Results demonstrated that UBM treat ment enhanced wound healing and reduced scar formation after injury via IL4 signaling predominantly from eosinophils in the cornea. Further, UBM particle size could be tuned to modulate the level of this response. Instead of suppressing the immune response to cornea damage via traditional steroid treatment, we use the immune response and direct it toward a desired regenerative phenotype.

UBM treatment reduces corneal scarring
Application of biological ECM scaffolds to the cornea presents a number of challenges including the need to maintain vision and ad equate retention in the context of the tear film and fluid flow. We tested formulations of UBM in the ocular environment, including micro and ultrafine (UF)scaledscaled UBM materials.
We first evaluated the physical properties and degradation kinet ics of the microUBM and UFUBM particulate. Morphologically, the microUBM particulate appeared crystalline by scanning elec tron microscopy (SEM) imaging. The UFUBM particulate had a more granularlike shape (Fig. 1A). The microUBM material had a relatively consistent size distribution, with most of the particulate ranging between 100 and 1000 m (Fig. 1B). Most of the UFUBM particulate ranged between 1 and 50 m. We examined the biode gradability of the UBM scaffolds in an enzymerich controlled envi ronment in vitro, to assess potential correlation between particulate size and expected resorption time in vivo. The microUBM showed a lower degradation rate compared to the UFUBM as measured by loss of weight percentage loss (Fig. 1C).
We then investigated whether treatment with UBM could enhance wound repair and reduce scar formation in the cornea. First, we labeled the materials with Alexa Fluor 790 to track retention in the ocular space after injection into the subconjunctival space using near infrared imaging. Athymic (nude) mice were used for these studies so the dye could be imaged without an autofluorescent background from the fur. Consistent with the in vitro degradation assay, micro UBM displayed longer retention in the subconjunctival space com pared to UFUBM as measured by nearinfrared imaging (Fig. 1D). In addition, we found that the scaffolds remained highly localized in the injection site and did not diffuse.
To evaluate the impact on wound healing in the cornea, microUBM and UFUBM were applied to a corneal debridement wound model in mice. In this wound model, a 1.5mmdiameter region in the central cornea is scraped to remove the epithelium leading to scar formation. The region of scar formation after injury was identified by photography (Fig. 1E, red dashed line). After blinded quantification of the corneas, we observed that UBM treatments (both microUBM and UFUBM) significantly reduced the haze formation compared to a phosphatebuffered saline (PBS) control (P < 0.0001) (Fig. 1, E and F), suggesting a reduction in scar formation.

UBM injection inhibits myofibroblast differentiation
To further characterize the haze and scar formation after corneal injury, we evaluated vascularization and fibroblast development. Immunofluorescent staining of the injured cornea for CD31 re vealed the vasculature in the limbus area after injury and treatment. In comparison with the relatively dim staining of CD31 in no injury controls, PBStreated corneas demonstrated an increase of vascula ture in the limbus area at 1 and 2 weeks after surgery, consistent with chronic inflammation and possible neovascularization. Micro UBM-treated corneas showed some increase of vasculature in the limbus area at 1 week after surgery, but by week 2, the extent of vascularization decreased. The vasculature in the UFUBM group remained low at both 1 and 2 weeks after injury ( Fig. 2A). We next stained smooth muscle actin (ɑSMA) to assess the myofibroblast activation in the corneal stroma. During wound healing, the ɑSMA expressing myofibroblasts are the key contributors to pathological scar formation (29,30). At 2 weeks after surgery, PBStreated corneas showed a higher level of ɑSMA + staining (indicated by white arrows), suggesting an increase in myofibroblasts as compared to micro and UFUBM treatments (Fig. 2B).
To further investigate the myofibroblasts, we used flow cytometry to characterize different fibroblast subsets. We applied a gating scheme to identify three primary corneal stromal cell lineages using the markers CD29 + ɑSMA + , CD29 + S100 + , and CD29 + SCA1 + (fig. S1). CD29 (integrin) is a broad marker for multiple cell types that is highly expressed in stromal cells. Because of the lack of precision around fibroblastspecific markers, we used CD29 (as positive marker) in combination with CD45, CD31, CD11b, and CD34 as exclusion markers. Different subsets of fibroblast may drive different patho logical consequences, some of which are immune dependent. For example, ɑSMA is a classic marker for myofibroblasts, which func tion in fibrosis, and has been associated with excessive deposition of haze and scar formation (31). S100 is linked with angiogenesis and tumorigenesis (32,33), whereas Sca1 (Ly6a) is associated with im mature or progenitor mesenchymal stromal cells (34).
At 2 weeks after injury, we found a greater density (more than 50%) of corneal stromal cells (CD45 − CD31 − CD34 − CD29 + ) in the wounded cornea without treatment ( fig. S1). UFUBM treatment significantly reduced the CD29 + S100 + cell population (P < 0.01), and CD29 + ɑSMA + cells significantly decreased with both micro and UFUBM treatments (PBS versus microUBM P < 0.05, PBS versus UFUBM P < 0.01). CD29 + Sca1 + fibroblasts did not change with UBM treatment (Fig. 2C). To further investigate fibrotic changes of wounded corneas with different treatments, we performed NanoString analysis using the nCounter Fibrosis Panel. Consistent with our earlier findings with flow cytometry where fibroblastassociated cell surface markers decreased with treatment, the expression of fibrosisassociated genes also significantly decreased with UFUBM treatment compared to the PBStreated control (Fig. 2, D and E). We performed a differen tial expression analysis of the NanoString data and found that UBM treatments downregulated a large set of fibrosisassociated genes when normalized to PBStreated control (Fig. 2, C and D). Markedly, UFUBM treatment demonstrated statistical significance in downregulated genes including Col3a1and Acta2, which are closely associated with scarrelated extracellular deposition and myofibroblast activation (35) (P < 0.05). The stromal cells in both UBM treatment groups expressed lower levels of the fibroblast acti vation markers Fap and Acta2, as well as decreased expression of ECM proteins (Col3a1, Eln, and Col14a1) and angiogenesis mark ers (Tek and Pdgfb) (Fig. 2, C and D).

UBM treatment increases type 2 immune response
Biological scaffolds applied to the cornea wounds induced a type 2 immune response characterized by the presence of T H 2 cells, eosin ophils, and alternatively activated (M2) macrophages (23,36). This type 2 immune polarization is perpetuated by IL4 production, which initiates a program of tissue repair (37). Using flow cytometric analysis of cells isolated from the injured corneas, we found that IL4 expression significantly increased in corneal T cells and eosin ophils at 1 week after injury after both micro and UFUBM treatments (T cells: PBS versus microUBM P < 0.01, PBS versus UFUBM P < 0.05; eosinophils: PBS versus microUBM P < 0.001, PBS versus UFUBM P < 0.05) (Fig. 3, A and B). The response was more pro nounced in microUBM compared to UFUBM at the time point analyzed, which might be due to the degradation difference between the two materials. We did not observe any significant phenotypic changes in other immune cells in the wounded corneal environment (figs. S2 and S3).
We also evaluated the expression of cytokines in the draining lymph node (cervical) and found that IL4-producing T cells (T H 2) significantly increased with micro and UFUBM treatments com pared to PBStreated controls at 1 week after injury, highlighting a crosstalk between local immune cells and those residing in periph eral lymphoid tissues (PBS versus microUBM P < 0.01, PBS versus UFUBM P < 0.05) (Fig. 3, C and D). At 2 weeks after injury, the total number of IL4-producing T cells in the cervical lymph node decreased compared to levels found at 1 week after injury. In the UFUBM group, the IL4-producing T cell numbers reduced to levels similar to the PBS group, while in microUBM-treated cor neas, the IL4-producing T cells remained elevated (P < 0.001). (Fig. 3, E and F). UBM treatments decreased interferon-positive (IFN + )-producing T cells (T H 1) compared to the PBStreated con trol at 2 weeks after surgery (Fig. 3, D to F). UBM treatments did not show a significant impact on the number of IL17 + -producing T cells (T H 17) in the draining lymph nodes.

Eosinophils play critical roles in UBM-suppressed corneal scarring
Given that eosinophils were a major source of IL4 in the cornea with UBM treatment (Fig. 3), we sought to determine whether these cells played a role in corneal wound healing and biomaterial re sponse. UBM treatments were applied to cornea wounds in Gata1 −/− mice that lack eosinophils. The proregenerative effect of the UBM biomaterials was completely ablated in the Gata1 −/− mice with iden tical scar formation and haze development regardless of UBM treat ment (Fig. 4, A to C). Immunofluorescence analysis confirmed no reduction of vasculature in UMBtreated Gata1 −/− mice, suggesting that UBM enhanced corneal repair in an eosinophildependent  (Fig. 4B). Furthermore, there was no increase in IL4 +producing T cells in the draining lymph nodes in Gata1 −/− mice, sug gesting the importance of crosstalk between eosinophils residing in the local tissue and T cells in the peripheral secondary lymphoid organs (Fig. 4D). We further characterized UBMassociated myofi broblasts in eosinophildepleted mice using flow cytometry. There was no reduction in CD29 + ɑSMA + fibroblast activation in Gata1 −/− mice regardless of UBM treatments (Fig. 4E). Together, these ob servations supported the notion that UBM promotes an eosinophil mediated type 2 immune response, which subsequently enhances corneal wound healing.

DISCUSSION
The concept of immune privilege in the eye refers to minimal im mune infiltration and inflammatory responses to preserve vision and maintain ocular architecture (38). This immune privilege pro tects the eye from swelling or other tissue changes caused by minor inflammatory response, so visual function is maintained. However, there is the Yin and Yang of immune privilege in the eye (39). The lack of blood vasculature and lymphatics limits the local immune cell populations; the absence of major histocompatibility complex class II + antigen-presenting cells negates CD4 T cells (40); and the local production of immunosuppressive factors such as TGF suppresses the inflammatory responses further (41). In the con text of infection or injury, proregenerative immune cells such as alternatively activated macrophages and T H 2 cells are critical to tis sue regeneration (23,41). Lacking natural activation of these cells in the eye, injuries to the cornea without intervention may lead to scar formation that impairs vision (39,42).
Biological therapies have been developed to modulate corneal immune response and improve corneal regeneration after injury, including stem cell and ECM therapies (13,28). ECM components from decellularized tissues can influence the immune response locally and systemically to facilitate wound healing processes such as an giogenesis, stem cell proliferation, and stimulating antiinflammatory responses (43,44). UBM particulate in the present study has a highly complex compositional profile according to a previous proteomic analysis (36). Most ECM components such as collagen I, III, IV, VII, and laminin survived after decellularization processes. NonECM residuals in the UBM can act as damageassociated molecular pat terns (DAMPs) to modulate immune response and promote wound healing (45). Direct implantation of UBM materials drives type 2 immune response by actively recruiting alternatively activated mac rophages, IL4-producing CD4 + T cells, and eosinophils (24), cre ating a proregenerative microenvironment for tissue regeneration in, for example, muscle tissue (36). The natural degradation of UBM also releases bioactive components, including ligands, cryptic pep tides, and matrixbound nanovesicles (MBVs) (46,47). These com ponents are slowly released to the environment during degradation, actively presenting biological cues such as small RNAs and miRNAs that can have a prolonged proregenerative impact on the host (46).   Cryptic peptides promote in vivo recruitment of progenitor cells in a murine digit amputation model (48). MBVs protect retinal ganglion cell death and axon degeneration against ischemiainduced injury and downregulate proinflammatory gene expressions including IL1, TNF, and IL6 (49). In our study, we directly injected UBM particulate of micro and UFscales into the subconjunctival space. Proteinase in the tear film likely gradually degraded the UBM par ticulate, releasing bioactive factors that contributed to modulation of the immune response.
Particle size has been extensively studied in the context of bio materials development for drug delivery (50,51). One of the advan tages of UFUBM particulate over microUBM particulate in the cornea application is their small size which allows them to be easily applied to the eye without altering vision while still remaining immunologically active. The smaller size allows more diffusion and relatively faster degradation and is potentially more desirable for immune recognition. As compared to microUBM, UFUBM suspension presents an ef fective mode of drug administration and a reasonable residence time (about 7 days) on the ocular surface after subconjunctival injection, suggesting UFUBM potential for ophthalmic applications. We found that injection with UBM induced a potent type 2 immune response consisting of IL4-producing eosinophils in the local corneal tissue and T H 2 cells in the peripheral draining lymph node. This upregulation of type 2 immune response remained for more than 14 days in the microUBM group, while in the UFUBM group, this upregulation peaked at day 7 then decreased to pretreatment levels. Ablation of eosinophils using Gata1 −/− mice diminished the prohealing outcome regardless of UBM treatment, suggesting that eosinophils were re quired for UBMfacilitated corneal wound healing.
During the tissue repair process, stromal cells proliferate and synthesize new ECM to repair and replace damaged tissue (52). If these cells are not regulated, they develop into a pathological posi tive feedback loop where more and more ECM is deposited. Espe cially in the cornea, excessive deposition of ECM can cause severe haze and vision impairment (53). Our results suggest that myofi broblast differentiation in the cornea after injury was orchestrated by these immunological shifts induced by the UBM microenviron ment. Early studies of stromal cell activation often rely on cell mor phology, and it is challenging to define stromal cell phenotypes due to a lack of definitive markers (54,55). We subset stromal cells into three distinct populations: CD29 + ɑSMA + , CD29 + S100 + , and CD29 + SCA1 + . We found that UBM inhibited CD29 + ɑSMA + myofi broblasts and CD29 + S100 + fibroblasts, whereas CD29 + SCA1 + pop ulations remained mostly unchanged. Evaluating gene expression using the NanoString fibrosis panel enabled us to gain a physiolog ical understanding of corneal cell, including stromal and epithelial cell expression patterns after injury. Significantly reduced expres sion of fibrosis genes, such as Acta2, Col3a1, and Eln, suggests a less activated myofibroblastic phenotype of corneal stromal cells in the UFUBM-treated group.
A major endeavor in the field of regenerative immunology has been to understand how different immune cells respond and com municate locally and systemically after biomaterial implantation. Our data demonstrated an immune crosstalk between eosinophils in the local tissue and T cells in the peripheral lymphoid organ. Eo sinophils can be seen at the corneal periphery near the limbus area (56). Eosinophils are equipped with various tissue damage sensing mechanisms, including DAMPs, which allow them to release tissuerepairing molecules and stimulate the adaptive immune responses (57). Previous studies found eosinophil depletion-impaired muscle and liver repair in a murine model (58,59), revealing the potentially critical role of IL4-secreting eosinophils in tissue repair and regeneration. IL4 is a multifunctional, pleiotropic cytokine that is the signature of T H 2 response. Emerging evidence suggests that IL4 and T H 2 responses are essential for tissue regeneration (60). Although T H 2 response and eosinophils have been extensively re ported in ophthalmology, they are often associated with an allergic response, keratitis, or dry eye (61,62). To our knowledge, the pro regenerative role of eosinophils or type 2 immune response has not been reported in ocular repair and regeneration. In this study, we showed that eosinophil's involvement in the cornea was necessary for orchestrating the adaptive immunity in UBM microenvironment and polarization of T H 2 cells. Detailed analysis of immune cells showed that IL4 was highly upregulated in the cornea and draining lymph nodes in wildtype (WT) mice treated with UBM. However, ablation of eosinophils prevented the differentiation of T H 2 cells, suggesting that they play a critical role in the initiation of this process. The con nection between eosinophils and adaptive immunity in the cornea is unknown. There is evidence that eosinophils and dendritic cells act in concert to activate and polarize T cells in the draining lymph node (63). Another possible explanation is that eosinophils in the cornea produce the T cell-polarizing cytokine IL4, which is drained to the peripheral lymph node and directly induces the differentiation of T H 2 effector cells (64,65). Further research is needed to understand how T H 2 proregenerative and allergic responses differ. Moreover, the diversity of patient health and immune status may affect ther apeutic response. For example, in cases where a patient's immune system is compromised, such as in advanced age, underlying meta bolic disorder, or immunosuppressant drug therapies, the composi tion and quality of the therapeutic immune response will be altered. As a result, the response to UBM treatment in these patient popula tions may be diminished. Further research into how the underlying immune status of patients affects response to regenerative immuno therapies will be critical to understand as these technologies reach more widespread clinical use.

Characterization of UBM materials
The microUBM and UFUBM were provided by ACell Inc. UBM is derived from decellularized basal and tunica propria layers of porcine urinary bladder tissue and includes a complex mixture of intracel lular and extracellular proteins (36). Lamina propria and epithelial basement membrane layers were isolated from porcine bladder and processed according to proprietary internal ACell Inc. procedures. The UBM was then lyophilized and milled processed into microUBM or UFUBM particulate that are amenable to delivery into the cor nea by injection into the conjunctival space. The UBM materials were terminally sterilized using electron beam irradiation method. Scanning electron microscopy A thin layer of UBM particulate was mounted on adhesive metallic tape and sputter coated with 3.5 nm of gold. The samples were viewed with a JEOL JCM6000 (JEOL Ltd.) SEM at magnifications ranging from 20× to 500×. Particle size analysis The relative particle size distribution was measured for each sample over the particle size range of 0.1 and 1000 m (Mastersizer, Malvern Instruments Ltd.).

Material degradation
An in vitro single enzyme (Proteinase K) test model was developed to investigate potential differences in relative degradation rates be tween experimental groups. Approximately 20 mg of microUBM and UFUBM was weighed and then transferred to 2ml protein LoBind tubes. A working concentration of ProteinaseK (0.415 mg/ml) (SigmaAldrich) enzyme in 1× PBS was used for sample degrada tion. Samples were incubated in a dry oven at 37°C for 2 and 5 hours. At the designated time point, samples were centrifuged at 4°C at 1500 xg for 5 min. All fluids were extracted from the samples and discarded. The degraded sample fibers found at the bottom of the tube (pellet) were washed with 1 ml of water and centrifuged again at 1500 xg for 5 min to remove additional salts. The remaining fluid was then discarded, and samples were dried overnight for weight measurement using analytical balance.

Murine corneal debridement wound model and treatment
All surgical procedures were performed under the guidelines of the Johns Hopkins University Animal Care and Use Committee, protocol number: MO17M308. Male adult BALB/C mice (8 to 12 weeks old), Gata1 knockout (Gata1 −/− ) mice, and IL4IRESeGFP (4get) mice were purchased from the Jackson laboratory (ME, USA). All exper imental groups had a sample size of n ≥ 3.
The murine corneal wound model was adapted from a previous study (66). Mice were weighted and anesthetized with ketamine HCl (90 mg/kg) (Vetone, ID, USA) and xylazine HCl (10 mg/kg) (Vetone) injection. Proparacaine hydrochloride ophthalmic eye drops (Sandoz, Holzkirchen, Germany) were applied after the mice were sedated. The center area of the cornea was marked by a 1.5mm bi opsy punch, and the epithelium layer was removed within the area by a 1.5mm flat blade (Fine Science Tools, CA, USA). After epithe lium removal, extra force was used on the wounded area using the same flat blade to disrupt the basement membrane, and a vol ume of 50l PBS solution with either no UBM addition or with microUBM or UFUBM (10 mg/ml ) (Acell, MD, USA) suspended was injected to the subconjunctival space of the wounded eye. After injection, drops of PBS solution were applied to both wounded and unwounded eyes to keep the eyes moist until the mice recovered from anesthesia.

UBM resorption in vivo
We tagged the microUBM and UFUBM with Alexa Fluor 790 NHS Ester (Thermo Fisher Scientific, MA, USA). Briefly, UBM (10 mg/ml) was suspended in PBS and sonicated for 60 min. One hundredmicrogram dye was dissolved in 40 l of dimethyl sulfoxide and then added to each UBM suspension. The suspensions were gently shaken at room temperature for 1 hour and washed three times with PBS. After washing in PBS for another 18 hours at 4°C, the UBM was collected and resuspended in PBS, followed by injection into male adult nude mice (8 weeks old from the Jackson laboratory) subconjunctival space. The labeled UBM was imaged with LICOR nearinfrared scanner on days 0, 3, 7, 14, and 21 after injection.

Scar formation and quantification
At 14 days after surgery, the mice were euthanized, and the eye globes were collected. The picture of each globe was taken under a surgical microscope (Nikon, Japan), and the scar areas and cornea areas were determined with ImageJ. The ratio was quantified as: scar ratio = A s (scar area)/A c (whole corneal area).

Flow cytometry Immune profile in wounded corneas
Wounded corneas were collected from each experimental group (PBS, microUBM, and UFUBM); five corneas were pooled for one "flow cytometry sample." Corneas in each group were digested in RPMI 1640 media containing Liberase TM TL (Thermolysin Low) (0.5 mg/ml) (SigmaAldrich, MO, USA) and deoxyribonuclease I (0.2 mg/ml) (Roche, Basel, Switzerland) for 45 min. Digested tissues were grinded through a 70m filter. Cell suspensions were centri fuged and washed, and each cell pellet was resuspended in 200 l of PBS for staining. The antibodies used are listed in Table 1.

Immune profile in draining lymph nodes
Draining lymph nodes (submandibular lymph nodes) were collected and ground through a 70m filter. Cells were collected after centrifu gation and washing with PBS. Lymphocytes were stimulated for 4 hours with cell stimulation cocktail (plus protein transport inhibitors) (eBioscience, CA, USA), followed by staining of surface markers. After permeabilization and fixation of cells, cytokines IL17 and IFN were stained for 4get mice, and IL17, IL4, and IFN were stained for WT and Gata1 −/− mice. The antibodies used were listed in Table 2.

Fibroblast subsets in wounded corneas
Corneas were collected and digested following the methods listed in "Immune profile in wounded corneas". For fibroblast staining, two corneas were pooled as one flow cytometry sample. The antibodies used for fibroblast panel were listed in Table 3.

Immunostaining
Dissected corneas were fixed in 100% methanol at −20°C for 30 min and permeabilized with PBS containing 0.25% Triton X100 (PBST). The cornea samples were blocked with 1% goat serum and 1% bovine serum albumin (BSA) in PBST for 30 min and stained with rabbit antimouse SMA (Abcam, Cambridge, United Kingdom) overnight at 4°C. Following washing with PBST, corneas were stained with goat anti rabbit 633 (Abcam) for 2 hours at room temperature and mounted flat in SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific). Zeiss Apotome microscope was used for fluorescence imaging.

Light sheet fluorescence imaging
Mouse eyes were harvested at 1 and 2 weeks after operation and immediately fixed in 4% paraformaldehyde at 4°C for 16 to 24 hours. CUBIC2. The cleared, stained mouse eyes were wholemounted and visualized using a LaVision BioTec UltraMicroscope II light sheet fluorescent microscope equipped with a LVMIFluor objective at 1× optical zoom. Imaging occurred immediately after mounting in im mersion medium (SigmaAldrich) with a sheet thickness of 5 m (nu merical aperture = 0.154), an axial step size of 2.5 m, an excitation at 561 nm, and a 590/33 nm emission filter. The resulting three dimensional volumes were reconstructed using Imaris software (Oxford Instruments).

NanoString
At day 14 (2 weeks after injury), wounded corneas were harvested and homogenized in TRIzol reagent by an automated tissue homog enizer (Bead Ruptor 12, Omni International). All mRNA was isolated and purified using the RNeasy PLUS microkit system (Qiagen, Hilden, Germany) for NanoString analyses. Samples harvested on day 14 were evaluated by the NanoString Fibrosis panel (XTCSOMFIB212, NanoString Technologies Inc.). One hundred nanograms of mRNA from each sample was added to a barcoded probeset mixture and hybridized for 20 hours at 65°C. Hybridized samples were further processed using the NanoString Prep Station operating under high sensitivity mode, and mRNA target transcripts were counted using the nCounter digital analyzer system (NanoString).

Statistical analysis
The flow cytometry samples were tested using Attune NxT Flow cytometer (Thermo Fisher Scientific). The flow cytometry data were computed using FlowJo v10 software and were displayed as the means ± SD. Multiple t test was performed using GraphPad Prism v8, with P < 0.05 considered statistically significant. NanoString data were analyzed using nSolver software (v3.0, NanoString). The top 50 differentially regulated genes were uploaded into the DAVID functional annotation tool, and Gene Ontology was run to calcu late significantly enriched gene sets.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/7/16/eabe2635/DC1 View/request a protocol for this paper from Bio-protocol.