The histone methyltransferase mixed-lineage-leukemia-1 drives T cell phenotype via notch signaling in diabetic tissue repair

mediated signaling, and that loss of MLL1 in nondiabetic state impairs wound healing via loss of T H


Brief summary:
MLL1, a histone methyltransferase, regulates T-cell differentiation in diabetic wounds, causing pathologic inflammation, and may serve as a therapeutic target to improve diabetic wound healing.

Introduction:
A deliberate and orchestrated response must occur between immune and structural cells to promote optimal tissue repair and regeneration.Pathologic states where tissue repair is impaired are characterized by dysregulated responses in immune cells and/or structural cells.In the setting of diabetes, chronic wounds are in a persistent inflammatory state, unable to transition out of the inflammatory phase to the proliferative phase of wound repair (1)(2)(3)(4).The etiology of these pathologic changes in diabetic immune cells are multifactorial and not completely understood, but epigenetic alterations that occur in the diabetic condition have been shown to control macrophage phenotypes in diabetic wound tissue (2,(5)(6)(7).
Although not as well studied in the setting of wound repair, CD4 + helper T cells (TH) cells are classically thought to play a supportive role in wound healing, influencing other key immune and structural cells present in the wound, and guiding these cells through the phases of wound healing in a regulated fashion (8)(9)(10)(11).
Specifically, circulating CD4 + T cells that infiltrate into the wound tissue following injury are the primary driver of CD4 + T cell dynamics in wound repair (9,12,13).Existing studies are limited, but have demonstrated that depletion of CD4 + cells at days 5-7 in murine wounds decreases wound inflammation and impairs wound healing, which established a critical role of CD4 + cell in orchestrating tissue repair processes (10,(13)(14)(15)(16).The balance of two particular CD4 + phenotypes, the anti-inflammatory regulatory TH cells (Tregs) and proinflammatory IL-17A-producing CD4 + TH17 cells, can independently dictate inflammation in tissues (17,18), and be relevant to complications of diabetes, by which the chronic inflammatory state is often hallmarkedwith high proportions of TH17 differentiation (19)(20)(21).
Despite a well-documented understanding of CD4 + cells and their role in inflammation in many tissues/disease states, surprisingly little is known about these cells in the setting of wound repair, specifically in diabetic wound healing.In the setting of non-pathologic wound repair, Tregs have been shown to prevent proinflammatory macrophage accumulation, and Treg-specific ablation has been shown to delay wound closure in normal mice, but Tregs have not been well studied in diabetic wound tissue (12,22).It is known that diabetic wounds are characterized by delayed, prolonged inflammation, driven in part by high levels of interleukin-17A (IL-17A) secreted in part, by TH17 cells (23,24).The mechanism that controls this imbalance in TH cell subtypes that is often seen in diabetic tissues is not understood.
The fate of TH cell differentiation in tissue is determined via its T cell receptor and cytokine signals (18); however cell-to-cell Notch signaling is also known to play a critical role (25)(26)(27).In humans and mice, there are four Notch receptors (Notch 1-4) and 5 ligands (Delta-like 1, 3, 4, and Jagged 1 and 2).Once bound by its ligand, Notch receptors undergo proteolytic cleavage events that result in the release of Notch intracellular domain (NICD) which translocates to the nucleus and functions as a transcription factor to complex with the DNA binding protein CSL, Mastermind (MAML), and histone acetyltransferases.This, in turn, promotes transcription of downstream Notch target genes (i.e., Hes, Hey).Depending on the context and location, loss of Notch signaling has been shown to both inhibit TH17 differentiation (27)(28)(29)(30)(31) and drive Treg differentiation (32,33).In other instances, Notch signaling protects Tregs from apoptosis and enhances Treg functionality (34)(35)(36).These studies, which were primarily done in vitro, do not provide a succinct likely mechanism of how notch receptor mediated signaling influences CD4 + TH cell phenotypes in wound repair.Furthermore, although Notch signaling has been shown to be important in keratinocyte (37) and Mφ (7) functions in diabetic wounds, Notch signaling has not been studied in T cells in diabetic wounds.Given the prior literature examining other diabetic tissues, we theorized that increased Notch signaling in diabetic wound TH cells is disrupting a physiologic balance between TH17 and Treg cells.
Our group and others have found that epigenetic processes regulate immune cell phenotypes in various tissues/disease states.Mixed-lineage-leukemia-1 (MLL1) is a chromatin modifying enzyme (CME) that upregulates gene transcription via trimethylation of the fourth lysine of the third histone (H3K4me3) at promoter sites, (38) and has been shown to regulate Notch signaling pathways in oncogenesis research (39).The role of MLL1 in wound TH cells is unknown, however in Mφs it has been shown to drive dysregulated inflammation in diabetes (7).Furthermore, alterations to histone methylation by other CMEs have been shown to influence TH cell phenotype in vitro by directly altering key transcription factors for TH17 (RORγ) and Treg (FOXp3) differentiation (40,41).
Here we show that Tregs and TH17 cells both play an important role in physiologic wound repair, and that loss of Notch signaling impairs TH cell differentiation and subsequent healing.We found that MLL1 regulates expression of Notch1 and Notch2 receptors on wound TH cells and serves to increase Notch receptor mediated signaling, and that loss of MLL1 in nondiabetic state impairs wound healing via loss of TH cell differentiation.In human and murine diabetic wounds, we found that Notch signaling is upregulated in TH cells, and that the increased Notch signaling preferentially skews TH cell phenotype towards TH17.We identify that MLL1 is increased in diabetic CD4 + TH cells which increases the TH17 cell phenotype via upregulation of Notch receptor expression and direct regulation of the transcription factor associated with CD4 + TH17cell differentiation, RORγ.Lastly, we show that MLL1 is a potential therapeutic target for nonhealing diabetic wounds, where pharmacologic inhibition of MLL1 reduces TH17 cells and IL-17A based inflammation in the setting of diabetes.

:
Human and Murine Diabetic Wound CD4 + T cells Display Increased Th17 and IL17A.
Given that the balance of Treg/TH17 has been shown in diabetic retinopathy and nephropathy to modulate inflammation, and that TH17 cells have been shown to negatively influence wound repair (23,24) we

Notch signaling is increased in human and murine diabetic wound CD4 + TH cells
In order to define pathways that influence T H cell phenotypes, we analyzed our human skin wound single cell RNA sequencing (scRNA-seq) data for cell/cell interactions and the pathways involved in this signaling.Notch signaling was found to be significantly upregulated in diabetic woundsfor both outgoing and incoming signaling pathways (Fig 2A).Given that our group and others have identified increased Notch ligands in macrophages (Mϕs) from diabetic murine wounds (37,42) and that Notch signaling plays an important role in regulating CD4 We chose to focus on examining Notch1 given the prior work in other inflammatory disease states and tissues linking Notch1 activation and TH17/Treg differentiation (30,(33)(34)(35)41,46) and relative paucity of literature examining Notch2, Notch3, and Notch4 (30,32).In order to examine the role of specific Notch In tissues, Notch receptors are activated by a host of Notch ligands, specific to Notch1 is DLL4 (28,37).
Since it has been previously found that DLL4 enhances TH17 -mediated responses in lung Mϕs, we examined the role of DLL4 ligand in Mϕs, the primary APC in wound tissue, on CD4 + T cell phenotypes in wounds and wound repair following injury (47).To determine if Mϕs play a role as a source of Notch ligand signal for wound CD4 + TH cells, and if DLL4 is important for wound healing, mice with a myeloid cell-specific deficiency of DLL4 (DLL4 f/f Lyz2 cre+ ) were assessed for healing after 4mm punch biopsy as previously described (2).We found that mice with a myeloid cell-specific deficiency in DLL4 demonstrated an impaired rate of wound repair with over Given that we found Notch receptors and downstream notch target genes were impaired in wound CD4 + cells from MLL1 f/f CD4 cre+ mice, we examined wound repair in MLL1 f/f CD4 cre+ mice and found that, Discussion: In this study, we identified that CD4 + TH cell function is important for normal wound repair and that that Notch signaling is crucial for physiologic CD4 + TH cell differentiation in wound repair.We showed that Notch signaling is upregulated in CD4 + TH cell in both human T2D as well murine models of diabetes, which correlates to increased TH cell differentiation to TH17, and supraphysiologic levels of IL-17A.Inhibition of Notch signaling through various methods reduced CD4 + cell differentiation to both Tregs and TH17cells and resulted in impaired wound healing.The epigenetic enzyme, MLL1, was found to be a key regulator of Notch receptors and downstream signaling and thus, wound CD4 + TH cell differentiation.Increased MLL1 in diabetic wound CD4 cells was found to drive TH17 differentiation through Notch signaling.Through treatment of diabetic CD4 + TH cells with a small molecule inhibitor targeting MLL1, pathologic IL-17A inflammation can be reversed.This promises an interesting avenue for further study into a cell-specific therapeutic target to improve wound healing in patients with diabetes.
It has been previously established that TH cell function is relevant in for normal wound repair, (22) and that depletion of CD4 + TH cells in wild type mice causes substaintail changes in wound inflammation and alters wound healing rates.However, the available literature is extremely limited and the regulation of TH cell function/phenotype in the setting of tissue injury has not been examined.Further, although it is known that human and murine diabetic wounds display increased IL17A and an imbalanced TH17/Treg ratio, the mechanism(s) responsible for this have not been identified.Although it is well known that Notch signaling can drive TH cell differentiation and function in other tissues and disease contexts, (25,26,49) the role of Notch signaling on CD4 + TH cell function following tissue injury in a normal or diabetic setting is unknown.Here, we showed that disruptions of Notch signaling to wound TH cells via depletion of MAML, the Notch1 receptor, and adjacent DLL4 ligand on wound Mφs led to significant loss of TH cell differentiation towards Treg and TH17 phenotypes, resulting in impaired healing.This was somewhat expected given that both TH17 and Tregs are likely essential for proper tissue repair (7,44).It was important to establish that loss of Notch signaling, and specifically DLL4/Notch1 mediated, led to the loss of both Treg and TH17 populations in the wound, given how perturbations of Notch can be tissue and cell dependent (32,33,49).It was also valuable to confirm that disruption of TH17 cells due to Notch signaling depletion in the wound correlated strongly to loss of whole wound IL-17A protein; given that other lymphocytes, including neutrophils, have been shown to produce IL-17A (50) and could have potentially maintained IL-17A levels (51).This is the first study to specifically examine Notch signaling in wound CD4 + TH cells following injury and to establish a link between Notch signaling and CD4 + TH cell differentiation in wounds.
Our group has a long history of investigating the pathophysiology behind prolonged pathologic inflammation in diabetic wound repair that contributes to delayed healing.Most of our prior work, as well as the work of many other in this field, has focused on the impact of myeloid cells as well as other structural cells in contributing to dysregulated inflammation and impaired immune responses in diabetic wound tissue that leads to impaired healing (2,6,44).Further, although it is known that dysregulated TH cell mediated inflammation drives other inflammation-associated complications in diabetes, such as retinopathy and nephropathy (20,52,53), the role of CD4 + cell differentiation on inflammation and wound repair has not been established.It has been previously found that IL-17A mediated inflammation is pathologic in diabetic wound healing, however the drivers of this excess IL-17A had not been known (23,24,54).The pathogenesis behind IL-17A mediated inflammation in diabetes is incompletely understood, and here we examined if this increased IL-17A in diabetic wound tissue was driven in part by notch-mediated increased TH17.Some prior research has suggested that Notch signaling is increased in diabetic wounds (7,37), but has not specifically looked at wound CD4 + TH cells before.We found that Notch signaling was increased in human and murine diabetic wounds.
Specifically in wound CD4 + TH cells we found that Notch1 and Notch2 receptors were upregulated and subsequent downstream expression of Notch target genes (Hes1, Hey1, and Hey2) were also increased.
Diabetic TH cells also were found to be exposed to increased Notch ligand; as human diabetic Mφs were found to express increased DLL4 ligand.
Given the increase in Notch signaling seen in diabetic wound TH cells, and prior work showing increased IL-17A in diabetic wounds (23,24,54) as well as increases in TH17 populations in other diabetic tissues (20,52,53), we expected that there would be increased TH17 cells seen in diabetic wounds.We did find this to be true in both human and murine wounds, and we also replicated the expected increase in IL-17A in diabetic wounds.However, despite our findings that loss of Notch signaling corresponds to loss of Tregs in nondiabetic wounds, we found that conversely, the increase in Notch signaling in diabetes did not increase Treg populations in diabetic wounds.In fact, we found that in diabetic wounds, Treg populations were decreased compared to nondiabetic controls.This is not entirely surprising given that prior literature showed an inverse relationship between Notch signaling on Tregs, depending on the context (32,33,49).This may be due, in part to the extensive cross-regulation that occurs between Treg and TH17 cell populations (55).It may be that in diabetic wound TH cells, the increase in Notch signaling drives such an increase in TH17 cells and IL-17A production that this in turn suppresses the Treg population.Given that Notch was increased in diabetic wound CD4 + TH cells, we examined whether inhibiting Notch signaling in diabetic wound TH cells, could reduce the pathologic increase in TH17 cells and IL-17A, thus improving diabetic wound healing.However, we found that total Notch depletion via DNMAML caused a loss of both Treg and TH17 cell populations and actually impaired wound healing.Thus, we wanted to see if we could find a way to reduce Notch signaling but without depleting it to the point it would interfere with physiologic TH cell function and differentiation.
To this end, we examined the regulation of CD4 + TH cell differentiation in the context of notch in wound CD4 + TH cells.Limited prior research in oncology had shown that pathologic changes in Notch signaling were regulated by the histone methyltransferase MLL1 (39); however this relationship had not been evaluated in TH cells or in wound tissue.Since similar CMEs are known to drive TH lineage via direct regulation of the key transcription factors FoxP3 (Treg) and RORγ (TH17), we decided to test MLL1 as a potential regulator of TH cell differentiation in the setting of tissue repair (40).We also knew from our own prior work that MLL1 was increased in diabetes and contributed to pathologic inflammation in diabetic wound Mφs, making it a good candidate to examine in CD4 + TH cells (7).We showed that MLL1 does regulate Notch signaling in wound CD4 + TH cells and thus also the Treg/TH17 ratio.Depletion of MLL1 leads to loss of Notch receptors in wound CD4 + TH cells, via loss of pro-transcription H3k4me3 on the promoters of notch1 and notch2 genes.This correlated strongly with loss of Notch activity as measured by downstream Hes and Hey1.We did identify that ex vivo treatment of CD4 + cells from wounds with an MLL1 inhibitor can reverse pathologic Th17 differentiation, making this a viable therapeutic target in diabetes to reverse TH17 mediated pathologic inflammation.Of note, our research specifically addresses Treg/ TH17 ratios in diabetic skin, but cannot comment on TH cell differentaion and ratios in lymph nodes.Further research is needed in the in vivo setting where a cell-specific targeting method will likely be needed, such as conjugation to CD3 + nanoparticles (56).
In summary, we found that Notch signaling regulates TH cell differentiation in wounds and is necessary for physiologic wound repair.Loss of Notch signaling decreases differentiation of both Tregs and TH17 cells and impairs healing.In diabetic wounds and specifically wound TH cells, Notch signaling is increased; with a corresponding increase in TH17 cells but not Tregs.Broadly inhibiting Notch signaling in diabetes did reduce TH17 populations, but also depleted Tregs, and did not rescue wound repair.Importantly, we identified that the  Sex as a biological variable.Sex was not considered as a biological variable in all reported human data.
Only males were used in the murine studies, since female C57BL/6 mice do not exhibit the DIO phenotype when placed on an HFD.
Murine Model of Diabetes.To induce a 'prediabetic' state, male C57BL/6 mice were maintained on a standard high-fat diet (60% kcal saturated fat, 20% protein, 20% carbohydrate, Research Diets, Inc) for 12-18 weeks to induce the DIO model of T2D as previously described (2).After the appropriate period, high-fat dietfed (DIO) mice developed obesity and insulin resistance with fasting blood sugars in the mid-200s and elevated insulin levels.All animals underwent procedures at 20-32 weeks of age with IACUC approval.For these experiments only male mice were used because female mice do not develop DIO.Number of mice used per experiment can be found in the figure legend of each corresponding experiment.
Murine Wound Healing Model.Mice were anesthetized, dorsal hair was removed with Veet (Reckitt Benckiser) and rinsed with sterile water, and two full-thickness back wounds were created by 6-mm punch biopsy with or without wound splinting.The initial wound surface area was recorded, and digital photographs were obtained daily using an 8 mm iPad camera as previously described (57).Photographs contained an internal scale to allow for standard calibration.The wound area was quantified using ImageJ software and calculated as a percentage of initial wound area.
Magnetic Activated Cell Sorting (MACS) of murine CD4 + T Helper Cells.Briefly, following tissue morcellation, wounds were digested in a suspension of Liberase (50 mg/mL; Sigma-Aldrich, cat 5101020001) and DNase I (20 U/mL; Sigma-Aldrich cat 9003-98-9) solution at 37 °C for 30 min.RPMI + FBS were then added to stopped the reaction, and the cells were filtered through a 100 micron filter and subsequently run through a CD4 + MACS Selection Kit (cat 18952, StemCell Technologies), achieving a 98.4% purity measured by FACS.For splenocytes, no digestion was performed and the morcellated samples were simply filtered through a 40 micron filter before running MACS.For naïve CD4 + cells, a negative selection kit was utilized (cat 19765, StemCell Technologies).
ex vivo naïve CD4 + culture and treatment with MLL1 inhibitor.Wound cell suspensions underwent MACS for naïve CD4 + cells, and subsequent cell suspensions were plated in H-2 media (RPMI + 10% FBS + 1 P/S + 1:1000 BME) with TH17 conditions (1 μg/mL of CD28, 2.5 μg/mL of IFNγ, 2.5 μg/mL of IL-4, 20ng/mL of IL-6, 2ng/mL of TGFβ) on 24 well plates precoated with CD3e (3 μg/mL for 3 hours).Experimental groups were also given 1 μg/mL of MLL1 inhibitor MI-2 (444825, Millipore Sigma).Cells and cell-free supernatants were harvested after 3 days of treatment for analysis of RNA expression via qPCR and IL-17A protein concentration via ELISA.After washing, samples were then acquired on a 3-Laser Novocyte Flow Cytometer (Acea Biosciences).Data were analyzed using FlowJo software version 10.0 (Treestar) and compiled using Prism software 9.2 (GraphPad).Regulatory T cells were initially identified in diabetic and nondiabetic wounds as CD3 + CD4 + CD25 + FoxP3 + cells, however to improve the specificity of our gating strategy we alter our identification of regulatory T cells to include cells that are CD3 + CD4 + CD25 + CD127 -FoxP3 + which is in agreement with the most recent scientific description of specific regulatory T cell populations (58,59).To verify gating and purity, all populations were routinely back-gated.

RNA isolation.
Total RNA extraction was performed with Trizol (Invitrogen, Thermo Fisher Scientific) using the manufacturer's directions.RNA was extracted using chloroform, isopropanol, and ethanol.iScript (Bio-Rad) or Superscript III Reverse Transcriptase (Thermo Fisher Scientific) kits were used to synthesize cDNA from extracted RNA.We used cDNA primers for Notch1 (Mm00627185_m1), Notch2 (Mm00803077_m1), Notch3 Inflammatory cytokine ELISA.Either whole wound homogenates or ex vivo isolated CD4+ cells had IL-17A levels measured by ELISA kits for IL-17A (R&D systems) per the manufacturer's protocol.
Western Blot.Cell suspensions were lysed in RIPA buffer (SIGMA) and standardized for protein concentrations using a Bradford protein assay (BioRad) to generate a standard curve.Equal amounts of protein were then loaded onto to 4%-12% SDS gel electrophoresis under reducing conditions.
Protein bands were then transferred to polyvinylidene difluoride (PVDF) membranes and probed with primary antibodies (anti-mouse MLL1 (14197, Cell Signaling), anti-mouse NICD (Val1744 D3B8, Cell Signaling) at 4 o C for 12 hours.All primary antibodies were diluted 1:500 in 5% BSA in 0.1 % Tween -Tris-buffered saline (TBS-T) solution.PVDF membranes were then washed and incubated with Horse radish peroxidase (HRP)labeled secondary antibody (Invitrogen PI32460) for 1 hour at room temperature and visualized with chemiluminescence (Themofisher Scientific).Blot images were analyzed using NIH ImageJ software to obtain sample densitometry readings normalized to beta-actin.
ChIP Assay.ChIP assay was performed as described previously.(2)Briefly, cells were fixed in 1% paraformaldehyde and lysed and sonicated using a Bioruptor Pico (Diagenode) to generate 300-to 500-bp fragments.Samples were then incubated overnight in anti-H3K4me3 antibody (ab8850, Abcam) or isotype control (rabbit polyclonal IgG ab171870, Abcam) in parallel followed by addition of protein A-Sepharose beads (Thermo Fisher Scientific).Beads were washed and bound; DNA was eluted and purified using phenol/chloroform/isoamyl alcohol extraction followed by ethanol precipitation.H3K4me3 deposition was measured by qPCR using 2X SYBR PCR mix (Invitrogen, Thermo Fisher Scientific) and primers targeting NF-κB-binding sites in the Notch1, Notch2, Rorγ, and Foxp3 promoters.Primers were designed using the Ensembl genome browser to search the Notch1, Notch2, Rorγ, and Foxp3 promoters for NFκB within the promoter region and then NCBI Primer-BLAST was used to design primers that flank this site.The following primers were used to amplify DNA in samples: Notch1: 5′ -AATGGAGGGAGGTGCGAAGT -3′ and 5′ -AAGGCTCCGCTGCAGACA -3′.
Ranger software.The samples were then merged into a single expression matrix using the cellranger aggr pipeline.For bulk RNA sequencing and scRNA-seq data accession, the numbers include GSE154556 and GSE154557 (Gene Expression Omnibus).
For bulk RNA sequencing, we computed the Principal components (PC) using the topmost 2,000 variable genes, and we calculated the Uniform Manifold Approximation and Projection (UMAP) with the top 30 topmost variable PC after batch effect correction with harmony.Cells were then clustered using shared nearest neighbor modularity optimization-based clustering, and marker genes were identified with FindAllMarkers.Cell typing was conducted by comparing marker genes to curated cell-type signature genes.NOTCH signaling was identified to be one of the most significant pathways enriched among the up-regulated genes in our full control gender matched analysis (p=5.78E-3 based on hypergeometric test).We have highlighted all the genes that were consistently altered in diabetic skin compared to controls belonging to the Notch signaling pathway.

Cell clustering and cell type annotation
The R package Seurat (v3.1.2)(60)was used to cluster the cells in the merged matrix.Cells with less than 500 transcripts or 100 genes, or more than 10% of mitochondrial expression were first filtered out as low-quality cells.The NormalizeData was used to normalize the expression level for each cell with default parameters.
The FindVariableFeatures function was used to select variable genes with default parameters.The The clusters were obtained using the FindNeighbors and FindClusters functions with the resolution set to 0.5.
The cluster marker genes were found using the FindAllMarkers function.The cell types were annotated by overlapping the cluster markers with the canonical cell type signature genes.To calculate the disease composition based on cell type, the number of cells for each cell type from each disease condition were counted.The counts were then divided by the total number of cells for each disease condition and scaled to 100 percent for each cell type.Differential expression analysis was carried out using the FindMarkers function.

Cell type sub-clustering
analyzed data.WJM, TMB, and KAG wrote the manuscript.All authors reviewed, edited, and accepted the manuscript in its final form.
our human single cell transcriptional profiling of diabetic and nondiabetic wounds to identify Treg/TH17 CD4 + cell phenotypes in wound tissue.This demonstrated increased CD4 + TH17 cells and decreased Treg cells in diabetic wound tissue as compared to non-diabetic control wounds (Fig 1A).When we examined murine wound CD4 + cells isolated on day 5 post-injury from diet induced obesity (DIO), a murine model for diabetes, and normal diet control mice and performed IC flow cytometry (Supplemental Fig 1), we found that DIO CD4 + wound T cells demonstrated an increased TH17 phenotype with fewer FoxP3 positive Treg cells (Fig 1B).Importantly, this imbalance between wound CD4 + Treg and T H17 phenotypes in diabetic wound tissue resulted in an increase in wound IL-17A as measured by ELISA in two murine models of diabetes (DIO and db/db mice) (Fig 1C).
receptor-ligand interactions, mice with CD4 + TH cell-specific deficiency in Notch1 receptor (Notch1 f/f CD4 cre+ ) were wounded.Compared to littermate controls, Notch1 f/f CD4 cre+ decreased Tregs and TH17 cells (Fig 3C), and decreased IL-17A protein in wound tissue from these mice (Fig 3D).These results suggest that Notch1 receptor plays a role in wound TH cell differentiation and inflammatory signaling following cutaneous tissue injury.Additionally, Notch signals activated in TH cells via other Notch receptors (Notch 2, 3 and 4) do not attenuate or fully compensate for the loss of Notch1 in wound CD4+ TH cells.
half of the mice failing to heal by day 10 (Fig 3E)as well as decreased Tregs and TH17 cells in wound tissue and decreased IL-17A protein in wound tissue (Fig3 F, G).These results suggest that DLL4 plays a role in facilitating physiologic TH cell phenotypes and the DLL4-Notch receptor interaction is important for normal wound repair to occur.Since the both murine and human diabetic wounds have increased Notch receptors, TH17 cells, and IL-17A levels, we hypothesized that by inhibiting Notch signaling in diabetic wound TH cells, we could improve wound healing by reducing CD4+ differentiation to TH17 and therefore lowering Il-17A levels in wounds by restoring a homeostatic balance to Treg and TH17 phenotypes.Surprisingly, diabetic mice deficient in CD4+ TH cell Notch signaling (DIO DNMAML f/f CD4 cre+ ) demonstrated decreased wound repair after injury with less than 50% of the wound area healed by day 7 (Fig3H) compared to Notch competent, diabetic littermate controls which we have previously demonstrated have completed robust wound healing by this timepoint(2,6,7,48).In addition, decreased numbers of Tregs (CD4 + CD25 + CD127 -FoxP3 + ) and TH17 cells (CD3 + CD4 + RORγ + ), as a percentage of total CD4 + cells, were seen in the wounds from diabetic mice lacking Notch signaling (DIO DNMAML f/f CD4 cre+ ) compared to the Notch-competent littermate controls (Fig3I).Taken together, these data suggest that both Tregs and TH17 cells are necessary for physiologic wound repair, that Notch inhibition and the resultant decrease in Tregs and TH17 cells, mediated through Mϕ DLL4, is harmful to the wound healing process.Despite supraphysiologic expression of TH17 cells and Il-17A in diabetic wounds, non-specific inhibition of Notch signaling and the resultant decrease in TH17 cells, did not improve diabetic wound healing.Given that we identified that Notch signaling is critical for TH cell differentiation in cutaneous wounds and necessary for wound repair where complete abrogation of this pathway impairs healing in both a normal and diabetic setting, we next examined the regulatory mechanisms that may control increased notchrelated gene expression in diabetic wound tissue as well as control wound CD4 + Th cell phenotypic differentiation in an effort to not block, but skew differentiation away from the pathologic TH17 cells without reducing the necessary Treg cells that are vital for tissue repair.MLL1 in wound CD4 + TH cells regulates Notch1 and Notch2 receptor expression and downstream Notch signalingSince we identified that Notch signaling can regulate TH cell differentiation and Notch signaling is increased in diabetic wound CD4 + TH cells, we sought to examine the regulation of Notch receptor genes in wound CD4 + TH cells.Since our group and others have identified that chromatin modifying enzymes (CMEs) can control immune cell differentiation and are relevant in wound repair, and since MLL1 has been shown to regulate Notch signaling in human cancer cell lines(7,39), we examined the role of the histone methyltransferase MLL1 in wound CD4 + TH cells.Using our Mll1 floxed mice crossed to CD4 + Cre mice, we obtained mice with a CD4 + TH cell-specific deficiency in MLL1 (MLL1 f/f CD4 cre+ ) and examined wound tissue for Notch-related genes and CD4 + T cell phenotypes.We performed flow cytometry on whole wounds isolated 3 days after wounding and found that the receptors Notch1 and Notch2 were decreased in MLL1-deficient CD4 + cells compared to their littermate controls (Fig 4A), whereas no differences were noted in the presence of receptors Notch3 and Notch4 (data not shown).Further, MLL1-deficient CD4 + TH cells isolated from wounded mice at multiple time points post wounding also demonstrated decreased Notch1 and Notch2 gene expression (Fig 4B), and Notch target gene hey1 (Fig 4C), compared to littermate controls.Next, in order to determine if MLL1 may potentially regulate Notch 1 and 2 receptor expression, we isolated CD4 + cells from MLL1-deficient mice and performed a chromatin immunoprecipitation (ChIP) assay for H3K4me3, the activating chromatin mark set by MLL1.Increased H3K4me3 was seen on the promoters of Notch1 and Notch2 in wound CD4 + cells isolated from MLL1 f/f CD4 cre-as compared to the MLL1 f/f CD4 cre+ (MLL1-deficient) CD4 + TH cells (Fig 4D).These data suggest that MLL1 regulates Notch1 and Notch2 receptor expression and that loss of MLL1 in wound CD4 + cells contributes to loss of Notch signaling/downstream Notch target gene expression in wound CD4 + TH cells.
especially at early time points, wound healing was impaired in MLL1 f/f CD4 cre+ mice compared to littermate controls (Fig 4E).Whole wounds from these mice were then analyzed via IC flow cytometry for TH cell phenotypes.Wound CD4 + TH cells from MLL1-deficient mice displayed decreased TH17 cells, with a smaller decrease in Tregs (Fig 4F).In effect, these results demonstrate that MLL1 plays a role in dictating Notch signaling in wound CD4 + TH cells and thus, CD4 + TH cell differentiation in wounds and that MLL1 in wound CD4 + T cells is important for tissue repair.MLL1 is increased in diabetic wound CD4 + T cells and skews wound CD4 + T cells towards TH17 by increasing Notch1 and Notch2 expression and by directly regulating RORγ Since MLL1 was shown to regulate Notch receptor expression and downstream Notch signaling in wound CD4 + cells and that we found that Notch signaling was increased in diabetic wound CD4 + cells, we examined the role of MLL1 in diabetic CD4 + T cells on Notch receptor expression and downstream Notch signaling.Murine wound CD4 + TH cells were isolated from wounds of DIO mice and control littermate controls and examined for Mll1 expression.Diabetic CD4 + TH cells demonstrated increased Mll1 expression as compared to control CD4 + cells (Fig 5A).To determine if the increased Mll1 expression resulted in a functional change in the CD4 + T cells, a chromatin precipitation assay was performed on DIO and control CD4 + T cells to examine MLL1-mediated H3K4me3 on the promoters of Notch1 and Notch2.Increased H3K4me3 was found at the promoters of Notch1 and Notch2 in DIO CD4 + T cells (Fig 5B), suggesting that increased Notch receptors in DIO CD4 + TH cells may be in part due to upregulated MLL1.Given that the diabetic wound CD4 + TH cells demonstrate a skewed differentiation toward the TH17 phenotype or away from the Treg phenotype (see Fig 1), we examined if genetic blockade of MLL1, specifically in CD4 + TH cells could reverse the increased Notch signaling in diabetic wound tissues to more physiologic levels and reduce TH17 differentiation in diabetic tissue.Wounds were isolated from mice subjected to a HFD for 12-16 weeks that were deficient in CD4 + TH cell-specific MLL1 (DIO MLL1 f/f CD4 cre+ ) and littermate controls and were analyzed by IC flow cytometry.Wounds from mice with an MLL1 deficiency in CD4 + TH cells demonstrated decreased RORγ + in TH17 cells as compared to littermate controls, suggesting that depletion of MLL1 in diabetic CD4 + cells can reduce TH17 differentiation.Phenotypically, IL-17A protein was also decreased by ELISA in CD4 + cells isolated from DIO MLL1 f/f CD4 cre+ mice as compared to littermate controls (Fig 5C).Given the reduction in TH17 cells with loss of MLL1, we also investigated whether MLL1 directly regulates expression of the TH17 associated transcription factor, RORγ.We found increased H3K4me3 on the promoter of RORγ in diabetic CD4 + T cells as measured by chromatin immunoprecipitation (Fig 5D).We next sought to determine if MLL1 had an effect on FoxP3 expression.Despite increased MLL1 in diabetic CD4 + TH cells, there was no increase in H3K4me3 on the promoter of Foxp3 (Fig 5E).This differential regulation of MLL1 on RORγ and not FoxP3 gene expression in the setting of diabetes may be contributing to the imbalance and skewed differentiation of diabetic wound CD4 TH cells seen towards a TH17 phenotype and away from the Treg phenotype in diabetic wound tissue.To examine the therapeutic potential of these findings, we isolated diabetic CD4 + T cells from wounds and treated them ex vivo with a small molecule inhibitor of MLL1, MI-2, and found this reduced IL-17A from the CD4 + T cells, as measured by ELISA (Fig 5F).Diabetic CD4 + TH cells treated with MI-2 also demonstrated decreased expression of Notch receptors Notch1 and Notch2, in addition to the downstream Notch target genes hey1 and hey2 (Fig 5G).Taken together, these results suggest that increased MLL1 in diabetic CD4 + TH cells drives pathologic TH17 differentiation via addition of its activating H3K4me3 mark at RORγ promoter and indirectly through increased Notch receptor expression/signaling, and that inhibition of MLL1 with therapeutic targeting can reverse this process and reduce TH17 mediated inflammation in diabetic wounds (Fig 6).
histone methyltransferase MLL1, regulated Notch signaling by enhancing transcription of the Notch1 and Notch2 receptors.In diabetic TH cells, MLL1 was increased which thereby increased notch signaling and TH17 cell differentiation by, directly driving a TH17 phenotype via activation of RORγ transcription.By blocking MLL1 activity in diabetic TH cells, we reversed the TH17 phenotype and reduced pathologic IL-17A levels, making this a viable therapeutic target Methods: Mice.All mice were maintained at the University of Michigan in the Unit for Laboratory and Animal Medicine.Mouse experiments were conducted with approval from our IACUC, and all regulatory and safety standards were strictly adhered to.C57BL/6 mice were obtained at 6-7 weeks of age from Jackson Laboratory and maintained in breeding pairs at the ULAM facilities.Mice with the Dnmaml, Notch1, or Mll1 gene deleted in T helper cells (Dnmaml f/f CD4 Cre+ , Notch1

FindIntegrationAnchors
and IntegrateData functions were used to integrate the samples prepared using different 10X Chromium chemistries.The ScaleData function was used to scale and center the counts in the dataset.Principal component analysis (PCA) was performed on the variable genes, and the first 20 PCs were used for cell clustering and Uniform Manifold Approximation and Projection (UMAP) dimensional reduction.

Figure 2 :
Figure 2: Notch signaling is increased in human and murine diabetic wound CD4 + TH cells.(A) River plots depicting cell groups and ligand-receptor pairs affect outgoing (signal source) and incoming

Figure 4 :
Figure 4: MLL1 in wound CD4+ TH cells regulates Notch1 and Notch2 receptor expression and downstream Notch signaling

ligand DLL4 in diabetic patients compared to nondiabetics (Fig 2F). We also found an increase
+ TH cell phenotypes in various tissues, we hypothesized that Notch signaling may dictate CD4 + TH differentiation in both normal and pathological diabetic wound healing.First, to determine if Notch signaling in the Notch ligand, DLL4, in DIO wound Mϕs compared to nondiabetic littermate controls (

Notch signaling in cutaneous wounds regulates CD4 + Treg/ TH17 cell phenotypes.
Cre mice.Animals were housed in a barrier facility on a 14-hour light/10-hour dark cycle (ambient temperature of 22 o C) with free access to water, food (Lab Supply Lab Diet Rodent 5001), and bedding (Andersons Lab Bedding Bed-o'Cobs combo).The number of animals per group in all the experiments was determined on the basis of prior literature, power calculation, and experience from our previous studies to ensure sufficient sample sizes to allow the detection of statistically significant differences.No animals were excluded from analysis.The number of replicates for each experiment is labeled in all figures or legends.
(6,45)4 Cre+ , Mll1 f/f CD4 Cre+ ) were generated by mating gene f/f mice with CD4 Cre mice (The Jackson Laboratory)(6,45).Mll1 deletion efficacy in the Mll1 f/f CD4 Cre+ was 87% (data not shown).Mice with the DLL4 gene deleted in myeloid cells (Dll4 f/f Lyz2 Cre+ ) were generated by mating Dll4 f/f mice with Lyz2 Cytometry.Wound cells were first stained with a LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Data are presented as the mean ± SEM.All data are representative of 2-4 independent experiments.Data were first analyzed for normal distribution, and if data passed the normality test, two-tailed Student's t test was used.