Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss

SUMMARY Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.


INTRODUCTION
With advancing age, hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) progressively acquire somatic mutations, some of which may confer a proliferative advantage to the mutant cell, enabling disproportionate clonal expansion relative to normal clones.2][3] In the absence of apparent hematologic disease, this condition is designated clonal hematopoiesis of indeterminate potential (CHIP).5][6][7] CHIP development may also be driven by selective pressure from cytotoxic chemotherapy or smoking. 86][17] Similarly, in DNMT3A-driven CHIP in mice, Dnmt3a-mutant HSPCs expand without apparent bias toward the production of particular types of mature hematopoietic cells. 18This model is based on the use of mice harboring a heterozygous mutation (R878H) that causes dominant negative loss of DNMT3A methylation activity 18 and is equivalent to the human hotspot heterozygous mutation R882H. 19ince chronic inflammatory diseases are largely driven by inflammatory immune cells, CHIP-driven alterations to their precursors in the BM are likely to influence multiple disorders.Periodontitis, a prevalent inflammatory disease of the tooth-supporting tissues, remains a significant public health burden [20][21][22][23] and is epidemiologically associated with rheumatoid arthritis, [24][25][26] which leads to progressive erosion of cartilage and bone in the joints. 279][30][31][32] We reasoned that CHIP may not merely correlate with aging 1,6,7 but may also contribute causally to inflammatory bone loss disorders, such as periodontitis and arthritis.[6]

RESULTS
Higher periodontitis prevalence in CHIP individuals with DNMT3A mutations In a community-based cohort of 4,946 adult participants of the Atherosclerosis Risk in Communities (ARIC) study 33,34 ages 52-74 (mean age 62 years), we identified 3.9% of individuals (n = 191) as CHIP carriers.Similar to published literature, [4][5][6] the most common mutations involved DNMT3A (61.8% of total carriers; n = 118), followed by TET2 and ASXL1 (Table S1).The majority of CHIP carriers (n = 108, 2.2% of sample, and 56.5% of total carriers) exhibited VAF > 10% (Table S1).CHIP increased with age and was more common among current (5.7%) vs. ever smokers (3.6%) (Table S2).CHIP due to DNMT3A mutations was significantly associated with a diagnosis of severe periodontitis (stage IV vs. stages I-III), as well as quantitative measures of ''clinical attachment loss'' and ''gingival inflammation'' (Table 1).The observed differences in periodontitis and clinical measures of periodontal disease were robust to adjustments for participants' age and sex.For example, when adjusted for age and sex, the prevalence of stage IV periodontitis in carriers with DNMT3A mutations was (average marginal effect [AME]) 11 percentage points (95% confidence interval [CI] = 0.0-21.5)higher than in those without CHIP (Table S3).In conclusion, DNMT3A CHIP is associated with higher prevalence and severity of periodontitis.
Young adult mice are periodontally healthy; however, disruption of periodontal tissue homeostasis, e.g., due to immunoregulatory defects, leads to uncontrolled inflammation and bone loss (in both mice and humans). 41,42To determine whether the clonal expansion of Dnmt3a R878H/+ cells and the associated systemic inflammation (Figures 1B, 1C, S1A, and S1B) affect periodontal tissue homeostasis, we assessed the alveolar bone levels by measuring the distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC).10% Dnmt3a R878H/+ BMT mice exhibited a significant increase over the mean CEJ-ABC distance of the control group (Figure 1D).Consistently, 10%Dnmt3a R878H/+ BMT mice displayed elevated gingival mRNA expression of pro-inflammatory cytokines (e.g., IL-23 and IL-17) and pro-osteoclastogenic factors (e.g., Tnfsf11/RANKL) (Figure 1E).Measurement of bone mass of the femur and spine revealed no significant differences between 10%Dnmt3a R878H/+ BMT and controls (Figures S1D-S1F), except for a modest decrease (8.9%) in trabecular bone volume to total bone volume fraction (BV/TV) in 10% Dnmt3a R878H/+ BMT mice (Figure S1D, left bar graph).Therefore, naturally occurring periodontal bone loss in 10%Dnmt3a R878H/+ BMT mice could not be attributed to general bone defects.
The finding that neutrophil depletion has a greater impact in mice that received mutant clones substantiates the disease-provoking effect of the R878H mutation in neutrophils.
Upon induction of CAIA in 10%Dnmt3a R878H/+ BMT mice and controls, the former exhibited significantly increased clinical arthritis score (Figure 3A) and ankle joint thickness (Figure 3B), as well as aggravated histopathology in knee joints, as revealed by hematoxylin and eosin (H&E) assessment of loss of joint architecture, inflammatory cell infiltrate, and synovial hyperplasia (Figure 3C).Moreover, safranin-O staining of knee joint tissue sections displayed increased cartilage loss and roughening of the articular surface in the mutant setting (Figure 3D).10% Dnmt3a R878H/+ BMT mice also showed elevated cellularity in the synovium (Figure 3E) with increased abundance of neutrophils (Figure 3F) and monocytes (Figure 3G).Moreover, 10% Dnmt3a R878H/+ BMT mice showed significantly increased frequency and total counts of CD45.2 + Dnmt3a R878H/+ neutrophils (Figure 3H), monocytes (Figure 3I), and T cells (Figure 3J).Overall, expansion of Dnmt3a-CHIP clones and infiltration of peripheral tissues with mutant leukocytes aggravates experimentally induced periodontitis and arthritis.
To further investigate this notion, we explored intercellular communication networks (interaction numbers and strength) in CD45.2 + (mutant) and CD45.1 + (WT) cells from 10% Dnmt3a R878H/+ BMT mice by performing comparative CellChat analysis. 51Differential interactions in cell-cell communication networks between CD45.2 + and CD45.1 + cells were determined and visualized, with red and blue color indicating, respectively, increased and decreased signaling in CD45.2 + mutant cells compared with CD45.1 + WT cells.The number and strength of putative signaling interactions within and between myeloid cell types (neutrophils, monocytes/macrophages, DC) generally increased in gingival mutant cells (Figure 4E), whereas only the strength of the signaling increased in synovial mutant cells (Figure S3D).By contrast, the number and strength of putative signaling interactions within T cells and between T cells and myeloid cells decreased in both gingival and synovial mutant cells (Figures 4E and S3D, respectively).There was a slight increase in putative signaling between myeloid cells (macrophages and DC) and osteoclasts in both gingiva and synovium (mutant setting) (Figures 4E and S3D).We moreover compared the information flow (overall communication probability) across CD45.2 + (mutant) and CD45.1 + (WT) cells for specific signaling pathways.Remarkably increased information flow in gingival mutant cells included resistin, midkine (MK), vascular cell adhesion protein (VCAM), receptor activator of nuclear factor-kB ligand (RANKL), and sialic acid-binding Ig-like lectin 1 (SN) signaling (Figure 4F).Among them, resistin is a potential biomarker in periodontitis, 52,53 MK and VCAM are putative risk factors for periodontitis, 54,55 and RANKL drives inflammatory bone loss. 56Remarkably increased information flow in synovial mutant cells included angiopoietin-like proteins (ANGPTL), lymphotoxin-a (LT), adiponectin, collagen, and IL-6 (Figure S3E).Of these, LT activates nuclear factor kB (NF-kB) signaling in chondrocytes and contributes to rheumatoid arthritis 57 ; adiponectin is increased in the synovial fluid and promotes arthritis 58,59 ; IL-6 is a therapeutic target in rheumatoid arthritis. 60Together, there appears to be increased autocrine and paracrine signaling probabilities between different cell types during experimental periodontitis and arthritis, and these inferences are consistent with a recent CellChat analysis of DNMT3A-mutant cells from the peripheral blood of heart failure patients. 61Mutant cells might thus promote inflammation not only in a cell-intrinsic manner but also via paracrine effects by activating neighboring WT cells, consistent with our finding that WT myeloid cells from the Dnmt3a-mutant setting expressed significantly higher levels of Il1b, Il6, Il23a, and Tnf than WT myeloid cells from the WT setting (Figure S1J).
Further analysis of the scRNA-seq data (with Seurat's DotPlot function) showed that-in the gingival mutant setting-CD45.2+ (mutant) neutrophils and macrophages expressed more Il1b,  (legend continued on next page) Tnf, and Il23a; monocytes expressed more Il6, Tnf, Il23a, and Tnfsf11 (RANKL); DC expressed more Tnf, Il23a, and Tnfsf11; and T cells expressed more Tnfsf11 and less Ctla4, as compared with their CD45.2+ (WT) counterparts in the WT setting (Figure 4G).Similarly, in the synovial mutant setting, CD45.2 + (mutant) cells in general expressed inflammatory/osteoclastogenic cytokines at higher levels than their CD45.2+ (WT) counterparts in the WT setting (Figure 4H).Moreover, in the gingival mutant setting, CD45.1 + (WT) macrophages expressed more Tnf; monocytes expressed more Il6 and Tnfsf11; and DC expressed more Tnfsf11, compared with their CD45.1 + (WT) counterparts in the WT setting (Figure S3A).In the synovial mutant setting, CD45.1 + (WT) DC expressed more Il23a relative to their CD45.1 + (WT) counterparts in the WT setting (Figure S3B).These observations are consistent with our above-described findings that the R878H mutation renders the affected leukocytes more pro-inflammatory than their WT counterparts and that WT cells in the mutant setting are more pro-inflammatory than WT cells in the WT setting.According to Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, the enriched upregulated genes in CD45.2 + (Dnmt3a R878H/+ ) cells in the mutant setting indicated enhanced activation of several Kyoto encyclopedia of genes and genomes (KEGG) inflammatory pathways (e.g., ''Th17 cell differentiation'' and ''NF-kB signaling pathway'') relative to CD45.2 + (Dnmt3a +/+ ) cells in the WT setting (Figures 4I and S3C, for gingiva and synovium, respectively).
3][64][65] GO enrichment analysis in gingival neutrophils showed that ''immune response,'' ''defense response,'' and related terms were enriched in the significantly upregulated genes in the 10%Dnmt3a R878H/+ BMT-CD45.2 group (vs.10%Dnmt3a +/+ BMT-CD45.2group) (Figure 4J).GO enrichment analysis in synovial neutrophils did not reveal any significantly enriched GO terms in the upregulated genes, whereas the ''defense response to other organism'' and related terms were among the top 10 enriched GO terms in the significantly downregulated genes (Figure 4K).GO enrichment analysis of gingival T cells showed that the GO terms ''lymphocyte differentiation'' and ''regulation of apoptotic process'' were among the enriched terms in the significantly upregulated genes in the 10%Dnmt3a R878H/+ BMT-CD45.2group, whereas ''immune system process'' and ''defense response'' were among the significantly enriched terms in the downregulated genes (Figure 4J).GO enrichment analysis of synovial T cells showed that ''cellular response to cytokine stimulus,'' and ''positive regulation of adaptive immune response'' were among the top 10 enriched GO terms in the significantly upregulated genes, whereas there were no enriched GO terms in the significantly downregulated genes (Figure 4K).
Analysis in all 15 clusters of the expression of Acp5 and Ctsk, which are major markers of osteoclasts, 66,67 revealed that these markers were most highly expressed in C15 (Figures S2B and  S2C), hence identified as osteoclasts.It should be noted that gingival tissues dissected from LIP-subjected mice do contain tartrate-resistant acid phosphatase (TRAP)-stained osteoclasts in areas adjacent to the underlying bone (Figure S2D); similarly, osteoclasts are found within the synovium at sites proximal to the bone. 68The frequency of osteoclasts (C15) in the 10% Dnmt3a R878H/+ BMT-CD45.2 group from gingiva and synovium was increased relative to the 10%Dnmt3a +/+ BMT-CD45.2control, especially in the gingiva (2.5-fold; Figure 4D, left).Because the influence of CHIP mutations on osteoclasts and T cells is less well understood than that on myeloid cells, we next investigated osteoclastogenesis and T cell function in the context of DNMT3A/CHIP-driven inflammatory disease.
The R878H mutation is associated with enhanced osteoclastogenesis and impaired Treg cell immunosuppressive activity Earlier work has identified a CD11b À/lo Ly6C hi monocytic population in the BM that is induced upon inflammation and is highly enriched in osteoclast precursors (OCPs). 69FACS analysis to identify this OCP-enriched population in the BM of LIP-subjected 10%Dnmt3a R878H/+ BMT mice and controls revealed no significant differences between the two groups regarding the frequency-among BM cells-of CD11b À/lo Ly6C hi cells or their total numbers in the BM (Figure S4A).However, the frequency and total numbers of CD45.2 + CD11b À/lo Ly6C hi cells in the mutant setting were significantly higher than the frequency and total numbers of their CD45.2+ counterparts in the WT setting (Figure S4B).Thus, although the presence of the hematopoieticspecific R878H mutation in the BM is not associated with expansion of the CD11b À/lo Ly6C hi population, within this population, the donor-derived (CD45.2+ ) mutant cells in 10% Dnmt3a R878H/+ BMT mice greatly outnumber their CD45.2+ WT counterparts in the WT setting.To determine whether the R878H mutation in this OCP-enriched population is associated with enhanced osteoclastogenesis, we performed a RANKLinduced osteoclast differentiation assay.CD11b À/lo Ly6C hi cells sorted from the BM of 10%Dnmt3a R878H/+ BMT mice yielded more osteoclasts (TRAP + multinucleated cells [MNCs]) than did CD11b À/lo Ly6C hi cells sorted from the BM of 10% Dnmt3a +/+ BMT controls (Figure 5A).

Rapamycin inhibits clonal expansion of Dnmt3a R878H/+ cells and bone loss
To better understand the impact of the Dnmt3a-R878H mutation on the DNA methylation status of mature leukocytes, we conducted whole-genome bisulfite sequencing (WGBS) in splenic CD11b + myeloid cells and CD4 + T cells from mutant (Dnmt3a R878H/+ ) mice and WT controls.Globally, the methylation levels across the genome in both CD11b + and CD4 + cells from Dnmt3a R878H/+ mice were significantly decreased relative to their counterparts from Dnmt3a +/+ controls (Figures 6A and 6B).The average methylation values of CD11b + and CD4 + cells from Dnmt3a R878H/+ mice were significantly lower than those from (G and H) Gene expression levels in distinctly defined CD45.2 + cell types from gingiva (G) or synovium (H).(I) List of gingival upregulated enriched genes in CD45.2 + (Dnmt3a R878H/+ ) cells in the indicated KEGG pathway terms analyzed by STRING.(J and K) Top 10 significantly enriched GO terms sorted by PANTHER based on significantly upregulated or downregulated genes (in 10%Dnmt3a R878H/+ BMT-CD45.2vs. 10%Dnmt3a +/+ BMT-CD45.2) in neutrophils and T cells from gingiva (J) and synovium (K) (Fisher test with FDR-correction, p < 0.05).See also Figures S2 and S3.
The results from our WGBS analysis revealing activation of mTOR signaling are consistent with a previous report that DNA hypomethylation associated with the R878H mutation in LSK cells leads to upregulation of mTOR, which in turn promotes overexpression of the cell-cycle regulator cyclin-dependent kinase-1. 75onsistently, scRNA-seq analysis of gingival cells (from LIP-subjected mice; Figure 4A) revealed elevated expression of Mtor and several mTOR-regulated genes in different cell types (especially osteoclasts) in the mutant setting, compared with the corresponding cell types in the WT setting (Figure 6E).Furthermore, BM cells from 10%Dnmt3a R878H/+ BMT mice at steady state have significantly increased expression of Mtor compared with 10%Dnmt3a +/+ BMT controls (Figure 6F).Also in line were our observations for upregulated mTOR expression in FACS-sorted Dnmt3a R878H/+ cells-namely, BM LSK cells, splenic CD11b + myeloid cells, and splenic CD4 + T cells-as compared with their Dnmt3a +/+ counterparts (Figure 6G).The Dnmt3a R878H/+ cells, especially LSK cells, also exhibited increased expression of mTOR-regulated genes (Pcna, Eif4ebp1, Eif4ebp2, Eif4ebp3, Hif1a, and Ccnd1 [75][76][77][78] ) (Figure 6G).

DISCUSSION
Our study has shown that DNMT3A-driven CHIP in mice causes naturally occurring periodontal inflammation and bone loss, as well as exacerbates experimentally induced periodontitis and arthritis.][81][82] CHIP is driven mostly by DNMT3A mutations and represents a significant public health risk with progressively increased prevalence in adults over 60, 7,83 when the severity and prevalence of chronic inflammatory diseases increase significantly. 84,85hrough depletion/intervention experiments, we causally implicated Tregs, IL-17, and neutrophils, a downstream cellular effector of IL-17, 86 in DNMT3A-driven inflammatory bone loss.IL-17 neutralization or neutrophil depletion diminished periodontal inflammation and bone loss in 10%Dnmt3a R878H/+ BMT mice to levels comparable to those seen in mice that received exclusively WT BM cells.Moreover, given that 10%Dnmt3a R878H/+ BMT mice contain dysfunctional Tregs that fail to restrain Th17 expansion, Treg depletion affected inflammatory bone loss selectively in the WT setting.The elevated Th17/Treg ratio in the periodontal tissue of 10%Dnmt3a R878H/+ BMT mice is in line with clinical observa-tions that human carriers of DNMT3A CHIP-driver mutations exhibit significantly elevated Th17/Treg ratio in peripheral blood. 16ur study overall shows that different hematopoietic cell types, which are produced downstream of DNMT3A-mutant hematopoietic progenitors, are affected in ways that aggravate bone loss.
DNMT3A-driven CHIP exacerbated periodontitis not only by enhancing inflammatory osteoclastogenesis but also by negatively impacting the host's capacity to resolve the periodontal lesion.Indeed, the R878H mutation compromised the ability of efferocytic macrophages to express key pro-resolving mediators, including Tgfb1.The diminished capacity of Dnmt3a R878H/+ macrophages to release TGF-b1 protein upon efferocytosis can be attributed directly to the defective enzymatic activity of mutant DNMT3A.In efferocytic macrophages, DNMT3A normally methylates and epigenetically represses the phosphatase DUSP4, thereby leading to prolonged extracellular signal-regulated kinase (ERK)1/2 activation and induction of Tgfb1 expression. 87Therefore, dysregulated efferocytosis signaling associated with the presence of mutant DNMT3A leads to reduced release of TGF-b1, which would otherwise contribute to inflammation resolution and bone regeneration. 88ur WGBS analysis revealed common hypomethylation phenotypes in CD11b + and CD4 + cells from Dnmt3a R878H/+ mice, consistent with a human WGBS study that examined DNA methylation in peripheral blood cells of patients with germline DNMT3A mutations. 89Their analysis revealed a focal canonical hypomethylation phenotype, which was most severe with the dominant negative DNMT3A R882H mutation.The authors concluded that the affected genes were dysregulated by mechanisms that were not specific to lineage or cell type. 89Collectively, these previous findings and our present data suggest that the origin of these changes in mature leukocytes lies in their common hematopoietic progenitors.
Our findings that DNMT3A-driven CHIP increases the severity of experimental periodontitis and arthritis and earlier mouse studies linking CHIP and cardiometabolic disorders 9, 39,90 suggest that CHIP may be a common mechanistic basis for inflammatory comorbidities in old age.Aging is considered a non-modifiable risk factor for periodontitis and comorbid chronic diseases. 91Our data, however, suggest that the effects of aging  (E) Relative mRNA expression of Mtor and mTOR-regulated genes in the indicated CD45.2 + cell types from gingiva (data from scRNA-seq; see Figure 4).(F and G) BMT was performed as in Figure 1A, and cells were analyzed 12 weeks post-BMT.(F) Relative Mtor expression in BM cells and (G) relative mRNA expression of indicated molecules in BM LSK cells (Lin À cKit + Sca1 + ), splenic CD11b + myeloid cells, and splenic CD4 + T cells from Dnmt3a +/+ or Dnmt3a R878H/+ mice.(H-J) BMT was performed as in Figure 1A, and recipient mice were treated with rapamycin or PBS control (see STAR Methods).12 weeks post-BMT, all groups were subjected to LIP. (H) Percentage of CD45.2 + white blood cells within total CD45 + cells (left), CD11b +  on periodontitis (and comorbidities) could be mitigated by targeting CHIP.Screening for CHIP may identify individuals with increased risk for severe periodontitis and comorbidities, including arthritis and cardiovascular disease.These individuals may benefit from therapeutic interventions aiming to block the aberrant expansion of CHIP clones.Specific inflammatory cytokines and mTOR signaling have been implicated in the selective expansion of hematopoietic stem cell (HSC) clones with CHIPdriver mutations 75,[92][93][94] (and this study).Enhanced mTOR expression and signaling is evidently a consequence of DNA hypomethylation in DNMT3A-driven CHIP 75 (and this study).Therefore, CHIP may be a reversible process if treated with inhibitors capable of interfering with the fitness advantage of CHIP-mutant clones.As a proof of concept, we have shown here that systemic treatment of 10%Dnmt3a R878H/+ BMT mice with an mTOR inhibitor (rapamycin) inhibits the clonal expansion of Dnmt3a R878H/+ hematopoietic cells and the development of periodontal inflammation and bone loss.
Rapamycin, an FDA-approved drug for transplantation indications, has been shown in experimental models to protect against aging-related pathologies. 95When administered orally to 20-month-old mice via the diet, rapamycin conferred protection against naturally occurring periodontal inflammation and bone loss. 96Here, systemic rapamycin did not have a significant effect on the bone levels of normal (10%Dnmt3a +/+ BMT) mice.However, there are important differences in the experimental designs of the two studies, including the age (20-to 22-week-old vs. 20-month-old mice) and route of rapamycin administration (systemic vs. oral).In old mice studied by An et al., 96 rapamycin could in principle inhibit inflammatory bone loss by suppressing the senescence-associated secretory phenotype that fuels aging-associated inflammation and/or by promoting clearance of senescent cells. 97The protective effect of rapamycin on naturally occurring and experimentally induced bone loss in this study could likely be attributed to the drug's ability to restrain DNMT3A-driven CHIP and thereby limit the infiltration of hyperinflammatory Dnmt3a-mutant leukocytes into the periodontium.
The epigenetic, transcriptomic, and phenotypic alterations in HSPCs and their mature progeny resulting from loss-of-function CHIP mutations affecting epigenetic modifiers 9,39,98-100 have mechanistic parallels with trained immunity. 3,1017][108] This maladaptive epigenetic rewiring associated with trained immunity can be long-lasting (for months) but is, in principle, reversible.On the other hand, an epigenetic state shaped by CHIP mutations on HSPCs and their leukocyte progeny drives a permanent state of maladaptive inflammation and hence mimics a ''fixed'' type of maladaptive trained immunity.
In conclusion, DNMT3A-driven CHIP integrates aging-related alterations in HSPCs and mature progeny with distinct inflammatory comorbidities.Our study further suggests that rapamycin may represent an effective intervention to mitigate the fitness advantage of DNMT3A-CHIP-mutant clones and suppress their impact on chronic inflammatory disease.

Limitations of the study
Our findings that CHIP due to DNMT3A mutations is associated with a higher prevalence and severity of clinically ascertained periodontal disease lack causality and directionality.However, a causal relationship is plausible given that the expansion of Dnmt3a R878H/+ hematopoietic cells in mice caused naturally occurring periodontitis and exacerbated experimentally induced periodontitis.Yet, we cannot exclude a bidirectional association between CHIP and periodontitis.This is because periodontitisassociated systemic inflammation could, at least in principle, exacerbate CHIP (by fueling the expansion of mutant clones or even influencing the acquisition of somatic mutations in HSPCs 93,109 ), thereby creating a vicious cycle linking CHIP and the disease.In our study, we modeled DNMT3A-driven CHIP using mice harboring a heterozygous mutation (R878H) in hematopoietic cells that is equivalent to the ''hotspot'' heterozygous mutation R882H in humans. 19Future studies in mice could investigate additional Dnmt3a mutations and whether their effects differ from those of the R878H mutation.Although through depletion/intervention experiments we confirmed the causal involvement in inflammatory bone loss of neutrophils, Tregs, and the IL-17 pathway, our bioinformatics analyses suggest that multiple cell types are probably affected by the R878H mutation.Thus, it is uncertain which population contributes the most to the observed disease phenotype.Although treatment of mice with rapamycin blocked the aberrant expansion of Dnmt3a-mutant clones and their adverse impact on inflammatory bone loss, we cannot safely extrapolate these preclinical findings to human DNMT3A-CHIP carriers.This possibility can be tested in clinical trials.Potential success is supported by findings that rapamycin suppresses the proliferation of mTOR-overexpressing human leukemic cell lines harboring DNMT3A mutations, including at the R882 hotspot, 75 based on which our preclinical model was developed.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: Ascertainment of CHIP carriage status in ARIC CHIP was previously determined using whole exome sequencing data using GATK Mutect2 118 and ANNOVAR 119 as reported by Bick et al. 6 and Uddin et al. 120 Consistent with previous investigations, CHIP was defined as the presence of pathogenic somatic mutations with variant allele frequency (VAF) >2% in specific hematologic cancer-driver genes (e.g., DNMT3A, TET2, ASXL1, JAK2) among participants without hematologic malignancy or clonal disease.CHIP data were generated using DNA that was collected in ARIC visits 1-4, prior to the conduct of the dental ancillary study.

Mice
WT C57BL/6 and congenic C57BL/6.SJL CD45.1 + mice (B6.SJL-Ptprc a Pepc b /BoyJ), as well as B6(Cg)-Dnmt3a tm1Trow /J (referred to as Dnmt3a fl-R878H/+ mice) and B6.Cg-Commd10 Tg(Vav1-icre)A2Kio /J (Vav1-iCre mice) were purchased from the Jackson Laboratory.Loberg et al. modelled DNMT3A-driven clonal hematopoiesis in mice engineered to express the heterozygous R878H mutation, which resulted in pronounced expansion of mutant HSPC. 18The mice in this previous work were generated by crossing Dnmt3a fl-R878H/+ mice to Mx1-Cre mice and expression of the Dnmt3a R878H mutant allele was induced with polyinosinic-polycytidylic acid (pIpC). 18ystemic administration of pIpC induces type I interferons, 121,122 which in turn can induce activation and/or innate immune training in hematopoietic progenitors, 104 thereby potentially confounding CHIP-driven effects in our inflammatory disease models.To prevent this potential issue, we crossed Dnmt3a fl-R878H/+ mice to Vav1-iCre mice and generated Cre + Dnmt3a R878H/+ mice with constitutive expression of the iCre recombinase (an optimized variant of Cre) and hematopoietic-specific expression of the heterozygous Dnmt3a R878H mutation (referred to as Dnmt3a R878H/+ mice).Cre + Dnmt3a +/+ or Cre -Dnmt3a fl-R878H/+ (collectively referred to as Dnmt3a +/+ ) mice were used as controls for BMT.In addition, we generated Dnmt3a R878H/+ GFP + mice (as well as Dnmt3a +/+ GFP + controls) by using the UBC-GFP transgenic mice 71,72 (Jackson Labs) as well.Mice were maintained in individually ventilated cages under specific pathogen-free conditions on a standard 12-h light/dark cycle.Food and water were provided ad libitum.Sex-and agematched mice were used for experiments at 8-10 weeks of age.Because there were no significant differences in the results obtained with male and female mice, the respective data were pooled per treatment group.Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania and were performed in compliance with institutional, state, and federal policies.

Ligature-induced periodontitis and bone measurements
4][125][126][127][128] LIP induces bone loss in conventional (but not germ-free) mice. 126,129To induce LIP, a 5-0 silk ligature was tied around the left maxillary second molar tooth for 5 days while keeping the contralateral tooth (right maxillary second molar) unligated to serve as baseline control. 130efleshed maxillae were used to measure bone heights, that is, the distances from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC).Measurements were at 6 predetermined points involving the ligated molar and affected adjacent regions as well as the corresponding points of the unligated contralateral molar, by means of a SMZ800 stereoscope with digital imaging measurement system (Nikon).To calculate bone loss in LIP experiments, the six-site total CEJ-ABC distance for the ligated site of each mouse was subtracted from the six-site total CEJ-ABC distance of the contralateral unligated site.The results were presented in millimeters, and negative values indicate bone loss relative to the baseline (unligated control).For naturally occurring bone loss (in experimental and control groups that were not ligated), the distance between the CEJ and the ABC was measured at 6 predetermined points in both sides of the maxilla.The results were presented as total CEJ-ABC distance in experimental mice as compared to that of the controls.In '10% Dnmt3a R878H/+ BMT' mice, which develop natural bone loss (Figure 1D), the ligature-induced bone loss was calculated against the unligated site that only had natural bone loss (0 baseline; Figure 2A).This normalization allows a meaningful and fair comparison with ligature-induced bone loss in '10% Dnmt3a +/+ BMT' controls (in other words, the natural bone loss in '10% Dnmt3a R878H/+ BMT' mice does not confound the comparison).To enable resolution of periodontitis, which leads to bone regeneration, 47 in some experiments the ligatures were removed after 10 days and the mice were sacrificed 5 days later (day 15).Periodontal bone measurements were performed as described above and the CEJ-ABC distance data were further transformed to indicate bone gain (or loss, if negative value) relative to the bone levels of control mice that were sacrificed at day 10, as we previously described. 47llagen antibody-induced arthritis Collagen antibody-induced arthritis (CAIA) was induced in mice by i.v.(retro-orbital) injection of 1.5 mg arthritogenic monoclonal antibodies (5-clone collagen antibody cocktail; Chondrex).131,132 Three days later, mice were injected i.p. with 50 mg of LPS.Clinical symptoms of arthritis were daily evaluated visually for each paw using a semiquantitative scoring system graded on a scale of 0-4 per paw by a blinded procedure: 0 for normal; 1 for mild redness, slight swelling of ankle or wrist; 2 for moderate swelling of ankle or wrist; 3 for severe swelling, including some digits, ankle and foot; 4 for maximally inflamed joint.The clinical score for each mouse was the sum of the 4 paw scores for a maximum score of 16.Hind ankle joint thickness was measured by using a Ka ¨fer pocket dial thickness gauge J 15.

Tissue processing and cell preparations
For BM single-cell suspension preparation, mouse femoral bones were flushed with ice-cold PBS (Gibco) supplemented with 5% FBS (Gibco).Cells were forced through 70-mm nylon cell strainer to get single-cell suspension for further flow cytometric analysis.To collect BM extracellular fluid, mice femurs were flushed with 500 ml ice-cold PBS (Gibco) and the supernatant was harvested after centrifugation at 300 x g for 5 min at 4 C. To prepare mouse BM-derived macrophages (BMDM), upon lysis of erythrocytes with RBC lysis buffer (eBioscience), BM cells were plated and cultured in the presence of recombinant murine granulocyte macrophage colonystimulating factor (GM-CSF; 20 ng/ml, R&D Systems).Culture medium was replaced every two days, and after seven days, differentiated BMDM were used in efferocytosis experiments. 49To isolate myeloid cells from the spleen, splenocytes were incubated with biotinylated anti-mouse CD11b antibody (clone M1/70; Biolegend) followed by anti-biotin microbeads from Miltenyi Biotec.Myeloid cells were positively selected using LS columns on the magnetic field of QuadroMACS separator according to the manufacturer's instructions (Miltenyi Biotec).For isolating splenic T cells, myeloid cells were first removed by negative selection for CD11b + cells and then T cells were obtained by positive selection for CD4 + (clone GK1.5; Biolegend) cells.To isolate splenic neutrophils, splenocytes were incubated with biotinylated anti-mouse Ly6G antibody (clone 1A8; Biolegend) followed by anti-biotin microbeads from Miltenyi Biotec.Neutrophils were positively selected using LS columns on the magnetic field of QuadroMACS Separator according to the manufacturer's instructions (Miltenyi Biotec).For isolating splenic monocytes, neutrophils were first removed by negative selection for Ly6G + cells and then monocytes were obtained by positive selection for Ly6C + (clone HK1.4; Biolegend) cells as we previously described. 104Neutrophils were also isolated from the peripheral blood by using EasySepÔ Mouse Neutrophil Enrichment Kit according to the manufacturer's directions (STEMCELL).Gingival tissues around the area of ligature placement (and the contralateral control area) were harvested on day 5 for analysis.On day 5, gingiva was dissected around the area of ligature placement and digested for 1h at 37 o C with RPMI 1640 medium (Gibco) supplemented with collagenase IV (3.2 mg/ml, Worthington) and DNase (0.15 mg/ml, Roche). 133Single-cell suspensions were obtained by mashing the tissue against a strainer using plungers and filtered for staining and flow cytometry.Synovial tissues from joints were digested with 2 mg/mL collagenase type IV (Worthington) and 0.1 mg/mL DNase I (Roche) in DMEM containing 10% fetal bovine serum and penicillin/streptomycin for 30 min at 37 C. Singlecell suspensions were obtained by mashing the tissue against a strainer using plungers and filtered for staining and flow cytometry.

ROS induction and measurement
Peripheral blood neutrophils were stimulated with 10 -7 M PMA for 10 min at 37 C and stained using the CellROX Green flow cytometry assay kit according to manufacturer's instructions (Thermo Fisher).The cells were then washed and ROS production was analyzed by flow cytometry.Mean fluorescence intensity (MFI) was calculated.

Chemotaxis assay
The chemotaxis of neutrophils towards 10 ng/ml of CXCL1 or CXCL2, as well as the chemotaxis of monocytes to 20 ng/ml MCP-1, were tested using a Transwell system with 5-mm pores (Corning) according to a previously described protocol. 137The number of migrated cells in the absence of chemokine was assigned an average value of 1 and the experimental results were expressed as a ratio (chemotactic index) relative to this value.

Cytokine measurements
For ex vivo stimulation of splenic monocytes and neutrophils with LPS, isolated cells were seeded into 96-wells plates and stimulated with 10 ng/ml of E. coli O111:B4 LPS (InVivogen) for 24h.The culture supernatants were collected for measuring IL-1b, IL-6, and TNF concentrations using mouse ELISA kits, according to the manufacturer's instructions (Invitrogen).ELISA was also used to measure cytokine concentrations in the BM extracellular fluid and the serum.
(legend continued on next page) Figure S4.Clonal expansion of Dnmt3a R878H/+ hematopoietic cells is not associated with increased numbers or frequency of CD11b -/lo Ly6C hi OCPs and effect of IL-17 neutralization and Treg depletion on inflammatory responses, related to Figure 5 Clonal expansion of Dnmt3a R878H/+ hematopoietic cells is not associated with increased absolute numbers or frequency of CD11b À/lo Ly6C hi OCPs among BM cells (A and B) and effect of IL-17 neutralization and Treg depletion on inflammatory responses (C and D).(A and B) Lethally irradiated CD45.1 mice were transplanted with either 10%Dnmt3a R878H/+ CD45.2 + BM cells and 90% WT CD45.1 + BM cells (10% Dnmt3a R878H/+ BMT group) or with 10%Dnmt3a +/+ CD45.2 + BM cells and 90% WT CD45.1 + cells (10%Dnmt3a +/+ BMT group).12 weeks post-BMT, mice were subjected to LIP for 5 days, and femur BM cells were collected from the two groups for FACS analysis of osteoclast precursors.(A) Representative FACS plots (left) and percentage within total BM cells (middle) and total numbers (right) of osteoclast precursors (OCPs), defined as CD11b À/low Ly6C hi , in BM. (B) Representative FACS plots (left) and percentage (middle) and total numbers (right) of CD45.2 + CD11b -/low Ly6C hi OCP in BM. (C and D) BMT was performed as described above (A and B), and, after 12 weeks, recipient mice were subjected to LIP after local treatment with anti-IL-17A (or isotype control) (C) or after systemic treatment with anti-GITR (or isotype control) to deplete Tregs (D).Relative gingival mRNA expression of the indicated molecules after IL-17A neutralization (C) or after Treg cell depletion (D).

IFigure 3 .
Figure 3. Clonal expansion of Dnmt3a R878H/+ cells increases the severity of inflammatory arthritis BMT was performed as in Figure 1A, and after 12 weeks, recipient mice were subjected to CAIA.(A and B) (A) Clinical arthritis scores and (B) joint thickness at indicated time points.(C and D) (C) Representative images of H&E and (D) of safranin-O staining of tissue sections from knee joints harvested on day 7. Scale bars, 500 mm.(E-G) (E) Total cell numbers and (F) percentage (left) and numbers (right) of neutrophils (CD45 + CD11b + Ly6G + ) and (G) of monocytes (CD45 + CD11b + Ly6C + Ly6G À ) in the synovium of knee joints on day 7.

Figure 4 .
Figure 4. Single-cell RNA sequencing analysis of gingival and synovial cells (A) Experimental design.(B and C) Two-dimensional UMAP representation of 69,509 cells, according to (B) sample origin and results of clustering and (C) results of annotation.(D) Heatmap visualization of the distribution of sequenced cells in gingiva (top left) and synovium (top right) and cluster analysis of cell types in gingiva (bottom left) and synovium (bottom right), normalized for the number of cells per sample in the dataset.(E and F) CellChat analysis of intercellular communication networks in CD45.2 + (mutant) and CD45.1 + (WT) cells from 10%Dnmt3a R878H/+ BMT mice.(E) Heatmap of differential number and strength of possible interactions between any two of the indicated analyzed cell populations in gingiva (red, increased interaction in mutant cells; blue, increased interaction in WT cells).(F) Visualization of the overall information flow of each indicated signaling pathway by calculating the sum of communication probability among all pairs of synovial cell groups in the inferred network.The red and green colors indicate increased enrichment in mutant or WT cells, respectively.(Gand H) Gene expression levels in distinctly defined CD45.2 + cell types from gingiva (G) or synovium (H).(I) List of gingival upregulated enriched genes in CD45.2 + (Dnmt3a R878H/+ ) cells in the indicated KEGG pathway terms analyzed by STRING.(J and K) Top 10 significantly enriched GO terms sorted by PANTHER based on significantly upregulated or downregulated genes (in 10%Dnmt3a R878H/+ BMT-CD45.2vs. 10%Dnmt3a +/+ BMT-CD45.2) in neutrophils and T cells from gingiva (J) and synovium (K) (Fisher test with FDR-correction, p < 0.05).See also FiguresS2 and S3.
(A) Two-dimensional UMAP representation of 69,509 cells, according to sample origin and results of clustering.(B) The expression of top 5 marker genes in distinctly defined cell types.(C) Expression of specific makers to define different cell types.Ly6c2 and S100a4 define monocytes; Ly6g, neutrophils; C1qa and C1qb, macrophages; H2-Ab1, DC; CD19, B cells; Acp5 and Ctsk, osteoclasts; Col1a1, fibroblasts; CD3g and Trbc1, T cells; and Klrd1 and Trbc1 define NKT cells.(D) Dissected gingival tissue contains osteoclasts.Gingival tissues were dissected from LIP-subjected mice and stained with tartrate-resistant acid phosphatase (TRAP).TRAP + cells (osteoclasts) can be observed in the areas overlying the alveolar bone.

Table 1 .
Periodontal disease diagnoses, clinical measures of periodontal health, and inflammatory markers' associations with DNMT3A-CHIP mutation carrier status among the dental ARIC participants b Obtained from chi-squared tests.cNon-parametrictest for trend(Cuzick).CAL, clinical attachment loss; SD, standard deviation.

TABLE
d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTIPANT DETAILS B The Dental ARIC study cohort B Human clinical data B Ascertainment of CHIP carriage status in ARIC B Mice B Ligature-induced periodontitis and bone measurements B Collagen antibody-induced arthritis B Tissue processing and cell preparations B Bone marrow transplantation B Cell depletion and intervention experiments B Treatment of transplanted mice with rapamycin d METHOD DETAILS B Quantitative real-time PCR (qPCR)