Macrophage transcription factor TonEBP promotes systemic lupus erythematosus and kidney injury via damage-induced signaling pathways

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients – in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN. Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients – in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN. Lay SummarySystemic lupus erythematosus (SLE) is a complex and clinically heterogeneous autoimmune disease caused by dysregulated innate and adaptive immune systems. Here, we show that tonicity-responsive enhancer-binding protein (TonEBP) is an autoimmune stress protein that mediates damage-associated molecular pattern–mediated signaling in macrophages, leading to skewing of T-cell populations in SLE and lupus nephritis (LN). LN is a common and severe manifestation of SLE associated with both acute kidney injury and chronic kidney disease. In patients with LN, TonEBP expression was elevated in both renal cells and infiltrating immune cells, including macrophages. Elevated renal TonEBP was associated with renal inflammation and local injury, suggesting that it may be an attractive therapeutic target. Systemic lupus erythematosus (SLE) is a complex and clinically heterogeneous autoimmune disease caused by dysregulated innate and adaptive immune systems. Here, we show that tonicity-responsive enhancer-binding protein (TonEBP) is an autoimmune stress protein that mediates damage-associated molecular pattern–mediated signaling in macrophages, leading to skewing of T-cell populations in SLE and lupus nephritis (LN). LN is a common and severe manifestation of SLE associated with both acute kidney injury and chronic kidney disease. In patients with LN, TonEBP expression was elevated in both renal cells and infiltrating immune cells, including macrophages. Elevated renal TonEBP was associated with renal inflammation and local injury, suggesting that it may be an attractive therapeutic target. Systemic lupus erythematosus (SLE) is a highly complex and heterogeneous chronic autoimmune disease. Lupus nephritis (LN), characterized by severe inflammation in the kidney with deposition of immune complexes, is a major risk factor for morbidity and mortality in SLE.1Maroz N. Segal M.S. 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Hoi A. Steinmetz O.M. et al.TLR9 and TLR4 are required for the development of autoimmunity and lupus nephritis in pristane nephropathy.J Autoimmun. 2010; 35: 291-298Crossref PubMed Scopus (102) Google Scholar, 12Savarese E. Steinberg C. Pawar R.D. et al.Requirement of toll-like receptor 7 for pristane-induced production of autoantibodies and development of murine lupus nephritis.Arthritis Rheum. 2008; 58: 1107-1115Crossref PubMed Scopus (116) Google Scholar, 13Patole P.S. Zecher D. Pawar R.D. et al.G-rich DNA suppresses systemic lupus.J Am Soc Nephrol. 2005; 16: 3273-3280Crossref PubMed Scopus (89) Google Scholar Genetic variations in these genes (TLR4, TLR7, TLR8, and TLR9) display a clear linkage to SLE in patients,14Rupasree Y. Naushad S.M. 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Wang H. et al.TLR7 gain-of-function genetic variation causes human lupus.Nature. 2022; 605: 349-356Crossref PubMed Scopus (136) Google Scholar Because most of our understanding of TLR functions in SLE is mainly based on B lymphocytes and dendritic cells, it is important to explore the function of TLRs in macrophages. Tonicity-responsive enhancer-binding protein (TonEBP), also known as nuclear factor of activated T cells 5, was initially identified as a central regulator of cellular responses to hypertonic stress.18Miyakawa H. Woo S.K. Dahl S.C. et al.Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity.Proc Natl Acad Sci U S A. 1999; 96: 2538-2542Crossref PubMed Scopus (484) Google Scholar, 19Go W.Y. Liu X. Roti M.A. et al.NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment.Proc Natl Acad Sci U S A. 2004; 101: 10673-10678Crossref PubMed Scopus (239) Google Scholar, 20Lee S.D. Choi S.Y. Lim S.W. et al.TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways.Am J Physiol Renal Physiol. 2011; 300: F707-F715Crossref PubMed Scopus (50) Google Scholar Recent studies in humans and mice show that upregulated expression of TonEBP mediates the pathogenesis of inflammatory and autoimmune diseases by contributing to the development and activation of immune cells in osmostress-dependent and osmostress-independent contexts.21Choi S.Y. Lee-Kwon W. Kwon H.M. The evolving role of TonEBP as an immunometabolic stress protein.Nat Rev Nephrol. 2020; 16: 352-364Crossref PubMed Scopus (36) Google Scholar In macrophages, TonEBP is induced by inflammatory signals, such as lipopolysaccharide (LPS) or hyperglycemia,22Lee H.H. Sanada S. An S.M. et al.LPS-induced NFkappaB enhanceosome requires TonEBP/NFAT5 without DNA binding.Sci Rep. 2016; 624921Google Scholar,23Choi S.Y. Lim S.W. Salimi S. et al.Tonicity-responsive enhancer-binding protein mediates hyperglycemia-induced inflammation and vascular and renal injury.J Am Soc Nephrol. 2018; 29: 492-504Crossref PubMed Scopus (23) Google Scholar driving differentiation into the M1 phenotype via stimulation of nuclear factor (NF)–κB. As a transcriptional suppressor for the IL-10 and HO-1 genes,24Choi S.Y. Lee H.H. Lee J.H. et al.TonEBP suppresses IL-10-mediated immunomodulation.Sci Rep. 2016; 625726Google Scholar,25Yoo E.J. Lee H.H. Ye B.J. et al.TonEBP suppresses the HO-1 gene by blocking recruitment of Nrf2 to its promoter.Front Immunol. 2019; 10: 850Crossref PubMed Scopus (12) Google Scholar TonEBP inhibits the M2 phenotype of macrophages. Given the diverse functions of TonEBP in macrophages, we asked whether TonEBP is involved in the pathogenesis of SLE. We found that patients with LN exhibited higher renal TonEBP expression in infiltrating immune cells, especially macrophages and CD4+ cells, as well as higher renal expression of proinflammatory cytokines, than controls. A mouse model of SLE revealed that TonEBP in macrophages contributes to SLE/LN by suppressing efferocytosis and promoting antigen presentation, thereby affecting CD4+ T-cell differentiation. Macrophage TonEBP is induced by damage-associated molecular patterns and, in turn, mediates activation of NF-κB and mammalian target of rapamycin–interferon regulatory transcription factor 3/7 (mTOR-IRF3/7), leading to expression of inflammatory cytokines and mediators. These data reveal that TonEBP may be an important regulator for the pathogenesis of SLE and LN. Among patients with LN who underwent kidney biopsy between 2010 and 2020 at Seoul National University Hospital, patients with type III, IV, or V lupus nephritis without a history of immunosuppressive medication before kidney biopsy were identified. A control group comprised patients who had biopsy-proven thin basement membrane disease. Renal biopsy specimens from patients with LN and controls were analyzed for tissue staining, enzyme-linked immunosorbent assay, and mRNA expression. All patients agreed to donate their kidney biopsy sample to the Korea Biobank with written consent at the time of biopsy. The biospecimens used for this study were provided by the Biobank of Seoul National University Hospital, a member of Korea Biobank Network. The medical records of patients with LN were retrospectively reviewed for demographic and laboratory data, including blood urea nitrogen, serum creatinine, urinalysis, urine protein/creatinine ratio, complement component 3 and 4, and C-reactive protein. Lupus classification, activity index, and chronicity index were determined according to the revised International Society of Nephrology/Renal Pathology Society classification.26Bajema I.M. Wilhelmus S. Alpers C.E. et al.Revision of the International Society of Nephrology/Renal Pathology Society classification for lupus nephritis: clarification of definitions, and modified National Institutes of Health activity and chronicity indices.Kidney Int. 2018; 93: 789-796Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar The Systemic Lupus Erythematosus Disease Activity Index score, an index of lupus disease activity, was calculated on the basis of medical records, as previously described.27Bombardier C. Gladman D.D. Urowitz M.B. et al.The Committee on Prognosis Studies in SLE. Derivation of the SLEDAI: a disease activity index for lupus patients.Arthritis Rheum. 1992; 35: 630-640Crossref PubMed Scopus (4196) Google Scholar This study followed the Declaration of Helsinki and was approved by Seoul National University Hospital Institutional Review Board (H-1806-176-956). TRIzol (Invitrogen) was used to extract total RNA from frozen kidney biopsy specimens that had been stored at –70°C in RNAlater solution (Invitrogen). Next, cDNA was synthesized from 1 μg of RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Real-time polymerase chain reaction (PCR) was performed using a QuantStudioTM3 PCR system with Power SYBR Green PCR master mix (Applied Biosystems). The PCRs were run for 20 cycles. All samples were analyzed in triplicate. mRNA expression of each target gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase, and the relative mRNA expression levels in the lupus nephritis group compared with those in the control group were calculated using the –ΔΔCt (cycle threshold) method. All experiments involving live animals were performed in accordance with approved guidelines. All experimental protocols were authorized by the Institutional Animal Care and Use Committee of the Ulsan National Institute of Science and Technology. Heterozygous TonEBP mice (TonEBP+/Δ; Nfat5+/tm1Snh), which had been back-crossed to the C57BL/6 strain,18Miyakawa H. Woo S.K. Dahl S.C. et al.Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity.Proc Natl Acad Sci U S A. 1999; 96: 2538-2542Crossref PubMed Scopus (484) Google Scholar and their wild-type littermates (TonEBP+/+) were used. Mice carrying loxP sites, targeting TonEBP gene (TonEBPfl/fl; Nfat5tm1.1Chku/tm1.1Chku), were reported previously.28Kuper C. Beck F.X. Neuhofer W. Generation of a conditional knockout allele for the NFAT5 gene in mice.Front Physiol. 2014; 5: 507PubMed Google Scholar Transgenic mice expressing Cre recombinase, specifically in myeloid lineage cell (LysM-cre), were purchased from The Jackson Laboratory. TonEBPfl/fl and LysM-cre mice were crossed to generate mice with myeloid-specific deletion of TonEBP (TonEBPfl/fl, LysM-cre). To induce SLE-like disease, 8-week-old female mice received a single i.p. injection of 0.5 ml pristane (2,6,10,14-tetramethyl-pentradecane; Sigma-Aldrich), whereas control mice received phosphate-buffered saline alone. Mice were analyzed after 4 or 8 months. Blood was obtained by retro-orbital bleeding, and spleens and kidneys were harvested for analysis. All data are expressed as the mean ± SEM. Categorical values were compared using χ2 test or Fisher exact test, as appropriate. Continuous values were compared by an unpaired t test for comparisons between 2 conditions, whereas Bonferroni correction was performed for multiple comparisons. The correlation between TonEBP expression and cytokine expression, proteinuria, serum levels of anti–double-stranded DNA (dsDNA) antibodies, and complements was analyzed using Pearson or Spearman correlation, as appropriate according to variables’ distribution. One-way analysis of variance and Tukey post hoc test were used for multiple comparisons of data from the pristane-induced murine model and the in vitro experiments. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 8.2 software (GraphPad Software Inc). First, we asked whether TonEBP plays a role in SLE and LN. In this case, we decided to investigate patients with LN. Twelve patients with type III, IV, or V LN were identified, along with 12 control patients with thin basement membrane disease. Although all patients displayed hematuria, the LN group showed higher levels of proteinuria and lower levels of serum complement than the control group (Table 1). The clinical characteristics of the patients with LN are summarized in Table 2. All had active LN with overt proteinuria, reduced complement levels, elevated anti-dsDNA antibodies, and normal serum creatinine levels, except for one patient who had a value of 1.61 mg/dl. The activity index ranged from 1 to 10, and the SLE disease activity index ranged from 15 to 27.Table 1Comparison of patients’ characteristics between the LN group and the control groupControl (N = 12)Lupus nephritis (N = 12)P valueAge, yr39.4 ± 17.728.8 ± 10.00.071Female, %58.391.70.155Creatinine, mg/dl0.8 ± 0.40.8 ± 0.30.982eGFR, ml/min per /1.73 m2104.0 ± 34.1101.2 ± 36.30.846Urine protein/creatinine, g/g0.7 ± 0.66.1 ± 3.9<0.001Hematuria, %aDefined as red blood cell count ≥ 5/HPF.100100>0.999Pyuria, %bDefined as WBC ≥ 5/HPF.16.783.30.003Complement component 3, mg/dl110.5 ± 14.752.6 ± 25.8<0.001Complement component 4, mg/dl22.6 ± 3.89.7 ± 7.3<0.001CRP, mg/dl0.5 ± 1.10.5 ± 1.10.651CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; HPF, high-power field; LN, lupus nephritis; WBC, white blood cell.Continuous values were expressed as mean ± SD.a Defined as red blood cell count ≥ 5/HPF.b Defined as WBC ≥ 5/HPF. Open table in a new tab Table 2Clinical features of lupus nephritis patients used in this studyClassificationActivity indexChronicity indexPrevious immunosuppressantAnti-ds DNAC3 (mg/dl)C4 (mg/dl)CRP (mg/dl)Serum creatinine (mg/dl)Urine protein/creatinineSLEDAI scorePatient 1aUsed both in immunohistochemistry and mRNA expression studies.IV and V104No703450.081.616.7727Patient 2aUsed both in immunohistochemistry and mRNA expression studies.IV50No59.23710.010.892.5626Patient 3aUsed both in immunohistochemistry and mRNA expression studies.IV71No44.53350.040.753.9225Patient 4bUsed only in mRNA expression studies.V21No29.55290.580.3110.6815Patient 5bUsed only in mRNA expression studies.III and V20No14.877180.010.8414.5321Patient 6bUsed only in mRNA expression studies.III and V10No63.1107240.230.704.2716Patient 7cUsed only in immunohistochemistry.III and V20No32074100.080.824.0125Patient 8cUsed only in immunohistochemistry.IV and V63No3.52220.051.199.1917Patient 9dUsed only in multiplex ELISA test.III21No2002731.800.665.7227Patient 10dUsed only in multiplex ELISA test.III10No42.03520.030.483.0712Patient 11dUsed only in multiplex ELISA test.IV30No60.13983.480.675.9620Patient 12dUsed only in multiplex ELISA test.IV1.50No31.568150.020.902.418Anti-dsDNA, anti–double stranded DNA antibodies; C3, complement component 3; C4, complement component 4; CRP, C-reactive protein; ELISA, enzyme-linked immunosorbent assay; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index.a Used both in immunohistochemistry and mRNA expression studies.b Used only in mRNA expression studies.c Used only in immunohistochemistry.d Used only in multiplex ELISA test. Open table in a new tab CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; HPF, high-power field; LN, lupus nephritis; WBC, white blood cell. Continuous values were expressed as mean ± SD. Anti-dsDNA, anti–double stranded DNA antibodies; C3, complement component 3; C4, complement component 4; CRP, C-reactive protein; ELISA, enzyme-linked immunosorbent assay; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index. Because upregulated expression of TonEBP, which drives transcription of proinflammatory cytokines,22Lee H.H. Sanada S. An S.M. et al.LPS-induced NFkappaB enhanceosome requires TonEBP/NFAT5 without DNA binding.Sci Rep. 2016; 624921Google Scholar,29Jeong G.W. Lee H.H. Lee-Kwon W. Kwon H.M. Microglial TonEBP mediates LPS-induced inflammation and memory loss as transcriptional cofactor for NF-kappaB and AP-1.J Neuroinflammation. 2020; 17: 372Crossref PubMed Scopus (8) Google Scholar is the key feature of inflamed tissues in rheumatoid arthritis30Yoon H.J. You S. Yoo S.A. et al.NF-AT5 is a critical regulator of inflammatory arthritis.Arthritis Rheum. 2011; 63: 1843-1852Crossref PubMed Scopus (68) Google Scholar,31Choi S. You S. Kim D. et al.Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis.J Clin Invest. 2017; 127: 954-969Crossref PubMed Scopus (59) Google Scholar and hepatitis,32Lee J.H. Suh J.H. Choi S.Y. et al.Tonicity-responsive enhancer-binding protein promotes hepatocellular carcinogenesis, recurrence and metastasis.Gut. 2019; 68: 347-358Crossref PubMed Scopus (32) Google Scholar we first analyzed mRNA expression encoding TonEBP and cytokines in frozen kidney biopsy samples. TonEBP mRNA expression was higher in the LN group than in the control group (Figure 1a). Expression of IL6, IL1B, MCP1, TNFA, and IFNB mRNA was also significantly higher in the LN group. In addition, expression of MCP1, IFNA, and IFNB mRNA correlated with that of TonEBP mRNA (Figure 1b), which is consistent with the role of TonEBP in transcription of proinflammatory cytokines. The protein levels of these cytokines also tended to be higher in the LN group than in the control group, although the difference was not statistically significant (Supplementary Figure S1). Furthermore, the level of TonEBP mRNA correlated significantly with the degree of proteinuria (Supplementary Figure S2A) and showed a trend toward a negative correlation with serum complement component 3 levels (Supplementary Figure S2C). Next, we performed immunohistochemistry to examine renal TonEBP. TonEBP was clearly detected in both nuclear and cytoplasmic regions (Figure 2a). TonEBP was localized to a variety of cell types, including podocytes, parietal epithelial cells, endothelial cells, and neutrophils, in the glomeruli of patients with LN. In the LN group, renal interstitial areas were heavily infiltrated with immune cells. Among renal tubules, TonEBP was prominently expressed in distal tubules with positive uromodulin staining and collecting duct with negative uromodulin staining, whereas the TonEBP expression was weak in proximal tubules (Figure 2a and Supplementary Figure S3). The number of TonEBP-positive cells was significantly higher in all areas of the kidney (glomerular, interstitial, and tubular regions) in the LN group than in the control group (Figure 2b). Thus, renal protein expression of TonEBP is clearly elevated in LN, in line with its mRNA levels. Next, we used multiplex immune fluorescence staining to localize TonEBP expression to various renal and immune cells. The number of TonEBP-positive cells was clearly elevated in the LN group (Figure 3 and Supplementary Figure S4A). The increase was observed in both epithelial (cytokeratin-positive) and endothelial (CD31-positive) cells. The same was seen with the immune cells (Figure 3 and Supplementary Figure S4B and C): CD4+ T cells, CD8+ T cells, CD68+ macrophages, and TonEBP+CD86+ M1 macrophages. However, we could not adequately assess renal TonEBP+CD20+ B cells, TonEBP+CD11c+ dendritic cells, and TonEBP+CD206+ M2 macrophages, because the numbers of these cells were too low. In sum, TonEBP expression is elevated in renal cells and infiltrating immune cells, including CD4+ T cells and macrophages, in LN. Next, we asked what triggers renal expression of TonEBP. To answer this question, we used a mouse model of SLE induced by a single injection of pristane (Figure 4a).33Zhuang H. Szeto C. Han S. et al.Animal models of interferon signature positive lupus.Front Immunol. 2015; 6: 291Crossref PubMed Scopus (52) Google Scholar,34Reeves W.H. Lee P.Y. Weinstein J.S. et al.Induction of autoimmunity by pristane and other naturally occurring hydrocarbons.Trends Immunol. 2009; 30: 455-464Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar We then compared TonEBP haplodeficient (TonEBP+/Δ) mice with their wild-type (TonEBP+/+) littermates; this is because haplodeficient animals are resistant to experimentally induced rheumatoid arthritis30Yoon H.J. You S. Yoo S.A. et al.NF-AT5 is a critical regulator of inflammatory arthritis.Arthritis Rheum. 2011; 63: 1843-1852Crossref PubMed Scopus (68) Google Scholar and diabetic nephropathy.23Choi S.Y. Lim S.W. Salimi S. et al.Tonicity-responsive enhancer-binding protein mediates hyperglycemia-induced inflammation and vascular and renal injury.J Am Soc Nephrol. 2018; 29: 492-504Crossref PubMed Scopus (23) Google Scholar Wild-type mice displayed a clear SLE/LN phenotype, which includes splenomegaly, elevated levels of circulating anti-dsDNA autoantibodies, decreased serum complement component 3 levels, and renal hypertrophy and glomerular injury in association with glomerular deposition of IgG and complement component 3 (Figure 4b–h). In addition, renal expression of TonEBP was markedly elevated (Figure 4h and Supplementary Figure S5A–C), similar to patients with LN. More important, this phenotype was absent from TonEBP haplodeficient animals, indicating that TonEBP may be involved in the development of SLE and associated renal injury. There was little immune complex deposition in the glomeruli of TonEBP halplodeficient animals, as well as the absence of anti-dsDNA antibodies, suggesting that systemic events important for the development of SLE are blocked. To examine the systemic events, we analyzed alveolar hemorrhage and immune cell profiles in the peritoneal cavity and lung at 2 weeks after pristane treatment (Supplemental Figure S6). There was clear alveolar hemorrhage, which was not affected by TonEBP haplodeficiency. By contrast, influx of Ly6Chi inflammatory monocytes/macrophages into the peritoneal cavity was less pronounced. Likewise, influx of CD11b+ cells, CD11b+Ly6G+ neutrophils, and CD11b+F4/80+ macrophages into lung was lower. Thus, we speculated that TonEBP in myeloid cells is associated with the pathogenesis of SLE and LN. Myeloid TonEBP is required for dendritic cell maturation35Ye B.J. Lee H.H. Yoo E.J. et al.TonEBP in dendritic cells mediates pro-inflammatory maturation and Th1/Th17 responses.Cell Death Dis. 2020; 11: 421Crossref PubMed Scopus (8) Google Scholar and macrophage activation31Choi S. You S. Kim D. et al.Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis.J Clin Invest. 2017; 127: 954-969Crossref PubMed Scopus (59) Google Scholar in rheumatoid arthritis. Given that we observed elevated TonEBP in macrophages in patients with LN (Figure 3 and Supplementary Figure S4C) and its role in systemic immune responses with reduced in myeloid cell populations in pristane-induced TonEBP haplodeficient mice (Supplementary Figure S6), we explored the role of myeloid TonEBP using mice with myeloid-specific deletion of TonEBP (TonEBPfl/fl, LysM-cre) and their TonEBPfl/fl littermates (Figure 5a). We found that mice with myeloid-specific deletion of TonEBP did not develop SLE and LN (Figure 5b–h), similar to mice with TonEBP haplodeficiency. Increased renal TonEBP