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ABSTRACT Transcription factors specialized to limit the destructive potential of inflammatory immune cells remain ill-defined. We discovered loss-of-function variants in the X-linked ETS

transcription factor gene _ELF4_ in multiple unrelated male patients with early onset mucosal autoinflammation and inflammatory bowel disease (IBD) characteristics, including fevers and

ulcers that responded to interleukin-1 (IL-1), tumor necrosis factor or IL-12p40 blockade. Using cells from patients and newly generated mouse models, we uncovered ELF4-mutant macrophages

having hyperinflammatory responses to a range of innate stimuli. In mouse macrophages, Elf4 both sustained the expression of anti-inflammatory genes, such as _Il1rn_, and limited the

upregulation of inflammation amplifiers, including _S100A8_, _Lcn2_, _Trem1_ and neutrophil chemoattractants. Blockade of Trem1 reversed inflammation and intestine pathology after in vivo

lipopolysaccharide challenge in mice carrying patient-derived variants in Elf4. Thus, ELF4 restrains inflammation and protects against mucosal disease, a discovery with broad translational

relevance for human inflammatory disorders such as IBD. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A DISEASE-ASSOCIATED GENE DESERT DIRECTS MACROPHAGE INFLAMMATION THROUGH ETS2 Article Open access 05 June 2024 INTERLEUKIN-22

REGULATES NEUTROPHIL RECRUITMENT IN ULCERATIVE COLITIS AND IS ASSOCIATED WITH RESISTANCE TO USTEKINUMAB THERAPY Article Open access 03 October 2022 TRANSCRIPTION FACTOR ELF-1 PROTECTS

AGAINST COLITIS BY MAINTAINING INTESTINAL EPITHELIUM HOMEOSTASIS Article Open access 08 March 2025 DATA AVAILABILITY RNA-seq data were deposited in the GEO database (accession no.

GSE175569). WES data will not be made publicly available because they contain information that could compromise research participant privacy/consent. Source data are provided with this

paper. Information on the WES raw data supporting the findings of the present study is available from the corresponding author, C.L.L., upon request. Mice harboring the Trp250Ser or KO

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  Download references ACKNOWLEDGEMENTS We thank the patients and their families for participating in the research and all clinical care staff for their contributions. We also thank P.

Schwartzberg, P.-P. Axisa and J.-M. Carpier for critical feedback. We thank Prometheus for providing recombinant IL-2 used in T cell culture experiments and the Yale Cancer Center for

support. We thank Yale New Haven Hospital and S. Bluell and J. Buell for their support of the Pediatric Genomics Discovery Program. C.L.L. is funded by the Mathers Foundation, National

Institute of Allergy and Infectious Diseases/National Institutes of Health (grant no. R01AI150913), Immune Deficiency Foundation, Hood Foundation and Yale University. A.M.M. is funded by a

Canada Research Chair (Tier 1) in Pediatric IBD, Canadian Institute of Health Research Foundation Grant, National Institute of Diabetes and Digestive and Kidney Diseases (grant no.

RC2DK118640) and the Leona M. and Harry B. Helmsley Charitable Trust. AUTHOR INFORMATION Author notes * These authors contributed equally: Paul M. Tyler, Molly L. Bucklin, Mengting Zhao.

AUTHORS AND AFFILIATIONS * Immunobiology Department, Yale University School of Medicine, New Haven, CT, USA Paul M. Tyler, Molly L. Bucklin, Mengting Zhao, Timothy J. Maher, Andrew J. Rice 

& Carrie L. Lucas * Pediatric Genomics Discovery Program, Yale University School of Medicine, New Haven, CT, USA Weizhen Ji, Saquib A. Lakhani & Carrie L. Lucas * Department of

Pediatrics, Yale University School of Medicine, New Haven, CT, USA Weizhen Ji, Paul McCarthy, Jason Catanzaro & Saquib A. Lakhani * SickKids Inflammatory Bowel Disease Center and Cell

Biology Program, Research Institute, Hospital for Sick Children, Department of Pediatrics and Biochemistry, University of Toronto, Hospital for Sick Children, Toronto, ON, Canada Neil

Warner, Jie Pan, Anne Griffiths & Aleixo M. Muise * Department of Pathology, Yale University School of Medicine, New Haven, CT, USA Raffaella Morotti * Department of Pediatrics, Division

of Pediatric Infectious Diseases and Immunology, Erasmus University Medical Center, Rotterdam, the Netherlands Annemarie M. C. van Rossum * Department of Clinical Genetics, Erasmus

University Medical Center, Rotterdam, the Netherlands Iris H.I.M. Hollink * Department of Internal Medicine, Division of Clinical Immunology and Department of Immunology, Erasmus University

Medical Center, Rotterdam, the Netherlands Virgil A.S.H. Dalm Authors * Paul M. Tyler View author publications You can also search for this author inPubMed Google Scholar * Molly L. Bucklin

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* Jason Catanzaro View author publications You can also search for this author inPubMed Google Scholar * Saquib A. Lakhani View author publications You can also search for this author

inPubMed Google Scholar * Aleixo M. Muise View author publications You can also search for this author inPubMed Google Scholar * Carrie L. Lucas View author publications You can also search

for this author inPubMed Google Scholar CONTRIBUTIONS P.M.T., M.L.B. and M.Z. performed experiments, analyzed the data and wrote the manuscript. T.J.M. and A.J.R. performed experiments and

analyzed the data. W.J. performed the analysis of the genomics data from family A. N.W. identified and evaluated the ELF4 variant in patient B.1. J.P. performed staining of biopsy samples

from patient B.1 and analyzed the data. R.M. provided pathology expertise for staining of biopsy samples from patient A.1. P.M. provided clinical care and insights for patient A.1. A.G.

provided clinical care and insights for patient B.1. A.M.C.v.R. provided clinical care and insights for patient C.1. I.H.I.M.H. performed genetic analysis of family C. V.A.S.H.D. recruited

and provided clinical care and insights for patient C.1. J.C. recruited and provided clinical care and insights for patient A.1. S.A.L. oversaw genetic analysis of family A. A.M.M. provided

clinical care and oversaw genomics analysis and histology staining of biopsies from family B. C.L.L. supervised overall research and data analysis, performed experiments and wrote/edited the

manuscript. All authors discussed and reviewed the manuscript. CORRESPONDING AUTHOR Correspondence to Carrie L. Lucas. ETHICS DECLARATIONS COMPETING INTERESTS S.A.L. is part owner of Qiyas

Higher Health and Victory Genomics, startup companies unrelated to this work. All other authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature

Immunology_ thanks the anonymous reviewers for their contribution to the peer review of this work. Ioana Visan was the primary editor on this article and managed its editorial process and

peer review in collaboration with the rest of the editorial team. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 EXTENDED DEX PATIENT CLINICAL AND CELLULAR FINDINGS AND GENERATION OF _ELF4_ KO AND TRP250SER MICE. A, NK cell, (B) NKT cell, (C) CD4+ and

CD8+ T cell, (D) monocyte, (E) B cell, (F) CD4+ memory and naïve, and (G) CD8+ memory and naïve flow cytometric immunophenotyping for the indicated markers on PBMCs from a healthy donor

(Ctrl) and patient A.1. H, NK cytotoxicity assay using PBMCs from patient A.1 (red) compared to the normal range (grey shading). I, Human IFNα ELISA in supernatants of LPS-stimulated PBMCs

from healthy donors (n = 3) and patient A.1 (n = 1). J, Western blot on THP1 lysates for ELF4. K, Histogram of missense variants in the gnomAD dabase in ELF4 gene. L, Western blot on 293T

cells overexpressing variants of ELF4 (myc-tagged) reported in gnomAD. M, Schematic of mouse _Elf4_. N, Western blot for Elf4 in mouse thymus. O, Sanger sequencing genotyping of Trp250Ser

mice. P, Relative allele usage of B.2 (X/Trp251Ser) PBMCs. Q, Relative allele usage (X/Trp250Ser) of mouse CD4+ or CD8+ cells. R, Percentage of perforin+ CD8+ T cells (WT n = 6, Trp250Ser n 

= 3, Elf4 KO n = 3) 4 days with after anti-CD3 and anti-CD28. S, Perforin gene expression in blasting CD8+ T cells isolated from healthy controls and patient A.1 determined by qRT-PCR (Ctrl

n = 3, A.1 n = 1). T, Histogram displaying perforin expression in NT and ELF4 CRISPR-edited human CD8 + T cells after 10 days of IL-2. U, Western blot showing CRISPR deletion of ELF4 from

human CD8 + T cells by CRISPR. V, Perforin expression determined by flow cytometry at 24-hour time point following overexpression of myc-tagged Trp251Ser and WT ELF4 mRNA in patient A.1, B.1

(pink), and C.1 (red) CD8 + T cells. Data are presented as mean + /- S.E.M. with two-tailed unpaired t-test (R) or paired t-test (V) *p < 0.05, **p < 0.01, ***p < 0.001, ****p <

 .0001, no marking indicates not significant. Source data EXTENDED DATA FIG. 2 EXTENDED SERUM ANALYSES IN PATIENT A.1. Concentrations of the indicated cytokine or chemokine in serum from

independent blood draws of unrelated healthy controls (n = 4–6), patient A.1 (n = 3), mom (blue circles, n = 3), and dad (green circles, n = 1–3). Data from three independent experiments is

presented as mean ± SEM. Statistical analysis was performed using two-tailed unpaired t-test. **p < 0.01, no marking indicates not significant. Source data EXTENDED DATA FIG. 3 EXTENDED

DATA ON T CELL DIFFERENTIATION AND GENE EXPRESSION. A, ELISA for IL-17A from human CD4+ cells (n = 1). B, ELISA for IL-17A from mouse CD4+ T cells (n = 1). (n = 1). C, Mouse IL-17A ELISA

following naïve CD4+ Th17 _in vitro_ differentiation under non-pathogenic conditions (TGFβ + IL-6) WT n = 3, Trp250Ser n = 3, Elf4 KO n = 3. D, E, Percentage of mouse or human naïve CD4 T

cells in spleen (WT n = 3, Trp250Ser n = 3, Elf4 KO n = 3) or PBMC (Ctrl n = 3, A.1 n = 2), respectively. F, Western blot of cytoplasmic (Cyto) and nuclear (Nuc) fractions of effector T

cells. G, Flow cytometry after treatment with anti-CD3 and anti-CD28 for 72 hours. H, List of gene sets and pathways associated with the differentially expressed genes in Elf4 KO naïve CD4+

T cells. I, Volcano plots of differentially expressed genes in Elf4 KO (1) or Trp250Ser (2) versus WT mouse naïve CD4+ T cells or Trp250Ser versus WT mouse _in vitro_ differentiation Th17

cells after 48 hours under non-pathogenic (3) or pathogenic (4) conditions. J, Top ten upregulated and downregulated genes in Elf4 KO or Trp250Ser CD4+ T cells. Values shown as log2(FC). K,

Naive CD4+ T cells differentiated _in vitro_ to Th17 cells. L, Heat map showing Z-score summary of naive CD4+ T cell ATAC-seq peak results filtered for genes with p-value < 0.01 and FC 

> 2. M, Venn diagram displaying overlap between ATAC-seq peaks in Elf4 KO and WT naive CD4+ T cells. N, Heatmap displaying genes involved in chromatin regulation that were differentially

expressed by RNAseq (WT vs Elf4 KO) and also display differences in accessibility by ATACseq. O, Reanalysis of DICE database 45. ELISA data are from a minimum of three experiments, each dot

representing one ELISA well with two wells/technical replicates per sample. A minimum of n = 3 mice (biological replicates) was used for each genotype in mouse experiments. DEX patient

samples represent blood from the same patient at different times. Data are presented as mean ± S.E.M. with two-tailed unpaired t-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p <

 .0001, no marking indicates not significant. Source data EXTENDED DATA FIG. 4 EXTENDED DATA ON MONOCYTE/MACROPHAGE CELLULAR RESPONSES. A, Indicated cytokine measured in culture supernatants

from LPS-stimulated human PBMCs. Data are combined from two independent experiments (patient A.1 vs 5 controls, and patient B.1 vs 5 controls) and expressed as fold change of patient values

normalized to the average of the controls (n = 13 healthy controls, n = 2 A.1 independent experiments, n = 1 B.1 experiment). B, PBMCs from patient A.1 and a healthy donor control were

treated with LPS alone or LPS and a titration of IL-10 for 12 hours, and IL-6 was measured in culture supernatants (n = 1 patient and n = 1 healthy donor control). C, RT-PCR analysis of

_ELF4_ gene expression in monocyte-derived macrophages from healthy donors after CRISPR targeting (NT: non-targeting gRNA, ELF4: _ELF4_ gRNA). D, IL-6 and CXCL1 measured in culture

supernatants from 24hrs MDP/PolyIC/β-glucan-stimulated BMDMs isolated from Elf4 KO, Trp250Ser, or WT mice. E, Endotoxic shock was induced in groups of male WT and age-matched Elf4 KO and

Trp250Ser mice by i.p. injection of 2 mg/kg ultra-pure (UP) LPS. Animals were scored for 0 h, 2 h, 4 h, 6 h and 16 h after LPS injection. F, Concentrations of the indicated cytokine or

chemokine in mouse serum 4 hr after i.p. LPS challenge. Analytes in red are significantly different between genotypes. G, Endotoxic shock was induced in groups of female WT (n = 3) and

age-matched heterozygous females (Elf4 KO n = 3 and Trp250Ser n = 3) by i.p. injection of 2 mg/kg ultra-pure (UP) LPS. H, Concentrations of the indicated cytokine or chemokine in mouse serum

4 hr after i.p. LPS challenge described in (G). Data are representative of three independent experiments and presented as mean ± SD. Statistical analysis was performed using two-tailed

unpaired t-test. *p < 0.05, **p < 0.01, ***/###p < 0.001, ****p < 0.0001, no marking indicates not significant. Source data EXTENDED DATA FIG. 5 EXTENDED DATA ON MACROPHAGE GENE

EXPRESSION AND RESPONSES TO TREM1 BLOCKADE. A-C, Volcano plots (-log10(FDR) vs fold change) of differentially expressed genes in Elf4 KO versus WT mouse BMDMs as indicated. D-F, Heatmaps

highlighting top 10 differentially expressed genes at each timepoint above. G, H, RT-PCR for _Il10_ and _Il1rn_ in WT, Elf4 KO, or Elf4 Trp250Ser BMDMs at 16 hours after stimulation with

LPS. For (G), (H), (K), and (L) n = 3 wt and n = 3 Trp250Ser mutant mice per group. (I) _IL1RN_ reporter data as in Fig. 5C but with three individual 5′-GGAA sites mutated to 5′-AAAA to

assess the contribution of each to ELF4-driven transcriptional activation of _IL1RN_ reporter, n = 1 experimental replicate, representative of three independent experiments, ±SD. J,

ChIP-sequencing traces for Elf4 bound near the indicated gene in mouse BMDM without (-) and with (+) 4 hr LPS stimulation. K, L, RT-PCR for _S100a8_ and _Trem1_ in WT, Elf4 KO, or Elf4

Trp250Ser BMDMs at 4 hours after stimulation with LPS. M, Functionally enriched gene ontology and KEGG pathways of upregulated differentially expressed genes in Elf4 KO compared to WT BMDMs

16 hrs after LPS stimulation. N-P, IL-6, IL-12p70, and IL-23 measured in culture supernatants at 24 hours after stimulation of indicated BMDMs with LPS or LPS and Trem1-Fc (n = 5/group). Q,

Endotoxic shock clinical score 16 hours after treatment (n = 8,7 WT/WT + Trem1-Fc; n = 3,4 Elf4 KO/Elf4 KO + Trem1 Fc; n = 5,4 Trp250Ser/Trp250Ser + Trem1 Fc). R, CXCL1 was measured in mouse

serum at 4 hr after _in vivo_ LPS challenge with the treatments indicated in (Q). Data are representative of three independent experiments and presented as mean ± SD. Statistical analysis

was performed using two-tailed unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, no marking indicates not significant. Source data SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Fig. 1 and Tables 1–4. REPORTING SUMMARY SUPPLEMENTARY TABLE 5 Oligonucleotide sequences for gRNAs and primers. SOURCE DATA SOURCE DATA FIG. 1

Statistical source data. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical

source data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data.

SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE

THIS ARTICLE Tyler, P.M., Bucklin, M.L., Zhao, M. _et al._ Human autoinflammatory disease reveals _ELF4_ as a transcriptional regulator of inflammation. _Nat Immunol_ 22, 1118–1126 (2021).

https://doi.org/10.1038/s41590-021-00984-4 Download citation * Received: 13 November 2020 * Accepted: 23 June 2021 * Published: 29 July 2021 * Issue Date: September 2021 * DOI:

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