Article https://doi.org/10.1038/s41467-023-42277-4 Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation Marlies Cortés 1 , Agnese Brischetto 1,12, M. C. Martinez-Campanario 1,12, Chiara Ninfali 1, Verónica Domínguez2, Sara Fernández3, Raquel Celis4, Anna Esteve-Codina5, Juan J. Lozano6, Julia Sidorova6, Gloria Garrabou3, Anna-Maria Siegert 7, Carlos Enrich8, Belén Pintado2, Manuel Morales-Ruiz 6,8,9, Pedro Castro 3, Juan D. Cañete4 & Antonio Postigo 1,6,10,11 Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drugMetformin inhibits acute and chronic inflammation throughmechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppres- sion by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immu- nosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macro- phages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression. Inflammation is a natural protective response to infection and tissue injury. It involves an acute inflammatory phase, which can be fol- lowed by an immunosuppressed (tolerogenic) state, wherein immune cells are unable to respond to a secondary challenge1–3. Immunosuppression protects tissues from excessive inflammation, but if prolonged over time, it can increase susceptibility to secondary infections. On the other hand, an incomplete or dysregulated reso- lution of acute inflammation and continuous proinflammatory sti- muli can override immune tolerance and lead to autoimmunity and chronic inflammation4. The transition of monocytes/macrophages from an inflammatory phenotype toward an immunosuppressed state involves their Received: 28 September 2022 Accepted: 5 October 2023 Check for updates A full list of affiliations appears at the end of the paper. e-mail: mcortesh@recerca.clinic.cat; idib412@recerca.clinic.cat Nature Communications | (2023) 14:7471 1 12 34 56 78 9 0 () :,; 12 34 56 78 9 0 () :,; http://orcid.org/0000-0002-9313-1194 http://orcid.org/0000-0002-9313-1194 http://orcid.org/0000-0002-9313-1194 http://orcid.org/0000-0002-9313-1194 http://orcid.org/0000-0002-9313-1194 http://orcid.org/0000-0002-7056-8721 http://orcid.org/0000-0002-7056-8721 http://orcid.org/0000-0002-7056-8721 http://orcid.org/0000-0002-7056-8721 http://orcid.org/0000-0002-7056-8721 http://orcid.org/0000-0002-2745-6505 http://orcid.org/0000-0002-2745-6505 http://orcid.org/0000-0002-2745-6505 http://orcid.org/0000-0002-2745-6505 http://orcid.org/0000-0002-2745-6505 http://orcid.org/0000-0002-5745-6119 http://orcid.org/0000-0002-5745-6119 http://orcid.org/0000-0002-5745-6119 http://orcid.org/0000-0002-5745-6119 http://orcid.org/0000-0002-5745-6119 http://orcid.org/0000-0002-8694-5894 http://orcid.org/0000-0002-8694-5894 http://orcid.org/0000-0002-8694-5894 http://orcid.org/0000-0002-8694-5894 http://orcid.org/0000-0002-8694-5894 http://orcid.org/0000-0002-9074-2272 http://orcid.org/0000-0002-9074-2272 http://orcid.org/0000-0002-9074-2272 http://orcid.org/0000-0002-9074-2272 http://orcid.org/0000-0002-9074-2272 http://orcid.org/0000-0002-6118-8970 http://orcid.org/0000-0002-6118-8970 http://orcid.org/0000-0002-6118-8970 http://orcid.org/0000-0002-6118-8970 http://orcid.org/0000-0002-6118-8970 http://orcid.org/0000-0003-4605-2634 http://orcid.org/0000-0003-4605-2634 http://orcid.org/0000-0003-4605-2634 http://orcid.org/0000-0003-4605-2634 http://orcid.org/0000-0003-4605-2634 http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-42277-4&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-42277-4&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-42277-4&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-42277-4&domain=pdf mailto:mcortesh@recerca.clinic.cat mailto:idib412@recerca.clinic.cat metabolic reprogramming5–7. During inflammation, there is a switch from mitochondrial oxidative phosphorylation (OxPhos) to glycolysis and lactate production. Mitochondria shift their role from ATP gen- eration to succinate oxidation, which in turn stimulates the production of reactive oxygen species (ROS) and inflammatory cytokines8,9. Lactate is not only a byproduct of glycolysis but it also promotes the epigenetic activation of anti-inflammatory genes, leading to their subsequent upregulation during immunosuppression10. Inhibition of mitochondrial translation by antibiotics of the tetracycline family and analogues of the anti-diabetic drug Metformin reduces both bacterial lipopolysacchar- ide (LPS)-induced production of inflammatory cytokines in vitro and tissue damage in vivo11,12. Alterations in themetabolism of immune cells also play a pathogenic role in the development of autoimmune chronic inflammatory diseases (e.g., psoriasis, arthritis, colitis)13–15. The mechanisms by which Metformin inhibits acute and chronic inflammation are cell- and context-dependent and are still being uncovered. Metformin exerts its anti-inflammatory effects through various pathways, including inhibiting the mitochondrial electron transport chain complex I (ETC-CI) and mTORC1 signaling, as well as reducing mitochondrial ROS production16,17. However, in some cancer cells and CD8+ tumor-infiltrating T cells, Metformin has been reported to stimulate ROS production18,19. Metformin promotes mitophagy to eliminate damaged mitochondria, thus dampening inflammasome activation20. Furthermore, Metformin can inhibit inflammasome acti- vation independently of AMPK- and NFkB signaling21,22. The transcription factor ZEB1 enables andmaintains cell plasticity in cancer cells and is best known for inducing an epithelial-to- mesenchymal transition (EMT) during embryonic development and cancer progression (reviewed in23–26). ZEB1 expression enhances the pro-tumoral effects of tumor-associatedmacrophages27, and induces a stem-like phenotype in macrophages upon viral infection28. This evi- dence prompted us to investigate the potential role of ZEB1 in the regulation of macrophage transcriptomic and metabolic plasticity during acute and chronic inflammation. Here, we find that ZEB1 expression is required for the inflamma- tory and immunosuppressive phenotypes of macrophages, playing opposing roles in both stages. In addition, we show that Metformin’s anti-inflammatory and ROS-inhibiting effects in models of sepsis and psoriatic disease are dependent on the expression of ZEB1 in macro- phages. Using mice lacking Zeb1 in their myeloid cells, as well as samples from human patients and mouse models of sepsis and psor- iasis, we show that the diverging effects of ZEB1 in inflammation and immunosuppression aremediated through its inhibition of amino acid transport, mitochondrial protein translation and content, and its induction of autophagy. Our results suggest that Metformin pre- treatment induces an immunosuppressive-like state in inflammatory macrophages and that ZEB1 is required for the inhibition of mito- chondrial translation in immunosuppressed macrophages. ZEB1 limits acute and chronic inflammation by reducing amino acid levels and consumption in macrophages thereby inhibiting mTORC1 signaling and mitochondrial translation. Altogether, these results identify a mechanism that regulates macrophage metabolic plasticity, presenting a potential target for modulating dysregulated inflammation and immunosuppression in sepsis and autoimmune diseases. Results ZEB1 has a dual role being required for both the induction and resolution of inflammation To study the immunogenic-to-immunosuppressive reprogramming of macrophages in the context of acute inflammation, we used amodel of acute inflammation triggered by lipopolysaccharide (LPS)29 (see Sup- plementary Fig. S1A and Supplementary Methods), where mice or macrophages were divided into different groups. One group was subjected to the acute inflammation protocol (referred to as “LPS”), wherein they received the vehicle (PBS) followed by a single dose of LPS after 24h. The other group underwent the immunosuppression protocol (referred to as “LPS + LPS”), wherein they received an initial dose of LPS, followed by a second dose of LPS 24 h later. These LPS- induced acute inflammation and immunosuppressive responses are primarily mediated by macrophages30. Accordingly, expression of the inflammatory cytokine IL6 in mouse peritoneal macrophages and human monocyte-derived macrophages increased during acute inflammation but to a lesser extent upon the second antigenic chal- lenge in the immunosuppressive protocol (Supplementary Fig. S1B and S1C). We also compared IL6 expression in human peripheral blood mononuclear cells (PBMC) in different conditions. Firstly, we com- pared PBMCs from septic patients at ICU admission (0 h, representing an acute inflammatory state) with the PBMCs of the same patients collected three days later (72 h, representing an immunosuppressed state) (Supplementary Fig. S1D). Additionally, we compared PBMCs from healthy donors with those from patients with a chronic inflam- matory disease, namely psoriatic arthritis (PsA), which affects approximately 30% of patients with psoriatic disease (Supplementary Fig. S1E). In all cases, PBMCs were either left untreated (PBS) or sub- jected to in vitro incubation with LPS for 2 hours. In line with their immunosuppressed state, the response of PBMCs from septic patients at 72 h to a new inflammatory challenge was only around 26% relative to the response of PBMCs from septic patients at 0 h (Supplementary Fig. S1D). In contrast, in the PBMCs of patients with PsA, IL6 increased around 500 times relative to PBMCs from healthy donors (Supple- mentary Fig. S1E). The transcription factor ZEB1 is best known for promoting cellular plasticity in epithelial cells during cancer initiation and progression24,25,31,32. We found that ZEB1 levels increased when both human and mouse macrophages transitioned to an immunosup- pressed state (Supplementary Fig. S1F–S1H). To examinewhether ZEB1 modulates the phenotype and function of macrophages during acute inflammation or immunosuppression, we generated a Zeb1fl/fl mouse (hereinafter referred to as Zeb1WT mouse) that was then crossed with LysmCre mice to delete Zeb1 in myeloid cells (hereafter referred to as Zeb1ΔM) (Supplementary Fig. S1I–S1K). Zeb1WT and Zeb1ΔM mice were each divided into two cohorts and subjected to the LPS-induced lethal endotoxemia and LPS + LPS- induced immunosuppression protocols (see Supplementary Fig. S1A and Supplementary Methods). In the LPS cohort (systemic acute inflammation protocol), Zeb1ΔM mice exhibited greater survival than Zeb1WT mice (Fig. 1a). Interestingly, in the LPS + LPS cohort (immuno- suppressive protocol), the reverse pattern was found; Zeb1ΔM mice exhibited lower survival than Zeb1WTmice. The composition ofmyeloid cells entering the peritoneal cavity varies during the LPS responsewith an increase in the proportion of monocytes during the course of sepsis33. However, we observe no difference in the distribution of myeloid subpopulations between Zeb1WT and Zeb1ΔM mice (Supple- mentary Fig. S1L). The above data suggest that ZEB1 plays opposing roles in the macrophage-mediated inflammatory and immunosuppressive responses to LPS. Todefine themechanismsbywhichZEB1 does so, we conducted a bulk RNA sequencing (RNAseq) of peritoneal macro- phages isolated from Zeb1WT and Zeb1ΔM mice subjected to the LPS and LPS + LPS protocols (Supplementary Fig. S1M, N). In the LPS condition (acute inflammation), Zeb1ΔM macrophages expressed lower levels of inflammatory genes (e.g., Il1a, Il6, Nfkb1) than their Zeb1WT counter- parts (Fig. 1b–f andSupplementary Fig. S1O). In theLPS + LPS condition (immunosuppression), inflammatory genes were expressed similarly in macrophages of both genotypes. However, a reverse pattern was observed with regard to several anti-inflammatory and homeostatic genes (e.g., Il4, Retnlg) (Fig. 1d, e, g). Although Zeb1WT and Zeb1ΔM Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 2 macrophages expressed similar levels of these anti-inflammatory and homeostatic genes during LPS, Zeb1ΔM macrophages expressed lower levels of those genes than Zeb1WT macrophages during LPS + LPS (Fig. 1e and g). Taken together, these findings suggest that ZEB1 plays dual roles inboth the induction and resolutionphases of inflammation. ZEB1 appears to modulate macrophage plasticity by upregulating inflammatorygenes during acute inflammation andhomeostatic genes during immunosuppression. ZEB1 increases glycolysis during acute inflammation The transition of macrophages from an inflammatory state to an anti- inflammatory state is accompanied by metabolic reprogramming, a b Untr LPS LPS LPS IL6 0 20 40 60 80 100 0 50 100 Time (hours) 0 10 20 30 40 0 50 100 Su rv iv al (% ) LPS (Acute inflammation) LPS + LPS (Immunosuppression) Time (hours) Zeb1 WT Zeb1 ΔM p = 0.02p = 0.032 Ze b1 W T Ze b1 ΔM β-ACTIN Ze b1 W T Ze b1 ΔM Ze b1 W T Ze b1 ΔM d 0 1000 2000 3000 4000 La ct at e co nc en tra tio n [μ M ] 0.065 e h k *** * ns Zeb1 WT Zeb1 ΔM LPS LPS LPS 0.5 1.0 1.5 m R N A R el at iv e ex pr es si on Untr LPS LPS LPS ns * * * * Slc2a1 0 2 4 6 8 m R N A R el at iv e ex pr es si on Untr LPS LPS LPS 0 1 2 3 4 m R N A R el at iv e ex pr es si on * ns ns ns ns ** Untr LPS LPS LPS Il4 Il6 Zeb1 WT Zeb1 ΔM Zeb1 WT Zeb1 ΔM 1 Phgdh Hk3 Myc Pfkm Hk2 Pdk3 Slc16a1 Slc2a1 Pfkp Eno2 Gapdh Pkm Ldha Pgam1 Slc16a3 Glycolytic Genes Ze b1 W T Ze b1 ΔM Ze b1 W T Ze b1 ΔM 2 3 1 2 3 1 2 1 2 3 LPS LPS LPS Zeb1 WT Zeb1 ΔM 1 Inflammation-related Genes Ze b1 W T Ze b1 ΔM Ze b1 W T Ze b1 ΔM 2 3 1 2 3 1 2 1 2 3 LPS LPS LPS Il6st Lbp Il27 Tnfsf9 Il15 Nfkb1 Irak2 Irf8 Ptgs2 Il6 Cxcl9 Tnfsf15 Cxcl1 Arg2 Il1a Cxcl10 Arg1 Mmp8 Cd209g Il4 Mmp25 Mmp9 Retnlg 2 1 0 -1 -2 2 1 0 -1 -2 Reactome Interleukin 10 signaling LPS LPS Zeb1 WTZeb1 ΔM NES Nominal p value - 2.06 0.01 LPS LPS Zeb1 WTZeb1 ΔM NES Nominal p value - 2.2 < 0.01 LPS LPS Zeb1 WTZeb1 ΔM NES Nominal p value - 2.09 < 0.01 Zeb1 WTZeb1 ΔM NES Nominal p value - 1.62 < 0.01 GOBP Positive regulation of acute inflammatory response GOMF Cytokine activity Reactome Interleukin 4 and Interleukin 13 signaling LPS LPS Zeb1 WTZeb1 ΔM NES Nominal p value - 1.93 0.01 WP Glycolysis and Gluconeogenesis LPS LPS LPS LPS LPS LPS c f g i j 0 2 4 6 8 IL 6 / β -A C TI N Zeb1 WT Zeb1 ΔM * ns ns Untr LPS LPS LPS Mouse macrophages 50 25 0 Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 3 shifting from glycolysis to oxidative phosphorylation (OxPhos)6. Consistent with the inflammatory signature linked to ZEB1 in acute inflammation, Zeb1WT macrophages expressed higher levels of genes associated with glycolysis, (e.g., Slc2a1, Hk2, Hk3, Pdk3) compared to Zeb1ΔM counterparts during acute inflammation (Fig. 1h–j). However, in the LPS + LPS condition, we found that enzymes associated with lower glycolysis that catalyze the conversion of glyceraldehyde-3-phosphate to pyruvate (e.g., Gapdh, Pkm, Pgam1, Ldha) were upregulated in both genotypes (Fig. 1h). In the same line, our analysis of a published array of the transcriptome of septic patients during the first week at the ICU (GSE131411)34 indicated that ZEB1 expression correlates with inflam- matory (IL1B) and glycolytic (SLC2A1) genes at the beginning of the septic process (16 h and 48 h) and with anti-inflammatory (IL4) and anti-oxidant (GSS) genes at the immunosuppressive state (Supple- mentary Fig. S1P). Lactate production by macrophages in the aftermath of acute inflammation is required for the subsequent activation of anti- inflammatory genes in immunosuppressed macrophages10,35. In that line, we found that Zeb1WT macrophages subjected to LPS + LPS pro- duced more lactate than those under LPS (Supplementary Fig. S1Q). Zeb1ΔM macrophages produced less lactate than Zeb1WT macrophages during immunosuppression (Fig. 1k), which may contribute to the impaired anti-inflammatory transition in Zeb1ΔM macrophages and the decreased survival of Zeb1ΔM mice following two doses of LPS. ZEB1 reduces mitochondrial content during immunosuppression It hasbeen reported that, during acute inflammation, the expression of mitochondrial DNA (mtDNA)-encoded genes in leukocytes correlates with the severity of sepsis36. Interestingly, our RNAseq analysis revealed that LPS-treated Zeb1WT macrophages exhibited higher expression of mtDNA-encoded genes compared to Zeb1ΔM macro- phages (Supplementary Fig. S2A). Thehigher induction of IL6 inZeb1WT macrophages compared to Zeb1ΔM macrophages was accompanied by an upregulation of TOMM20, a nuclear DNA (nDNA)-encoded mito- chondrial protein that we used as a proxy for mitochondrial content (Supplementary Fig. S2B). This suggests that the reduced inflamma- tory response of Zeb1ΔM macrophages may be related to their altered mitochondrial function. Peritoneal macrophages from mice either untreated (PBS) or fol- lowing treatment with one or two doses of LPS were assessed for their mitochondrial content by their staining for MitoTracker™ Green (MTG). Immunosuppressed macrophages from mice treated with LPS + LPS showed lower mitochondrial content than macrophages from mice injected with PBS or a single dose of LPS (Fig. 2a). A com- parable decrease in mitochondrial content—assessed by both MTG staining and MT-CO1 (mitochondrially-encoded Cytochrome C Oxi- dase I) expression—was also found in the immunosuppressed PBMCs of septic patients at 72 h relative to the immune-responsive PBMCs from the same septic patients at 0 h or healthy donors (Fig. 2b–d, and Supplementary Fig. S2C). These results prompted us to investigatewhether alterations in the mitochondria content can contribute to in vivo immunosuppression in mice.We conducted a transmission electronmicroscopy (TEM) analysis to examine the ultrastructure ofmacrophages isolated from Zeb1WT and Zeb1ΔM mice that had either been left untreated or treated with a single dose of LPS for different durations (30min, 3 h, and 12 h), aswell aswith LPS + LPS. Interestingly, macrophages isolated from Zeb1ΔM mice trea- ted with LPS at 3 h contained fewer mitochondria compared to mac- rophages from Zeb1WT mice. However, when mice were treated with LPS + LPS,macrophages from Zeb1ΔMmice hadmoremitochondria than macrophages from Zeb1WT mice (Fig. 2e, f). These results support the hypothesis that ZEB1 regulates mitochondria content in opposite directions in inflammation and immunosuppression. ZEB1 activates p62 and promotes autophagy during inflammation The accumulation of damaged mitochondria during inflammation increases ROS production and activates inflammasome signaling, highlighting the importance of mitophagy as an important anti- inflammatory self-limiting mechanism37,38. Compared to Zeb1WT coun- terparts, and particularly in the LPS condition, Zeb1ΔM macrophages expressed lower levels of autophagy/mitophagy-related genes (e.g., Sqstm1, Tbc1d17, Rab9, Cisd2) and higher levels of anti-autophagy/ mitophagy ones (e.g. Usp30) (Fig. 2g). p62 (encoded by Sqstm1) binds damaged mitochondria—as well as other damaged organelles and ubiquitinated proteins—and recruits them to autophagosomes, which subsequently fuse with autolysosomes for degradation (mitophagy) in a mTORC1-dependent manner39. Mitophagy prevents the release of inflammasome-activating signals and limits excessive ROS production during acute inflammation40–42. Treatment of Zeb1WT and Zeb1ΔM mice with LPS upregulated p62 mRNAandprotein expression inZeb1WTmacrophagesbut not inZeb1ΔM counterparts (Fig. 2h, i, and Supplementary Fig. S2D). Analysis of the SQSTM1 promoter identified several consensus sequences for ZEB1 whose capacity to recruit ZEB1 were tested in chromatin immuno- precipitation (ChIP) assays. ZEB1 bound to SQSTM1 promoter and to a larger extent in human monocyte-derived macrophages treated with LPS than in those treated with LPS + LPS (Supplementary Fig. S2E). In addition, compared to LPS-treated Zeb1ΔM peritoneal macrophages, LPS-treated Zeb1WT macrophages showed increased co-localization of lysosome staining (Lyso DyeTM) with the Mtophagy DyeTM, indicating enhanced lysosomal-mediated degradation (Supplementary Fig. S2F). Next, we examined macrophages from both genotypes treated with a single dose of LPS for signs of autophagy using TEM. Zeb1WT macro- phages, in comparison to Zeb1ΔM macrophages, exhibited a higher number of autolysosomes (Fig. 2J, yellow asterisks) containing cyto- solic material, including mitochondria, indicative of damaged Fig. 1 | ZEB1 plays a dual role being required for both the induction and reso- lution of inflammation. a Survival plots of 8–10 weeks old female Zeb1WT and Zeb1ΔM mice treated with LPS (15 Zeb1WT, 12 Zeb1ΔM) to induce acute inflammation, and treated with LPS + LPS (14 Zeb1WT, 12 Zeb1ΔM) to induce immunosuppression. b Peritoneal macrophages from Zeb1WT and Zeb1ΔM mice were untreated, or treated with LPS or LPS + LPS and blotted for IL6 along with β-ACTIN as a loading control. The blot shown is representative of five independent experiments. c quantification of IL6 levels relative to β-ACTIN levels in n = 5 biologically independent experi- ments as in (b). d Left Panel: GSEA plots for inflammatory signatures comparing macrophages from Zeb1WT and Zeb1ΔM mice treated with LPS. Right panel: GSEA plots for anti-inflammatory signatures comparing macrophages from Zeb1WT and Zeb1ΔM mice treated with LPS + LPS. e Heatmap of inflammation-related genes in peritoneal macrophages from Zeb1WT and Zeb1ΔM mice treated with either LPS or LPS + LPS. f Il6 mRNA levels in peritoneal macrophages from Zeb1WT and Zeb1ΔM mice either untreated, treated with LPS, or treated with LPS + LPS (n = 5,5,8,8,9,7). g as in (f), but for Il4 (n = 3,3,7,6,8,4). h Heatmap of glycolytic genes in peritoneal macrophages from Zeb1WT and Zeb1ΔM mice subjected to either LPS or LPS + LPS. i GSEA plots for Glycolysis and Gluconeogenesis signature comparing macro- phages from Zeb1WT and Zeb1ΔM mice treated with LPS. j As in (f), but for Slc2a1 (n = 4,3,5,4,5,3). k Lactate levels in macrophages from Zeb1WT and Zeb1ΔM mice treated with LPS (n = 8) or LPS + LPS (n = 7). Statistical analysis of Kaplan Meier survival plots was assessed by the Log-rank (Mantel-Cox) test. Graphbars represent mean values +/− SEM with two-tailed unpaired Mann-Whitney test. p ≤0.001 (***), p ≤0.01 (**) or p ≤0.05 (*) levels, or non-significant (ns) for values of p >0.05. Numerical values had been added for 0.05 < p <0.075. Raw data along p values for statistical analyses are included in the Source Data file. Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 4 mitochondria undergoing mitophagy (Fig. 2j and Supplementary Fig. S2G). These data suggest that ZEB1 promotes autophagy in the context of acute inflammation. Metformin depends on ZEB1 expression in macrophages for its anti-inflammatory effects Prompted by the above data suggesting that the decrease in mitochondria in macrophages under the LPS + LPS condition accounts for their compromised immune response, we investi- gated the effects of Metformin, known for inhibiting mitochon- drial function and the inflammatory response of macrophages to LPS17. We hypothesized that the anti-inflammatory effects of Met- formin mimic the immunosuppression observed in macrophages under the LPS + LPS condition. To test that hypothesis, we exam- ined the in vivo and in vitro effects of Metformin in the response to LPS. Mouseandhumanmacrophageswere treated in vitrowith a single dose of LPS in the presence or absenceofMetformin orwith two doses of LPS (Fig. 3a and Supplementary Methods). As expected, Metformin reverted the LPS-induced upregulation of IL6 in both human and mouse macrophages (Supplementary Fig. S3A and S3B). In human macrophages, pre-treatment with Metformin before LPS resulted in upregulation of ZEB1 and reduced expression of MT-CO1, which mir- rored the expression changes observed in the immunosuppressed PBMCs of septic patients at 72 h shown above or following two doses of LPS (Supplementary Figs. S3C and S3D). a c g h i j 0 10 20 30 40 N um be r o f m ito ch on dr ia / ce ll UNT LPS 30 min LPS 3 h LPS 12 h LPS LPS ns ** Zeb1 WT Zeb1 ΔM GAPDH 0.0 0.5 1.0 1.5 2.0 M T- C O 1 / G AP D H Human PBMC Healthy Donors Sepsis 0h Sepsis 72h 1 2 3 1 2 3 1 2 3 MT-CO1 e p62 Zeb1 WT Unt LPS LPS LPS Unt LPS Healthy donors Sepsis 0h Sepsis 72h Untreated Zeb1 WT Zeb1 ΔM Zeb1 WT Zeb1 ΔM Zeb1 WT Zeb1 ΔM LPS LPS + LPS 15 KX 40 KX LPS LPS 0.0 0.5 1.0 1.5 m R N A R el at iv e ex pr es si on Untr LPS LPS LPS M M Nucleus M ER * * * Zeb1 WT Zeb1 ΔM Zeb1 ΔM β-ACTIN LPS 3h Sqstm1 * ns ** **** ** **ns Zeb1 WT Zeb1 ΔM Pmaip1 Hsh2d Alpl Sqstm1 Rab9 Map1lc3b Tbc1b17 Zfyve1 Fundc2 Gabarap Mcl1 Cisd2 Usp30 Gabarapl1 Ulk1 1 Ze b1 W T Ze b1 ΔM Ze b1 W T Ze b1 ΔM 2 3 1 2 3 1 2 1 2 3 LPS LPS LPS Autophagy/Mitophagy Genes 50 ns ns ** ** * 2 1 0 -1 -2 d f b Mouse Macrophages 50 37 75 50 50 37 Human PBMC 104 105 Sepsis 0h Sepsis 72h Healthy Donors Mito Tracker TM Green LPS Mouse macrophages Zeb1 WT Mac LPS LPS 101 102 103 104 Untreated Mito Tracker TM Green Fig. 2 | ZEB1 regulates mitochondrial content and autophagy during inflam- mation. aMTG staining in peritoneal macrophages fromwild-typemice untreated, treated with LPS, or treated with LPS + LPS. The FACS plot is representative of at least 3 independent experiments. bMTG staining in the monocyte-enriched PBMC of healthy controls and septic patients at 0 h and 72 h. The FACS plot is repre- sentative of at least 5 independent experiments. c Western blot for MT-CO1 and GAPDH in PBMCs from three healthy donors and three septic patients at 0 and 72 h. The blot is representative of three independent experiments. d MT-CO1 protein levels in five healthy controls and five patients relative to GAPDH. e TEM of mito- chondria in sorted macrophages from Zeb1WT (labeled in blue) and Zeb1ΔM (orange) mice treated with PBS (Untreated), LPS for the indicated periods, or LPS + LPS. A representative macrophage from a 4–5mice pool for each genotype and condition at 15,000X and 40,000X magnification. Scale bar: 2μm. f At least 6 pictures were quantified for each genotype and condition. (n = 7,6,6,8,9,10,6,8,10,7) g Heatmap of autophagy-related genes in peritoneal macrophages from Zeb1WT and Zeb1ΔM mice treated with LPS or LPS + LPS. h Sqstm1mRNA in peritoneal macrophages from Zeb1WT and Zeb1ΔM untreated, treated with LPS or LPS + LPS. (n = 5,4,6,4,4,4). i As in Fig. 2c, but for p62/SQSTM1 and β-ACTIN. Blots are representative of four independent experiments. j Ultrastructure of autophagic vacuoles in LPS-treated macrophages. TEM images of Zeb1WT and Zeb1ΔM macrophages from mice treated with LPS for 3 h. Yellow asterisks: autophagic vacuoles in Zeb1WT macrophages, two with increased electron density (autolysosomes). N nucleus, M mitochondria, ER endoplasmic reticulum. Scale bar: 1000nm. A representative macrophage from a 4–5 mice pool for each genotype and condition. At least 6 pictures were analyzed for each genotype and condition. Graph bars in Fig. 2 represent mean values +/− SEM with two-tailed unpaired Mann–Whitney test. p ≤0.001 (***), p ≤0.01 (**) or p ≤0.05 (*) levels, or non-significant (ns) for values of p >0.05. Raw data along p values for statistical analyses are included in the Source Data file. Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 5 Metformin reduces the oxygen consumption rate (OCR) of macrophages treated with LPS21. To determine whether ZEB1 mod- ulates the effect of Metformin on the overall metabolic profile of macrophages during inflammation and immunosuppression, Zeb1WT and Zeb1ΔM macrophages were subjected to the protocols in Fig. 3a and assessed for their OCR by Seahorse cell metabolic flux analysis (Fig. 3b, c). We found that the pre-treatment of Zeb1WT macrophages with Metformin prior to LPS resulted in a reduction in basal OCR and Spare respiratory capacity to levels comparable to those found in macrophages treated with LPS + LPS. Additionally, Metformin did not alter OCR in Zeb1ΔM macrophages, suggesting that the metabolic effects of Metformin on OCR require the presence of ZEB1. To test whether the anti-inflammatory effects of Metformin also depended on ZEB1, peritoneal macrophages from Zeb1WT and Zeb1ΔM Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 6 mice were subjected to the experimental conditions in Fig. 3a, and their inflammatory status was assessed by their Il6 expression. As expected, Metformin alone had no effect on Il6mRNA levels of Zeb1WT and Zeb1ΔM macrophages (Fig. 3d). However, pre-treatment with Met- formin reduced the induction of Il6 by LPS in Zeb1WT macrophages but had no effect in LPS-treated Zeb1ΔM macrophages (Fig. 3d). Further- more, the effect of Metformin on the systemic inflammatory status in mice of both genotypes was evaluated bymeasuring their serum levels of IL6. Consistent with the findings in the septic shock model, Zeb1WT mice exhibited higher levels of IL6 in response to LPS compared to Zeb1ΔM mice (Fig. 3e). Remarkably, Metformin effectively inhibited the LPS-induced upregulation of IL6 in Zeb1WT mice, while no significant effect was observed in Zeb1ΔM mice (Fig. 3e). Similar results were observed in the analysis of IL6 intracellular staining by FACS in mac- rophages of both genotypes (Fig. 3f, g). To gain a comprehensive understanding of the inflammatory response of Zeb1WT and Zeb1ΔM macrophages, we evaluated the protein levels of a panel of inflammatory markers using a quantitative bead- based cytokine/chemokine multiplex array, which was analyzed by FACS (Fig. 3h). As expected, Metformin alone did not have any effect on macrophages from either genotype. Under the LPS condition, Zeb1ΔM macrophages exhibited reduced levels of IL1β, IL6, TNFα, and CXCL1 compared to LPS-treated Zeb1WT macrophages. Interestingly, the pretreatment with Metformin brought the expression levels of these inflammatorymarkers inZeb1ΔMmacrophages to a similar level as that of Zeb1WT macrophages (Fig. 3h). In contrast, under the LPS + LPS condition, Zeb1ΔM macrophages exhibited higher levels of IL6 and CXCL1, whereasnodetectable levels of IL1βor TNFαwereproducedby macrophages of either genotype (Fig. 3h). These findings suggest that the anti-inflammatory effects of Metformin in macrophages are dependent on and, at least partially, mediated by ZEB1. Comparing our RNAseqdatawith a publishedRNAseq (GSE98731) of Metformin-regulated genes in alveolar macrophages during an inflammatory response to air pollution in wild-type mice22, we found that like the alveolar macrophages in the aforementioned study, our Zeb1WT peritoneal macrophages, in contrast to Zeb1ΔM peritoneal macrophages, exhibited enrichment of Metformin-induced genes during LPS-induced immunosuppression (Supplementary Fig. S3E). This led us to explore by RNAseq the gene signature regulated by Metformin in vivo in peritonealmacrophages isolated from Zeb1WT and Zeb1ΔM mice that had been pre-treated with Metformin before the administration of LPS. Metformin differentially induced a “negative regulation of the inflammatory response” signature in Zeb1WT macro- phages relative to Zeb1ΔM macrophages (Fig. 3i). Of all the DEGs between the conditions LPS and Metformin + LPS, Metformin regu- lated 2,034 genes in the same direction in Zeb1WT and Zeb1ΔM macro- phages. However, 1,628 and 1,668 genes were specifically regulated by Metformin in Zeb1WT and Zeb1ΔM macrophages. Interestingly, within gene signature specific for Zeb1WT macrophages, Metformin increased genes associated with an anti-inflammatory response (e.g., Arg1, Mrc1, Il4) and gene annotations related to the induction of tolerance (e.g., Acod1), positive regulation of mitochondrial depolarization (e.g., Tspo), wound healing, metalloproteinase activity (e.g. Timp1, Cldn1), reactive oxygen species (ROS)metabolic processes (e.g., Sod2). In turn, in the specific signature of Zeb1ΔM macrophages, Metformin-regulated genes were associated with a signature of maturation of SSU-rRNA (e.g., Wdr3) (Fig. 3j). These analyses suggested that dependence on ZEB1 forMetformin’s anti-inflammatory effects is at least in part due to the regulation of mitochondrial function. ZEB1 mediates the immunosuppression-mimicking effect of Metformin by reducing mitochondrial content and ROS levels The late stages of an acute systemic inflammatory response are char- acterized by the apoptosis of the majority of immune cells, which contributes to the subsequent immunosuppression stage43. The accumulation of ROS and oxidative stress trigger apoptosis, while autophagy serves as an adaptive mechanism to counteract oxidative stress to overcome apoptosis. At the end of the LPS protocol, Zeb1ΔM mice exhibited a decreased macrophage count compared to Zeb1WT mice (Supplementary Fig. S4A). Zeb1ΔM macrophages exhibited higher levels of apoptosis during acute inflammation as assessed by Annexin V (Supplementary Fig. S4B) and a lower expression of anti-apoptotic genes (e.g., Bcl2 and Mcl1) (Supplementary Fig. S4C). During acute inflammation, there is a reduction in ATP generation from mitochondria, leading to an increase in the membrane potential (ΔΨm), which is necessary for the generation of ROS9. In response to LPS, Zeb1WT macrophages exhibited lower ATP levels compared to Zeb1ΔM macrophages (Supplementary Fig. S4D). In contrast, pre- treatment with Metformin or exposure to LPS + LPS resulted in the opposite effect. In macrophages from Zeb1WT mice, treatment with Metformin + LPS or LPS + LPS resulted in the upregulation of an anti- oxidant signature—as many of the genes in the GSEA annotation “superoxide metabolic process”—compared to Zeb1WT mice treated with LPS alone. This signature was also upregulated in Zeb1WT mice treated with Metformin + LPS or LPS + LPS compared to Zeb1ΔM mice with the same treatments. In order to assess the in vivo antioxidant effect of pre-treatment with a previous dose of LPS, we measured ROS production using the luminescence probe L-012 (8-amino-5-chloro-7-phenyl-pyrido[3,4-d] pyridazine-1,4(2H,3H)dione). It was found that Zeb1WT mice exhibited higher levels of ROS compared to Zeb1ΔM mice during treatment with LPS, but not during LPS + LPS (Supplementary Fig. S4E). Furthermore, we also evaluated ROS production, mitochondrial content, and ΔΨm of macrophages from Zeb1WT and Zeb1ΔM mice during LPS and LPS + LPS using staining with 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (CH2-DCFDA),MTG, and tetramethylrhodaminemethyl ester perchlorate (TMRM), respectively. In line with the transcriptomic Fig. 3 | Metformin depends on ZEB1 expression in macrophages for its anti- inflammatory effects. a Experimental design for the in vivo and ex vivo treatment withMetformin (MET).bOxygen consumption rates (OCR)ofZeb1WTmacrophages, either untreatedor subjected to the LPS,Metformin + LPS or LPS + LPS protocols as in (a), were assessed by Seahorse XF Cell Mito Stress Test Kit. Average from at least two independent experiments each including two mice per genotype and condi- tion, each in triplicate. Quantification of basal OCR and Spare Respiratory Capacity of Zeb1WT macrophages (n = 5,6,4,7). Untreated is set to 100. c As in (b), but for Zeb1ΔMmacrophages.d Il6mRNA levels in peritonealmacrophages from Zeb1WT and Zeb1ΔM untreated (PBS), treated with Metformin, LPS or Metformin + LPS (n = 4,5,4,5,4,5,4,5). e Zeb1WT and Zeb1ΔM mice were injected i.p. with PBS or LPS, Metformin + LPS or LPS + LPS. IL6 serum levels were measured by ELISA 3 h after the last LPS injection. (n = 6,4,7,3,4,4,5,4 mice). f Intracellular IL6 was assessed by FACS in F4/80+ peritoneal macrophages from Zeb1WT and Zeb1ΔM subjected to the indicated treatments. A representative plot of at least three independent experiments. g Quantification of IL6+ macrophages in (f) (n = 7,5,2,2,8,7,8,8,5,4). h Cytokine/chemokine production was assessed in the supernatant of peritoneal macrophages from Zeb1WT and Zeb1ΔM mice subjected to the indicated treatments. n = 4 with a pool of two mice per sample. i GSEA plots for negative regulation of inflammatory response signature comparing LPS versus Metformin + LPS in Zeb1WT or Zeb1ΔM macrophages or Metformin + LPS in Zeb1WT versus Zeb1ΔM macrophages. j Venn diagram and gene ontology Cytoscape analysis on the effect of Metformin (MET) in the gene signature of LPS (MET+ LPS versus LPS) for each genotype. Specific Metformin signatures for Zeb1WT (blue) Zeb1ΔM (orange) or shared sig- natures (green). Each node shows selectedDEGs associatedwith its ownGOcluster. Graph bars in Fig. 3 represent mean values +/− SEM with two-tailed unpaired Mann–Whitney test. p ≤0.001 (***), p ≤0.01 (**) or p ≤0.05 (*) levels, or non- significant (ns) for values of p >0.05. Numerical values had been added for 0.05 < p < 0.075. Raw data along p values for statistical analyses are included in the Source Data file. Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 7 analysis shown in Fig. 4a, in LPS, pre-treatment with Metformin or a first sublethal dose of LPS resulted in reduced ROS production, mito- chondrial content, andΔΨm inZeb1WTmacrophages (Fig. 4b–d). These effects were not observed in Zeb1ΔM macrophages under the same treatment. Additionally, Metformin pre-treatment decreased TMRM and CH2-DCFDA staining in human macrophages treated with LPS (Supplementary Fig. S4F and S4G). Similar reductions in MTG and TMRM staining were observed in CD14+ PBMCs from septic patients at 72 h compared to baseline levels at 0 h (Fig. 4e). Based on the above results, we can draw three main conclusions. Firstly, Zeb1WT macrophages under acute inflammation exhibited higher ROS production compared to Zeb1ΔM macrophages. Secondly, Zeb1ΔM macrophages under immunosuppression showed higher mitochondrial content, ΔΨm, and ROS production compared to Zeb1WT macrophages. Lastly, Metformin pre-treatment reduced mito- chondrial content, ΔΨm, and ROS levels in Zeb1WT macrophages, while no such reduction was observed in Zeb1ΔM macrophages. ZEB1 inhibitsmitochondrial protein translation in inflammatory macrophages Metformin pre-treatment resulted in a reduction of phosphorylated p65 (P-p65) and MT-CO1 protein expression in inflammatory human macrophages (LPS) to levels comparable to those found in immuno- suppressed human macrophages (LPS + LPS) (Fig. 4f, g, and Supple- mentary Fig. S4H). Analysis of our RNAseq data showed that Zeb1ΔM macrophages treated with LPS had higher expression of a signature related to “positive regulation ofmitochondrial translation” compared to Zeb1WTmacrophages (Fig. 4h). To explore the potential involvement of ZEB1 in mitochondrial translation in macrophages under LPS treatment, we examined the incorporation and tracing of L-homo- propargylglycine (HPG), an analogue of methionine that enables the quantification of newly synthesized proteins. We used emetine to specifically inhibit cytosolic translation and doxycycline to inhibit mitochondrial translation44,45. As expected, the combination of doxycycline and emetine more effectively reduced the incorporation of HPG (as a measure of new protein synthesis) in Zeb1WT macrophages compared to emetine alone (Supplementary Fig. S4I). While there was no difference in mitochon- drial protein synthesis between Zeb1WT and Zeb1ΔM macrophages under basal conditions, treatment of mice from both genotypes with LPS or LPS + LPS resulted in higher mitochondrial translation in Zeb1ΔM mac- rophages compared to Zeb1WT macrophages (Fig. 4i). These findings suggest that ZEB1 modulates the macrophage response during acute inflammation and immunosuppression by influencing mitochondrial translation. Inhibition of mitochondrial mRNA translation by tetracyclines reduces in vitro LPS-induced macrophage upregulation of inflamma- tory cytokines and ameliorates lung and liver damage in endotoxin- induced systemic inflammation11. We found here that doxycycline inhibition of ROS production and Il6 expression in inflammatory macrophages treated is also dependent on ZEB1 expression; in con- trast to Zeb1WT macrophages, pre-treatment with doxycycline did not have an effect in LPS-treated Zeb1ΔM macrophages (Fig. 4j, k, and Supplementary Fig. S4J). In human monocyte-derived macrophages, doxycycline exhibited a similar immunosuppressive effect as LPS + LPS, resulting in the reduction of MT-CO1 expression Supplementary Fig. S4K). Lactate and the inhibition of mitochondrial translation inhibit chronic inflammation in a psoriasis model We questioned whether the role and mechanism of action of ZEB1 in the regulation of acute inflammation and immunosuppression are conserved in the context of chronic autoimmune inflammation. To that effect, we selected the psoriatic disease, an immune-mediated chronic inflammatory condition affecting not only the skin but also other tissues like joints, where myeloid cell metabolism plays a key pathogenic role13,46. While psoriasis is primarily characterized by ery- thematous and indurated skin plaques, it is considered a systemic inflammatory disease that is associated with increased levels of inflammatory markers in the serum, and about a third of patients will develop psoriatic arthritis (PsA)47,48. We investigated whether elevated lactate levels or decreased mitochondrial translation can limit chronic inflammation, similar to their effects in acute inflammation. Topical application of imiquimod (IMQ), a TLR7/8 activator, on the ear ofmice leads to the development of psoriasiform skin lesions and epidermal thickening (acanthosis), resembling milder forms of human psoriatic skin lesions49,50. The ears of Zeb1WT mice were left untreated or treated with imiquimod, with or without prior and concurrent systemic treatment of PBS, lactate, or doxycycline (Fig. 5a). Doxycycline and lactate exert their effects through different mechanisms. Doxycycline reduces inflammation by suppressing mitochondrial translation, while lactate exerts its effects by triggering anti-inflammatory and reparative responses10,11. We explored whether inhibition of mitochondrial translation by doxycycline has an anti-inflammatory effect not only in acute inflammation but also in chronic inflammation. Treatment with lactate and doxycycline in Zeb1WT mice, improved psoriasiform lesions, resulting in reduced ear and epidermal thickening (Fig. 5b, c), and increased lactate production by their macrophages (Fig. 5d). Doxycycline increased ZEB1 expression while reducing MT-CO1 protein levels, whereas lactate had the opposite effects (Fig. 5e and Supplementary Fig. S5A and S5B). Additionally, compared to lactate, doxycycline exhibited greater effectiveness in reducing sys- temic inflammation, as evidenced by its effects on imiquimod- induced splenomegaly and ROS production (Supplementary Fig. S5C and S5D). Metformin requires ZEB1 expression inmacrophages for its anti- inflammatory effects in psoriasis We then investigated whether the reliance on ZEB1 for the anti- inflammatory effect of Metformin observed in the context of acute inflammation also applies to psoriasis. imiquimod treatment resulted in reduced Zeb1 expression in the ear sections of Zeb1WT mice (Sup- plementary Fig. S5E), and it led to greater erythema and epidermal thickening in the ears of Zeb1ΔM mice compared to Zeb1WT mice (Fig. 5f, g). IMQ-treated Zeb1ΔM mice also exhibited greater systemic inflammation than Zeb1WT mice as evidenced by the larger spleens in the former (Supplementary Fig. S5F). Pre-treatment of mice with Metformin prior to imiquimod resulted in a reduction of macroscopic skin lesions, histological acanthosis, and macrophage infiltration in Zeb1WT mice, while it had no effects in Zeb1ΔM mice (Fig. 5f–h). These findings further support the dependence of Metformin’s anti- inflammatory effects on the expression of ZEB1 in macrophages. ZEB1 was found in scattered cells in the healthy skin but it was nearly absent in psoriatic skin lesions (Supplementary Fig. S5G). As reported, ZEB1 is upregulated in melanoma skin lesions51 (Supplementary Fig. S5G). We identified ZEB1 in CD68+ macrophages in healthy skin that are negative for ZEB1 in the psoriatic skin (Fig. 5i and Supplementary Fig. S5I). In addition, our analysis of a published gene microarray (GSE14905)52 also revealed the downregulation of ZEB1 in psoriatic skin lesions (Supplementary Fig. S5H). ZEB1 was also expressed in CD68+ macrophages in the synovial membrane of PsA patients but it was nearly absent in the synovium of osteoarthritis patients (Fig. 5i and Supplementary Fig. S5I). We also found that in contrast to the immunosuppressed PBMCs of septic patients at 72 h, the monocyte-enriched PBMCs of PsA patients exhibited higher mitochondrial content and ROS production compared to PBMCs from healthy donors (Fig. 5j, k, and Supplementary Fig. S5J). As in endotoxin-induced immunosuppression (LPS + LPS), ZEB1 Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 8 expression by macrophages is also necessary to restrict chronic inflammation and Metformin was dependent on ZEB1 expression for its anti-inflammatory role. In summary, our findings suggest that ZEB1 expression in mac- rophages is required for limiting both acute and chronic inflammation, as well as for the anti-inflammatory effects of Metformin in both forms of inflammation. ZEB1 inhibits mitochondrial protein translation by restricting amino acids transport After showing that ZEB1 regulates the inflammatory and immunosup- pressive responses of macrophages through inhibiting mitochondrial translation, we sought to further investigate the underlying molecular mechanism. Analysis of our RNAseq indicated that Zeb1ΔM macro- phages expressed higher levels of gene signatures associated with a b Untreated LPS MET LPS LPS LPS Untreated LPS MET LPS LPS LPS N or m al iz ed to m od e Untreated LPS MET LPS LPS LPS N or m al iz ed to m od e LPS Doxycycline + LPS N or m al iz ed to m od e N or m al iz ed to m od e 101 103102 104 105 101 103102 104 105 101 103102 104 105 101 103102 104 105 102 104103 105 106 102 104103 105 106 102 104103 105 102 104103 105 106 101 103102 104 105 101 103102 104 105 101 103102 104 105 101 103102 104 105 N or m al iz ed to m od e 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 101 103102 104 101 103102 104 101 103102 104 60 40 20 0 M ito Tr ac ke rTM G re en R el at iv e M FI c d Te tra m et hy lrh od am in e R el at iv e M FI H 2-D C FD A R el at iv e M FI e f g LPS LPS NES Nominal p value 1.96 < 0.01 GOBP Positive regulation mitochondrial translation Zeb1 WTZeb1 ΔM j k Untreated LPS LPS LPS Zeb1 WT Zeb1 ΔM Zeb1 WT Zeb1 ΔM Zeb1 WT Zeb1 ΔM Zeb1 WT Zeb1 ΔM 0 50 100 150 200 M ito ch on dr ia l t ra ns la tio n R el at iv e M FI Untr LPS LPS LPS ns ns* ns * ns Zeb1 WT Zeb1 ΔM ** Untr LPS Doxy LPS Zeb1 WT Zeb1 ΔM 75 100 50 37 MT-CO1 P-P65 Untr LPSMET 37 GAPDH Human macrophages MET LPS LPS LPS Hea lth y Sep sis 0 Sep sis 72 R el at iv e M TG M FI ** * R el at iv e TM R M M FI TMRM Septic Patients Hea lth y Sep sis 0 Sep sis 72 ns * MTG Septic Patients Untr MET LP S MET LP S LP S LP S 0.0 0.5 1.0 1.5 m tC O 1/ G AP D H 0.0 0.5 1.0 1.5 2.0 P- P6 5/ G AP D H Untr MET LP S MET LP S LP S LP S * * * ns **ns ns ns h i TetramethylrhodamineMito TrackerTM Green N or m al iz ed to m od e 0 20 40 60 80 100 101 103102 104 103102 104 0 20 40 60 80 100 Human CD14+ PBMC Il6 ns 0 300 200 100 0 50 100 150 0 50 100 150 * m R N A R el at iv e ex pr es si on ns * ** 101 103102 101 103102104 104 80 100 Zeb1 WT NES Nominal p value 1.6 0.01 GOBP Superoxide metabolic process Zeb1 WT Zeb1 WT NES Nominal p value 1.9 < 0.01 GOBP Superoxide metabolic process Zeb1 WT Zeb1 WTZeb1 ΔM NES Nominal p value - 1.8 < 0.01 GOBP Superoxide metabolic process Zeb1 WTZeb1 ΔM NES Nominal p value - 1.4 0.03 GOBP Superoxide metabolic process LPS LPS LPS MET LPS LPS MET LPS MET LPS LPS LPS LPS LPS H2-DCFDA Mito TrackerTM Green Tetramethylrhodamine HPG H2-DCFDA 0 50 100 150 200 Zeb1 WT Zeb1 ΔM MET LPS Untr LPS LPS LPS ns *** *** ** ** * * MET LPS Untr LPS Zeb1 WT Zeb1 ΔM 0 5000 10000 15000 20000 LPS LPS ns * * ns ns** *** ns nsns 0 10000 20000 30000 40000 50000 MET LPS Untr LPS LPS LPS Zeb1 WT Zeb1 ΔM *** ns ns*** ns ns ns ns * ** * Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 9 mitochondrial tRNA aminoacylation and tRNA modification (Supple- mentary Fig. S6A). The methylthiotransferase Cdk5rap1 catalyzes 2- methyl-2-thio (ms2) modifications of several mt-tRNAs and its defi- ciency hampers mitochondrial protein synthesis and OxPhos activity and increases ROS production53. LPS treatment resulted in the down- regulation of Cdk5rap1 expression in Zeb1WT macrophages but not in Zeb1ΔM macrophages (Supplementary Fig. S6B). In contrast, ms2 tRNA modifications were reduced in Zeb1ΔM macrophages after one dose of LPS but increased after two doses of LPS (Supplementary Fig. S6C). Given its opposing effects on the regulation of Cdk5rap1 expression and ms2 tRNA modifications, we concluded that ZEB1 inhibits mito- chondrial protein translation through mechanisms independent of these processes. Both cytoplasmic and mitochondrial protein translation, as well as autophagy, are regulated by mTOR, which is activated by the uptake and metabolism of amino acids54,55. Analysis of our RNAseq revealed that when Zeb1WT macrophages were pre-treated with Metformin (MET+ LPS) or exposed to a previous dose of LPS (LPS + LPS), there was a decrease in the expression of GSEA annotations associated with amino acid transport (Fig. 6a). Furthermore, Zeb1WT macrophages in the LPS + LPS condition exhibited reduced levels of an amino acid transport signature compared to Zeb1ΔM macrophages (Supplementary Fig. S6D). The L-type bidirectional amino acid trans- porter SLC7A8/LAT2 is required for the uptake of glutamine and branched-chain amino acids (BCAA), which are critical for the activation of mTORC1 signaling56,57. Treatment of Zeb1WT macrophages with Met- formin + LPS and LPS + LPS led to a decrease in the mRNA and protein levels of SLC7A8 compared to LPS treatment, whereas this effect was not observed in Zeb1ΔM macrophages (Fig. 6b, c, Supplementary Fig. S6E). In line with the upregulation of SLC7A8 during LPS, we observed that Zeb1WT macrophages but not Zeb1ΔM macrophages dis- played higher glucose and glutamine consumption specifically in the LPS condition (Fig. 6d, e). We then aimed to explore the potential alterations in intracellular levels and consumption of BCAAs as well as glutamine and its derived amino acid glutamate in inflammatory mac- rophages. We observed that, similar to glucose and glutamine, a single dose of LPS resulted in increased levels of BCAAs in Zeb1WT macro- phages, but not in Zeb1ΔM macrophages (Fig. 6f). Conversely, BCAAs levels were comparable between Zeb1WT and Zeb1ΔM macrophages treated with Metformin + LPS and LPS + LPS. Furthermore, we found a reduction in the intracellular levels of glutamate in LPS-treated Zeb1ΔM macrophages compared to their Zeb1WT counterparts. Of note, no sig- nificant difference was observed in intracellular glutamine levels between LPS-treated Zeb1ΔM and Zeb1WT macrophages, suggesting that glutamine may have already been metabolized to glutamate at the analyzed time point (Fig. 6g). We hypothesized that if ZEB1 inhibits mitochondrial translation by downregulating SLC7A8 expression, restricting glutamine availability would alleviate this effect. To test this, we treated Zeb1WT and Zeb1ΔM macrophages with LPS or LPS + LPS in the presence or absence of glu- tamine. In the absence of glutamine, there was a decrease in MTG staining in both genotypes and across all treatments. Consequently, the decrease in mitochondrial content observed in Zeb1WT macrophages treated with LPS + LPS compared to Zeb1ΔM macrophages, which was observed in the presence of glutamine, was prevented in the glutamine- free condition (Supplementary Fig. S6F). These results suggest that ZEB1 plays a role in restricting amino acid consumption, which is essential for mTORC1 activation and mitochondrial translation. We also assessed mitochondrial DNA (mtDNA) copy number (MDCN) in Zeb1WT and Zeb1ΔM macrophages under our different experimental conditions and in the presence or absence of glutamine. In the presence of glutamine and compared to a single dose of LPS, pre-treatment with Metformin (Metformin + LPS) or treatment with LPS + LPS reduced MDCN in Zeb1WT macrophages but not in Zeb1ΔM macrophages (Fig. 6h). However, in the absence of glutamine, MDCN was comparablebetween LPS-treated Zeb1WT and Zeb1ΔMmacrophages. Additionally, the treatments of Metformin + LPS or LPS + LPS did not alterMDCN in either Zeb1WT and Zeb1ΔMmacrophages compared to LPS treatment. Taken together, the abovedata suggest that in the presence of glutamine, treatmentwithMetformin or LPSbefore adding a second dose of LPS (Metformin + LPS or LPS + LPS) results in a reduction in mitochondrial content andMDCN in LPS-treated Zeb1WTmacrophages, but not in Zeb1ΔM macrophages. Based on these findings, two conclu- sions can be drawn: (1) MDCN in inflammation (LPS) is dependent on the availability of glutamine, and (2) glutamine levels in immunosup- pressed (LPS + LPS as well as Metformin + LPS) macrophages are regulated by ZEB1 expression. ZEB1 inhibitsmTORC1/p70S6K signaling in immunosuppression mTORC1 serves as an energy sensor with pleiotropic functions, including the regulation of nuclear DNA-encoded mitochondrial pro- tein translation44. Activation of mTORC1 is primarily driven by growth factors and nutrient availability, particularly amino acids56,57. Regula- tion of protein synthesis by mTORC1 is mediated through its phos- phorylation and activation of p70 ribosomal protein S6 kinase (p70S6K, encoded by Rps6kb1)44. Given the inhibitory effect of Met- formin on mTORC1 signaling58, we aimed to investigate whether the mechanism by which ZEB1 regulates immunosuppression and med- iates the anti-inflammatory effects of Metformin is through the reduction of amino acid uptake and metabolism, leading to the downregulation of mTORC1. Compared to Zeb1WT and Zeb1ΔM macrophages treated with a sin- gle dose of LPS,Metformin pre-treatment (MET + LPS) or a subsequent dose of LPS (LPS + LPS) resulted in reduced P-p70S6K levels in Zeb1WT macrophages, but not in Zeb1ΔM macrophages (Fig. 6i and Supple- mentary Fig. S6G). By examining the levels of phosphorylated p70S6K (P-p70S6K), IL6, and SLC7A8 in PBMCs from healthy controls, septic patients at 0 h (immunogenic), and septic patients at 72 h Fig. 4 | ZEB1 mediates Metformin’s immunosuppression-mimicking effect by reducing mitochondrial content and ROS levels. a GSEA plots for “Superoxide metabolic process” signature comparing macrophages from Zeb1WT and Zeb1ΔM mice subjected to the indicated treatments.b Zeb1WT and Zeb1ΔMmicewere injected i.p. with PBS, LPS, Metformin + LPS, or LPS + LPS and ROS production was assessed by FACS for CH2-DCFDA staining in F4/80+ cells. A representative plot of 6–15mice per genotype and condition in four independent experiments. CH2-DCFDA MFI in macrophages (n = 7,3,13,15,12,13,4,5 mice). c As in (b), macrophages were assessed for MTG staining (n = 7,7,13,13,9,10,6,7 mice). d As in (b), macrophages were assessed for TMRM (n = 9,6,15,14,11,11,6,8 mice). e At the indicated time, human CD14+ PBMCs from a healthy donor and septic patients were assessed forMTG and TMRM staining. Representative FACS plots (n = 5 individuals per condition) and MFI quantification. fA representative blot of at least four independent experiments to assess MT-CO1 and P-p65 in human macrophages treated as indicated. g Quantification of MT-CO1 (n = 5,4,5,5,5) and P-P65 expression relative to GAPDH (n = 5,4,4,4,5) in all experiments for (f). h GSEA plot for “Positive regulation of mitochondrial translation” annotation comparing macrophages from LPS-treated Zeb1WT and Zeb1ΔM mice. i Mitochondrial translation in macrophages from Zeb1WT and Zeb1ΔM mice either untreated or treated with LPS or LPS + LPS was assessed by FACS for L-HPG with Alexa Fluor® 647 picolyl azide. Representative plots of n = 7 independent experiments. Quantification analysis of all experiments. j CH2-DCFDA staining of macrophages from Zeb1WT and Zeb1ΔM mice treated with LPS or dox- ycycline + LPS. The FACS plots shown are representative of a total of 4–5 mice per genotype and condition assessed in two independent experiments. k Il6mRNA in macrophages from Zeb1WT and Zeb1ΔM mice either untreated or treated with LPS or doxycycline + LPS. Average of 5–6 mice for each genotype assessed in two inde- pendent experiments (n = 4,5,5,5,6,7). Graph bars in Fig. 4 represent mean values +/− SEMwith two-tailed unpairedMann–Whitney test. p ≤0.001 (***), p ≤0.01 (**) or p ≤0.05 (*) levels, or non-significant (ns) for values of p >0.05. Raw data along p values for statistical analyses are included in the Source Data file. Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 10 (immunosuppressed), we observed decreased levels of all three in the PBMCs of sepsis 72 h, indicating an immunosuppressive state (Fig. 6j and Supplementary Fig. S6H and S6I). In contrast to the down- regulationofMT-CO1 inPBMCs fromseptic patients at 72 h, the PBMCs from PsA patients exhibited an upregulation of MT-CO1 (Supplemen- tary Fig. S6H). Additionally, PBMCs from septic patients at 72 h showed reduced mitochondrial respiratory capacity compared to healthy donors (Supplementary Fig. S6J). Collectively, these results suggest a Zeb1 WT Zeb1 ΔM b c d e f g h i j Untreated Ze b1 W T Ze b1 ΔM IMQ MET + IMQUntreated IMQ MET + IMQ Ze b1 W T Ze b1 ΔM Ps or ia tic A rth rit is O st eo ar th rit is Human Skin Human Synovium CD68 / ZEB1 CD68 / ZEB1 H ea lth y sk in Ps or ia tic s ki n Untreated PBS LactateDoxycycline IMQ IMQ Untreated IMQ Doxycycline + IMQ Lactate + IMQ 1 72 3 4 5 6 PBS Doxy Lact Lact Lact Lact Lact Lact Lact Doxy Doxy Doxy Doxy Doxy Doxy PBS PBS PBS PBS PBS PBS IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ IMQ Euthanasia Days GAPDH PBS IMQ Doxy IMQ Lact IMQ Unt ns Mouse macrophages MT-CO1 ZEB1 TOMM20 0 20 40 60 80 100 Ep id er m is T hi kn es s (μ m ) Untr PBS Doxy Lact IMQ 0 Untr IMQ MET IMQ 0.0 0.2 0.4 0.6 0.8 Ea r t hi ck ne ss (m m ) Untr PBS Doxy Lact IMQ 0 500 1000 1500 2000 L- La ct at e co nc en tra tio n (μ M ) Untr PBS Doxy Lact IMQ * ns ns * * ns * * * ns ** * Ep id er m is th ic kn es s (μ m ) R el at iv e M FI Mito TrackerTM Green ** 50 100 150 * * ns * ns 0 100 200 300 400 Healthy Donor PsA Patient Human CD14+ PBMC Human CD14+ PBMC Mito TrackerTM Green k 0 50 100 150 R el at iv e R O S M FI C ou nt s * Healthy Donor PsA Patient MET CH2-DCFDA CH2-DCFDA PBS PBS PBS PBS PBS PBS PBS 0.057 15 37 200 37 50 Fig. 5 | Metformin requires ZEB1 expression in macrophages for its anti- inflammatory effects in psoriatic disease lesions. a Schematic of the protocols used in the Imiquimod (IMQ) mouse model. Ten-to-twelve weeks age male mice treated with Imiquimod and either PBS, doxycycline, or lactate. b H&E staining of histological ear sectionsofmice either untreated and treatedwith Imiquimod, Lactate + Imiquimod, or Doxycycline + Imiquimod. Scale bar, 50μm. c Quantification of the epidermal ear thickness assessed in fourpictures fromat least fourmiceper condition as in (b) (n= 3,5,4,4 mice for each condition). Quantification of the ear thickness of four mice per condition. d Lactate levels in the supernatant of peritoneal macro- phages from wild-type mice left untreated or treated with PBS + Imiquimod, Dox- ycycline + Imiquimod, or Lactate + Imiquimod (n= 3,4,3,4). eWestern blot for ZEB1, MT-CO1, TOMM20 andGAPDH in peritoneal wild-typemacrophages treated as in (b). f Representative pictures from three independent experiments of Zeb1WT and Zeb1ΔM mice after 7 days of treatment with PBS, Metformin, Imiquimod or Metformin + Imiquimod.gAs in (f), but ear sectionswere counterstained forH&E. Scale bar: 50μm. h As in (f), epidermal ear thickness in four mice per genotype and condition in three independent experiments was quantified by ImageJ. Four separate areas in two pic- tures were quantified for eachmouse ear (n=4). ICD68 and ZEB1 staining along with DAPI in the skin samples fromhealthydonors andpsoriatic patients aswell as synovial membrane samples from osteoarthritis and PsA patients. Representative captions of at least two independent experiments. Scale bar: 25μm. jMFI quantification and representative FACS plots for MTG staining in CD14+ PBMCs from six PsA patients relative to the MTG’s MFI of six healthy controls (n=6). k As in (j), but CH2-DCFDA staining inCD14+ PBMCs fromfourhealthydonors and fourPsApatients (n=4).Graph bars in Fig. 4 representmean values +/− SEMwith two-tailed unpairedMann–Whitney test. p≤0.001 (***), p≤0.01 (**) or p≤0.05 (*) levels, or non-significant (ns) for values of p>0.05. Numerical values had been added for 0.05

0.05. Raw data along p values for statistical analyses are included in the Source Data file. Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 13 levels as those observed in LPS + LPS macrophages. Although the mechanism by which ZEB1 mediates the effect of Metformin to pro- mote an immunosuppressive status in macrophages remains to be elucidated, Metformin inhibits mTORC1, whose genetic and pharma- cological ablation upregulates ZEB1 in cancer cells5,58,59,66. We found that relative to LPS, treatment ofmousemacrophages with Metformin + LPS or LPS + LPS reduced the phosphorylation of p70S6K as in the immunosuppressed PBMC of septic patients. ZEB1 suppressed mTORC1 activation by inhibiting amino acid transport, which resulted in reduced mitochondrial translation and ROS production, as well as lower inflammatory cytokine production. Interestingly, we found that the role of ZEB1 mediating the anti-inflammatory and ROS-inhibiting effects of Metformin were independent of Metformin’s ability to increase glucose uptake. Our results suggest that Metformin is dependent on ZEB1 for the regulation of amino acid transport. How- ever, we can not rule out that ZEB1 may also be involved in other pathways contributing to the anti-inflammatory effects of Metformin, such as autophagy or AMPK signaling. In contrast to its pro-inflammatory effects in sepsis, ZEB1 had a protective anti-inflammatory role in psoriasis. Its expression was downregulated in the psoriatic disease macrophages. The anti- inflammatory role of Metformin in the IMQ model also depended on the expression of ZEB1. Inhibition of mitochondrial mRNA translation by tetracycline family antibiotics inhibits LPS-induced production of inflammatory cytokines bymacrophages and ameliorates lung and liver damage in endotoxin-induced systemic inflammation11. Although the mechanism by which the inhibition of mitochondrial translation ame- liorates inflammation is not known, it is likely to involve alterations in the mitochondrial electron transport chain and ROS production. It has been reported that Metformin, by blocking mitochondrial complex I, increases the levels of lactate,which in turnpromotes anti-inflammation through lactylation of histones in anti-inflammatory genes.17,20. We found here that lactate and the inhibition of mitochondrial translation with doxycycline reverted ROS production and the inflammatory effects of imiquimod. Therefore, we propose a model in which ZEB1 triggers and self-limits the inflammatory responses of macrophages by modulating their metabolism. ZEB1 expression in macrophages increases lactate levels, triggering a homeostatic response through histone lactylation in anti-inflammatory and reparative genes. Addi- tionally, both ZEB1 and metformin reduce macrophage amino acid levels and consumption, thereby inhibiting mTORC1 activity, essential for mitochondrial translation. Overall, our results uncovered a mechanism regulating the inflammatory and tolerogenic responses ofmacrophages and set ZEB1 Il6 Slc2a1 Slc7a8 Il4 ZEB1ZEB1 mitochondrial translation ROS Lactate Glycolysis Metformin mitochondrial number nDNA SLC7A8 Amino acids mTORC1 p70S6K Amino acids ZEB1ZEB1 Metformin Inflammation Immunosuppression Mitochondrial translation Mitochondrial content ROS / IL6 P mitochondrial number P P P Glucose SLC2A1 Fig. 7 | Schematic summarymodel. ZEB1 regulates inflammation and immunosuppression inmacrophages and is required for the anti-inflammatory and ROS-inhibiting effects of Metformin. Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 14 as a potential target in acute and chronic inflammation to prevent hyperinflammation and immunoparalysis. Methods Human samples The use of human samples in this study was approved by the local Ethics Research Committee under protocols HCB/2017/0767, HCB/2019/1012, HCB/2020/0100, had the informed consent of patients and conformed with the principles of the Helsinki Declaration. Skin samples from psoriatic and melanoma patients, synovial membranes from psoriatic arthritis and osteoarthritis patients, and peripheral blood from healthy donors, septic patients, and patients with psoriatic arthritis were obtained as detailed in Supplementary Methods. Mouse models and isolation of mouse macrophages The use of mice in this work followed the guidelines established by the Animal Experimental Committee at the University of Barcelona School of Medicine (Barcelona, Spain) and by the Generalitat de Catalonia that reviewed and approved under references 396/18 and 133/19 and 1041, respectively. The Zeb1fl/fl mouse (herein referred to as Zeb1WT) was generated by CRISPR as detailed in Supplementary Methods. The Zeb1WT mouse was then crossed with a mouse carrying the Cre recom- binase selectively in myeloid cells under the control of the endogenous lysozyme 2 (Lyz2, also referred as LysM) promoter/enhancer (official name: B6.129P2-Lyz2tm1(cre)Ifo/J), (The Jackson Labs, Bar Harbor, ME, USA), to generate the myeloid conditional Zeb1 knockout (Zeb1fl/fl/LysMCre, referred in the manuscript as Zeb1ΔM) mice. The mice were housed in a temperature-controlled barrier room maintained at 21-22 °C with a 12- hour light/dark cycle. They were provided with standard rodent chow (RM1-P, SDS, Dietex, Argenteuil, France) and had access to water ad libitum. All mice were euthanized by cervical dislocation. The setting of the LPS- and imiquimod-induced models of sepsis, and psoriasis, respectively, as well as the isolation of macrophages, are detailed in the Supplementary Information. Determination of mRNA expression and tRNA modifications, and RNA sequencing mRNA expression and analyses of tRNA modifications were deter- mined by quantitative real-time PCR (qRT-PCR) as described in Sup- plementary Methods. RNA sequencing was conducted as described in Supplementary Methods. Determination of protein expression by FACS, Western blot, and ELISA Determination of cell surface and intracellular protein expression by FACS and Western blot and of cytokines/chemokines by an enzyme- linked immunosorbent assay or using a beads-based multiplex array are detailed in Supplementary Methods. Determination of lactate and amino acids The intracellular levels of glutamine, glutamate and BCAAs and the uptake of glucose and glutamine were assessed as detailed in Sup- plementary Methods. Assessment ofmitochondrial content, ROS andATPproduction, mitochondrial membrane potential, lysosome/mitophagy, mitochondrial protein translation, and mtDNA copy number These parameters were assessed in vitro and/or in vivo as detailed in Supplementary Methods. Immunostaining and electron transmission microscopy Immunostaining and morphological analysis of macrophages by optical and electron transmission microscopy were conducted as in Supplementary Methods. Extracellular flux and high-resolution respirometry Analyses of oxygen consumption and extracellular acidification were assessed in a Seahorse XFe96 Extracellular Flux Analyzer. High- resolution respirometry was carried out in an Oroboros Oxygraph- 2k. See Supplementary Methods for details. Statistics and reproducibility All replicates in this study are biologically independent human samples, mice, and peritoneal macrophages. No prior sample size calculation was performed and sample sizes in the experiments were set based on our previous experience and similar studies in the lit- erature. All experimental results were included in the figures, encompassing all data points without any selection or exclusion. Each experiment was independently repeated at least twice with similar results. Blinding was not technically feasible in this study because transgenic mice were cohoused, and therefore, they had to be marked for identification. For all in vivo experiments, after being genotyped, age- and sex-matched mice were distributed in roughly equal numbers to the different treatments. To minimize cage effects, mice of different genotypes and under different treatment condi- tions were housed together in mixed cages. For in vitro experiments, macrophages from age- and sex-matched mice of each genotype were isolated and randomly assigned to either treatment or control groups. The RNAseq was performed at an external facility and staff were blinded to the genotype or treatment condition of the samples, which were identified by a code. Most data collected and analyzed in the study were quantitative in nature rather than qualitative in nat- ure. Except for RNA-seq experiments, statistical analysis of the data was conducted using Prism for Mac 9.3.1 (GraphPad Software, La Jolla, California). Bar graphs throughout the manuscript represent the mean with standard errors in which the statistical significance was assessed with a non-parametric Mann-Whitney U test. Survival curves in Kaplan Meier plots were compared by the Log-rank (Man- tel-Cox) test. Where appropriate, relevant comparisons were labeled as either significant at the p ≤0.001 (***), p ≤0.01 (**) or p ≤0.05 (*) levels, or non-significant for values of p > 0.05, and with specified numerical values for 0.05 < p < 0.075. 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Rios (IDIBAPS, Hospital Clinic, and Autonomous University of Barcelona, Barcelona, Spain) for their expert guidance on the statistical analyses of the data in the study.We also thank Dr. L. Ribas de Pouplana (BIST-IRB, Barcelona, Spain) for advice on mitochondrial translation experiments. We acknowledge technical assistance by staff in the Flow Cytometry Unit at IDIBAPS, the Molecular Interactions Ser- vices Unit at the Biomedical Research Institute of Bellvitge (IDIBELL), and the Transmission ElectronMicroscopyUnit at theUniversity of Barcelona School of Medicine. We also thank A Téllez (Hospital Clinic, Barcelona, Spain) for his help in collecting samples from septic patients, and Dr. MJ Fernández-Aceñero (Hospital Clinico SanCarlos, Madrid, Spain) for help in collecting skin samples from healthy controls, psoriatic patients, and melanoma patients. We are also thankful to Dr. DC Dean (University of Louisville, KY, USA) for his generous gift of an anti-ZEB1 polyclonal antibody.We thank Dr. A. Garcia for the artistic drawing of schematics in the article. IDIBAPS is partly funded by the CERCA Programme of Gen- eralitat de Catalunya. The study was conducted at IDIBAPS’ Centre de Recerca Biomèdica Cellex building, which was partly funded by the Cellex Foundation. The different parts of this study were independently funded bygrants to AP from the Leo Foundation (LF-OC-19-000166), the Catalan Agency for Management of University and Research Grants (AGAUR) (2017-SGR-1174 and 2021-SGR-01328), and the Spanish State Research Agency (AEI) of the Ministry of Science and Innovation (MICINN) (PID2020-116338RB-I00) aspart ofMICINN’sNationalScientific and Technical Research and Innovation 2021-2023 Plan, which is co- financed by the European Regional Development Fund (ERDF) of the European UnionCommission. AB is a recipient of a PhD scholarship from AGAUR (FI Program, 2021 FI_B 00514). Author contributions M.C. and A.P. conceived, designed and interpreted experiments. M.C. also performedmost of the experimental work in the study. A.B., M.C.M.- C. and C.N. conducted some experiments in the study. V.D. and B.P. generated the Zeb1WT mouse. S.F. and P.C. assisted us in procuring human samples from septic patients. R.C. and J.D.C. assisted us in procuring human samples from PsA patients. A.E.-C. conducted bioin- formatics analyses of the raw data from the RNAseq. J.J.L. and J.S. per- formed the Cytoscape analysis. G.G. and A.M.S. provided advice on metabolic experiments. C.E. contributed in the capture and analysis of TEM pictures. MMR conducted the liquid chromatography–mass spec- trometry analysis of glutamine levels. A.P. also supervised and obtained funding for the study and wrote the manuscript. All authors provided critical comments to the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-42277-4. Correspondence and requests for materials should be addressed to Marlies Cortés or Antonio Postigo. Peer review information Nature Communications thanks Philipp Hen- neke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available. Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional affiliations. 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To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2023 Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 17 https://doi.org/10.1038/s41467-023-42277-4 http://www.nature.com/reprints http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ 1Group of Gene Regulation in Stem Cells, Cell Plasticity, Differentiation, and Cancer, IDIBAPS, 08036 Barcelona, Spain. 2National Center of Biotechnology (CSIC-CNB) and Center for Molecular Biology Severo Ochoa (CSIC/UAM-CBMSO) Transgenesis Facility, Higher Research Council (CSIC) and Autonomous University of Madrid (UAM), Cantoblanco, 28049 Madrid, Spain. 3Medical Intensive Care Unit and Department of Internal Medicine, Hospital Clínic of Barcelona, Group of Muscle Research and Mitochondrial Function, IDIBAPS, and CIBERER, 08036 Barcelona, Spain. 4Arthritis Unit, Dept. of Rheumathology, Hospital Clínic and IDIBAPS, 08036 Barcelona, Spain. 5National Center for Genomics Analysis (CNAG), 08028 Barcelona, Spain. 6Biomedical Research Networking Centers in Digestive and Hepatic Diseases (CIBERehd), Carlos III Health Institute, 08036 Barcelona, Spain. 7MRC Metabolic Diseases Unit, University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB1 0QQ, UK. 8Department of Biomedicine, University of Barcelona School of Medicine and Health Sciences, 08036 Barcelona, Spain. 9Department of Bio- chemistry and Molecular Genetics, Hospital Clínic of Barcelona and IDIBAPS, 08036 Barcelona, Spain. 10Molecular Targets Program, Division of Oncology, Department ofMedicine, J.G. BrownCancerCenter, Louisville, KY40202, USA. 11ICREA, 08010Barcelona, Spain. 12These authors contributed equally: Agnese Brischetto, M. C. Martinez-Campanario. e-mail: mcortesh@recerca.clinic.cat; idib412@recerca.clinic.cat Article https://doi.org/10.1038/s41467-023-42277-4 Nature Communications | (2023) 14:7471 18 mailto:mcortesh@recerca.clinic.cat mailto:idib412@recerca.clinic.cat Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation Results ZEB1 has a dual role being required for both the induction and resolution of inflammation ZEB1 increases glycolysis during acute inflammation ZEB1 reduces mitochondrial content during immunosuppression ZEB1 activates p62 and promotes autophagy during inflammation Metformin depends on ZEB1 expression in macrophages for its anti-inflammatory effects ZEB1 mediates the immunosuppression-mimicking effect of Metformin by reducing mitochondrial content and ROS levels ZEB1 inhibits mitochondrial protein translation in inflammatory macrophages Lactate and the inhibition of mitochondrial translation inhibit chronic inflammation in a psoriasis model Metformin requires ZEB1 expression in macrophages for its anti-inflammatory effects in psoriasis ZEB1 inhibits mitochondrial protein translation by restricting amino acids transport ZEB1 inhibits mTORC1/p70S6K signaling in immunosuppression ZEB1 expression in inflammatory PBMCs from septic and PsA patients is associated with differential expression of glucose and amino acid transporters Discussion Methods Human samples Mouse models and isolation of mouse macrophages Determination of mRNA expression and tRNA modifications, and RNA sequencing Determination of protein expression by FACS, Western blot, and ELISA Determination of lactate and amino acids Assessment of mitochondrial content, ROS and ATP production, mitochondrial membrane potential, lysosome/mitophagy, mitochondrial protein translation, and mtDNA copy number Immunostaining and electron transmission microscopy Extracellular flux and high-resolution respirometry Statistics and reproducibility Reporting summary Data availability References Acknowledgements Author contributions Competing interests Additional information