Cell-extrinsic autophagy in mature adipocytes regulates anti-inflammatory response to intestinal 1 tissue injury through lipid mobilization 2

Cell-extrinsic autophagy in mature adipocytes regulates anti-inflammatory response to intestinal 1 tissue injury through lipid mobilization 2 Felix Clemens Richter1, Matthias Friedrich1,2, Mathilde Pohin1,*, Ghada Alsaleh1,*, Irina Guschina3, 3 Sarah Karin Wideman4, Errin Johnson5, Mariana Borsa1, Klara Piletic1, Paula Hahn1, Henk Simon 4 Schipper1,6, Claire M. Edwards7,8, Fiona Powrie1, Anna Katharina Simon1,# 5 6 *Authors contributed equally. 7 #Corresponding author: katja.simon@imm.ox.ac.uk 8 9 10 Affiliations 11


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Autophagy is an essential cellular recycling pathway that engulfs cellular contents, including organelles 43 and macromolecules, in a double membraned autophagosome and directs them towards lysosomal 44 degradation. The released nutrients can then be used for both biosynthetic building blocks and energy 45 generation (Riffelmacher et al., 2018). Immune cells are cell-intrinsically reliant on autophagy during 46 their differentiation and for their immune functions (Clarke and Simon, 2019). For instance, neutrophils 47 require autophagy for the liberation of free fatty acids (FFA) from their intracellular lipid droplet stores 48 in order to generate energy through oxidative phosphorylation (Riffelmacher et al., 2017). Similarly, 49 autophagy-deficient macrophages are arrested in a glycolytic metabolic program promoting expression 50 of pro-inflammatory cytokines and reactive oxygen species (Kang et al., 2016;Stranks et al., 2015).

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While the cell-intrinsic need for recycled nutrients is evident, whether these mobilized nutrients can also 52 be provided to immune cells in an autophagy-dependent manner remains less well understood. In 53 plants, nutrients recycled via autophagy are mobilized to other plant organs or stored in seeds 54 (Guiboileau et al., 2013;Guiboileau et al., 2012). In animals, only a few pioneering studies have looked 55 at the mobilization of nutrients, in particular amino acids. For instance, autophagy in non-cancer cells 56 control amino acid availability and thus tumour growth (Katheder et al., 2017;Poillet-Perez et al., 2018; 57 Sousa et al., 2016). Activation of autophagy in hepatic stellate cells by the cancer cell induces the 58 release and provision of alanine to the cancer cell (Sousa et al., 2016). Despite mounting evidence for 59 autophagy-dependent amino acid mobilization, it remains unclear whether this also exists for other 60 nutrients such as FFA.

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Adipocytes are highly specialized cells responsible for the conversion and storage of energy-rich 62 nutrients in form of lipids and for their release during times of high nutrient demand. In addition, the 63 adipose tissue represents an important immunological organ harbouring a variety of immune cells, 64 which are highly adapted to live in lipid-rich environments, such as macrophages (Grant and Dixit, Cytofix/CytoPerm (BD Bioscience) following manufacturer protocol. Samples were acquired on LSRII 152 or Fortessa X-20 flow cytometers (BD Biosciences).

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Quantitative PCR 155 Adipocytes and adipose tissue RNA were extracted using TRI reagent (T9424, Sigma). Colon tissue 156 RNA were extracted in RLT buffer containing 1,4-Dithiothreitol. Tissues were homogenised by lysis in 157 2mL tubes containing ceramic beads (KT03961-1-003.2, Bertin Instruments) using a Precellys 24 158 homogenizer (Bertin Instruments). RNA was purified following RNeasy Mini Kit (74104,Qiagen) 159 manufacturer instructions. cDNA was synthesized following the High-Capacity RNA-to-cDNA™ kit 160 protocol (4388950, ThermoFischer). Gene expression was assessed using validated TaqMan probes 161 and run on a ViiA7 real-time PCR system. All data were collected by comparative Ct method either 162 represented as relative expression (2 -ΔCt ) or fold change (2 -ΔΔCt ). Data were normalized to the two most 163 stable housekeeping genes; for adipose tissues Tbp and Rn18s and for colon Actb and Hprt.

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Bulk RNA sequencing 166 Visceral adipocytes were isolated as floating fraction upon digestion. RNA was extracted and converted 167 to cDNA as described above. PolyA libraries were prepared through end reparation, A-tailing and 168 adapter ligation. Samples were then size-selected, multiplexed and sequenced using a NovaSeq6000.

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The oven temperature was programmed as follows: 170°C for 3min, increased to 220°C at 4°C/min), 210 and then held at 220°C for 15min. FAMEs were identified routinely by comparing retention times of 211 peaks with those of G411 FA standards (Nu-Chek Prep Inc). TotalChrom software (Perkin-Elmer) was 212 used for data acquisition and quantification.

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Immunoblotting 215 Autophagic flux in adipose tissues was measured by incubating adipose tissue explants from 216 experimental animals in RPMI in the absence or presence of lysosomal inhibitors 100nM Bafilomycin 217 A1 and 20mM NH4Cl for 4 hours. DMSO was used as 'vehicle' control. Adipose tissues were collected 218 and snap frozen. Protein extraction was performed as previously described (An and Scherer, 2020

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Transmission electron microscopy 231 Mice were sacrificed by increasing concentrations of CO2. Adipose tissues were excised, cut into small 232 1-2mm pieces and immediately fixed in pre-warmed (37 ˚C) primary fixative containing 2.5% 233 glutaraldehyde and 4% formaldehyde in 0.1M sodium cacodylate buffer, pH7.2 for 2 hours at room 234 temperature and then stored in the fixative at 4 ˚C until further processing. Samples were then washed 235 for 2x 45 min in 0.1M sodium cacodylate buffer (pH 7.2) at room temperature with rotation, transferred 236 to carrier baskets and processed for EM using a Leica AMW automated microwave processing unit.

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Briefly, this included three washes with 0.1M sodium cacodylate buffer, pH 7.2, one wash with 50mM 238 glycine in 0.1M sodium cacodylate buffer to quench free aldehydes, secondary fixation with 1% osmium 239 tetroxide + 1.5% potassium ferricyanide in 0.1M sodium cacodylate buffer, six water washes, tertiary 240 fixation with 2% uranyl acetate, two water washes, then dehydration with ethanol from 30%, 50%, 70%, 241 90%, 95% to 100% (repeated twice). All of these steps were performed at 37 ˚C and 15-20W for 1-2 242 mins each, with the exception of the osmium and uranyl acetate steps, which were for 12 min and 9 243 min respectively. Samples were infiltrated with TAAB Hard Plus epoxy resin to 100% resin in the AMW 244 and then processed manually at room temperature for the remaining steps. Samples were transferred 245 to 2ml tubes filled with fresh resin, centrifuged for ~2mins at 2000g (to help improve resin infiltration), 246 then incubated at room temperature overnight with rotation. The following day, the resin was removed 247 and replaced with fresh resin, then the samples were centrifuged as above and incubated at room 248 temperature with rotation for ~3 hrs.

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To investigate whether autophagy in mature adipocytes is altered in response to intestinal inflammation,

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we deployed a mouse model of intestinal inflammation evoked by the administration of 1.5-2% DSS in 284 drinking water ( Figure 1A). As expected, treatment with DSS damaged the colonic epithelial architecture 285 and triggered intestinal inflammation, as measured by body weight loss ( Figure 1B), increased 286 histopathological inflammation score ( Figure S1A), shortened colon length ( Figure S1B) and enlarged 287 mesenteric lymph nodes ( Figure S1C). In addition, DSS treatment resulted in a significantly higher 288 infiltration of immune cells in the inflamed colon, which appeared to be predominantly of myeloid origin 289 (Figure S1D-E). Next, we assessed the impact of DSS-induced colitis on the adipose tissue. In line with 290 body weight loss, visceral adipose tissue mass was reduced ( Figure 1C), as were serum FFA levels 291 seven days after initial DSS administration ( Figure 1D).

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To assess changes in autophagy levels, adipose tissue explants from water-or DSS-treated animals 293 were cultured in the absence or presence of lysosomal inhibitors and the accumulation of the lipidated 294 autophagosomal marker LC3 protein (LC3-II) was quantified. DSS-induced intestinal inflammation 295 substantially increased autophagic flux in mesenteric and in gonadal white adipose tissue (mWAT and 296 gWAT, respectively) ( Figure 1E), indicating that both adipose tissues proximal and distal to the intestine 297 are responsive to the inflammation. Although this data suggests that autophagy in the adipose tissue is 298 induced during colitis, several cell types, including adipose tissue immune cells, could be responsible 299 for this change. To validate that adipocytes contribute to the increased autophagic flux in the adipose 300 tissue, we first prepared adipose tissue for transmission electron microscopy. Autophagosomal double 301 membrane structures were identified in adipocytes, predominantly from DSS-treated mice ( Figure 1F).

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Additionally, we digested the adipose tissues and enriched for a floating adipocyte fraction and a 303 pelleted stromal vascular fraction (SVF). Adipocyte fractions showed increased transcript levels of 304 several Atg8 homologues in DSS colitis, further demonstrating an increase in autophagic flux in this cell 305 type ( Figure 1G). In contrast, SVF containing adipose tissue-resident immune cells showed no is induced in adipocytes in response to DSS-induced colitis.

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Next, we addressed whether loss of autophagy in adipocytes affects immune development and 311 intestinal inflammation at steady state. Given the crucial role of adipocyte autophagy during 312 adipogenesis (Singh et al., 2009;Zhang et al., 2009) and to ensure normal adipocyte tissue formation,

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we established a tamoxifen-inducible knockout mouse model to ablate the essential autophagy gene 314 Atg7 specifically in mature adipocytes (Atg7 Ad ) ( Figure 2A). We first confirmed efficient deletion of Atg7 315 and disruption of autophagic flux. Activation of Cre nuclear translocation by tamoxifen administration 316 led to the significant reduction of Atg7 transcript levels in visceral adipocytes ( Figure 2B). This deletion 317 was further confirmed on the protein level ( Figure 2C). Importantly, the adipocyte-specific loss of ATG7   sought to determine the effects of autophagy loss in adipocytes during colitis. Since we were unable to 344 find differences at homeostasis between the genotypes, we grouped both wild type and Atg7 Ad 345 tamoxifen-treated homeostatic mice an untreated control group. Upon DSS-treatment ( Figure 3A),

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Atg7 Ad mice showed increased body weight loss in comparison to littermate controls ( Figure 3B). In  Intestinal inflammation induced by DSS is self-resolving (Ho et al., 2021). Therefore, we assessed the impact of adipocyte autophagy loss two weeks after DSS induction ( Figure S3A). At this timepoint, we 361 were unable to find any differences in colon length between Atg7 Ad and littermate controls and equally 362 there were no significant histopathological differences observed between the groups ( Figure S3B-C).

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While induction of colitis is lymphocyte-independent, it has been suggested that its resolution is also 364 controlled by lymphocytes (Wang et al., 2015). Interestingly, frequencies and total numbers of colonic classified into three distinct subsets based on co-expression of TH2 and TH17 transcription factors 368 GATA3 + and RORgt + , respectively (Whibley et al., 2019). While all populations tended to be diminished 369 in Atg7 Ad mice, only RORgt -FOXP3 + Tregs were significantly reduced ( Figure S3E). This suggests that 370 adipocyte autophagy does not interfere with the resolution of intestinal inflammation.

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To further delineate the impact of adipocyte autophagy on intestinal inflammation, we next assessed its 372 impact in another model of colitis. To this end, mice were treated with the pathobiont Helicobacter 373 hepaticus (Hh) by oral gavage and co-administration of IL-10 receptor blocking antibody allowed for 374 breakage of intestinal immune tolerance to Hh (Danne et al., 2017). We measured the effects of 375 adipocyte autophagy loss at both peak inflammation and resolution stages, at 2 and 6 weeks post-Hh inflammatory pathways and cytokine production (Mustain et al., 2013). For example, adipocytes 389 produce pro-inflammatory cytokines such as IL-6 and TNFα upon inflammation (Hotamisligil, 2017). We 390 therefore hypothesized that autophagy may impact the transcriptional inflammatory profile of visceral 391 adipocytes during intestinal inflammation, thus promoting inflammation. In order to analyse autophagy-392 dependent differences in adipocyte transcription profiles, visceral adipocytes were collected from mice 393 treated with DSS or water and subsequently sequenced. Since we anticipated sex-specific differences 394 in adipocyte transcription profiles (Oliva et al., 2020), we included the same number of male and female 395 mice in each experimental group ( Figure 4A). As expected, sex-specific transcriptional changes 396 explained ~33% of the dataset variance ( Figure 4B), in line with previous reports. While the treatment clearly separated the experimental groups in the principal component analysis (PCA), there was no 398 major impact of the genotype on the transcriptomic dataset ( Figure 4B). Reassuringly, visceral 399 adipocytes from Atg7 Ad mice had a strong reduction in Atg7 levels and an increase in estrogen receptor 400 1 (Esr1) expression, due to the Cre transgene expression ( Figure S5A-B), when compared by 401 differential gene expression analysis. In line with the PCA analysis, across the treatment groups only 402 17 genes were significantly upregulated and 15 genes were downregulated in Atg7-deficient visceral 403 adipocytes compared to wild-type ( Figure S5A-B). The limited effect of autophagy loss has previously 404 been observed in other contexts (Cadwell et al., 2008). Importantly, this data suggests that the loss of 405 adipocyte autophagy does not alter the transcriptional regulation of inflammatory pathways in the 406 adipocytes themselves, neither during homeostasis nor DSS treatment.

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Having confirmed that autophagy loss does not substantially affect the transcriptional profile of 408 adipocytes in either homeostasis or DSS-induced colitis, we next compared non-inflamed to inflamed 409 adipocytes from wild-type animals. More than 4700 genes were differentially regulated between these 410 states ( Figure S5C), among which 2415 were significantly upregulated and 2333 downregulated. Gene 411 ontology analysis using clusterProfiler further revealed an enrichment in several gene sets ( Figure 4C).

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Confirming our earlier results that adipocyte autophagy is affected by DSS-induced colitis ( Figure 1G to β-adrenergic receptor-mediated lipolysis (Cai et al., 2018;Son et al., 2020). To confirm the 429 importance of adipocyte autophagy for optimal lipolytic output, supernatant FFA levels were measured 430 upon exposure of autophagy-deficient and -sufficient adipocyte tissues to isoproterenol. Strikingly, FFA 431 secretion was reduced upon lipolysis stimulation in autophagy-deficient adipocytes ( Figure S6A). TNFα, 432 a crucial cytokine for human and murine IBD pathologies (Friedrich et al., 2019), can affect adipose 433 tissue through inhibition of lipogenesis and by promoting FFA secretion (Cawthorn and Sethi, 2008).

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Furthermore, TNFα has also been implicated as a necessary component to elicit anti-inflammatory 435 pathways during intestinal inflammation (Kojouharoff et al., 1997;Noti et al., 2010). Since we found  Figure 5B). Furthermore, TNFα is a potent inducer of adipocyte lipolysis 442 (Green et al., 1994;Ryden et al., 2002), therefore we used adipose tissue explants from Atg7 Ad mice 443 or littermate controls and stimulated them with recombinant TNFα. In the presence of TNFα, adipocytes 444 turn on FFA secretion, however strikingly, autophagy-deficient adipocytes showed a significant 445 reduction in FFA secretion upon TNFα stimulation ( Figure 5C). Consistent with the decreased lipolytic 446 activity of autophagy-deficient adipocytes, Atg7 Ad mice exhibit reduced serum FFA levels compared to 447 wild-type littermates upon DSS colitis ( Figure 5D). While autophagy is well-known to be a potential 448 source of FFA, it remains unclear whether autophagy can affect specific FFA species more than others.

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To investigate this, serum samples from water and DSS-treated animals were analysed by  Interestingly, the proportion of individual serum FFAs was unaffected by the loss of adipocyte 451 autophagy ( Figure 5E). However, confirming our initial findings, the serum concentration of several FFA 452 species was reduced upon adipocyte autophagy loss, indicating that adipocyte autophagy controls 453 overall FFA levels rather than specific FFAs ( Figure 5F). The expression of cytosolic lipases in visceral 454 autophagy regulates FFA secretion on a post-transcriptional level ( Figure S6B).

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In addition to controlling circulating FFA levels, adipocytes take part in the control of other serum lipid 457 species. Therefore, using an unbiased approach, the serum lipidome of Atg7 Ad and wild-type littermate

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Next, we tested whether the autophagy-dependent decrease in adipose tissue lipolysis resulted in DSS-treated wild type mice, their expression was diminished in adipocyte autophagy-deficient mice 484 ( Figure 6A). The difference in IL-10 levels in the serum did not arise from the colon, since only Il27 485 transcript levels, but not Il10, were increased in the lamina propria of Atg7 Ad mice upon colitis induction 486 ( Figure 6B).

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It has been previously described that adipose tissue immune cells can increase expression of IL-10 488 upon intestinal inflammation (Kredel et al., 2013) . Unbiased cytokine screening of secreted cytokines 489 from wild-type mice revealed that several cytokines are released from the mesenteric adipose tissue in 490 response to both DSS-and Hh-induced colitis ( Figure S7A-D). Importantly, and validating previous 491 reports, IL-10 secretion from the mesenteric adipose tissue was significantly up-regulated at peak 492 inflammation of DSS-induced colitis ( Figure 6C). We therefore tested whether IL-10 secretion from the

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FFA restriction impairs IL-10 production in macrophages 507 Next, to determine which cells are the main source of IL-10 in adipose tissues, immune cells were 508 isolated and their cytokine production capacity was measured. We found that, upon DSS-induced 509 colitis, F4/80 + macrophages are the main producers of IL-10 in mesenteric and visceral adipose tissues, 510 although CD4 + T cells appear to contribute as well in mesenteric WAT ( Figure 7A). To directly confirm 511 whether local FFA availability can modify macrophage-derived IL-10 production, we sought to restrict 512 FFA availability from bone marrow derived macrophages. Charcoal treatment has previously been used 513 to deplete FFAs from serum (Chen, 1967). First, we confirmed that FFA concentrations in medium ( Figure 7C), which correlated with medium FFA concentration ( Figure 7D). Importantly, limiting FFA 517 availability led to an increased proportion of TNFα-producing macrophages after 16h hours, while IL-518 10 producing cells were drastically reduced ( Figure 7E). This indicated that FFA restriction leads to a 519 sustained pro-inflammatory macrophage phenotype. In line with this, supernatant concentrations of IL-520 10 were clearly reduced upon FFA restriction ( Figure 7F), thus indicating that local FFA availability 521 impairs the production of anti-inflammatory IL-10 from macrophages.

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Inflammation and activation of immune cells promote intracellular metabolic adaptation, which is 525 important to govern pro-and anti-inflammatory pathways (O'Neill et al., 2016). However, immune cells 526 reside within distinct tissue environments and the impact of local nutrient availability on inflammatory 527 processes remains incompletely understood . In this study, we demonstrate that 528 autophagy in adipocytes promotes a cell-extrinsic effect on the secretion of IL-10 by adipose-tissue 529 resident ATMs. Our results further indicate that autophagy in mature adipocytes is crucial for optimal 530 cellular release and availability of FFA during inflammation. Autophagy-dependent IL-10 secretion from 531 adipose tissue contributes to systemic levels, and limits inflammation at a distant tissue site, the 532 intestine. Therefore, our study provides novel insights into a cross-tissue anti-inflammatory mechanism, 533 enabling the development of alternative therapeutic approaches to treat inflammatory diseases.

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Autophagy genes are well established as genetic risk factors for IBD susceptibility. Yet, little is known 536 about the role of adipocyte autophagy in this disease. We found that autophagy is increased in visceral 537 adipocytes upon DSS-induced colitis and showed that adipocytes transcriptionally increased the 538 expression of several Atg8 homologues during peak inflammation. These observations parallel findings during muscle atrophy, where the expression of Map1lc3b, Gabarapl1, Bnip3, Bnip3l and Vps34 is 2007). It appears plausible that a similar FOXO3-dependent cachexia occurs in adipocytes.

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Interestingly, Foxo3-deficient mice develop more severe DSS-induced colitis than wild-type littermates 543 (Snoeks et al., 2009) (Mustain et al., 2013). Of note, the transcriptome of visceral adipocytes did not reveal changes in 555 inflammatory cytokine expression upon DSS-treatment, thus indicating that the expression of these 556 cytokines is likely derived from adipose tissue-resident immune cells. In addition to inflammatory 557 cytokines, the secretion of IL-10 was observed in visceral adipose tissues upon intestinal inflammation, 558 confirming findings from the Siegmund lab that mesenteric ATMs upregulate expression of IL-10 during 559 intestinal inflammation in both human and mouse (Batra et al., 2012;Kredel et al., 2013). Importantly, 560 this study underscores the importance of adipose-tissue derived IL-10 in controlling disease severity.

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Interestingly, we were unable to find differences in the Hh-induced colitis model. This may be explained 562 by the administration of the IL-10R blocking antibody which neutralizes the anti-inflammatory effects of 563 adipose tissue-derived IL-10 in this model of colitis, especially since we noted that IL-10 is actively 564 secreted from Hh and IL10R-adipose tissues, thus suggesting that a similar pathway may also be 565 present in this model of colitis. Recent single cell transcriptomic analysis of immune cells resident in 566 creeping fat tissues revealed an important anti-inflammatory and pro-repair role of ATMs, further 567 supporting their beneficial role during intestinal inflammation (Ha et al., 2020). Intriguingly, their study 568 also identified a significant upregulation of IL-10 in ATMs in creeping fat tissue, suggesting that the 569 adipocyte-immune cell pathway identified here is relevant for human IBD.

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Early studies found that autophagy is crucial for adipogenesis and the normal differentiation of adipose 572 tissues in vivo (Singh et al., 2009;Zhang et al., 2009). However, the significance of autophagy in mature 573 adipocytes remained unexplored until recently. We propose that autophagy in mature adipocytes fine-574 tunes lipolytic output of adipocytes upon metabolic and/or inflammatory stress conditions. Supporting 575 this view, post-developmental ablation of autophagy in mature adipocytes decreased β-adrenergic 576 receptor-induced lipolysis (Cai et al., 2018;Son et al., 2020). Conversely, disruption of mTOR by genetic 577 deletion of Raptor increases lipolytic output via autophagy . While we were unable 578 to observe signs of lipophagy by electron microscopy, it is possible that adipocyte autophagy controls 579 lipolytic output via the degradation of key proteins involved in the lipolytic machinery such as described 580 for perilipins in fibroblasts and adipocytes (Ju et al., 2019;Kaushik and Cuervo, 2016).

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Macrophages accumulate in creeping fat tissues of CD patients and in the mesentery of mice upon 583 DSS-induced colitis (Batra et al., 2012). We show that local FFA availability can dictate functional 584 macrophage responses, such as the secretion of IL-10. In line with this, M2-type macrophages require 585 uptake of lipid substrates through CD36 to engage OXPHOS-dependent cell activation (Huang et al., (Odegaard et al., 2007;Odegaard et al., 2008). The resulting reduction in 590 systemic IL-10 levels due to lipid restriction prolongs pro-inflammatory programs at the inflammation 591 site. Especially, IL-10 signalling is required for intestinal macrophages to prevent excessive glycolytic 592 and pro-inflammatory activity during DSS-induced colitis by inhibiting mTOR activity (Ip et al., 2017).

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The importance of cell-extrinsic autophagy becomes increasingly apparent for intercellular and inter-595 tissue communication. While the exchange of nutrients, especially amino acids, has been 596 predominantly characterized in the context of cancer (Poillet-Perez and White, 2019), it remained 597 unclear whether it occurs in immunity. This report presents to our knowledge the first demonstration of 598 an autophagy-dependent mobilization of lipids during inflammation. This is supported by an elegant study suggesting that, during organ wasting, autophagy cell-extrinsically mobilizes stored nutrients to 600 cancer cells to sustain their growth (Khezri et al., 2021). Despite its central function in bulk degradation 601 and nutrient recycling, parts of the autophagic machinery have been implicated in other cell-extrinsic 602 processes such as secretory autophagy and the release of extracellular vesicles (Kuramoto et al., 2021; 603 Leidal et al., 2020;Nicolas-Avila et al., 2020). While we found that regulation of autophagy-dependent 604 FFA levels can control IL-10 production in macrophages, we cannot exclude that other cell-extrinsic 605 processes of autophagy may contribute to the observed phenotype.

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Overall, this study reveals that metabolically healthy adipose tissues are important regulators of 608 excessive inflammation during colitis. While visceral adipose tissues can adapt both pro-and anti-609 inflammatory properties, its impact on the pathology may depend on the overall disease state, genetic 610 predispositions and co-morbidities. In this context, the expansion of the mesentery during CD may 611 initially be beneficial through prevention of bacterial translocation and signalling pathways poised to 612 promote anti-inflammatory and pro-fibrotic pathways (Batra et al., 2012;Ha et al., 2020). However, 613 sustained inflammation may ultimately subvert the function of the mesentery and ultimately lead to 614 adipose tissue fibrosis and intestinal strictures (Mao et al., 2019). Here, we demonstrate that adipocyte  (A) Schematic of experimental design. Sex-matched and age-matched wild-type mice were treated for 5 days with 1.5-2% DSS in drinking water, before switched to water for two more days. Mice were sacrificed at day 7 post-DSS induction. (B) Body weight development upon DSS treatment; n = 10-11/group, pooled from three independent experiments. (C) Tissue weights measured in mesenteric (mWAT) and collective visceral white adipose tissue (visWAT), consisting of gonadal (gWAT), retroperitoneal and omental white adipose tissue at day 7 after start of DSS regime; n = 7-8/group pooled from two independent experiments.  (A) Schematic of experimental design. Sex-matched and age-matched littermates were treated at 8-12 weeks of age with tamoxifen for five consecutive days before tissues were analysed 14 days after the last tamoxifen administration. (B) Representative quantification of knock-out efficiency measured on Atg7 transcript level by qRT-PCR in purified primary visceral adipocyte at two weeks post-tamoxifen treatment (n = 4-11/group).      (B) Expression of cytokines in lamina propria measured by qRT-PCR at 7 days post-DSS induction; n = 20-24/group pooled from three independent experiments. (C) Colitis was induced in mice for 7 days and mesenteric adipose tissue explants were cultured with FBS. IL-10 secretion into the supernatant was measured after 24h of culture; n = 4-12/group pooled from two independent experiments. (D and E) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of (D) IL-10 and ( Tamoxifen  -14   0 Collect tissues